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Cigarette smoke-induced oxidative stress in COPDHoffmann, Roland Frederik

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Cigarette smoke-induced oxidative stress in COPDEffects on mitochondrial function, the lipidome and glucocorticoid

responsiveness in airway epithelium

Roland Hoffmann

This research was partially performed within the framework of the Top Institute Pharma project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, with partners University Medical Center Groningen, University of Groningen (RUG), GRIAC Research institute Groningen, University Medical Center Utrecht, University Medical Center Maastricht, Nycomed BV (now Takeda), GlaxoSmithKline, Danone, AstraZeneca and Foundation TI Pharma. This research was financially supported by Stichting Astmabestrijding (SAB 2012/039).

Printing of this Thesis was financially supported by: University of Groningen, University Medical Center Groningen, Stichting Astmabestrijding (SAB), Chiesi Pharmaceuticals bv en Boehringer Ingelheim bv.

ISBN: 978-94-6299-301-3 Cover: Venice by Night - Ingrid LeegteLayout: Nikki Vermeulen - Ridderprint BV - www.ridderprint.nlPrinting: Ridderprint BV - www.ridderprint.nl

© Roland Hoffmann, 2016, the Netherlands.All rights reserved. No parts of this thesis may be reproduced in any form, by any means, without prior written permission of the author.

Cigarette smoke-induced oxidative stress in COPDEffects on mitochondrial function, the lipidome and glucocorticoid

responsiveness in airway epithelium

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen

op gezag van de rector magnificus prof. dr. E. Sterken

en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op

maandag 29 februari 2016 om 14.30 uur

door

Roland Frederik Hoffmann

geboren op 1 oktober 1979 te Stadskanaal

Promotores Prof. Dr. H.I. Heijink Prof. Dr. A.J.M. van Oosterhout

Copromotor Dr. N.H.T. ten Hacken

Beoordelingscommissie Prof. Dr. H.G.D. Leuvenink Prof. Dr. L. Koenderman Prof. Dr. I.M. Adcock

Voor Fayenn en Finn

Paranimfen Harold de Bruin

Dennie Rozeveld

Table of contentsChapter 1 General introduction. 9

Chapter 2 Prolonged cigarette smoke exposure alters mitochondrial structure 25 and function in airway epithelial cells. Respiratory Research. 2013.

Chapter 3 Glycogen synthase kinase-3β modulation of glucocorticoid 51 responsiveness in COPD. American Journal of Physiology-Lung Cellular and Molecular Physiology. 2015.

Chapter 4 Mitochondrial dysfunction increases pro-inflammatory cytokine 77 production and impairs regeneration and glucocorticoid responsiveness in lung epithelium. Manuscript in preparation

Chapter 5 Untargeted Lipidomic Analysis in Chronic Obstructive Pulmonary 97 Disease. Uncovering Sphingolipids. American Journal of Respiratory and Critical Care Medicine. 2014

Chapter 6 Untargeted lipidomic analysis in long-term cigarette smoke exposed 145 bronchial epithelial cells. Manuscript in preparation.

Chapter 7 Summary, general discussion, conclusions and future perspectives. 161

Chapter 8 Nederlandse Samenvatting. 181

Appendices Dankwoord 191 List of publications 197

CHAPTER 1

General Introduction

11

General introduction

Chapter

1Pathogenesis of Chronic Obstructive Pulmonary Disease Chronic obstructive pulmonary disease (COPD) is a chronic disease predominantly affecting the lungs, with an increasing rate in morbidity and mortality, especially in the elderly, and high healthcare costs (43). Estimates from the world health organization (WHO) suggests that 65 million people worldwide have moderate to severe COPD. It is currently the fourth leading cause of death (39, 43), and it is estimated to be the third leading cause of death worldwide by 2030 (WHO, 2014 http://www.who.int). COPD is characterized by chronic lung inflammation, resulting in aberrant lung tissue repair and remodelling, not fully reversible airflow limitation and accelerated lung function decline. COPD includes chronic bronchitis, with increased deposition of structural proteins, thickening of the small airway walls and mucus production on one hand, and emphysema, with degradation of structural proteins in the alveoli, loss of alveolar tissue and loss of lung elastic recoil on the other. These features can occur separately and within the same patient (6, 8). The most prominent risk factors for COPD are cigarette smoking, environmental cigarette smoke exposure or exposure to other noxious particles and gases, including other tobacco smoke, fuels and wood smoke (42, 43). Tobacco smoking is a major problem in the western world and upcoming industrialized countries and reaches an almost epidemic proportion. The `World Health organization` (http://www.who.int/tobacco/global_report/2011/en/) reports that worldwide, the use of tobacco causes more than 5 million deaths per year, and current trends show that tobacco smoke will cause more than 8 million deaths annually by 2030. Also, smoking is the fourth most common risk for developing other serious diseases, including various cancers, stroke and cardiovascular disease. Crucially, non-tobacco related risk factors of COPD include genetic deficiencies, in particular alpha1-antitrypsine deficiency, and occupational exposure to dusts and chemicals. Important to note is that 95% of the COPD patients are smokers, but only 10-15% of the smokers actually develop COPD, indicating that genetic susceptibility contributes to the development of COPD (6, 15, 28, 39, 42, 43, 56, 57, 68). Inhaled cigarette smoke induces an aggravated inflammatory response and abnormal tissue damage and repair responses in the lungs of COPD patients. The chronic inflammatory response in the airways of COPD patients is characterized by infiltration of inflammatory cells, e.g. CD8+T-cells, neutrophils and macrophages. These cells release a variety of proteases, including neutrophil elastase, known to be responsible for the degradation of the elastin and thus emphysema development (8, 41, 70). Inhaled cigarette smoke first encounters the airway epithelium, continuously lining the airways and forming a tightly regulated barrier against environmental insults. The airway epithelium is also a source of pro-inflammatory cytokines, especially when damaged. Inhaled cigarette smoke induces oxidative stress and epithelial damage, leading to activation of epithelial cells in the lungs and the release of several cytokines and chemokines, including Interleukin 6 (IL-6), CXC-chemokine ligand 1 (CXCL1), CC-chemokine ligand 2 (CCL2), Chemokine C-X-C-Motif Ligand 8 (CXCL8) and

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Chapter 1

Granulocyte-macrophage colony-stimulating factor (GM-CSF) (7, 8, 10, 18, 29, 36, 49). CCL2, CXCL1 and CXCL8 attract monocytes and neutrophils to the lungs, while GM-CSF promotes the survival, proliferation and differentiation of neutrophils (7). In addition, epithelial cells produce CXCL9, CXCL10 and CXCL11, which are responsible for the recruitment of T helper 1 (Th1) cells and type 1 cytotoxic T (Tc1) cells to the lungs (7, 10, 18). Th1 cells produce cytokines that promote the recruitment and activation of inflammatory cells, while the production of proteolytic enzymes by Tc1 cells induces apoptosis or necrosis of epithelial cells (7).Next to chronic inflammation of the lungs, systemic alterations have been observed in COPD patients, with systemic inflammation, a decrease in body weight and loss of skeletal muscle mass (wasting) as common manifestations of the disease. It is well known that skeletal muscle cells of COPD patients are more dependent on the glycolytic metabolism than oxidative metabolism. As a result, less adenosine triphosphate (ATP) is produced per mole of glucose and more rapid acidification occurs in muscle tissue of COPD patients (24, 59, 60). This phenomenon is thought to be an indication for a mitochondrial dysfunction in these cells.

Treatment of COPD: Glucocorticoid responsivenessCurrent treatment with long-acting bronchodilators and inhaled glucocorticoids (IGC) provides relatively little therapeutic benefit in COPD, despite the broad anti-inflammatory effects of IGC. They reduce exacerbations, but do not effectively change the course of neither the disease nor the tissue damage that results from chronic airway inflammation. The GLUCOLD study demonstrated beneficial glucocorticoid (GC) effects on airway wall inflammation and decline in lung function; however, there were great inter-individual differences (38). The poor response of COPD patients to IGC may stem from the development of GC insensitivity, in which oxidative stress has been implicated (5, 19). GCs are the first choice of medication used to suppress an inflammatory response and belong to a class of steroid hormones that bind to and activate the cytosolic glucocorticoid receptor (GR) α, which is present in almost all eukaryotic cells. Upon its activation, GR translocates to the nucleus to regulate the expression of anti-inflammatory genes with a glucocorticoid response element (GRE) in their promoter. In addition, GR activation induces recruitment of histone deacetylases, leading to reduced access of various transcription factors, e.g. nuclear factor (NF)-κB, to promoter regions in pro-inflammatory genes (5, 63). In vitro studies have shown that the ability of dexamethasone to suppress cytokine release (e.g. CXCL8) from alveolar macrophages is impaired in COPD patients compared to healthy smokers (21). Furthermore, macrophages from healthy smokers are more resistant to GCs than alveolar macrophages from non-smokers (62). We have previously shown that GC insensitivity also exists in bronchial epithelial cells from COPD patients, and may be the

13

General introduction

Chapter

1result of excessive oxidative stress (29), leading to reduced suppressive effects of IGC on pro-inflammatory cytokine production. Indeed, oxidative stress has been linked to GC unresponsiveness. Oxidative stress can induce PI3K-dependent post-translational histone deacetylase 2 (HDAC2) modifications, leading to reduced expression and activity of HDAC2 (1, 32, 45, 61). In addition to the regulation of NF-κB response elements, HDAC2 can deacetylate the GRα (31–33). In line with a role for reduced HDAC2 expression in the observed GC unresponsiveness in COPD, reduced HDAC2 expression has been observed in the lungs and alveolar macrophages of COPD patients and has been implicated in GC insensitivity in COPD (32, 33). This suppression may also play a role in reduced GC responsiveness of the airway epithelium, which is in first contact with cigarette smoke, inducing oxidative stress (60, 74). GC insensitivity may be gradually acquired by smoking, and because of the increased burden of oxidative stress in COPD patients, we propose that smokers with COPD are more prone to develop GC insensitivity than smokers with normal lung function.

Mitochondria and oxidative stressMitochondria are thought to play an important role in pathologies associated with oxidative stress, including COPD. Mitochondria are oval to rod shaped cellular organelles that have a double membrane structure, where the internal membrane is lobular shaped to increase its surface area for proper energy production. Mitochondria are present in most eukaryotic cells with a wide variety in numbers ranging from a none (mature erythrocytes) to several thousand mitochondria per cell (liver and muscle cells), depending on the cell type. Lung cells contain relatively high numbers of mitochondria, although exact mitochondrial numbers are hard to determine per cell type because their occurrence correlates with the cell’s level of metabolic activity. The mitochondrial inner membrane is composed of lipids, including cardiolipin similar to those found in prokaryotic cells and it is the cell’s only organelle that contains its own circular shaped DNA (mitochondrial DNA; mtDNA) (14, 26). The specified lipid and DNA profile in mitochondria led to the `endosymbiotic theory`, which proposes that prokaryotic cells were taken up inside a eukaryotic cell as an endosymbiont, and that most of the prokaryotic genes have been transferred to the host cell genome over time (25). Mitochondria are often referred to as the `energy factories` of the cell and they are composed of various compartments that carry out multiple specialized functions involving energy metabolism. These compartments include the outer membrane, the inter-membrane space, the inner membrane, the cristae and matrix (Figure 1). The matrix of the mitochondrion contains enzymes of the tricarboxylic acid cycle (TCA) or Krebs cycle and other structures and compounds including ribosomes, matrix granules and mitochondrial DNA. Energy is derived from oxidative phosphorylation (OXPHOS), a process involving electron transfer over the inner-membrane and proton pumps that drive

14

Chapter 1

the synthesis of chemical energy in the form of ATP. Next to producing energy in the form of ATP, the mitochondria are responsible for programmed cell death, calcium storage and signaling, roles in cellular proliferation and differentiation, hormone and haem synthesis and cellular signaling processes (4). Together, this makes the mitochondria key regulators of cellular function, which is pivotal for cellular homeostasis in healthy tissue. Depending on the cell type, mitochondrial dysfunction can lead to the loss of various crucial processes, including ATP production, apoptosis regulation, cellular proliferation, differentiation and signaling processes, calcium storage and signaling.

CristaeInter‐membrane Space

Outer membrane

MatrixInner membrane

mtDNA RibosomesMatrix Granule

Function:‐ Cellular Metabolism‐ Programmed cell death (Apoptosis)‐ Calcium storage and signaling‐ Cellular proliferation and differentiation‐ Hormone and Heme synthesis ‐ Cellular signaling processes

OXPHOS Complexes

CytC

Porins

Mitochondrion structure and function

Figure 1: Overview of a mitochondrion and important matrix located components which all serve various important functions in the cells. Cytochrome C; CytC, Oxidative Phosphorylation; OXPHOS, Mitochondrial DNA; mtDNA.

For the production of ATP, ~85% of the cell’s oxygen is consumed, while a small fraction of this is converted to superoxide (O2-), a highly reactive radical that needs to be neutralized as quickly as possible (55, 66). Thus, while electron leakage and subsequent oxidant production is a normal consequence of the OXPHOS process, oxidative stress should be controlled. Mitochondria have their own quality control and mechanisms to ensure a healthy mitochondrial population within the cell, including endogenous anti-oxidant defense mechanisms and exchange of mtDNA through fission and fusion processes with neighboring mitochondria. When anti-oxidant enzymes, e.g. superoxide dismutase, catalase and glutathione cannot neutralize the superoxide radical to H

2O fast enough,

oxidative damage to components will occur and accumulate within the mitochondria (23, 55). Mitochondria that are damaged beyond repair are normally cleared through a process called mitophagy. This process is initiated by the recruitment of the proteins peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PPARGC1α), PTEN induced putative kinase 1 (PINK1) and Parkin, proteins involved in mitochondrial biogenesis, repair

15

General introduction

Chapter

1or clearance (71). In addition, telomerase, which was initially thought only to have a nuclear function, has been shown to have an protective effect on mitochondrial DNA as it migrates to the mitochondria during times of excessive oxidative stress (27). Furthermore, the energy metabolism protein Glycogen-synthase kinase (GSK3)β, has recently been shown to be present in mitochondria, protecting the mitochondria against oxidant induced responses (27, 54). Increased oxidative stress, e.g. upon long-term cigarette smoking and/or impaired anti-oxidant responses, can cause mtDNA mutations and damage, excessive electron leakage and increased production of reactive oxygen species (ROS). In turn, mitochondrial dysfunction may lead to excessive ROS production in a vicious circle. ROS are known to stimulate inflammation by activating the pro-inflammatory transcription factor NF-κB. Indeed, it has been shown that mitochondrial dysfunction, induced by different chemical compounds, can increase or induce inflammatory responses by increasing the expression of CXCL8 and cyclooxygenase 2 (COX-2) (77, 78). Additionally, mitochondrial dysfunction may lead to cellular damage and impaired regeneration responses. Mitochondrial dysfunction has been linked to cellular ageing, which also results in accumulation of mtDNA mutations and impairment of oxidative phosphorylation. Importantly, cigarette smoke has been shown to induce oxidative stress in mitochondria of lung epithelial cells (74, 75), and may thus induce mitochondrial dysfunction in these cells, potentially leading to accelerated ageing, impaired repair and ongoing pro-inflammatory responses (Figure 2).

Inflammation

Oxidative Stress

Mitochondrial Dysfunction

Risk factors:• Metabolic syndrome• Inadequate antioxidant response • Genetics• Aging• (Ischemic) Injury• Smoking• Occupational hazards

Mitochondrial role in COPD inflammation

Figure 2: Possible model of a vicious circle sustaining inflammatory response in COPD. Various risk factors may increase the production of ROS and/or induce mitochondrial dysfunction, stimulating pro-inflammatory responses and accelerating the onset of the disease.

Mitochondria and diseaseAs outlined above, mitochondrial dysfunction can hamper multiple functions within the cell and depending on the cell type, this can give rise to the onset of various (often age-related) diseases, including diabetes, Alzheimer`s and Parkinson`s disease. Mutations in mtDNA, impaired energy supply, Ca2+ buffering, increased ROS production and dysregulation of cellular apoptosis by mitochondria may all contribute to the progressive decline in longevity of the cell (71). The cell types that are often affected by mitochondrial dysfunction are those that relies the most on proper mitochondrial function and often have the most

16

Chapter 1

mitochondria. Cells of the muscle, heart, brain, liver, kidneys, nerves, pancreas, eyes and lungs have high numbers of mitochondria in their cytosol and rely strongly on proper mitochondrial function. Known inherited conditions in which a (partial) mitochondrial dysfunction has been implicated include diseases that result either from mutations/high levels of heteroplasmy in the mtDNA or mutations in the genomic DNA (gDNA) encoding for important mitochondrial components (55, 64, 72). All these inherited conditions affect the multitude of the previous mentioned organs and include Kearns–Sayre syndrome, Leber hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like syndrome, Myoclonic epilepsy and ragged-red fibers, Leigh syndrome sub-acute sclerosing encephalopathy, neuropathy, ataxia, retinitis pigmentosa, and ptosis and myoneurogenic gastrointestinal encephalopathy (12, 37, 55, 64, 65, 72, 76). Therefore, most research is focused on these diseases, while relatively little is known about mitochondrial (dys)function and its contribution to lung disease.

Role of mitochondrial dysfunction in COPDThe role of mitochondria in lung disease has not yet intensively been investigated, although several studies have already demonstrated that cigarette smoke can disrupt the mitochondrial membrane potential (Δψ

m) in macrophages of the lungs and epithelial

cells (2, 74, 75). Cigarette smoke can directly damage mitochondria in lung cells, however, excessive damage to mitochondria can also occur by reactive oxygen species (ROS) generated by the mitochondria themselves, for instance when their antioxidant response is insufficient (55). Of interest, the production of various anti-oxidant enzymes, including Mn-SOD, catalase and glutathione peroxidase (GPx), is reduced in cells/lung tissue of COPD patients (35, 59, 61). Recently, polymorphisms in Mn-SOD have been linked to the development of bronchial hyperresponsiveness in COPD (67). Possibly, cells from COPD patients are deficient in their compensatory mechanisms that protect from mitochondrial damage. When these mechanisms fail, this may result in excessive oxidative damage, which may also extend to mtDNA and proteins of the OXPHOS system, eventually hampering mitochondrial function. As described above, mitochondrial damage can lead to increased production of ROS and activity of NF-κB, thus sustaining an inflammatory response seen in COPD (Figure 2). Mitochondria that are damaged beyond repair are cleared through a process called autophagy, an intracellular degradation process. Mitophagy is an autophagic degradation pathway specific for mitochondria, a process initiated by the recruitment of the proteins PINK1 and Parkin (17, 22, 53), as described above. There are indications that the autophagy processes is disrupted in COPD patients (13, 48). In mitophagy deficient cells or when the mitochondrial clearance capacity is exhausted, defective mitochondria will accumulate (3, 34). In this case, mitochondria can fall apart, leading to the release of their lipid and protein

17

General introduction

Chapter

1components, which are capable to trigger a localized (lung) inflammatory or apoptotic response (Figure 3).

CytC

mtDNA

CytC

ROS

H2O2O2ˉ

OHˉ

Bacterial `like` lipids

Cardiolipin

O2ˉ

ROS

O2ˉ

TLR‐4

TLR‐9

CpG DNA repeats and and formyl peptides

LPS like lipids

APAF

‐1AP

AF‐1

Pro‐caspase9

Pro‐caspase9

NFκBIκB

NFκB

NFκB activation

IκB

Mitochondrial damage associated molecular patterns

FPR1

mtDNA

lipids

H2O2

1

2 3

4 5

CXCL‐8

Figure 3: Model of mitochondrial damage associated with various inflammatory and apoptotic responses. 1) When extensive mitochondrial damage occur various proteins, nucleic acids, reactive oxygen species (ROS) and lipid compounds will be released into the cytosol which all can contribute to a pro-inflammatory response and cell death. 2) ROS can activate the pro-inflammatory transcription factor. 3) Binding of cytochrome-C can lead to the activation of caspase-9 and start the induction of apoptosis. 4) The release of CpG DNA repeats and formyl peptides can trigger Toll like receptor (TLR)-9 and formyl peptide receptor 1 (FPR1) and induce a pro-inflammatory response through MyD88 signaling leading to the transcription of pro-inflammatory genes (e.g. CXCL8). 5) Lipids of the inner-membrane, which are similar in composition to bacterial lipids, can also induce a pro-inflammatory response through TLR-4-MyD88 signaling.

These components are also referred to as mitochondrial damage associated molecular patterns (mDAMPs), acting on so-called pattern recognition receptors on innate immune cells to induce inflammatory responses. In this respect, CpG DNA (mtDNA) or N-formyl-peptides released from mitochondria can activate Toll-like receptor (TLR)9 and Formyl Peptide Receptor 1 on neutrophils and epithelial cells. This induces the activation of pro-inflammatory signaling molecules, e.g. MyD88, leading to the release of pro-inflammatory mediators like CXCL8 (58). In addition, release of ROS from damaged mitochondria has been proposed to induce the activation of NF-κB by degrading its inhibitor (40). It has also been suggested that the secretion of the pro-inflammatory cytokine IL-1β, which is elevated

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Chapter 1

in the airways of COPD patients (6), can be induced by cytosolic release of mtDNA in autophagy deficient cells (3, 11, 52). Lipids released from mitochondria, such as cardiolipin which especially in oxidized form resembles bacterial lipopolysaccharide (LPS), can trigger an inflammatory responses through the activation of TLR4 (3, 46, 73). Both TLR4 and TLR9 activation can lead to CXCL8 production and secretion, resulting in neutrophil accumulation (50, 51). Thus, besides from impaired respiration, damaged mitochondria and circulating mitochondrial components can inflict inflammatory responses in the lungs. In addition to these inflammatory responses, the sudden release of high quantities of cytochrome-C from the mitochondria upon mitochondrial damage can initiate (3, 46, 73) apoptosis through the activation of caspase 9 (3).

Oxidative stress induces signaling events leading to mitochondrial dysfunction in COPDAs described above, oxidative stress may induce mitochondrial dysfunction, with important consequences for COPD. Various signaling processes may contribute to oxidative stress induced mitochondrial dysfunction. In particular, the regulation of the redox-responsive kinase GSK3β activity may be of interest for mitochondrial dysfunction in COPD. GSK3β is considered to be predominantly localized in the cytosol of the cell, however, localization in the nucleus and mitochondria has been reported and is linked to a higher activity of the enzyme (9). The exclusive function of mitochondrial GSK3β activity is not yet fully understood. There are studies indicating an important role in apoptosis regulation by GSK3β, changing the mitochondrial outer membrane permeabilization by destabilization of Myeloid Cell Leukemia 1 MCL-1 (47). Another group has shown that GSK-3β is able to mediate phosphorylation of Dynamin-1-like protein (DRP1), which is an important member of superfamily that regulate mitochondrial fission, inducing an elongated mitochondrial morphology against oxidative stress (16). Furthermore, GSK3β mediates phosphorylation of the Voltage-Dependent Anion Channel (VDAC), which controls the outer mitochondrial membrane permeability (44). In all cases, GSK3β activity is subject to dynamic regulation by various kinases, which are commonly involved in oxidant-mediated responses, including Akt, indicating that GSK3β may represent an important downstream effector of oxidant-mediated signaling. Akt-mediated phosphorylation of GSK3β at Ser9 inhibit its activity (20). In cardiomyocytes, phosphorylation of GSK3β at Ser9 protects against oxidant induced apoptosis (54). In addition, GSK3β activity has been implicated in glucocorticoid-induced apoptosis in lymphoma cells, its inactivation resulting in GC insensitivity (69). Two other studies showed that pharmacological inhibition of GSK3β by SB216763 in a guinea pig model of LPS-induced pulmonary inflammation did not affect inflammatory cell influx, but prevented small airway remodeling and LPS-induced muscle mass decline and myofiber atrophy, which is normally

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General introduction

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1observed in COPD (30, 79). Thus, inactivation of GSK3β by phosphorylation at Ser9 may be involved oxidant-induced GC insensitivity, although it may also exert beneficial effects and protect from thickening of the airway walls. Together, we hypothesize that alterations in GSK3β may have important implications for mitochondrial function and development of airway inflammation in COPD.

Scope of this thesisWe hypothesize that the damaging effects of cigarette smoke-induced oxidative stress in lung epithelial cells affect the mitochondria. We speculate that these dysfunctional mitochondria contribute to the (onset) of the disease, leading to pro-inflammatory responses of epithelial cells and impaired regeneration upon damage. In addition, oxidative stress and mitochondrial dysfunction may lead to GC insensitivity of airway epithelial cells. In chapter 2, we aimed to assess the dose depend effects of long-term cigarette smoke exposure on mitochondria in human bronchial epithelial cells. In particular, we studied the effect of long-term exposure to cigarette smoke extract on mitochondrial morphology, function, fission and fusion in the human bronchial epithelial cell line BEAS-2B. Additionally, we explored whether and how mitochondrial morphology and function is altered in bronchial epithelial cells from COPD patients. In chapter 3, we studied the mechanisms involved in cigarette smoke-induced GC insensitivity, a pinnacle in the treatment of COPD patients, and whether GSK3β is involved in this effect. This was studied in human bronchial epithelial 16HBE cells as well as monocytes. Furthermore, we compared steroid responsiveness in primary bronchial epithelial cells from non-smokers, smoking controls and COPD patients. In chapter 4 of this thesis, after demonstrating the influence of long-term cigarette smoke extract on mitochondria, we investigated whether mitochondrial dysfunction leads to changes in epithelial pro-inflammatory responses, repair and GC (in)sensitivity. In order to do so, we used A549 Rho-0 cells, which are depleted of mitochondrial DNA (mtDNA), and studied effects on IL-8 release, wound healing capabilities and GC responsiveness. In

chapter 5, we explored the potential of lipidomics to monitor cigarette smoke-induced, and/or disease related-changes in sputum from COPD patients, which we compared to sputum from smokers without COPD and never-smokers. In chapter 6, we studied whether similar changes were observed in lipid compounds of long-term cigarette-smoke-exposed bronchial epithelial BEAS-2B cells that were used in chapter 2. With these studies, we aimed to improve insight in the role of cigarette smoke-induced mitochondrial dysfunction in the development of COPD, and whether mitochondria can serve as future therapeutic target.

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CHAPTER 2

Prolonged cigarette smoke exposure

alters mitochondrial structure and function

in airway epithelial cells

Roland F. Hoffmann, Sina Zarrintan , Simone M. Brandenburg, Arjan Kol, Harold G. de Bruin, Shabnam Jafari, Freark Dijk, Dharamdajal Kalicharan, Marco Kelders,

Harry R. Gosker, Nick H.T. ten Hacken, Johannes J. van der Want, Antoon J.M. van Oosterhout, Irene H. Heijink

Hoffmann et al. Respiratory Research 2013

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Abstract Background: Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis.

Methods: We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls.

Results: We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1.

Conclusion: The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.

Keywords: Mitochondria, Primary bronchial epithelial cells, Smoking, Reactive oxygen species, COPD

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2

IntroductionChronic Obstructive Pulmonary Disease (COPD), a chronic respiratory disease, is one of the leading causes of death today with a worldwide increase in incidence. COPD is characterized by accelerated lung function decline and a chronic inflammatory response in the lungs in response to cigarette smoke, the largest risk factor for COPD. Inhaled cigarette smoke first encounters the airway epithelium, where it may induce oxidative stress by both acute effects of its reactive components and by the intracellular generation of endogenous reactive oxygen species (ROS) by mitochondria (1-4,21,22). Mitochondria can protect themselves and the cell from oxidative damage in several ways, e.g. by producing anti-oxidant scavengers, regulating the oxidative phosphorylation (OXPHOS) process responsible for ATP generation and exchanging mitochondrial DNA (mtDNA) through fusion and fission events (4-7). Excessive oxidative stress and/or an imbalance or depletion of key mitochondrial fission and fusion markers, including Dynamin-related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), Mitofussion (Mfn1 and Mfn2), Optic Atrophy 1 (OPA1) and mitochondrial transcription factor A (Tfam) can lead to mitochondrial damage and disorganized and aberrant cristae formation (8). Furthermore, it will augment ROS production and cellular apoptosis through cytochrome-C release (6,9-14). Normally, damaged mitochondria are repaired or cleared by a process called mitophagy, which is induced by PTEN-induced putative kinase 1 (PINK1). PINK1 regulates mitochondrial turnover and thereby protects mitochondria from stress, in concert with peroxisome proliferator-activated receptor gamma co-activator 1-alfa (PPARGC1α), which controls mitochondrial biogenesis (15-18). We hypothesize that an aberrant response to cigarette smoke and excessive oxidative stress, e.g. due to an inefficient anti-oxidant response as observed in COPD lungs (19,20), may lead to impaired mitochondrial structure and function due to impaired clearance or repair. This may contribute the COPD pathology, inducing apoptosis, tissue damage as well as airway inflammation, since airway epithelial cells respond to increased ROS levels by producing cytokines/chemokines that attract inflammatory cells to the area of damage (19,21-24). Although oxidative stress is known to induce post-translational modifications/damage to mitochondrial proteins (25,26), it is currently unknown whether mitochondrial structure and function in airway epithelial cells are affected by cigarette smoke (27,28), and whether these changes are present in COPD epithelium. Since COPD is a gradually acquired disease that develops upon chronic cigarette smoke exposure, we anticipated that a long-term cigarette smoke exposure model reflects the in vivo situation better than single exposure experiments. Therefore, we studied long-term (6 months) cigarette smoke extract (CSE)-induced changes in the human bronchial epithelial cell line BEAS-2B to mimic the condition of continuous smoking for prolonged periods, and investigated whether these changes are persistent upon CSE depletion. Additionally, we investigated whether similar changes

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are observed in mitochondria from primary bronchial epithelial cells (PBECs) of ex-smoking GOLD stage IV COPD patients with age-matched never-smoking and smoking controls.

Methods

Long-term CSE exposed human bronchial epithelial cell cultureBEAS-2B cells were grown in RPMI 1640 (Lonza, Walkersville, MD) supplemented with 15% heat-inactivated Fetal Bovine Serum (FBS), penicillin (100 U/ml) (Lonza) and streptomycin (100 μg/ml) (Lonza) on collagen-coated plates. Cells were grown to >90% confluence and passaged by trypsin. Cigarette smoke extract (CSE) was prepared fresh (used within 1 hr) from filter-less research-grade cigarettes (3R4F, Tobacco Research Institute, University of Kentucky, Lexington, KY) as described previously (21). BEAS-2B cells were cultured in medium without 0% (control), 1%, 2.5%, 10% or 12.5% CSE for 6 months. For 10% CSE and 12.5% CSE the concentration was increased stepwise by 0.5-1% after each passage every 3–4 days starting from 2.5% CSE. Additionally, cells were exposed to 10% CSE for 3 months followed by 3 months culture in 0% CSE (referred to as −10% CSE). Cell viability was tested by trypan blue staining.

Primary epithelial cell culturePrimary bronchial epithelial cells (PBECs) were isolated from ex-smoking GOLD stage IV COPD patients and age-matched never-smoking and smoking controls. Patient characteristics are described in Table 1. The study was approved by the Medical Ethics Committee of the University Medical Center of Groningen and all subjects gave their written informed consent. PBECs were cultured in bronchial epithelium growth medium (Lonza) in flasks coated with collagen and fibronectin for at least 3 weeks in total as described (29). Cells were plated in 24- or 6-well plates (EM), grown to confluence and placed overnight in BEBM containing transferrin, insulin, gentamicin and amphotericin B (Sigma-Aldrich).

Table 1: Characteristics of the subjects: primary bronchial epithelial cells (PBECs)

Subject control non-smoker control smoker GOLD stage IV

n 15 15 24

Age 55,5 (43–76) 51.6 (40–70) 59,7 (48–71)

Gender M (%) 7(46.7%) 12(80%) 10 (41.7%)

Pack-years 0 (0–0) 31.3(11–60) 39.3(11–80)

All PBECs included in the study were provided by Lonza or the NORM and TIP study (50) within the University Medical Center Groningen. COPD patients were included on a basis of FEV

1 < 50% of predicted, FEV

1/FVC < 70%

and ≥20 pack-years for GOLD stage IV. All control subjects had FEV/FVC > 70% and FEV1 > 90% of predicted. FEV/

FVC, FEV1 predicted and pack-years from Lonza cells are unknown.

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Chapter

2

RT-qPCRRNA isolation, Primer Sequences, RT-PCR on Tfam, OPA1, Drp1, Fis1, Mfn1, Mfn2 using MyiQ single-color Real-Time thermal cycler (31) and RT-PCR for IL-6, IL-8, IL-1β, Mn-SOD, PINK1, PPARGC1α, OPA1 and TFAM using Taqman® were performed as described in the Supplemental Figure 1.

IL-8 releaseUpon 3 days of culturing, levels of IL-8 were analyzed in cell-free culture supernatants by sandwich ELISA (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions.

Western blottingCell lysates were prepared and immunodetection was performed as described (29). MitoProfile total OXPHOS antibody cocktail (Mitosciences, Eugene OR) or antibodies to mitochondrial loading control COXIV (clone 3E11) and anti-oxidant Mn-SOD (EMD Millipore Corporation, Billerica MA). Anti-GAPDH and Anti-β-actin (Cell Signalling Technology, Danvers MA, USA) were used for loading control.

Electron microscopyCultured cells were fixed on 6-wells plates using 0.1 M phosphate buffered glutaraldehyde (2%) overnight at 4°C. Cells were pre-washed in 0.1 M cacodylate buffer and post-fixed in 1% OsO4 supplemented with 1.5% K3Fe(Cn6) for 2 hours, dehydrated and embedded in epoxy resin according to routine procedures. Sections were collected on copper grids, counterstained and inspected in a Philips CM 100. Data sampling is explained in Supplemental Figure 2.

StatisticsANOVA and Chi-square was used to compare data sets indicated in Tables 2 and 3. Differences in protein/gene expression in BEAS-2B cells were tested by One-way ANOVA with Bonferroni post-hoc test or unpaired t-test. Differences between subject groups were analyzed by the Mann–Whitney test.

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Results

Morphological ultrastructure and matrix changes in mitochondria of long-term CSE exposedBEAS-2B cellsBEAS-2B cells exposed to different concentrations of CSE did not display differences in viability or morphology compared to cells that were not exposed to CSE, and were able to form a confluent layer (Supplemental Figure 3). Moreover, expression of the cellular senescence marker p21 was not affected (Supplemental Figure 3). Analysis of mitochondrial ultrastructure in long-term CSE exposed BEAS-2B cells revealed significant changes in morphological structure (branching/fragmentation), cristae formation and matrix density of mitochondria compared to the 0% CSE control cells (Figure 1). At 1% CSE, we observed an increased number of branched mitochondria when compared to the absence of CSE as well as to higher CSE concentrations (10% and 12.5% CSE, Figure 1 and Table 2, p < 0.001). Fragmentation, however, was more prominent at higher concentrations (10% and 12.5% CSE). Interestingly, although mitochondrial fragmentation was reduced upon depletion of CSE (−10% CSE) when compared to 10% CSE, CSE-depleted cells still showed more branched mitochondria compared to control conditions. This indicates that CSE-induced fragmentation, but not branching, is reversible upon its depletion. In addition to these morphological changes, the mitochondrial matrix was significantly affected by 2.5%, 10% and 12.5% CSE (Table 2, p < 0.001), which remained present upon CSE depletion (−10%). The density of the “dark” lipoid matrix also increased with higher CSE concentrations and this increased density also remained present upon depletion of CSE (−10% CSE).

Table 2: Mitochondrial shape variation and density of the matrix

Shape Status of Matrix

Rod Branching Fragmented Clear Dense (Lipoid)

Control 28 (93.3%) 0 (0.0%) 2 (6.7%) 30 (100.0%) 0 (0.0%)

1% CSE 25 (80.6%) 6 (19.4%) 0 (0.0%) 31 (100.0%) 0 (0.0%)

2.5% CSE 31 (96.9%) 0 (0.0%) 1 (3.1%) 28 (87.5%) 4 (12.5%)

10% CSE 24 (70.6%) 2 (5.9%) 8 (23.5%) 23 (67.6%) 11 (32.4%)

12.5% CSE 24 (69.6%) 3 (8.6%) 8 (22.9%) 29 (82.9%) 6 (17.1%)

−10% CSE 19 (63.3%) 9 (30.0%) 1 (3.1%) 22 (73.3%) 8 (26.7%)

*** ***

***Estimated by chi-square test (p < 0.001). BEAS-2B cells were exposed to various concentrations of cigarette smoke extract (CSE) for 6 months and subjected to EM analysis. BEAS-2B cells are exposed to: 0% CSE (control), 1% CSE, 2.5% CSE, 10% CSE, 12.5% CSE and −10% CSE (depletion). In total of 30–35 mitochondria were analyzed per experimental condition. Significance was determined by chi-square test.

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Chapter

22

3 4

1

0% CSE

10% CSE

1% CSE

12.5% CSE

2.5% CSE

-10% CSE

5 6Figure 1: Differences in mitochondrial shape, the status of matrix, swelling, visibility of double membranes, fragmentation, branching and cristae depletion upon long term CSE exposure in BEAS-2B cells. BEAS-2B cells were exposed to various concentrations of cigarette smoke extract (CSE) for 6 months and subjected to EM analysis. Representative images of BEAS-2B cells exposed to: 0% CSE (control), 1% CSE, 2.5% CSE, 10% CSE, 12.5% CSE and −10% CSE. Scale bar indicates 2 μm and 1 μm (Top left).

Subsequently, we analyzed the relative frequency of mitochondrial cristae that were observed within a single mitochondrion. We observed that the numbers of cristae were strongly reduced upon long-term CSE exposure, which was already observed at 1% CSE further diminished with increasing concentrations of CSE and remained present after CSE depletion (−10% CSE) (Table 2, p < 0.001) compared to the 0% CSE treated cells. Together, these data show that long-term CSE exposure induces strong and persistent structural mitochondrial changes. These data are further confirmed by fluorescent 3D imaging of

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mitochondrial distribution in BEAS-2B cells upon long-term 10% CSE exposure, showing increased numbers of branched mitochondria (Supplemental Figure 4).

Table 3: Frequencies and percentages of number of cristae and number of cristae divided by length

Number of cristae per mitochondrion per section FRQ/L**

0 < 3 3-6 >6 Mean ± SD Mean ± SD

Control 0 (0.0%) 0 (0.0%) 2 (6.7%) 28 (93.3%) 2.9 ± 0.3 2.2 ± 0.8

1% CSE 0 (0.0%) 2 (6.5%) 9 (29.0%) 20 (64.5%) 2.6 ± 0.6 1.9 ± 0.6

2.5% CSE 4 (12.5%) 4 (12.5%) 8 (25%) 16 (50%) 2.1 ± 1.1* 1.7 ± 1.0*

10% CSE 4 (11.8%) 5 (14.7%) 19 (55.9%) 6 (17.6%) 1.8 ± 0.9* 1.3 ± 0.8*

12.5% CSE 0 (0.0%) 5 (14.3%) 18 (51.4%) 12 (34.3%) 2.2 ± 0.7* 1.6 ± 0.6*

−10% CSE 1 (3.3%) 4 (13.3%) 12 (40%) 13 (43.3%) 2.2 ± 0.8* 1.6 ± 0.7*

*** **** ****

* p < 0.05 pairwise comparison to 0% CSE (Control). **Frequency divided by length. ***Estimated by chi-square test (p < 0.001). ****Estimated by One-Way ANOVA test (p < 0.001). BEAS-2B cells were exposed to various concentrations of cigarette smoke extract (CSE) for 6 months and subjected to EM analysis. Representative images of BEAS-2B cells exposed to: 0% CSE (control), 1% CSE, 2.5% CSE, 10% CSE, 12.5% CSE and −10% CSE are shown in Figure 1. In total of 30–35 mitochondria were analyzed per experimental condition. Significance was determined by chi-square test and One-Way ANOVA.

Transcriptional increase in OPA1 expression upon long-term CSE exposure in BEAS-2B cellsNext, we performed qRT-PCR to examine whether the observed changes may result from altered mRNA expression of various genes involved in fission and fusion, e.g. Mfn1, Mfn2, Fis1, Drp1, OPA1 and the mitochondrial transcription factor Tfam (Figure 2). We observed a significant 1.9 (±0.1) fold increase in the mRNA expression of the profusion related protein OPA1 upon exposure to 10% CSE (Figure 2). Surprisingly, we observed no significant transcriptional differences in other mitochondrial fission (Fis1 and Drp1) or fusion markers (Mfn1 and Mfn2) nor in Tfam expression (Figure 2).

Increased mitochondrial capacity in long-term CSE exposed BEAS-2B cellsTo assess whether CSE exposure induces differences in the total mitochondrial fraction, we studied the expression of the mitochondrial COXIV protein (which is generally continuously expressed at a high level). Although exposure to 2.5%, 10% and 12.5% slightly decreased the COXIV levels, this was not significant when compared to the 0% CSE control cells (Figure 3A). However, upon CSE depletion, mitochondrial mass showed a marked and significant increase compared to the presence of 10% CSE, indicating a recovery process upon CSE depletion (Figure 3A). Next, we studied whether the observed structural changes may affect mitochondrial function by studying ATP levels after 10% CSE exposure as a proof of concept. Contradictory to the unaltered mitochondrial mass and reduced cristae

33

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number, however, ATP levels were increased in 10% CSE exposed cells by approximately 1.6 fold when compared to the control (0% CSE) condition (Figure 3B), which may reflect an increase in mitochondrial OXPHOS capacity. In line with this, exposure to 10% CSE and 12.5% CSE induced a ~2-3 fold increase protein levels of the anti-oxidant protein Manganese Superoxide Dismutase (Mn-SOD, Figure 3C), a marker of oxidative stress, with a similar increase in Mn-SOD expression upon exposure to 10% CSE and 12.5% CSE at the mRNA level (data not shown). Interestingly, Mn-SOD protein levels were restored in −10% CSE exposed cells indicating a reduction in oxidative stress levels upon CSE depletion. To study whether oxidative stress causes mtDNA damage, we assessed mtDNA damage by fragment PCR (51) and observed that hydrogen peroxide, gas phase cigarette smoke and CSE induced mtDNA damage in BEAS-2B cells (1,26,32,33)( Supplemental Figure 5).

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Figure 2: Expression of fission and fusion markers in long-term CSE exposed BEAS-2B cells. Cells were exposed to 0% CSE, 2.5% CSE, 10% CSE, 12.5% CSE (n = 5 for each group) for 6 months. RNA was isolated and fission and fusion markers were detected by qPCR. Ct values were obtained for the standard curve and each sample. Relative DNA starting quantities of samples were derived from the standard curve based on the Ct values using the iQ5 optical system software version 2.1. Genes of interest expression was normalized to a GeNorm factor obtained from the housekeeping genes RPLPO and RPL13a. Median interquartile ranges (IQR) are indicated. * = p < 0.05 between the indicated values.

Regulation of oxidative phosphorylation is important for mitochondrial capacity and the protection against oxidative stress. Therefore, we analyzed the expression of three major components of the OXPHOS system i.e., Complex II, Complex III core 2 and (the ATPase subunit) Complex V F1α by western blotting. Interestingly, Complex II (Figure 3D), Complex III (Figure 3E) and Complex V (Figure 3F) were significantly increased in cells cultured

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in 10% CSE, but not 2.5%, when compared to the other concentrations. Surprisingly, expression of all complexes was again lower upon 12.5% CSE when compared to 10% CSE exposure, although the expression of Complex II, but none of the other complexes, was still significantly increased at 12.5% CSE. CSE depletion resulted in the restoration of complex II and III protein levels, similar to the non-persistent effects of CSE on mitochondrial mass and Mn-SOD, while complex V levels were not significantly reduced upon CSE depletion (Figure 3F).

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Figure 3: Mitochondrial capacity is increased in long-term cigarette CSE exposed BEAS-2B cells. Cells were exposed to 0% CSE, 2.5% CSE, 10% CSE 12.5% CSE (n = 4-5) for 6 months. A) Mitochondrial mass was determined by COXIV as a mitochondrial loading control using western blotting. GAPDH was used as loading control. B) ATP levels were measured by bioluminescence. C) Mn-SOD protein levels were detected by western blotting, related to actin and medians are indicated. Western blot analysis of, D) Complex II, E) Complex III and F) Complex V of the oxidative phosphorylation, related to GAPDH (medians are indicated). p = 0.06, p = 0.08, * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001 between the indicated values.

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Increased inflammatory response in long-term CSE exposed cellsTo investigate whether these mitochondrial changes induced by long-term CSE exposure were accompanied by increased pro-inflammatory activity of bronchial epithelium, we measured expression of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8, which are known to be elevated in the airways of COPD patients (34,35). We found a significant increase in IL-1β and IL-6 mRNA expression in cells exposed to 10% CSE compared to control cells (Figure 4A). Additionally, IL-8 mRNA (Figure 4B) and protein levels (Figure 4C) were significantly increased in cells exposed to 10% and 12.5% CSE. CSE in a concentration of 2.5% did not to affect the mRNA nor on protein expression of pro-inflammatory cytokines, in line with the absence of effects on mitochondrial function (Figure 4A, 4C).

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Figure 4 Increased inflammatory response in long-term CSE exposed BEAS-2B cells. Cells were exposed to 0% CSE, 2.5% CSE, 10% CSE 12.5% CSE as indicated for 6 months. A) IL-6, IL-1β mRNA expression (n = 5) and B) IL-8 mRNA expression was detected by qPCR and related to the housekeeping genes (n = 4). ΔCt values are shown (lower values reflect higher expression) and median interquartile ranges (IQR) are indicated. C) IL-8 protein levels were measured in cell-free culture supernatants (n = 4) and mean (±SEM) levels are shown. Significance is as indicated, * = p < 0.05, ** = p < 0.01, ***p = <0.001.

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A B

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Figure 5 Primary bronchial epithelial cells (PBECs) from COPD patients display elongated and swollen mitochondria, fragmentation, branching and cristae depletion and altered expression of mitochondrial markers compared to control PBECs. PBECs were isolated from ex-smoking COPD patients with GOLD stage IV (n = 10), control smokers (n = 7) and never-smokers (n = 6) A) Complex V ATPase protein and Mn-SOD were detected by western blotting. GAPDH was used as loading control. B) OPA1, Tfam, PINK1 and PPARGC1α mRNA expression was detected by qPCR and related to housekeeping genes. ΔCt values are shown and median interquartile ranges (IQR) are indicated. C) Panel 1 shows a representative picture of PBECs from 4 never-smokers displaying normal mitochondria. Panel 2 shows a representative picture from 9 ex-smoking COPD patients displaying increased numbers of mitochondria with cristae depletion and ’club shaped’ ends. Panel 3 COPD patient shows severe branching. Panel 4 COPD patient shows swollen structures. Scale bar indicates 5 μm (bottom) and 2 μm (Top). p = 0.07, * = p < 0.05 and ** = p < 0.01 between the indicated values.

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Mitochondrial changes in PBECs from COPD patients compared to PBECs from (never-) smoking individualsIn order to assess whether long-term smoking also results in (persistent) structural changes in epithelial mitochondria in COPD, we studied mitochondrial alterations in PBECs derived from severe ex-smoking COPD GOLD stage IV patients and age-matched never-smoking and smoking controls. First, we investigated OXPHOS levels and observed that protein levels of Complex V F1α were not significantly different in PBECs from non-smokers compared to smokers. Interestingly, levels were significantly increased in PBECs from COPD patients when compared to non-smoking control PBECs, while a trend was visible when compared to PBECs from smokers, indicating a disease-related effect (Figure 5A). Similar to the data in BEAS-2B, increased Complex V levels were accompanied by increased Mn-SOD protein levels in PBECs from COPD patients, which may reflect a compensatory mechanism in response to increased energy demand and/or elevated oxidative stress (Figure 5A).

In contrast to the elevated levels in CSE-exposed BEAS-2B cells, we found no evidence for altered mRNA expression of OPA1 or Tfam in PBECs from COPD patients or control smokers compared to never-smokers (Figure 5B). We also studied the expression of PINK1 and PPARGC1α, genes that are activated upon mitochondrial damage and play a key role in mitochondrial turnover. Of interest, the mRNA expression of both PINK1, a marker not significantly different in CSE exposed BEAS-2B cells (Supplemental Figure 6), and PPARGC1α, a marker that was not detectable in CSE exposed BEAS-2B cells, was significantly increased in PBECs from COPD patients when compared to non-smoking controls (Figure 5B). In line herewith, we observed that PBECs from COPD patients show remarkable abnormalities in mitochondrial structure, similar to the changes observed in BEAS-2B cells upon long-term CSE-exposure, albeit more severe. These observations include excessive branching of the mitochondria and cristae depletion, but also mitochondrial swelling and elongation, when compared to mitochondria in control PBECs (Figure 5C). Together, our data indicate that epithelium from severe COPD patient’s displays persistent mitochondrial damage.

DiscussionFor the first time, we show that long-term CSE exposure induces robust and persistent changes in mitochondrial structure and function in human bronchial epithelial cells, including increased fragmentation, branching, density of the matrix and reduced numbers of cristae. All of these changes, except for fragmentation, remained present after depletion of CSE, suggesting that these are persistent upon smoking cessation. In line with the observed changes, mRNA expression of OPA1, a critical regulator of cristae formation and fission/fusion, was increased at the transcriptional level in long-term CSE-exposed BEAS-2B cells. Furthermore, we observed increased protein levels of OXPHOS components and increased

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levels of the oxidative stress marker and anti-oxidant Mn-SOD, indicating augmented energy demand and oxidative stress. These mitochondrial changes were accompanied by increased pro-inflammatory activity in long-term CSE-exposed cells, as reflected by higher levels of the pro-inflammatory cytokines IL-1β, IL-6 and IL-8. Moreover, our data show that similar mitochondrial changes are present in bronchial epithelium from severe ex-smoking COPD patients in comparison to healthy controls, with depletion of cristae, increased branching, elongation and swelling of the mitochondria, increased Mn-SOD, PINK1 and PPARGC1α, but not OPA1 mRNA expression and increased OXPHOS Complex VF1α levels. These changes in Mn-SOD, PINK1, PPARGC1α and Complex VF1α were not observed in epithelial cells from control smokers.It is tempting to speculate that an attenuated anti-oxidant response with elevated ROS in COPD (19,20,28,36) may lead to an increased oxidant burden, possibly contributing to the observed mitochondrial defects in bronchial epithelial cells from COPD patients (22,30,32). Cigarette smoking induces both oxidative stress and anti-oxidant responses in airway epithelium and, with great variability amongst individuals (38). An increased oxidant burden due to lower levels of anti-oxidant defense has been reported in COPD, which has been associated with decreased lung function (39,40). Of importance, oxidative stress exceeding the anti-oxidant response may also lead to accelerated ageing and thus accelerated lung function decline (8,20,27). Recent studies suggest that due to increased oxidative stress, damaged mtDNA induces premature ageing in human cells (7,27,37). Importantly, similar morphological changes of mitochondria as observed here are regarded as biomarkers of mitochondrial aging. For example, senescent endothelial cells showed degenerated cristae and swollen regions devoid of cristae (8). Of note, the ageing lung shows morphological changes that resemble emphysema in COPD, including alveolar enlargement and a reduction in elastic recoil, contributing to lung function decline (41). Although we have not studied effects in alveolar epithelial cells, our findings may also have important implications for lung emphysema. ROS are potent inducers of cellular apoptosis by promoting the release of cytochrome-C, which may lead to alveolar cell apoptosis, lung tissue damage, inflammation and emphysema (27,42,43,46). Thus, we anticipate that lung epithelium from COPD patients may be more prone to cigarette smoking-induced mitochondrial damage/dysfunction and premature ageing, which may eventually affect lung function. Future studies will be of interest to assess whether the airway epithelial structure and function of mitochondria differs between in young non-smoking individuals who are non-susceptible and susceptible to develop COPD.In line with the hypothesis that epithelium from control smokers is better protected against cigarette smoke-induced changes in mitochondrial function, OXPHOS complex levels were not (persistently) higher in PBECs from smokers with normal lung function than from never-smokers, although OXPHOS complex II, III and VF1α were increased upon long-term

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CSE exposure in BEAS-2B. With respect to PBECs from ex-smoking COPD patients, we only observed an increase in complex VF1α, but not complex II and III, in line with the finding that the levels of complex II and complex III returned back to baseline levels upon CSE depletion in BEAS-2B cells.Our data strongly suggest that cigarette smoking induces structural and functional changes in mitochondria resulting in a pro-inflammatory phenotype of airway epithelium in severe COPD patients, and that these are persistent, as they are still present upon smoking cessation. In line with this, all CSE-induced morphological changes in BEAS-2B mitochondria that remained present upon CSE depletion were also observed in PBECs from COPD patients. Furthermore, PBECs from COPD patients displayed increased levels of Mn-SOD and OXPHOS complex VF1α, in agreement with the findings in BEAS-2B cells. These changes indicate increased cellular energy demand that is still present in cultured airway epithelium from ex-smoking COPD patients. Persistent mitochondrial damage in airway epithelium of COPD patients is further evidenced by the increased expression of PINK1, a sensor for damaged mitochondria, and PPARGC1α, a regulator of mitochondrial biogenesis (18,44). Both PINK1 and the fission/fusion protein OPA1 are known to be involved in the regulation of cristae formation (45), and increased expression of these proteins may thus reflect cristae damage. Since we did not observe significant changes in PINK1 and PPARCG1α expression in control smokers, nor in the CSE-exposed BEAS-2B cells, we anticipate that additional factors, e.g. susceptibility gene expression, may be involved in their aberrant expression in COPD epithelium. Vice versa, we did not observe significant differences in OPA1 expression between the subject groups, although OPA1 expression was increased in the long-term CSE-exposed BEAS-2B cells. We did not observe significant differences in other important mitochondrial genome and fusion markers, including Tfam and Drp1, between the subject groups or upon CSE exposure in BEAS-2B. However, we cannot exclude the possibility that these markers are modified at the posttranslational level by cigarette smoke-induced oxidative stress, leading to the observed changes in mitochondrial morphology, and this needs further investigation.To summarize, we propose that mitochondrial structure changes in COPD epithelium may derive from a sustained oxidative stress due to cigarette smoke exposure, which results in an inflammatory response (Figure 6). In line with our findings on the increased expression of pro-inflammatory cytokines upon long-term epithelial CSE exposure, bronchial epithelial cell from COPD patients have been shown to produce higher levels of IL-8 than controls individuals (34,35). Excessive oxidative stress may induce oxidative modifications, including peroxidation of mitochondrial lipids, oxidative damage of mitochondrial proteins including OXPHOS components, cristae remodeling, mutations and severe damage in mitochondrial DNA (mtDNA) (20-22,39-42). This may result in a further increase in oxidative stress, with important consequences, since ROS are additionally known to increase the NF-κB response.

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Eventually, this pro-inflammatory activity of the airway epithelium (2,23,47-49) may contribute to the development of chronic airway inflammation in COPD.

ROS

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Figure 6: Proposed model on the effect of cigarette smoke induced mitochondrial changes. Cigarette smoke is able to generate intracellular ROS production of which mitochondria are the main producers. ROS can damage important cellular components like nuclear DNA and organelles. Additionally, endogenous ROS affect mitochondrial DNA, membrane lipids and proteins, including fission/fusion proteins and PINK1, and induce oxidative modification or blocking of the OXPHOS system. Upon persistent mitochondrial damage or oxidative stress exceeding the anti-oxidant response, mitochondrial dysfunction will be introduced. Mitochondrial dysfunction and ROS from cigarette smoke may eventually induce inflammation, premature ageing and the onset of COPD.

In conclusion, our study demonstrates that long-term exposure to CSE leads to structural and functional changes in mitochondria that persist upon smoking cessation and may contribute to the onset pathogenesis of COPD. Herewith, we propose a new pathophysiological concept in the development of the COPD, which opens novel avenues for therapeutic strategies aimed towards the improvement of mitochondrial function and protection against ROS/damage.

AbbreviationsCSE, Cigarette smoke extract; Drp1, Dynamin-related protein 1; Fis1, Mitochondrial fission 1 protein; Mfn1 and Mfn2, Mitofussion1 and 2; Mn-SOD, Manganese Superoxide Dismutase; Opa1, Optic atrophy type 1; OXPHOS, Oxidative phosphorylation; PBEC, Primary Bronchial Epithelial Cells; PINK1, PTEN-induced putative kinase 1; PPARGC1α, Proliferator-activated receptor gamma co-activator 1-alfa; ROS, Reactive Oxygen Species; Tfam, Mitochondrial transcription factor A

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45. Sämann J, Hegermann J, Von Gromoff E, Eimer S, Baumeister R, Schmidt E: Caenorhabditits elegans LRK-1 and PINK-1 act antagonistically in stress response and neurite outgrowth. The J Biol Chem 2009, 284:16482–91.

46. Henry-Mowatt J, Dive C, Martinou J-C, James D: Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 2004, 23:2850–60.

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47. White SR: Apoptosis and the airway epithelium. Journal of allergy 2011, 2011:948406.

48. Slebos D-J, Ryter SW, Van der Toorn M, Liu F, Guo F, Baty CJ, et al: Mitochondrial localization and function of heme oxygenase-1 in cigarette smoke-induced cell death. Am J Respir Cell Mol Biol 2007, 36:409–17.

49. Aguilera-Aguirre L, Bacsi A, Saavedra-Molina A, Kurosky A, Sur S, Boldogh I: Mitochondrial dysfunction increases allergic airway inflammation. J Immunol (Baltimore, Md: 1950) 2009, 183:5379–87.

50. Lo Tam Loi AT, Hoonhorst SJM, Franciosi L, Bischoff R, Hoffmann RF, Heijink I, et al: Acute and chronic inflammatory responses induced by smoking in individuals susceptible and non-susceptible to development of COPD: from specific disease phenotyping towards novel therapy. Protocol of a cross-sectional study. BMJ Open. 2013; 3(2): e002178.52.

51. Santos JH, Mandavilli BS, Van Houten B: Measuring oxidative mtDNA damage and repair using quantitative PCR. Methods Mol Biol 2002, 197:159–176.

Supplemental Information

46

Chapter 2

Target Bronseq Fw primer Rev primer

RPLPO NM_001002 TCTACAACCCTGAAGTGCTTGATATC GCAGACAGACACTGGCAACATT

RPL13A NM_012423 CCTGGAGGAGAAGAGGAAAGAGA TTGAGGACCTCTGTGTATTTGTCAA

B2M ENSG00000166710 CTGTGCTCGCGCTACTCTCTCTTTGAGTAAACCTGAATCTTTGGAG-TACGC

GAPDH ENST00000229239 GCACCACCAACTGCTTAGCA TGGCAGTGATGGCATGGA

Cyclo NM_021130 CATCTGCACTGCCAAGACTGA TTCATGCCTTCTTTCACTTTGC

Actin NM_001101 AAGCCACCCCACTTCTCTCTAA AATGCTATCACCTCCCCTGTGT

Mfn1 ENSG00000171109 CTGAGGATGATTGTTAGCTCCACG CAGGCGAGCAAAAGTGGTAGC

Mfn2 ENSG00000116688 TGGACCACCAAGGCCAAGGA TCTCGCTGGCATGCTCCAC

Opa1 ENSG00000198836 TACCAAAGGCATTTTGTAGATTCT-GAGTT

GCATGCGCTGTATACGCCAA

Fis1 ENSG00000214253 CCTGGTGCGGAGCAAGTACAA TCCTTGCTCCCTTTGGGCAG

Drp1 ENSG00000087470 CGACTCATTAAATCATATTTTCT-CATTGTCAG

TGCATTACTGCCTTTGGCACACT

MTP18 ENSG00000242114 TCTCTACCGGGACACGTGGGT GAAAGCCTCGCCCACCTCA

PGC1a ENSMUSG00000029167 ATGCCTTTAGATGTGAGCTAACAG-TAGGTAATGA

CGTACAGCCATCAAAAAGGGACAC

Tfam ENSG00000108064 GAAAGATTCCAAGAAGCTAAGG-GTGATT

TCCAGTTTTCCTTTACAGTCT-TCAGCTTTT

Supplemental Figure 1. Primer sequences and RT-qPCR methods. RNA was extracted using TriReagent, Applied Biosystems/Ambion (Foster City, CA), reverse transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad, Herculus, CA). Primers (Sigma Genosys, Haverhill, UK) were designed using Primer Express 2.0 (Applied Biosystems, Foster City, CA). Real-time PCRs on Tfam, OPA1, Drp1, Fis1, Mfn1, Mfn2 were performed in a MyiQ single-color Real-Time thermal cycler (Bio-Rad, Herculus, CA). Normalization was performed to a GeNorm factor obtained from reference genes RPLPO, RPL13A, GAPDH, Cyclophilin and β-actin (Freeware, Gent University, Belgium) (31). IL-6, IL-8, IL-1β, histon deacetylase (HDAC)2, Mn-SOD, PINK1, PPARGC1α, OPA1 and TFAM expression was analyzed by real-time PCR using Taqman® (Applied Biosystems, Foster City, CA). Validated probes and housekeeping genes, B2M and PP1A and TaqMan Master Mix were purchased from Applied Biosystems. Amplification efficiencies were similar between the target and housekeeping genes. Samples were measured in duplicates, which never diverged more than 0.5 Ct.

47

Mitochondrial changes in lung epithelium

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2

Supplemental Figure 2. Image criteria; analysis of mitochondrial ultrastructure in in long-term CSE- exposed BEAS-2B cells. We studied mitochondrial ultrastructural changes in long-term (6 months) CSE exposed BEAS-2B cells at different CSE concentrations (0%, 1%, 2.5%, 10% and 12.5%). Additionally, we exposed cells to 10% CSE for 3 months followed by 3 months control conditions (-10%) in order to determine whether or not the induced changes are permanent. For electron microscopy analysis, mitochondria were randomly selected from cells with intact cytoplasm and nuclei. Other parameters that were taken into account include distribution of mitochondria, either in clusters or dispersed. Images were taken of 3-5 mitochondria in a clear visible field at different magnification (4500x, 17500x) to a total of 30-35 mitochondria per experimental condition. Shape was measured with respect to length, or in case of branching with bifurcating extensions and a third category of fragmented mitochondria. The mitochondrial matrix was distinguished on the basis of electron lucent “transparent matrix” or an electron dense “dark” lipoid matrix. Since most mitochondria will not be viewed in their full extent due to their unknown orientation, we have calculated the frequency of the cristae per unit length; these values were also significantly different from a random distribution (Table 2, last column). Because mitochondrial cristae are equally distributed in a randomly oriented cell, we compared categories with respectively 0, <3, 3-6 and more than 6 cristae. Representative images of the mitochondrial changes are shown, including increased branching, fragmentation, density of the mitochondrial matrix and cristae depletion.

48

Chapter 2

 

BEAS-2B Control BEAS-2B 2.5% CSE BEAS-2B 10% CSE A) B)

Control

2.5% C

SE

10% C

SE0

2

4

6

8

10de

lta C

t p21

Supplemental Figure 3. Long-term CSE-exposed BEAS-2B do not show abnormal morphology or senescence. BEAS-2B cells grown for 6 months in 0%, 2.5% or 10% CSE. A) Bright-field microscopy images of BEAS-2B cells cultured for 6 months in the absence (0%=control) or presence of 2.5% or 10% CSE showed no abnormal cell growth during cigarette smoke exposure. B) mRNA expression of the senescence marker p21 does not significantly differ between control- and long-term CSE exposed BEAS-2B cells. ΔCt values are shown (lower values reflect higher expression) and median interquartile ranges (IQR) are indicated

 

 

BEAS-2B 10% CSE

63 x 1.4 Leica AOBS 63 x 1.4 Leica AOBS

BEAS-2B 0% CSE

Supplemental Figure 4. Increased numbers of branched mitochondria in long-term CSE exposed BEAS-2B cells. BEAS-2B cells grown for 6 months in 10% CSE or 0% CSE (control). Cells were stained with Mitotracker DeepRed and (3D) images were taken on a Leica AOBS confocal microscope.

49

Mitochondrial changes in lung epithelium

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2

 

Supplemental Figure 5. Oxidative stress induces mtDNA damage in BEAS-2B. BEAS-2B were untreated (Basal) or treated with hydrogen peroxide (25, 50 and 100 µM), gas phase (Somborac-Bacura et al. Exp Physiol. 2013) cigarette smoke (CS: 30s , 45s and 60s) or cigarette smoke extract (CSE: 25%, 50%, 75% and 100%) for 5 hours. After total DNA isolation, two mitochondrial fragments were amplified using PCR and the short fragment (220bp) was used for normalization of mtDNA, and the large fragment (8,9 kb) was used for quantification of mtDNA damage upon analysis by gel electrophoresis (51). A dose-dependent damage of mtDNA after short-term treatment with hydrogen peroxide, gas-phase CS and CSE was observed.

 

Bas2.5

%10

%12

.5%0

5

10

15PINK1

delta

Ct

Supplemental Figure 6. PINK1 expression is not altered in long-term CSE-exposed BEAS-2B cells . BEAS-2B cells grown for 6 months in 0%, 2.5% or 10% CSE. mRNA expression of PINK1 IL8 (n=4) mRNA expression was detected by qPCR and related to the housekeeping genes. ΔCt values are shown (lower values reflect higher expression) and median interquartile ranges (IQR) are indicated.

CHAPTER 3

Glycogen synthase kinase-3β modulation of

glucocorticoid responsiveness in COPD

Anta Ngkelo*, Roland F Hoffmann*, Andrew L Durham, John A Marwick, Simone M

Brandenburg, Harold G de Bruin, Marnix R Jonker, Eleni Tsitsiou, Gaetano Caramori, Marco

Contoli, Paolo Casolari, Francesco Monaco, Filippo Andò, Giuseppe Speciale, Iain Kilty, Kian F

Chung, Alberto Papi, Mark A Lindsay, Nick HT ten Hacken, Maarten van den Berge, Wim Timens,

Peter J Barnes Antoon J van Oosterhout, Ian M Adcock*, Paul A Kirkham*, Irene H Heijink**These authors contributed equally to this work

Ngkelo, Hoffmann et al. AJP-lung. 2015

52

Chapter 3

AbstractIn Chronic Obstructive Pulmonary Disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. GSK3β inactivation plays a key role in mediating signalling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3β is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of p-GSK3β-ser9, a marker of GSK3β inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers and non-smokers. We observed increased levels of p-GSK3β-ser9 in monocytes, alveolar macrophages and bronchial epithelial cells from COPD patients and control smokers compared to non-smokers. Pharmacological inactivation of GSK3β did not affect CXCL8 or GM-CSF expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3β inactivation is a common effector of oxidative stress induced activation of the MEK/ERK-1/2 and PI3K/Akt signalling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3β inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3β, acting as a ROS-sensitive hub.

Chapter

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53

Effect of GSK3β on glucocorticoid function in COPD

IntroductionCOPD is characterized by chronic lung inflammation, airway remodelling and pulmonary emphysema, which leads to airflow limitation and accelerated lung function decline. Current therapies fail to prevent either disease progression or mortality. Glucocorticoids are widely used because of their broad anti-inflammatory effects, but they provide relatively little therapeutic benefit in COPD (3). The reduced responsiveness to the anti-inflammatory effects of glucocorticoids is a major barrier to effective management of COPD patients. Therefore, there is an urgent need to understand the underlying molecular mechanisms. The increased oxidant burden in the lungs of COPD patients, derived from cigarette smoke (CS) and the respiratory burst from inflammatory cells, plays a significant role in the reduced glucocorticoid responsiveness (4, 10, 25, 33, 41). Oxidative stress has a profound impact on inflammation by inducing pro-inflammatory mediators that attract and activate neutrophils, including CXCL8 and GM-CSF. This induction is driven by activation of redox sensitive kinase pathways (including MAPK and PI3K/Akt signalling) and pro-inflammatory transcription factors such as the nuclear factor-κB (NF-κB) (8, 26, 27, 31, 32, 37). In addition, oxidative stress can induce PI3K-dependent post-translational histone deacetylase 2 (HDAC2) modifications, including phosphorylation, resulting in proteasomal HDAC2 degradation (1, 22, 27, 33). HDAC2 can deacetylate the glucocorticoid receptor (GRα) as well as histones at NF-κB response elements within promoter regions of inflammatory genes (21–23). Reduced HDAC2 expression has been observed in the lungs and alveolar macrophages of COPD patients and has been implicated in glucocorticoid insensitivity in COPD (22, 23). In addition to alveolar macrophages, we recently observed that bronchial epithelial cells from COPD patients are less responsive to glucocorticoids than those from healthy controls (16). Here, pro-inflammatory cytokine production was effectively suppressed by glucocorticoids in cells from healthy controls, while this response was compromised in COPD-derived cells.The constitutively active serine/threonine kinase glycogen synthase 3β (GSK3β) is regulated by oxidative stress and has been linked to several inflammatory diseases (6, 20, 24). GSK3β activity is negatively regulated by phosphorylation on serine 9, which can be mediated by ERK1/2 MAPK and Akt (15). As these kinase pathways are commonly involved in oxidant-mediated responses, GSK3β may represent an important downstream effector of oxidant-mediated signalling during COPD inflammation. We hypothesized that GSK3β is involved in oxidative stress-induced glucocorticoid responsiveness in COPD. We demonstrate that levels of inactive GSK3β are enhanced in monocytes, macrophages and bronchial epithelial cells from COPD patients compared to smokers and non-smokers, and that oxidative stress-induced GSK3β inhibition regulates glucocorticoid responsiveness in both monocytes/macrophages and bronchial epithelial cells.

54

Chapter 3

Methods

Human studiesPeripheral lung sections, peripheral venous blood, primary bronchial epithelial cells (PBECs) and tissue sections were isolated from age-matched non-smokers, smokers with normal lung function and COPD patients. For GSK3β phosphorylation and total staining analysis, peripheral lung sections were collected from 21 patients with COPD, 19 smokers, and 14 non-smokers subjects (Table 1) and from 12 COPD patients, 12 smokers and 10 non-smokers (Table 2) respectively. Phospho-GSK3β was also detected in tissue macrophages of severe COPD patients (Table 1). Peripheral venous blood was collected from 10 patients with COPD, 6 smokers with normal lung function and 7 non-smokers (Table 3). Primary bronchial epithelial cells (PBECs) were obtained from 14 current and ex-smoking COPD patients with GOLD stage II-IV, 16 age-matched control smokers and 14 non-smokers (Table 4)(17). With the exception of the COPD stage IV patients, subjects did not use ICS, long-acting β-agonists and long-acting anticholinergics for at least 4 weeks preceding the study. The study protocol for this part was consistent with the Research Code of the University Medical Center Groningen (http://www.rug.nl/umcg/onderzoek/researchcode/index) and national ethical and professional guidelines (“Code of conduct; Dutch federation of biomedical scientific societies”; htttp://www.federa.org).

Table 1. Characteristics of subjects for the immunohistochemical study of phospho-GS3Kβ

Non-Smokers Smokers COPD Severe COPD

Age 67.7± 8.1 70.0± 6.7 69.1± 6.6 70.3 ± 2.8

Sex (M/F) 0/14 18/1 18/3 7/0

Current/former smokers N/A 9/10 7/14 3/4

Pack years N/A 49.4± 32.3 40.5± 20.1 50.6 ± 11.6

FEV1 (L) 2.1± 0.4 2.5± 0.7 2.03± 0.5 1.13 ± 0.073

FEV1 (% pred) 101.5± 22.5 91.8± 14.6 75.3± 16.6 41.3 ± 3.0

FEV1/FVC Ratio (%) 76.4± 3.5 75.5± 4.6 56.1± 9.1 51.7 ± 4.9

GOLD Stage N/A N/A 8 grade 1,11 grade 2,2 grade 3

All grade 3

Peripheral lung tissue sections were collected from patients recruited from the Section of Respiratory Diseases of the University Hospital of Ferrara. Data are presented as means ± SDs; FEV

1: Forced expiratory volume in one

second; FVC: Forced vital capacity. The FEV1/FVC ratio is after bronchodilator for subjects with COPD but not for

smokers or non-smokers; GOLD, Global Initiative for Chronic Obstructive Lung Disease guideline classification of patients with COPD; % pred: % predicted; M: Male, F: Female.

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55

Effect of GSK3β on glucocorticoid function in COPD

Table 2. Characteristics of subjects for the immunohistochemical study of total GS3Kβ

Non-smokers Control smokers COPD

Age 69.1 ±2.5 65.4 ±1.9 69.5±2.1

Sex (M/F) 2/8 12/0 12/0

Current/former smokers N/A 6/6 7/5

Pack-years N/A 49.1±12.1 37.9±3.3

FEV1 (% pred) 111.2±6.2 90.5±4.8 70.4±3.8

FEV1/FVC ratio (%) 78.1±1.4 76.8±1.2 59.4±2.1

Peripheral lung tissue sections were collected from patients recruited from the Section of Respiratory Diseases of the University Hospital of Ferrara. Data are presented as means ± SDs; FEV

1: Forced expiratory volume in one

second; FVC: Forced vital capacity. The FEV1/FVC ratio is after bronchodilator for subjects with COPD but not for

smokers or non-smokers; % pred: % predicted; M: Male, F: Female.

Table 3. Characteristics of subjects: peripheral blood monocytes

Non-Smokers Controls smokers COPD

Age 57 ± 4.4 55.5 ± 3.2 65.6 ± 4.4

Sex M/F 3/4 4/2 8/2

Current/former smokers N/A 5/1 2/8

Pack years N/A 31 ± 12.3 33.1 ± 14.2

FEV1 (% pred) 105.6 ± 7.5 92.2 ± 14.5 68.65 ± 16

FEV1/FVC Ratio 72.54 ± 3.9 73.02 ± 8.8 58.79 ± 15.7

Peripheral venous blood was collected from patients at the Royal Brompton hospital of London. Data are presented as means ± SD; FEV

1: Forced expiratory volume in one second; FVC: Forced vital capacity. The FEV

1/FVC ratio is after

bronchodilator for subjects with COPD but not for smokers or non-smokers; % pred: % predicted. M: Male, F: Female.

Table 4. Characteristics of the subjects: primary bronchial epithelial cells (PBECs)

Non-Smokersn=11*

Control smokers n=12*

COPDn=8*

Age 56 (43-76) 54 (43-70) 56.5(50-65)

Sex M/F 5/6 9/3 4/4

Pack years 0(0-0) 39.5 (19-50) 33.5(11-54)

Medians (range) or number*All experimental controls included

Primary bronchial epithelial cells included in the study were obtained from Lonza or the NORM and TIP study within the University Medical Center Groningen. COPD patients were included on a basis of FEV

1<50% of predicted,

FEV1/FVC<70% and ≥10 pack-years for GOLD stage IV. All control subjects had FEV

1/FVC>70% and FEV

1>90% of

predicted. PBECs obtained from Lonza are not indicated with FEV1/FVC and FEV

1 predicted nor pack-years.

56

Chapter 3

All participants gave informed consent to a protocol approved by the ethics committee of the Royal Brompton and Harefield NHS Trust/National Heart and Lung Institute and the University Medical Center Groningen. The Section of Respiratory Disease at the University Hospital of Ferrara and the Pneumology Unit at the University Hospital of Messina have ethics committee approval for the collection and analysis of specimens from lung resection surgery.

Cell culturePeripheral blood mononuclear cells (PBMCs) were isolated from whole venous blood by Histopaque (Sigma, Dorset, UK), as previously described (27). Monocytes were isolated from PBMCs by MACS with the Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany). PBECs were cultured in bronchial epithelium growth medium (BEGM, Lonza) in flasks coated with collagen and fibronectin as described previously (19). Human bronchial epithelial cells (16HBE) were kindly provided by Dr. G. Gruenert and grown in EMEM/10% FCS (UCSF, USA) (18).

Cell culture and treatmentsIsolated monocytes and human monocyte-macrophage cells (MonoMac6) were cultured in RPMI 1640 GlutaMAX phenol red free media (Invitrogen, Paisley, UK) with 1% FCS, 2mM L-glutamine, 1% nonessential amino acids and 1% sodium pyruvate. Primary monocytes were pre-treated with U0126 (MEK/ERK-1/2 inhibitor), MK-2206 (Akt inhibitor) or IC87114 (PI3K-δ inhibitor) all at 1µM for 30 minutes and were stimulated with H

2O

2 (100µM) for 30

minutes. Primary monocytes were pre-treated with the GSK3β inhibitor CT99021 (100 nM and 1 µM) for 15-120 minutes as indicated. To study the function of glucocorticoids, primary monocytes or transfected MonoMac6 cells were pre-treated with dexamethasone (10nM, 100nM, 1µM) for 30 minutes before being stimulated with LPS (10ng/ml) for 16 hours. Cells or cell-free supernatants were harvested for RNA isolation, cell lysate preparation or measurement of cytokines.PBECs were cultured for at least 3 weeks and used at passage 3. PBECs and 16HBE cells were passaged by trypsin, plated in 24-well plates and grown to ~90% confluence. Subsequently, PBECS were hormone/growth factor-deprived using basal medium (BEBM, Lonza, Breda, The Netherlands) supplemented with transferrin and insulin (PBECs) and 16HBE were serum-deprived overnight.PBECs and 16HBE cells were pre-treated with or without 7.5% cigarette smoke extract (CSE) for 6 hours. NAC (5mM) was added 90 min prior to CSE exposure. CT99201 (10µM) was added 30 min prior to CSE treatment for 6 hours or budesonide (1, 10 and 100nM) treatment for 2 hours and cells were subsequently stimulated with TNF-α (10ng/ml) for 24 hours. Cells or cell-free supernatants were harvested for RNA isolation, cell lysate preparation or measurement of cytokines.

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Effect of GSK3β on glucocorticoid function in COPD

TransfectionsGSK3β on-target siRNA (Dharmacon, Colorado, USA) was used according to the manufacturer’s instructions. The HA-GSK3β-S9A-pcDNA3 and HA-GSK3β-K85A-pcDNA3 expression vectors were kindly provided by Dr. J. Woodget (Toronto, Canada). The following plasmids were obtained from Addgene: plasmid 14754 - GSK3β S9A mutant pcDNA3 (36); Addgene plasmid 14755 - GSK3β K85A mutant pcDNA3.1 or the negative control pcDNA3 construct lacking the GSK3β insert (36).

Cigarette smoke extract (CSE)Two 3R4F research cigarettes (Tobacco Research & Development Center, Lexington, KY) bubbled at 70 rpm through 25 ml EMEM, using a high flow peristaltic pump (Watson Barlow, Rotterdam, the Netherlands) represents 100% CSE. The extract was prepared freshly for each experiment.

RT-qPCRRNA was isolated and HO-1 mRNA expression was analyzed by real-time PCR using Taqman® (Applied Biosystems, Foster City, CA) as described (16). Validated probe and housekeeping genes, β-2-microglobulin (B2M) and peptidylprolyl isomerase A (PP1A) and TaqMan Master Mix were purchased from Applied Biosystems.

Lung tissue processing and immunohistochemistryLung tissue processing and immunohistochemistry were performed as previously described (44). The anti-p-GSK3β-ser9 (sc-11757-R) and anti-total GSK3β (sc-9166) antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Biotinylated horse anti-rabbit IgG secondary antibody was used (Vector BA 1000) at 1:200 and staining revealed using a Vectastain ABC kit (Vector, PK-6100) according to the manufacturer’s instructions.

HDAC2 activity assay Cell lysates were prepared and subjected to HDAC immunoprecipitation as previously described (28). Immunoprecipitation was conducted with anti-HDAC2 antibody (Sigma, UK). HDAC activity in the immunoprecipitates was assessed using a fluorometric assay kit (Biovision, Mountain View, CA). Phosphorylated levels of HDAC2 were measured using an anti-p-Ser394-HDAC2 (Abcam, Cambridge, UK).

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Western BlotWhole cell lysates were subjected to western blotting as previously described (28). p-Ser473-Akt, GSK3β, p-GSK3β-ser9, ERK1/2 and p-ERK1/2 antibodies were purchased from Cell Signalling Technology (Herts, UK). Anti-human β-actin was obtained from Santa Cruz Biotechnology and anti-human GAPDH antibody from Abcam (Cambridge, UK).

GM-CSF and CXCL8 cytokine releaseLevels of GM-CSF, and CXCL8 were analysed in cell-free supernatants by sandwich ELISA (R&D Systems, Abingdon, UK) according to the manufacturer’s instructions.

Genome wide mRNA expression profileMonocytes were treated with CT99021 (1 µM), dexamethasone (10-8 M, 30 min), and LPS (10 ng/ml) as described above and RNA (0.5 µg) was extracted using the RNeasy Mini Kit (Qiagen, Crawley, UK). The mRNA expression profile was determined using the Agilent SurePrint G3 Human microarrays v2 following the manufacturer’s instructions. Differential gene expression was determined using the Partek Genomics Suite using a false discover rate <0.05. Differences> 1.2- fold on mRNA expression were taken into consideration for our analysis. Gene sets significantly enriched in Partek were transferred to the Database for Annotation, Visualisation and Integrated Discovery (DAVID) version 6.7 (http://david.abcc.ncifcrf.gov/). Pathway analysis was performed by Kyoto Encyclopaedia of Genes and Genomes (KEGG).

Statistical analysisData were analysed by Friedman or Kruskal-Wallis ANOVA and the Mann-Whitney test to determine statistical significance of non-parametric data. For parametric data, ANOVA and Dunnett’s post-test were used for tests between groups and the student’s t-test was used for tests within groups.

Chapter

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59

Effect of GSK3β on glucocorticoid function in COPD

ResultsIncreased P-GSK3β-ser9 levels in COPD alveolar macrophages, monocytes and bronchial epithelial cellsWe first assessed the levels of phosphorylated/inactive GSK3β in peripheral lung tissue, peripheral blood monocytes and PBECs from COPD patients, non-smokers and smokers with normal lung function. Phospho-GSK3β-Ser9 staining was higher in lung tissue macrophages of COPD patients (70.5±4.1% positive) than in control smokers (46.5±6.2, p<0.01) and non-smokers (19.3±3.8, p<0.001) and was also higher in control smokers than non-smokers (Fig. 1a and 1b). The levels of phospho-GSK3β staining were even higher in macrophages (84.9±9.8% positive, p<0.01) from severe COPD compared to moderate COPD patients (Fig. 1b). We were unable to obtain sufficient BAL samples to confirm these results in BAL macrophages using Western blotting. The ratio of phosphorylated to total GSK3β was increased in peripheral blood monocytes from COPD patients compared to healthy subjects with no smoking history as determined by Western blot analysis (0.81±0.17 vs 3.48±0.74, p<0.05)(Fig. 1c). Furthermore, phospho-GSK3β-Ser9 levels were significantly increased in PBECs from COPD stage GOLD IV patients (1.57±0.10, p<0.05) and control smokers (1.57±0.10, p<0.05) compared to non-smoking individuals (1.05±0.05) (Fig. 1d) without significant differences in total GSK3β levels (Fig. 1e). Immunohistochemical staining also indicated strong phospho-GSK3β-Ser9 staining in bronchial epithelium of smokers with and without COPD, although all bronchial epithelial cells in lung tissue stained positive for phospho-GSK3β-Ser9 and we did not detect clear differences compared to non-smoking controls using this method (Fig. 1f ). Immunostaining for total GSK3β, detecting both phosphorylated and non-phosphorylated forms, indicated similar, albeit less strong, increases in staining in macrophages from smokers with and without COPD, without a significant difference between these two groups (Figs. 1g, h). All bronchial epithelial cells were stained, and we did not observe differences between the groups (Fig. 1g). Having shown inactivation of GSK3β in different airway cells and blood monocytes in COPD patients, we examined the functional consequences of this altered activation state in monocytes/macrophages and airway epithelial cells.

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Chapter 3

Non-smokers Smokers COPD Severe COPD0

20

40

60

80

100

** p=0.0029

*** p<0.0001

** p=0.0053

*** p<0.0001

** p=0.0019

% o

f P-G

SK3

(ser

9) +

vePe

riph

eral

Lun

g M

acro

phag

es

Non-smokers Smokers COPD 0

1

2

3** p=0.0014

** p=0.0031

P-G

SK3

(ser

9)fo

ld in

crea

se in

PB

EC

s

A Macrophages

Non-smokers Smokers COPD0

2

4

6

8 *p=0.013

*p=0.03

P-G

SK3

(ser

9)fo

ld in

crea

se in

Mon

ocyt

es

Non-Smoker

Smoker COPD

Isotype control

p-GSK3β-ser9

GAPDH

B

Monocytes

Epithelial cellsMacrophages D

CSmokers

P-GSK3β (Ser9)Total GSK3ββ-actin

Non-smokers COPD

Smokers COPDNon-smokers

x1000 x1000 x1000

x1000x1000x1000

Non-smokers smokers COPD 0

1

2

3

Tot

al G

SK3

in P

BE

Cs

Total GSK3β

GAPDH

E

F

G Epithelial cells

Macrophages

Epithelial cells

H

Smoker COPD

Non-smoker

Epithelial cells

Isotype control

Chapter

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61

Effect of GSK3β on glucocorticoid function in COPD

Figure 1. P-GSK3β-ser9 levels are elevated in peripheral lung alveolar macrophages, peripheral blood monocytes and primary bronchial epithelial cells (PBECs) from COPD patients. Representative images (a) and percentage of macrophages (b) positively stained for p-GSK3β-ser9 in peripheral lung sections from non-smokers (n=14), smokers (n=19), mild-moderate COPD (n=21) and severe COPD patients (n=7). Ratio of p-GSK3β/total GSK3β in primary monocytes (n=6-10) with representative blots (c) and p-GSK3β/GAPDH (d) and total GSK3β/GAPDH (e) with representative blots in PBECs from non-smokers, smokers and GOLD stage IV COPD patients (n=7-8). Representative images of staining of p-GSK3β in large airway epithelial cells in peripheral lung sections from non-smokers, smokers and COPD patients are shown beneath (f ) along with an isotype-stained section as a control. (g) Total GSK3β staining in macrophages of COPD subjects and controls. (h) Percentage of GSK3β-positive macrophages. *=p<0.05 and **=p<0.01 as tested by Kruskal-Wallis ANOVA.

GSK3β inhibition abrogates glucocorticoid responsiveness in primary human blood monocytes We next investigated whether GSK3β inactivation affects the regulation of inflammatory cytokines. Treatment of monocytes from healthy subjects with the selective GSK3β inhibitor CT99021 had no effect on basal GM-CSF and CXCL8 release, nor on the release upon treatment with the pro-inflammatory stimulus LPS (Fig. 2a and 2b).Previously, we showed that the glucocorticoid dexamethasone was less effective at repressing LPS-induced GM-CSF and CXCL8 release in blood monocytes from patients with COPD compared with age-matched smokers (27). Therefore, we also investigated the effect of GSK3β inactivation on glucocorticoid responsiveness upon LPS stimulation in monocytes. Of interest, dexamethasone (10nM) dependent inhibition of LPS-induced GM-CSF (63.9±6.7%, p<0.05) and CXCL8 (24.42±6.5%, p<0.05) release was completely abrogated

by CT99021 (Fig. 2c and 2d).

Oxidative stress induces PI3K/Akt-dependent inhibition of GSK3β activity in monocytes Because of our hypothesis that oxidative stress induces glucocorticoid responsiveness, we next examined how GSK3β activity is modulated in response to exogenous reactive oxygen species (ROS) in primary monocytes, as both hydrogen peroxide (H

2O

2) and cigarette smoke

extract (CSE) exposure reduce glucocorticoid sensitivity in monocytes (7, 30, 31). H2O

2

increased phospho-GSK3β-ser9 levels in a time-dependent manner (Fig. 3a). Exposure to H

2O

2 also activated PI3K as measured by increased levels of phospho-Akt-Ser473, at earlier

time points than GSK3β-ser9 phosphorylation (Fig. 3a). Inhibition of Akt (MK-2206) reversed the oxidant-induced inactivation of GSK3β (Fig. 3b). Similarly, GSK3β phosphorylation was reduced by the MEK/ERK-1/2 inhibitor U0126, indicating involvement of the ERK1/2 pathway in the oxidant-induced effect on GSK3β in primary monocytes (Fig. 3a and 3b). We previously showed that PI3Kδ is responsible for the oxidant-induced activation of Akt in monocytes (27). However, selective inhibition of PI3Kδ with IC87114 did not affect oxidant

62

Chapter 3

induction of phospho-GSK3β-ser9 in monocytes (Fig. 3b), indicating involvement of other PI3K isoforms or signaling mediators.

+ + +0

50

100

150

200

** p=0.001

*** p=0.0006

GM

-CSF

rel

ease

(% o

f con

trol

)

+ + +

- + + - - +

LPS (10ng/ml)

CT99021 (1µM)Dex (10nM)

0

50

100

150

200

** p=0.003

**p=0.008

CX

CL

8 re

leas

e (%

con

trol

)

+ + +

- + + - - +

LPS (10ng/ml)

CT99021 (1µM)Dex (10nM)

- - + +- + - +

LPS (10ng/ml)CT99021 (1µM)

- - + +- + - +

LPS (10ng/ml)CT99021 (1µM)

0

200

400

600

800 *p=0.015

GM

-CSF

(pg/

ml)

0

5000

10000

15000 **p=0.002

CX

CL

8 (p

g/m

l)

A C

B D

Figure 2. GSK3β inhibition attenuates the anti-inflammatory action of glucocorticoids in monocytes. CT99021 does not affect baseline or LPS-induced GM-CSF or CXCL8 secretion in primary monocytes from non-COPD individuals. GM-CSF (a) and CXCL8 (b) levels were measured in supernatants of primary monocytes upon pre-treatment with CT99021 for 30 minutes followed by 24 hours of LPS. Treatment of monocytes isolated from healthy subjects with CT99021 inhibits dexamethasone-induced suppression of LPS-stimulated (c) GM-CSF and (d) CXCL8 release (mean±SEM, n=6-7). P values are indicated.

GSK3β protein knockdown and overexpression of inactive GSK3β reduces glucocorticoid function in monocytesTo gain further mechanistic insight in the role of GSK3β in oxidant-induced glucocorticoid unresponsiveness in monocytes, we used siRNA to knock-down total GSK3β levels and analyse the anti-inflammatory actions of dexamethasone. Transfection of MonoMac6 cells with GSK3β on-target siRNA significantly reduced GSK3β total protein levels compared with scrambled control siRNA (Fig. 4a). In line with the CT99021 effect, knockdown of GSK3β significantly inhibited the ability of dexamethasone to suppress CXCL8 expression, and the dexamethasone EC

50 was increased from 22 to 100nM. In addition, the inhibitory effect of

dexamethasone on LPS-stimulated CXCL8 expression was decreased from 52.5±4.8% to 75.8±7.1% (Fig. 4b, left panel). Similar effects were seen with GSK3β knockdown on the dexamethasone suppression of LPS-induced GM-CSF secretion (Fig. 4b, right panel).

Chapter

3

63

Effect of GSK3β on glucocorticoid function in COPD

p-GSK3β-ser9

Total GSK3β

p-ERK1/2 -thr202/tyr204

Total ERK1/2

0 5 10 15 30 60 min (100M H2O2)

p-Akt-ser473

Total Akt

p-GSK3β-ser9

Total GSK3β

- + + + + H2O2 (100µM) - - MEK AKT PI3K-δ Inhibitor

NS H2O2 +MEK +AKT +ICOS0

1

2

3

4* p=0.01

* p=0.008

* p=0.008

p-Se

r9 /

tota

l GSK

3 fo

ld in

crea

se

- + + + + - - + - -- - - + -- - - - +

H2O2 (100µM)U0126 (1µM)

IC87114 (1µM)MK-2206 (1µM)

A

B

Figure 3. Oxidative stress induced inactivation of GSK3β is mediated via PI3K/Akt in primary monocytes. (a) Primary monocytes from non-COPD individuals were exposed to H

2O

2, which time-dependent increase in

PI3K, ERK1/2 and GSK3β phosphorylation as detected by western blotting. Representative blots of 4 independent experiments are shown. (b) GSK3β phosphorylation is PI3K/Akt and ERK1/2-dependent in monocytes as indicated by pre-treatment of the cells with MEK/ERK-1/2 inhibitor U0126, the Akt inhibitor MK-2206 and PI3Kδ inhibitor IC87114. Densitometry was performed and phospho-ser9-GSK3β levels are expressed as ratio of total GSK3β (mean±SEM, n=5).

To validate our findings, we overexpressed the K85A kinase dead GSK3β mutant and analysed dexamethasone function. MonoMac6 and primary cells produce similar levels of inflammatory mediators following stimulation with LPS (Fig. 4c, left panel). Dexamethasone significantly reduced LPS-induced GM-CSF release by 33.1±2.8% in MonoMac6 cells transfected with the control pcDNA3.1. Dexamethasone had no significant inhibitory effect on LPS-induced GM-CSF release when the K85A GSK3β mutant was overexpressed (91.3± 12.2% versus 111.4±15.5%, Fig. 4c).

64

Chapter 3

-LP

S-

LPS

0

500

1000

1500

GM-CSF release (pg/ml)

**p=0

,007

*p=0

,01

Moc

k -v

e pc

DN

A3.1

-13-12-11-10

-9-8

-7-6

020406080100

120

140

GM-CSF release(% of LPS induced release)

050100

150

Scra

mbl

e co

ntro

lsi

RN

A 2

00nM

GSK

3ßsi

RN

A 2

00nM

Gsk3 protein levels/-actin(% of scramble)

** P

=0.0

07

GSK

3ββ-actin

Con

trol

siR

NA

GSK

3βsi

RN

A20

0

4

00

200

400

nMsi

RN

A

050100

150

* p=0

.03

ns

GM-CSF (% of control)

+

+

+

+

-+

-

+ D

ex(1

0nM

)L

PS (1

0ng/

ml)

-ve

cont

rol p

cDN

A3.

1K

85A

pcD

NA

3.1

GSK

3βsi

RN

A

Con

trol

siR

NA

-11

-10

020406080100

120

140

-9-8

-7-6

EC

50:2

2.3n

M

EC

50:1

00nM

dex

[M]

CXCL8 release(% of LPS induced release)

LPS

Dex

amet

haso

ne (l

og[M

])

406080100

120

*p=0

.02

ns

*p=0

.03

GM-CSF (% of control)

+

+

+

+

+

+

-+

-+

-+

--

+

+

+

+

LPS

(10n

g/m

l)D

ex(1

0nM

)H

2O2

(100

µM)

-ve

cont

rol p

cDN

A3.

1

S9A

pcD

NA

3.1

AC

BD

Dex

amet

haso

ne (l

og[M

])LP

S

Figu

re 4

. GSK

3β m

odul

ates

dex

amet

haso

ne fu

nctio

n in

Mon

oMac

6 ce

lls. (

a) G

SK3β

leve

ls a

re re

duce

d by

GSK

3β s

iRN

A a

fter

24

hour

s of

tran

sfec

tion.

Den

sito

met

ry

was

per

form

ed a

nd G

SK3β

leve

ls a

re e

xpre

ssed

as

ratio

of β

-act

in a

s lo

adin

g co

ntro

l. (b

) GSK

3β s

iRN

A k

nock

-dow

n (2

4 ho

urs)

inhi

bits

the

con

cent

ratio

n-de

pend

ent

supp

ress

ion

of L

PS-in

duce

d C

XCL8

(le

ft p

anel

) an

d G

M-C

SF (

right

pan

el)

rele

ase

by d

exam

etha

sone

(m

ean±

SEM

, n=

6). (

c) L

PS-in

duce

d G

M-C

SF r

elea

se i

n m

ock

tran

sfec

ted

mon

ocyt

es a

nd c

ells

tran

sfec

ted

with

the

posi

tive

cont

rol p

cDN

A3.

1 (p

g/m

l, mea

n±SE

M, n

=4)

, and

effe

ct o

f ove

rexp

ress

ion

of th

e in

activ

e m

utan

t GSK

3βK8

5A

on d

exam

etha

sone

sup

pres

sion

of L

PS-in

duce

d G

M-C

SF re

leas

e (%

, mea

n±SE

M, n

=4)

. (d)

Ove

rexp

ress

ion

of t

he c

onst

itutiv

ely

activ

e G

SK3β

S9A

mut

ant

rest

ores

H2O

2-in

duce

d de

xam

etha

sone

unr

espo

nsiv

enes

s of

GM

-CSF

rele

ase

(mea

n±SE

M, n

=4)

.

Chapter

3

65

Effect of GSK3β on glucocorticoid function in COPD

To confirm that GSK3β mediates oxidant-induced glucocorticoid insensitivity in monocytes, we overexpressed a constitutively active S9A GSK3β mutant in MonoMac6 cells and analysed dexamethasone function during H

2O

2 exposure. In line with previous studies (27),

the anti-inflammatory effect of dexamethasone was significantly attenuated by H2O

2 pre-

treatment (Fig. 4d). In the presence of the active S9A mutant, H2O

2-induced dexamethasone

insensitivity was suppressed, leading to a significant (27.2±1.3%) inhibition of LPS-stimulated GM-CSF release, similar to that observed in control cells (26.7±2.5% inhibition, Fig. 4d).

GSK3β-regulated glucocorticoid responsiveness is HDAC2-dependent in human monocytesSince HDAC2 has been implicated in oxidative stress-induced glucocorticoid unresponsiveness (4), we investigated whether ROS-induced inactivation of GSK3β may lead to modulation of HDAC2 activity in primary monocytes. Inhibition of GSK3β activity by treatment with CT99021 reduced the activity of immunoprecipitated HDAC2 (Fig. 5a). This reduction in HDAC2 activity correlated with increased phosphorylation of ser394 (Fig. 5b), while HDAC2 mRNA and protein levels were not affected by GSK3β inactivation (data not shown).

Effect of GSK3β inhibition on dexamethasone-regulated inflammatory gene expression in human monocytesWe also investigated the effect of CT99021 on dexamethasone regulation of LPS-induced gene expression using gene arrays. Partek analysis identified 17 genes that were differentially expressed (FDR <0.05) upon CT99021 exposure. CT99021 specifically affected genes involved in the Wnt/β-catenin signalling pathway (P=0.05) in LPS-stimulated MonoMac6 cells (data not shown), thereby confirming the specificity of CT99021 action. We next investigated the effect of CT99021 on LPS/dexamethasone-treated monocytes. 164 known genes were differentially expressed upon GSK3β inhibition in the presence of LPS/dexamethasone (P<0.05). KEGG analysis showed key pathways that these genes regulate (Table 5). 13 out of the 164 genes encode for inflammatory chemokines (such as CXCL6, CXCL3, CXCL2 and CXCL1), cytokines (such as GM-CSF, G-CSF) and cytokine/chemokine receptors involved in the cytokine-cytokine receptor interaction pathway (P=2.4×10-5). 10 more genes encode for proteins involved in chemokine signalling pathways (P=2×10-4) and 7 genes encode for proteins that regulate the neuro-active receptor-ligand interaction (P=6.5×10-2).

66

Chapter 3

- 100nM 1M

0

50

100

150

CT99021(30 min)

* p=0.03IP

- HD

AC

2 ac

tivi

ty/ W

B(%

con

trol

)

- 15 30 60 1200.0

0.5

1.0

1.5

2.0

2.5

CT990211(1M)

*p=0.011

time (minutes)

P-se

r394

HD

AC

2/ to

tal H

DA

C2

fold

incr

ease

p- HDAC2 (Ser394)

Total HDAC2

0 15 30 60 120 (min) CT99021 (1µM) B

A

Figure 5. GSK3β-regulated glucocorticoid function is HDAC2-dependent in monocytes. (a) Treatment of primary monocytes with CT99021 inhibits the enzymatic activity of HDAC2 (mean±SEM, n=4) (b) CT99021 treatment induces p-HDAC2-ser394 in primary monocytes. Densitometry was performed and phospho-HDAC2 levels are expressed as ratio of total HDAC (mean±SEM, n=4).

Table 5. Pathways affected by differential gene expression in response to CT99021 treatment in MonoMac6 cells.

Category TermGenes

(out of 164)% of total number

genesP value

KEGG_PATHWAYCytokine-cytokine receptor

interaction13 7.9% 2.4E-5

KEGG_PATHWAY Chemokine signaling pathway 10 6.1% 2.0E-4

KEGG_PATHWAYNeuroactive ligand-receptor

interaction7 4.2% 6.5E-2

KEGG analysis showed 164 known genes that were differentially expressed due to GSK3β inhibition in the presence of LPS/dexamethasone. 13 out of the 164 genes encode for inflammatory chemokines. 10 genes encode for proteins involved in chemokine signalling pathways and 7 genes encode for proteins that regulate the neuro-active receptor-ligand interaction.

Chapter

3

67

Effect of GSK3β on glucocorticoid function in COPD

GSK3β inhibition abrogates glucocorticoid responsiveness in primary human bronchial epithelial cellsWe previously reported that TNF-α-induced GM-CSF production in PBECs from GOLD stage II COPD patients was less responsive to the clinically used inhaled glucocorticoid budesonide compared to non-smoking controls, with an intermediate effect of budesonide in PBECs from control smokers (16). In a similar manner to monocytes, CT99021 had no effect on GM-CSF and CXCL8 release in PBECs from non-COPD individuals at baseline or upon stimulation with TNF-α, a relevant mediator of inflammation in COPD (Fig. 6a and 6b). In further line with our findings in monocytes, pre-treatment of PBECs from non-COPD individuals with CT99021 resulted in complete abrogation of the anti-inflammatory effect of budesonide on TNF-α-stimulated GM-CSF and CXCL8 release (Fig. 6c and 6d).

Oxidative stress leads to reduced glucocorticoid responsiveness, activation of the PI3K/Akt pathway and inhibition of GSK3β activity in bronchial epithelial cellsBecause of the limited cell numbers of primary cultures, further mechanistic studies were performed in the human bronchial cell line 16HBE. Bronchial epithelial cells are in direct contact with inhaled cigarette smoke, which known to induce oxidative stress in these cells (4, 33, 41). Therefore, we studied the effect of CSE, which is known to exert similar effects in 16HBE and PBECs at least in part through oxidative stress mechanisms (18). Exposure of 16HBE cells to CSE (7.5%) for 6 hours led to a significant up-regulation of heme-oxygenase-1 (HO-1) mRNA, a marker of oxidative stress, which was blocked by the oxidant scavenger N-acetyl-cysteine (NAC), confirming that CSE exposure induces oxidative stress in epithelial cells (data not shown). In addition, CSE exposure reduced the ability of budesonide to suppress TNF-α-induced CXCL8 release (Fig. 7a and 7b), which was reversed by NAC, confirming the involvement of oxidative stress (Fig. 7c). Similar to the effects of oxidative stress in monocytes, CSE (7.5%) induced activation of PI3K/Akt signaling and inactivation of GSK3β in 16HBE, which was also reversed by NAC although this did not reach significance (p=0.0692)(Fig. 7d). Together, our data show that CSE-derived ROS result in GSK3β inactivation in bronchial epithelial cells and that GSK3β inhibition reduces glucocorticoid sensitivity of pro-inflammatory responses in both human monocytes and bronchial epithelial cells.

68

Chapter 3

TNF

TNF+

BU

DC

T+TN

F+B

UD

050100

150

***

p=0.

0008

** p

=0.0

011

CXCL8 release (% control)

--

+

+

-+

-+

TN

FT

NF+

BU

DC

T+T

NF+

BU

D050100

150

***

p=0.

0007

***

p=0.

0006

GM-CSF release(% of control)

+

+

+

-+

+

--

+

TNF-α

(10n

g/m

l)

CT9

9021

(10µ

M)

Bud

(10n

M)

TN

F-α

(10n

g/m

l)

CT

9902

1 (1

0µM

)B

ud (1

0nM

)

+

+

+

-

+

+

-

-+

T

NF-α

(10n

g/m

l)

CT

9902

1 (1

0µM

)B

ud (1

0nM

)

TN

F-α

(10n

g/m

l)C

T99

021

(10µ

M)

--

+

+

TN

F-α

(10n

g/m

l)C

T99

021

(10µ

M)

-+

-

+

0

1000

2000

3000

*p=0

.041

CLCX8 (pg/ml)0

200

400

600

**p=

0.00

47

GM-CSF (pg/ml)

BAC

D

Figu

re 6

. GSK

3β in

hibi

tion

atte

nuat

es th

e an

ti-in

flam

mat

ory

actio

n of

glu

coco

rtic

oids

in p

rim

ary

bron

chia

l epi

thel

ial c

ells

(PBE

Cs).

CT9

9021

doe

s no

t aff

ect

GM

-CSF

or C

XCL8

sec

retio

n in

PBE

Cs.

GM

-CSF

(a) a

nd C

XCL8

(b) l

evel

s w

ere

mea

sure

d in

cel

l-fre

e su

pern

atan

ts o

f PBE

Cs

from

non

-CO

PD in

divi

dual

s up

on p

re-t

reat

men

t w

ith C

T990

21 fo

r 30

min

utes

follo

wed

by

24 h

ours

TN

F-α

stim

ulat

ion

of P

BEC

s (m

ean±

SEM

, n=

4-6)

. Pre

-tre

atm

ent

with

CT9

9021

(30

min

) rev

erse

s bu

deso

nide

(Bud

)-in

duce

d su

ppre

ssio

n of

TN

F-α-

stim

ulat

ed G

M-C

SF (c

) and

CXC

L8 (d

) rel

ease

in P

BEC

s fro

m n

on-C

OPD

indi

vidu

als

(mea

n±SE

M, n

=8)

.

Chapter

3

69

Effect of GSK3β on glucocorticoid function in COPD

-+

-+

7.5

% C

SE (6

hrs)

--

+

+

NA

C

p-G

SK3β

-ser

9p-

Akt

-ser

394

GA

PDH

-+

-+

7

.5%

CSE

(6hr

s)-

-+

+

N

AC

5075100

125

150

*p=0

.0131

ns

p=0.0692

p-GSK3:GAPDH

+

+

+

+

TN

F-α

(10n

g/m

l)+

+

+

+

B

ud (1

0nM

) -

+

-

+

C

SE (7

.5%

)-

-+

+

N

AC

(5m

M)

5075100

**p=

0.0027

**p=

0.0063

% of inhibition by budesonideTNF--induced CXCL8 release

0

1000

2000

3000

*p=0

.011

**p=

0.001

**p=

0.0079

CLCX8 release (pg/ml)

-+

+

+

+

--

+

-+

--

-+

+

TN

F-α

(10n

g/m

l)B

ud (1

0nM

) C

SE (7

.5%

)

+

+

+

+

TN

F-α

(10n

g/m

l)-

+

-

+

B

ud (1

0nM

) C

SE (7

.5%

)-

-+

+

AC D

B

0255075100

125

**p=0.0027

CXCL8 release (%)

Figu

re 7

. Cig

aret

te sm

oke

extr

act (

CSE)

-indu

ced

oxid

ativ

e st

ress

indu

ces b

udes

onid

e un

resp

onsi

vene

ss in

TN

F-α-

stim

ulat

ed 1

6HBE

cel

ls. (

a, b

, c) P

re-t

reat

men

t w

ith C

SE re

duce

s bu

deso

nide

(Bud

)-ind

uced

sup

pres

sion

of T

NF-

α-st

imul

ated

CXC

L8 re

leas

e an

d pr

e-tr

eatm

ent

with

NA

C (3

0 m

in) r

esto

res

CSE

-indu

ced

bude

soni

de

unre

spon

sive

ness

(mea

n±SE

M, n

=5)

. Abs

olut

e va

lues

(a),

valu

es re

late

d to

the

TN

F-α-

indu

ced

cont

rol (

b) a

nd p

erce

nt in

hibi

tion

by b

udes

onid

e (c

) are

sho

wn

(d) C

SE

indu

ces

PI3K

and

GSK

3β p

hosp

hory

latio

n ce

lls, a

s in

dica

ted

by W

este

rn b

lott

ing.

Rep

rese

ntat

ives

of 3

inde

pend

ent e

xper

imen

ts a

re s

how

n. T

he C

SE-in

duce

d in

crea

se in

G

SK3β

pho

spho

ryla

tion

is a

brog

ated

by

NA

C p

re-t

reat

men

t. D

ensi

tom

etry

was

per

form

ed a

nd p

hosp

ho-s

er9-

GSK

3β le

vels

are

exp

ress

ed a

s ra

tio o

f GA

PDH

as

load

ing

cont

rol (

mea

n±SE

M, n

=6)

.

70

Chapter 3

Together, our data indicate that GSK3β inhibition reduces glucocorticoid sensitivity of pro-inflammatory responses in both human monocytes and bronchial epithelial cells without modulating inflammatory mediator expression per se. The mechanism for the effect differs between cell types.

DiscussionThe increased oxidant burden derived from cigarette smoking in the lungs of COPD patients has been associated with reduced glucocorticoid responsiveness. The molecular mechanisms of oxidative stress-induced glucocorticoid unresponsiveness have remained unknown to date. Our data show, for the first time, that levels of inactive phosphorylated GSK3β are higher in lung macrophages, peripheral blood monocytes and bronchial epithelial cells from COPD patients compared to control subjects. In both monocytes and bronchial epithelial cells, pharmacological inactivation of GSK3β resulted in reduced responsiveness of inflammatory mediators to glucocorticoids. We observed a difference in glucocorticoid insensitivity between non-smoking controls and COPD patients, but not between current smokers and COPD patients or current smokers and healthy controls. This indicates that smokers have an intermediate state of sensitivity, and that individuals with COPD are more susceptible to develop steroid insensitivity upon smoking. Furthermore, we previously observed more pronounced glucocorticoid unresponsiveness in epithelial cells from severe compared to moderate COPD patients (16). Because severe COPD patients are dependent on the use of inhaled or oral glucocorticoids, it is of importance to elucidate the mechanisms of glucocorticoid unresponsiveness in COPD order to improve the treatment of patients with severe symptoms.Since we observed that phospho-GSK3 levels were still increased in PBECs from severe, ex-smoking COPD patients, even after 2-3 weeks of culture, we anticipate that there may be persistent alterations in the regulators of GSK3 phosphorylation, resulting from rewiring of the intracellular inflammatory pathways rather than an effect of recent exposure to the local inflammatory milieu in the COPD lung. The antibody used to detect GSK3β also detects GSK3α, thus although we cannot discount a role of GSK3α here, they have identical functions. With respect to therapeutic intervention, it will be important to further elucidate the downstream mechanisms involved in the reduced glucocorticoid responsiveness upon GSK3β inactivation. GSK3β is involved in numerous intracellular pathways, and preventing its inactivation, e.g. by pharmacological inhibition of the PI3K/Akt or MAPK pathways, may lead to serious side effects. In line with our results, the activation of GSK3β has been previously implicated in glucocorticoid-induced apoptosis in lymphoma cells, its inactivation resulting in glucocorticoid resistance, although effects on inflammatory responses were not studied

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(35). In monocytes, GSK3β inactivation reduced glucocorticoid suppression of pro-inflammatory responses by inhibition of the enzymatic activity of HDAC2. This reduction in activity was not associated with a change in expression but an increase in HDAC2 phosphorylation at serine 394. Casein kinase 2 (CK2) phosphorylates HDAC2 at ser394 and is a direct target of GSK3β, negatively regulating its function (38). Therefore, inactivation of GSK3β in monocyte-macrophages may increase CK2-induced phosphorylation of HDAC2. This may be involved in the observed glucocorticoid unresponsiveness towards NF-κB-induced pro-inflammatory cytokine production upon GSK3β inactivation in monocytes/macrophages. Although the functions of individual HDAC2 phosphorylation sites and the responsible kinases are unclear, the activity of this important GRα co-repressor may deprive GRα of a key mechanism by which to control inflammatory gene expression.In line with our findings in monocytes, our data indicate that glucocorticoid unresponsiveness is induced upon GSK3β inactivation in airway epithelial cells, and that cigarette smoke-induced oxidative stress may be responsible for this effect. Our combined data from monocytes and epithelial cells suggest that GSK3β may be an important common redox sensing effector molecule for a number of signalling pathways including MEK/ERK-1/2 and PI3K/Akt, regulating NF-κB activation and the subsequent inflammatory mediator release and inflammatory cell recruitment (12, 29). These redox sensitive p38 MAPK, ERK-1/2 and PI3K/Akt pathways can all induce GSK3β phosphorylation (8,(15) and have been implicated in glucocorticoid insensitivity in COPD (5). This further corroborates the role of GSK3β in oxidative stress-induced glucocorticoid unresponsiveness.The monocyte microarray data show that the anti-inflammatory effects of dexamethasone are prevented in the presence of CT99021. Not surprisingly, therefore, the effect of GSK3β inhibition on enhancing the expression of LPS-induced inflammatory genes was more marked in the presence of dexamethasone. These genes, mostly chemokines and cytokines, regulate signalling pathways that induce neutrophil, lymphocyte and macrophage activation indicating that GSK3β activity is important for regulating multiple inflammatory pathways modulated by glucocorticoids. This supports our hypothesis that aberrant GSK3β activity is involved in driving chronic inflammation through enhancing glucocorticoid insensitivity in COPD. GSK3β inhibition also resulted in significant hits in the neuropeptide/neurotransmitter receptor interacting pathways, including those for the 5-hydroxytryptamine (5-HT) receptors 2b and 6 (HTR2b and HTR6), the protease-activated receptor (PAR) family, galanin receptor 2 (Galr2), the glutamate receptor delta 2 (Grid2) and GABA B receptors 1 and 2. The neurotransmitter serotonin (5-HT) is also released from the neuroendocrine cells of the human lung, increasingly recognized for their immunomodulatory effects outside the CNS and contributing to the pathogenesis of autoimmune and chronic inflammatory diseases (40). The serum concentration of the tryptophan, the amino acid precursor of the

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5-HT is increased in patients with COPD (42), whereas plasma 5HT levels are elevated in smokers with normal lung function but reduced in COPD patients (39). In addition, cigarette smoke affects airway hyper-responsiveness through 5-HT in precision-cut lung slices (11). Finally, recent evidence indicates that 5-HT suppresses efferocytosis in human alveolar macrophages, although this appears to be independent of HTR2B (40). COPD patients have higher maximum thrombin levels, rates of thrombin generation and total thrombin formation although this was not linked to severity or inflammatory mediator expression (43). PAR-1 is activated by the thrombin and it is overexpressed in the alveolar macrophages from smokers with normal lung function (34). PAR-4 is activated by thrombin and trypsin. PAR-4 methylation and altered expression may be important in the enhanced risks associated with cigarette smoking that continue even after cessation (45). GABA is produced by the bronchial epithelium and contributes to the relaxation of airway smooth muscle tone (14). GABA A receptors are known to be expressed on bronchial epithelial cells, mediating mucus production in response to nicotine (13), while the loss of GABA B receptors modifies the biochemical and behavioral responses to nicotine withdrawal (2). However, the function of GABA B receptors in the human airways and in COPD patients is unknown. It is increasingly evident that there are neural-like transmitter interactions in human bronchial epithelial cells which may link CNS-active drugs to the increased inflammation and mucin production seen in COPD.Taken together, our study shows that reduced GSK3β activity in COPD monocytes-macrophages and airway epithelial cells may contribute to cigarette smoke-induced glucocorticoid insensitivity in the airways of COPD patients. The key nodal function of GSK3β in integrating various ROS-induced upstream and downstream signalling pathways in different cell types suggests that reversing this inactivation may constitute a novel therapeutic strategy to improve glucocorticoid function and thereby suppress airway inflammation in COPD.

AcknowledgementsThis research was partially performed within the framework of the Top Institute Pharma project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, with partners University Medical Center Groningen, University Medical Center Utrecht, University Medical Center Maastricht, Nycomed BV, GlaxoSmithKline, Danone, AstraZeneca, and Foundation TI Pharma. This project was supported by the NIHR respiratory Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation and Imperial College London. AN is the recipient of a BBSRC CASE award with Pfizer. IMA and PJB are supported by a Wellcome Trust programme grant and through COPD-MAP. GC is supported by unrestricted educational grants from AstraZeneca Italy, Boehringer Ingelheim Italy, GSK Italy and Menarini.

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CHAPTER 4

Mitochondrial dysfunction increases

pro-inflammatory cytokine production and

impairs regeneration and glucocorticoid

responsiveness in lung epithelium

Hoffmann R.F, Brandenburg S.M, ten Hacken, N.H.T, Van Oosterhout A. J.M, Heijink I.H.

Manuscript in preparation

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AbstractNoxious gases, including cigarette smoke, are the major risk factor for COPD, leading to chronic lung inflammation. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced glucocorticoid (GC) sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA, epithelial barrier function (electrical resistance) and wound repair using ECIS, in the presence and absence of the inhaled GC budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired recovery of epithelial integrity upon wound-healing. Furthermore, budesonide strongly suppressed cytokine production, and increased epithelial barrier function in wild-type cells, while A549 Rho-0 displayed reduced GC sensitivity compared to wilt-type A549 cells with respect to the pro-inflammatory response and barrier function. This steroid unresponsiveness is likely mediated by glycolysis and the associated activation of PI3K signaling, as the specific PI3K inhibitor LY294002 restored GC sensitivity in A549 Rho-0. In conclusion, mitochondrial defects as observed in COPD patients may lead to increased lung epithelial pro-inflammatory responses, reduced regeneration and reduced GC responsiveness in lung epithelium, and thus contribute to the pathogenesis of COPD.

AbbreviationsCOPD, Chronic Obstructive Pulmonary Disease; ECIS, Electric Cell substrate Impedance Sensing; Mn-SOD, Manganese Superoxide Dismutase; mtDNA, mitochondrial DNA; OXPHOS, Oxidative Phosphorylation; ROS, Reactive Oxygen Species.

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IntroductionChronic obstructive pulmonary disease (COPD) is a complex disease with increasing morbidity and mortality, characterized by irreversible airflow limitation and accelerated lung function decline. COPD is mainly caused by noxious environmental factors, including cigarette smoke, resulting in exaggerated lung inflammatory responses. Abnormal tissue repair and remodeling upon cigarette smoke-induced inflammation and damage is an important pathophysiologic feature of COPD, leading to fibrosis in the small airways and/or destruction of lung tissue (emphysema). Despite their broad anti-inflammatory effects, glucocorticoids (GC) provide relatively little therapeutic benefit in COPD. They reduce exacerbations, but do not effectively change the course of the disease nor the tissue damage that results from chronic airway inflammation (1, 2, 16, 22, 34). Furthermore, we recently observed that airway epithelial cells of COPD patients display reduced responsiveness to corticosteroids (15).Inhaled cigarette smoke first encounters the epithelial lining of the lungs, where it induces inducing oxidative stress. This inflicts lung epithelial cell death and damage, promoting the release of cytokines that attract and activate neutrophils (14). In addition, oxidative stress has been implicated in corticosteroid unresponsiveness (2, 6). Mitochondria are major intracellular targets of oxidative stress. Our previous findings have indicated that bronchial epithelial cells from COPD patients display mitochondrial abnormalities, with depletion of cristae and persistent mitochondrial damage. Similar mitochondrial changes were observed in epithelial cells that were exposed to cigarette smoke in vitro for 6 months (19). These abnormalities were accompanied by increased pro-inflammatory activity. However, it is currently unknown what the consequences of this mitochondrial dysfunction are and whether this may contribute to aberrant epithelial pro-inflammatory and repair responses in COPD. Of interest, Islam et al have recently shown that transfer of intact mitochondria by mesenchymal stem cells can contribute to lung tissue repair in a mouse model of lethal lung injury, suggesting that intact mitochondria may play be crucial for lung regeneration loss of mitochondrial function may impair this process (21). Furthermore, loss of mitochondrial function is known to lead to compensatory secondary metabolism, glycolysis, which has been implicated in glucocorticoid resistance in lymphoblastic leukemia (3, 20).We hypothesize that mitochondrial dysfunction has important implications for epithelial responses, leading to increased pro-inflammatory activity, altered GC responsiveness and impaired epithelial barrier and repair responses. To address our hypothesis, we compared wild-type human alveolar A549 cells and A549 cells with depleted of functional mitochondria, A549 Rho-0. We show that A549 Rho-0 produce higher levels of pro-inflammatory cytokines, are less responsive to GC and display impaired wound healing responses compared to wild-type A549 cells.

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Material and Methods

Cell culture The wild-type alveolar type-II carcinoma cell A549 and mitochondria-depleted A549 Rho-0 cells were kindly provided by Dr. Lodovica Vergani (Padova University, Padova, Italy) and created by Ian Holt (Cambridge University, Cambridge, United Kingdom) by culturing A549 in addition of 50 ng/mL Ethidium Bromide for 8-12 passages. Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, (D6429-500ML) supplemented with MEM Amino Acids (50x) solution (Sigma, M7020-100ML), MEM Non-essential Amino Acid Solution, (Sigma, M7145-100ML), Vitamins, (Sigma, M6895-100ML) sodium pyruvate, Uridine 50 ng/ml (Sigma, cat. n. U-3003), 2,5 µg/ml amphotericin (Sigma-A2942-100ML), 25% foetal bovine serum (FBS; Hyclone, Logan, UT), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen (Gibco), Breda, The Netherlands) in uncoated T25 flasks. Before experimentation, cells were grown ~90% confluence and serum-deprived overnight.

Western blotting Cell lysates were prepared and immunodetection was performed as described previously (17) using MitoProfile total OXPHOS antibody cocktail (Mitosciences, Eugene OR) anti-Mn-SOD (EMD Millipore Corporation, Billerica MA) and anti-GAPDH or anti-β-actin (Cell Signalling Technology, Danvers MA, USA) as loading control. Densitometry was performed using the gel-scan program QuantityOne.

ATP assayIntracellular ATP levels of A549 were measured after ATP extraction. To extract ATP, cells were lysed using 0,5% TCA. Subsequently, TCA was neutralized with TE-buffer. ATP was measured with the luciferin–luciferase method (Enliten ATP assay system, Promega). Briefly, 100 μl sample was added to 50 μl of ATP assay mix and the luminescence was measured with a Luminoskan® Ascent microplate luminometer (Thermo Scientific, Waltham, USA ) for 200ms per sample.

Cytokine levels Cell-free culture supernatants (24 hours) were collected and analysed for CCL20 and CXCL8 using a duo-set ELISA assay (R&D Systems Europe, Abingdon, UK) or analysed for CXCL8 (IL-8), CCL20 (MIP-3α), CCL3 (MIP-1α), CCL4 (MIP-1β), G-CSF, IL-6, IL-12, CCL5 (RANTES), CXCL10 (IP-10), VEGF, IL-7, CCL2, IL-15 and EGF using a multiplex ELISA (Millipore, Billerica, MD) following manufacturer’s instructions.

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Lactate assayLactate levels were measured in cell-free culture supernatants (24 hours) using a lactate assay kit (BioVision, Milpitas, USA) according to manufacturer’s protocol.

ECISElectrical resistance properties of confluent or wounded cells were measured using Electric Cell-substrate Impedance Sensing (ECIS, Applied Biophysics, Troy, NY, USA) as described previously (18). Upon inoculation in ECIS arrays, cells were incubated with/without 10-8M budesonide for 48 hours and resistance and capacitance were measured at 400Hz and 40kHz respectively. Cells were wounded by electroporation (5V, 40kHz, 30s) upon establishment of a confluent monolayer.

Confocal microscopyCells were stained with JC1, MitoTracker® DeepRed FM and Picogreen (Molecular Probes, Invitrogen) for 45 minutes in D-PBS (GIBCO, 14287-080) according to manufacture protocol. Subsequently, cells were washed twice with D-PBS and visualized using a Leica AOBS confocal microscope.

Statistical Analysis The unpaired t-test followed by a Welch correction or a Mann-Whitney test was performed to evaluate differences A549 WT and Rho-0 cells and P=<0.05 was considered statistically significant (Figures 1 and 2). The Wilcoxon signed rank test (n=6) or a repeated measures ANOVA including a Bonferroni`s multiple comparison post-test (n=5) was used to evaluate differences within cell lines (Figures 3A and 3B) respectively. A two-way ANOVA was performed in addition using a Dunnett’s post-test when comparing time curves (Figures 4 and 5).

Results

Depletion of mitochondrial (mt) DNA and mitochondrial dysfunction in A549 Rho-0 compared to wild-type A549 cellsFirst, we assessed whether the mtDNA was successfully depleted in the A549 Rho-0 cells. We analyzed both wild-type A549 and A549 Rho-0 cell lines for mtDNA and the presence of mitochondrial bodies. Co-localization of mtDNA (Picogreen) and mitochondrial bodies (Mitotracker DeepRed) were clearly visible in the wild type A549 but not in the A549 Rho0, confirming the lack of mtDNA (Fig. 1a). JC1 staining, which detects changes in the mitochondrial membrane potential, showed a drastic decrease in membrane potential the A549 Rho-0 cells (Fig. 1b).

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Surprisingly, the intra-cellular concentrations of ATP did not differ between the wild-type A549 and the A549 Rho-0 cells, indicating an alternative pathway for ATP generation in A549 Rho0 cells than the mitochondrial-mediated oxidative phosphorylation (OXPHOS) process (Fig. 1c). Loss of mitochondrial function may lead to compensatory secondary metabolism, glycolysis, to produce ATP as well as lactate. To confirm a switch to glycolysis in the A549 Rho-0 cells, we analyzed lactate production. Indeed, lactate levels in the culture medium of A549 Rho-0 cells were significantly increased when compared to wild-type A549 cells (Fig. 1d). This was accompanied by a significant reduction in the expression specific components of the OXPHOS system, complex III subunit core 2 and complex IV subunit 3, further suggesting a switch to glycolysis (Fig. 1e), although the ATPase subunit α was not affected. In addition, we studied the expression of the mitochondrial anti-oxidant protein Mn-SOD, and observed a trend towards an increase in the A549 Rho-0 cells compared to the WT A549 cells, suggesting increased ROS production (Fig. 1f ), although this failed to reach statistical significance.

Elevated pro-inflammatory cytokine/chemokine secretion in A549 Rho-0 cellsNext, we studied the effect of mitochondrial dysfunction on the pro-inflammatory cytokine response of lung epithelial cells. We measured a panel of pro-inflammatory cytokines/chemokines that have been associated with lung inflammation in COPD patients and/or show increased levels in COPD lungs (Table 1).

Table 1. Elevated cytokine/chemokine production and action in COPD

Cytokine/chemokine Effect in COPD Mechanism in Disease References

Il-6 Promotes disease T-cell/B-cell Proliferator

CXCL-8 (IL-8) Promotes disease Neutrophil Attractant and Activator

CCL5 (RANTES) Promotes disease T-cell/eosinophil Attractant

G-CSF Promotes disease Neutrophil Attractant

IL-12 Promotes disease Th1 differentiator/Stimulates IFNγ

CXCL-10 (IP10) Promotes disease Monocytes/macrophages and T-cell Attractant

CCL3 (MIP1α) Promotes disease Neutrophil Attractant/Stimulates IL1/IL-6 release

CCL-4 (MIP1β) Promotes disease Neutrophil Attractant/Stimulates IL1/IL-6 release

CCL20 Promotes disease Strong Lymphocyte Attractant

(27, 38)

(4, 40)

(41)

(29)

(12, 36, 39)

(13, 33, 39)

(37)

(5, 31)

(8, 32)

CCL20; Chemokine (C-C motif ) Ligand 20; IL: Interleukin; IFNγ: Interferon gamma IL-RA: IL-1 Receptor Agonist; CXCL-10 (IP-10): Interferon gamma-induced Protein 10; CCL-3 or 4 (MIP-1α/β): Macrophage Inflammatory Protein 1α/β; CCL-5 (RANTES): Regulated on Activation, Normal T-cell Expressed and Secreted; Th2: Type 2 T-helper cell; VEGF: Vascular Endothelial Growth Factor; G-CSF: Granulocyte Colony-Stimulating Factor.

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Of all the detected pro-inflammatory cytokines and chemokines, CXCL8 (IL-8), CCL20 (MIP-3α), CCL3 (MIP-1α), CCL4 (MIP-1β), G-CSF, IL-6 and IL-12, were significantly increased in Rho-0 compared to wild-type A549 cells, while a trend was observed for CCL5 (RANTES, p=0.0556) and CXCL10 (IP-10, p=0.09) (Fig. 2). These data suggest that mtDNA depletion results in an increased pro-inflammatory response in lung epithelial cells. We also found elevated levels of VEGF and IL-7 (Supplemental Figure 1a). Additionally, we found cytokines/chemokines that were unchanged between A549 WT and Rho-0, i.e. CCL2, IL-15 and EGF (Supplemental Figure 1b). INF-α, INF-γ, Eotaxin, IL-2, IL-13, FGF-basic, IL-1β, IL-4, IL-5, IL-10, IL-17, MIG, HGF and GM-CSF were hardly detectable in both cell types (data not shown).

100

1000

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100000

*

***

0.0952

*

*

*

0.0556

*

CXCL8 CXCL10CCL20 CCL3 CCL4 CCL5 G-CSF IL-6 IL-12

WT

Rho-0

pg/m

l (lo

g10)

Figure 2. Increased pro-inflammatory response in A549 Rho-0 compared to wild-type A549 cells. A549 wild-type and Rho-0 cells were grown to confluence and serum deprived overnight. (CXCL8, CXCL10, CCL3, CCL4, CCL5, CCL20, G-CSF, IL-6, IL-12 (mean ± SEM, n=5) were measured in cell-free supernatants. *=p<0.05 and, **=p<0.01 between the indicated values as analysed by a Mann-Whitney test.

A549 Rho-0 cells show reduced GC responsivenessTo investigate whether mitochondrial dysfunction may contribute to reduced GC sensitivity as observed in COPD (15), we studied the suppressive effect of budesonide on CXCL8 production in wild-type A549 and A549 Rho-0 cells. We observed that budesonide significantly reduced CXCL8 secretion by approximately 75-80% in the wild-type A549 cells. Of interest, budesonide failed to significantly suppress IL-8 secretion in the A549 Rho-0 cells (Fig. 3a). This suggests that a mitochondrial dysfunction triggers a pro-inflammatory response that is insensitive to corticosteroids. Next, we studied whether the observed switch to glycolysis and activation of the associated PI3K/Akt signaling pathway is involved in the observed GC unresponsiveness in A549 Rho-0 cells, as PI3K has previously been implicated in GC unresponsiveness in COPD (28). We blocked PI3K/Akt activity, using the specific inhibitor LY294002 (10 μM). In both wild-type

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A549 and A549 Rho-0 cells, we observed a marked reduction in IL-8 production when the PI3K pathway was blocked (Fig. 3b). Moreover, inhibition of the PI3K/Akt pathway restored corticosteroid sensitivity in A549 Rho-0 cells. In contrast to the absence of LY294002, budesonide significantly reduced CXCL8 production in A549 Rho0 cells in the presence of LY294002 (Fig. 3b). Thus, our data strongly suggest that the mitochondrial defect in A549 Rho-0 cells induces a PI3K/Akt-dependent GC insensitive pro-inflammatory response in lung epithelial cells.

Bas

BUD 10nM M

LY2940

02 10

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BUD 10nM/LY29

4002

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BUD 10nM*

M *

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02 10

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10

0

25

50

75

100

125

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***

******

******

***

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on (C

LCX8

)

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BUD 10nM

basal

BUD 10nM

0

1000

2000

3000

4000

*

**

ns

A549 wt A549 Rho-0

CXC

L8 (p

g/m

l)

a b

Figure 3. A549 Rho-0 cells are less sensitive to budesonide than wild-type A549 cells, which is restored by blocking of PI3K/akt signaling. A549 wild-type and Rho-0 cells were grown to confluence, serum deprived overnight and incubated with/without budesonide (BUD, 10 nM) for 24 hours. A) CXCL8 was measured in cell-free supernatant (mean ± SEM, n=6). B) LY294002 (10µM) was added 30 min before the exposure to BUD and CXCL8 levels (mean ± SEM, n=5) are expressed as percentage of the levels without BUD. *=p<0.05, **=p<0.01 ***=p<0.001 between the indicated values.

Reduced regenerative capacity in A549 Rho-0 cellsIn addition to pro-inflammatory responses, we studied whether mitochondrial dysfunction impacts on epithelial regeneration. We assessed the recovery of a confluent monolayer of A549 cells upon wounding by electroporation using the ECIS. Wild-type A549 were able to recover from this type of wounding within 4 hours, as observed by the return of resistance to values prior to wounding. This wound healing response was impaired in A549 Rho-0 cells, taking approximately 8 hours to recover, as reflected by stabilization of resistance values (Fig. 4). Moreover, resistance levels in A549 Rho-0 did not completely return to the values prior to wounding. This indicates that mtDNA depletion affects epithelial regenerative capacity. Together, A549 Rho-0 cells with defective mitochondria display abnormalities with respect

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to pro-inflammatory responses, corticosteroid responsiveness as well as the capacity to regenerate upon wounding.

0,00

0,20

0,40

0,60

0,80

1,00

1,20

-2,00 4,00 10,00 16,00 22,00Time (hrs)

Res

ista

nce

(ohm

)

A549 WTA549 Rho-0

Figure 4. A549 Rho-0 cells display reduced wound healing compared to wild-type A549 cells. A549 wild-type and Rho-0 cells were grown to confluence in ECIS arrays. Upon 2 days, cells were wounded by electroporation. Resistance was monitored for 22 hours at 40 Hz and levels were normalized to the values just prior to wounding (mean ± SEM, n=4). *** =p<0.001 between the indicated values as analyzed by 2-way ANOVA.

Corticosteroid effect on barrier formationIn addition to their suppressive effects on cytokines, we have previously reported that corticosteroids exert protective effects on epithelial barrier function (16). Therefore, we tested whether mitochondrial dysfunction also impairs the responsiveness to GC with respect to epithelial barrier function. Using ECIS, we measured low-frequency resistance and high-frequency capacitance as most sensitive parameters to measure epithelial cell-cell and cell-matrix contacts respectively, during cell growth in the presence and absence of budesonide (18). The stabilization of high-frequency capacitance at 20-30 hours indicates the formation of a confluent layer (Fig. 5a). After this, low-frequency resistance did not further increase, indicating that epithelial barrier function in A459 cells is mainly established by the formation of a monolayer, but not the formation of intracellular junctions. The presence of budesonide induced a two-fold increase in barrier function in wild-type A549 cells, which was observed for both high-frequency capacitance and low-frequency resistance (Fig. 5a and 5b). Although A549 Rho-0 cells showed a similar growth curve, A549 Rho-0 cells (Fig. 5a and 5b) were again less responsive to budesonide, as it did not significantly alter low-frequency resistance nor decreased high-frequency capacitance in these cells. Together, our results indicate that mitochondrial dysfunction impacts on epithelial pro-inflammatory responses, regeneration and corticosteroid responsiveness.

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b

0

10

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30

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DiscussionWe show for the first time that alveolar epithelial cells lacking functional mitochondria display increased production of pro-inflammatory cytokines and loss of responsiveness of CXCL8 production to GC. This is accompanied by increased pro-inflammatory responses, impaired regenerative responses and reduced responsiveness of epithelial barrier function and pro-inflammatory cytokine production to the GC budesonide, which may be mediated by activation of the glycolysis (Fig. 6). While we observed higher levels of lactate produced by A549 cells with dysfunctional mitochondria, inhibition of the glycolysis-associated PI3K pathway reduced pro-inflammatory cytokine production and restored GC sensitivity. We previously observed long-term cigarette smoke-exposed bronchial epithelial cells display mitochondrial abnormalities, and that these are accompanied by increased production of the pro-inflammatory cytokines CXCL8 and IL-6 (19). Moreover, cultured bronchial epithelial cells from COPD patients also show mitochondrial abnormalities (19) and produce higher levels of CXCL8 at baseline than epithelial cells from control donors (35). We now show that mitochondrial dysfunction leads to increased levels of CXCL8, CCL20, G-CSF, CCL3 and CCL4, IL-6 and IL-12, all of which are increased in lungs of COPD patients (4, 5, 8, 12, 27, 29, 31, 32, 36–40). Our current and previous (19) findings may also explain why bronchial epithelial cells from COPD patients are less responsive to corticosteroids with respect to both cytokine production as well as barrier function, as observed previously (18). Our current findings are limited to the use of a cell line, since the depletion of mtDNA for creating Rho-0 cells requires long-term exposure to a low dose of ethidium bromide. In future studies, it will be of interest to test the effect of mitochondrial function blockers/uncouplers on pro-inflammatory responses in primary epithelial cells. When depleted of mtDNA, cells are not able to perform normal electron transport or ATP synthesis and rely on ATP derived from glycolysis, where glucose is metabolized for survival and growth to produce lactate (7). This metabolic switch is dependent on the activation of PI3K signaling, as demonstrated in naive T cells and cancer cells (9, 10). Of interest, it has previously been reported that GC resistance in T-lineage acute lymphoblastic leukemia is associated with the upregulation of glycolysis and activation of PI3K/AKT/mTOR signaling (3, 20). We observed a switch to glycolysis in Rho-0 cells, as indicated by increased lactate production and reduced expression of specific OXPHOS components. Furthermore, we found that inhibition of PI3K reversed the GC insensitivity of CXCL8 production in A549 Rho-0 cells. PI3K inhibition itself also significantly reduced CXCL8 production, and the GC unresponsiveness of A549 Rho-0 cells could thus be a consequence of PI3K activity being insensitive to GC, resulting in GC-insensitive CXCL8 production. Alternatively, PI3K activity is known to reduce GC responsiveness, e.g. by mechanisms involving post-translational histone deacetylase 2 (HDAC2) modifications and proteasomal degradation of HDAC2, leading to altered acetylation of the GC receptor and/or histones within the promoter regions of

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pro-inflammatory genes (22, 23, 30). We have previously demonstrated that IL-17 reduces GC responsiveness of CXCL8 production in airway epithelial cells in a PI3K and HDAC2-dependent manner (24). Similar mechanisms may be involved in GC unresponsiveness upon mtDNA depletion, although further studies will be required to establish whether mitochondrial dysfunction also leads to a reduction in HDAC expression. It is currently unknown whether similar mechanisms may be involved in the inability of GC to improve the integrity of A549 Rho-0 monolayers. Since GC fail to efficiently suppress inflammation in the majority of COPD patients and to prevent the loss of alveolar tissue in emphysema, it will be of interest to elucidate the precise mechanisms of GC unresponsiveness in mitochondria-depleted epithelial cells.

Figure 6. Proposed mechanism of reduced glucocorticoid (GC) insensitivity upon mitochondrial dysfunction. Lung cells with mitochondrial dysfunction are more prone to pro-inflammatory responses through the PI3K/Akt-pathway upon a switch to glycolysis, leading to reduced GC sensitivity of cytokine production as well as barrier function.

In addition to the changes in GC responsiveness, our findings may have additional implications for COPD, as aberrant tissue repair responses are thought to contribute the pathogenesis of the disease. We observed that mitochondrial depletion impairs the ability of epithelial cells to close wounds. Although we did not further study the mechanisms by

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which functionally intact mitochondria may promote epithelial regeneration or modulate epithelial adhesion, it is conceivable that migration of epithelial cells to cover the wounded area requires active energy metabolism. Of interest, lung ageing is also associated with mitochondrial dysfunction and impaired repair capacity of lung epithelial cells, fibroblasts and stem cells, leading to emphysema-like features (11, 19, 25, 26). It will therefore be of interest to study the effect of mitochondrial function blockers on the regenerative response of primary bronchial epithelial cells in future studies. In conclusion, our data indicate that mitochondrial dysfunction leads to increased pro-inflammatory activity, inefficient wound healing and reduced responsiveness to GCs in alveolar epithelium. Our results suggest that novel strategies towards improved mitochondrial function may be promising in COPD. Furthermore, restoration of steroid responsiveness, e.g. by means of pharmacological inhibition of the PI3K pathway, may increase the suppressive effects of GC on lung inflammation and improve the integrity of lung epithelial cells in COPD.

AcknowledgementsThis research was partially performed within the framework of the Top Institute Pharma project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, with partners University Medical Center Groningen, University Medical Center Utrecht, University Medical Center Maastricht, Nycomed BV, GlaxoSmithKline, Danone, AstraZeneca, and Foundation TI Pharma.

This study was made possible by: Stichting Astmabestrijding (SAB 2012/039).

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Supplemental Information

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a

b

1

10

100

1000

10000

CCL2 IL-15 EGF

WTRho-0

pg/m

l (lo

g10)

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Supplemental Figure 1. Cytokine and chemokine secretion in A549 Rho-0 cells. A549 wild-type and Rho-0 cells were grown to confluence and serum deprived overnight. A) VEGF, IL-7, B) CCL2, IL-15 and EGF were measured in cell-free supernatants (mean ± SEM, n=5) *=p<0.05 between the indicated values as analysed by a Mann-Whitney test.

CHAPTER 5

Untargeted Lipidomic Analysis in COPD:

Uncovering Sphingolipids

Eef D. Telenga*, Roland F. Hoffmann*, Ruben t’Kindt, Susan J.M. Hoonhorst, Brigitte W.M. Willemse, Antoon J.M. van Oosterhout, Irene H. Heijink,

Maarten van den Berge, Lucie Jorge, Pat Sandra, Dirkje S. Postma, Koen Sandra, Nick H.T. ten Hacken

*both authors contributed equally

Telenga, Hoffmann et al, American Journal of Respiratory and Critical Care Medicine. 2014

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Abstract Rationale: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases like COPD.

Objective: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking.

Methods: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD. Measurements and Main Results: >1500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared to smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. 20 (glyco)sphingolipids and 6 tobacco-related compounds were higher expressed in smokers without COPD compared to never-smokers. Two-month smoking cessation reduced expression of 26 sphingolipids in smokers with and without COPD.

Conclusions: Expression of lipids from the sphingolipid pathway are higher in smokers with COPD compared to smokers without COPD. Considering their potential biological properties, they may play a role in the pathogenesis of COPD.

Key words: Lipidomics, Cigarette Smoke, COPD, Induced Sputum.

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IntroductionLipidomics is an emerging “omics” science, which is based on the analysis of all lipids present in a biological system known as the lipidome. The cellular lipidome consists of species that are of key importance to various cellular processes, including energy storage, membrane integrity and cellular signaling processes including cell proliferation, metabolism and apoptosis induction.(1-3) The complex composition and function of the lipidome in various diseases is currently poorly understood. The crucial role of lipids in cell, tissue and organ physiology has been demonstrated by a large number of genetic studies and by the existence of many human diseases that involve the disruption of lipid metabolic enzymes and pathways.(2) Examples of such diseases are cancer, diabetes, neurodegenerative diseases, infections and inflammation.(4, 5) To date, little is known about the role and the impact of the lipidome/lipid signaling in Chronic Obstructive Pulmonary Disease (COPD), a disease that affects millions of people worldwide and is associated with a high disease burden and mortality rate.

It is plausible that the lipidome plays a role in COPD since extracellular signals from (inflammatory) cytokines, which are abundantly present in the lungs of COPD patients, are able to influence the activity of lipid-modifying enzymes, such as phospholipases, sphingosine kinases and sphingomyelinases.(5) Lipids from the sphingolipid pathway, like ceramides, are major players in the induction of apoptosis and cellular senescence,(6, 7) and free fatty acids can activate Toll-like receptor 4, thereby inducing similar inflammatory responses as lipopolysaccharides do.(8) An imbalance of major lipid signaling caused by cigarette smoke may result in lipid accumulation and/or alterations that are also harmful to the cell by compromising cellular integrity of various cell/organelle membranes. This may contribute to disease progression seen in COPD by sustaining the underlying chronic inflammation.(5, 8)

To assess whether the lipidome could play a role in COPD and is affected by smoking, we investigated induced sputum in this explorative study. Induced sputum reflects epithelial lining fluid from the large and small airways, making this an interesting compartment to study since it represents the first line of defense against noxious particles such as cigarette smoke. We investigated the effect of COPD on lipid expression in chronic smokers with and without COPD. Furthermore, the effect of smoking on lipid expression was assessed in smokers and never-smokers without COPD and in a group of smokers with and without COPD before and after 2-month smoking cessation.

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Methods and materials

SubjectsInduced sputum supernatant samples from 3 studies were analyzed (study-I: clinicaltrials.gov NCT00848406, study-II: Lo Tam et al.(9), study-III: Willemse et al.(10)). We included 19 smokers with COPD, 20 smokers without COPD and 14 never-smokers. Additionally, induced sputum samples were collected at baseline and after 2-month smoking cessation from 7 smokers with and 10 smokers without COPD in study III. All studies were approved by the University Medical Center Groningen medical ethical committee and all subjects provided written informed consent.

All COPD patients were current-smokers and had severity stage-II or stage-III according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD).(11) Subjects without COPD were asymptomatic, had no history of pulmonary diseases and had normal spirometry (post-bronchodilator forced expiratory volume in 1 second (FEV

1) ≥80% predicted, post-

bronchodilator FEV1/forced expiratory volume (FVC) ≥ lower limit of normal and reversibility

to 400 µg salbutamol <10% of the predicted FEV1). Never-smokers were subjects who had

not smoked in the last year, had never smoked for ≥1 year and had a total cigarette exposure <0.5 packyears.

Sputum inductionSputum inductions and sputum processing was performed at the same facility, according to standard protocol and operating procedures. Fifteen minutes after inhalation of 400 μg salbutamol, hypertonic saline (4.5% in the first two studies and 3%, 4%, and 5% in the third study) was nebulized with an ultrasonic nebulizer (Ultraneb, DeVillbiss, Somerset, PA, USA) and inhaled for 5 minutes in the first two studies and 7 minutes in the third study. The output of the nebulizer was calibrated at 1.5 ml/min. After each concentration, patients were encouraged to cough and expectorate sputum. Whole samples were processed according to the method of Fahy et al. with some modifications.(12) An equal volume of dithiothreitol 0.1% (Sputalysin 10%, Behring Diagnostics Inc, Sommervillle, NY, USA) was added to the weight of the sputum and after 15 minutes filtered through a nylon (48 μm) gauze. The sputum sample was centrifuged (10 min, 450g, 4° Celsius) and the supernatant was stored at -80°C within 4 hours. The samples had not been thawed and refrozen before the current lipid analysis.

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Lipidomics analysisA detailed description of the lipidomics analysis, which is based on high-resolution liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS(/MS)), is available in the Supplemental methods.(13-15) In brief, lipids were extracted from sputum using liquid/liquid extraction incorporating methyl-tert-butylether (MTBE). Extracts were analyzed on a 6530 Q-TOF MS system (Agilent Technologies, Walbronn, Germany) combined with a 1200 RRLC system (Agilent Technologies). Lipid extracts were analyzed by both positive (+ESI) and negative (-ESI) electrospray ionization mass spectrometry, as some lipid species are only detected in one ionization mode (e.g. fatty acids in ESI- and glycerolipids in ESI+).(13, 14) Data were processed in an untargeted manner using the MassHunter Qualitative Analysis software (Agilent Technologies) and converted to a statistically accessible data matrix in MassProfiler Professional (Agilent Technologies). Data was normalized using total lipid abundance, correcting for sputum dilution. All experiments were quality controlled by analyzing quality control (QC) samples, representing an equal mixture of all study samples, through the different analytical sequences.

Lipid NomenclatureThe International Lipid Classification and Nomenclature Committee (ILCNC) has been used, i.e. ‘‘Comprehensive Classification System for Lipids’’.(16-18) Regarding glycosphingolipids, LC-MS cannot discriminate between galactose and glucose, or N-acetylglucosamine and N-acetylgalactosamine. Therefore, lipid nomenclature of these species contains hexose (Hex) instead of glucose or galactose.

Statistical analysesFirst, we analyzed if differences in the expression between different lipid classes. Peak areas of all identified lipid species were summed up per lipid class for each individual sample, both in +ESI and -ESI mode. After normalization for sputum dilution, relative quantities for each lipid class were calculated. The relative expression of classes between the subject groups was compared using Mann-Whitney unpaired analysis with Benjamini Hochberg false discovery rate correction for multiple testing. (19) Second, we compared the relative expression of classes of the sphingolipid subclass in a similar manner. Third, we investigated the expression of individual lipids with the Mann-Whitney-U test for cross-sectional data and the Wilcoxon signed-rank test for longitudinal data. In these analyses >1500 different lipids were investigated, therefore we corrected for multiple testing with the Benjamini Hochberg false discovery rate. All reported p-values for these analyses are corrected p-values. Finally, to assess associations between lipid expression and clinical outcome variables, we performed Spearman’s rank correlation test for the lipids that were differentially expressed (≥2.5 fold change) between smokers with and without COPD.

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Results

Subject characteristicsSmokers with COPD were older, more often male and had a higher number of packyears than smokers without COPD (Table 1). There were no significant differences in baseline characteristics between the smokers without COPD and never-smokers. Twelve COPD patients had COPD GOLD stage-II and 7 patients GOLD stage-III. For the smoking cessation study 10 smokers without COPD and 7 smokers with COPD were included. Their baseline characteristics are shown in Table 2. The total lipid abundance in induced sputum between the subject groups did not differ significantly (Supplemental Figure 1).

Table 1: Baseline characteristics of study subjects

Never-smokersSmokers without COPD

Smokers with COPD

n=14 n=20 n=19

Study (study I / II / III) 14 / 0 / 0 15 / 0 / 5 0 / 4 / 15

Age (years 54 (23 -58) 42 (20 – 51) 59 (54 – 65)

Gender (m/f ) 6 / 8 10 / 10 16/3

Cigarettes / day 0 17 (11 – 24) 20 (12 – 25)

Packyears 0 13 (3 – 25) 37 (31 – 46)

ICS use (y/n) 0 / 14 0 / 20 4 / 15

GOLD stage (II / III) 12 / 7

FEV1 post bd (%predicted) 108 (103 - 111) 110 (102 - 117) 70 (52 -79)

FEV1/FVC post bd (%) 82 (76 - 85) 83 (80 - 88) 56 (47 - 70)

Sputum*

Cell concentration (x 106/ml) 0.80 (0.27 – 1.14) 0.51 (0.31 – 1.96) 1.84 (0.46 – 3.62)

Neutrophils (%) 38 (31 – 52) 54 (41 – 68) 78 (74 – 89)

Eosinophils (%) 0.00 (0.00 – 0.15) 0.30 (0.00 – 1.00) 0.80 (0.30 – 2.60)

Macrophages (%) 62 (47 – 67) 43 (31 – 58) 18 (11 – 22)

Lymphocytes (%) 0.6 (0.2 – 0.9) 0.2 (0.0 – 0.8) 0.8 (0.5 – 1.6)

Bronchial epithelial cells (%) 1.8 (0.8 – 4.7) 2.5 (1.0 – 8.8) 0.8 (0.0 – 2.0)

Data are presented as medians with interquartile range or numbers; COPD = chronic obstructive pulmonary disease; ICS = inhaled corticosteroids; FEV

1 = forced expiratory volume in 1 second; FVC = forced vital capacity; bd =

bronchodilator. * Sputum cell concentrations do not include squamous cells. Bronchial epithelial cell percentages are expressed as percentage of the cell concentration, inflammatory cell percentages are expressed as percentage of only inflammatory cells (excluding bronchial epithelium).

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Table 2: Baseline characteristics of the smoking cessation study subjects

Smokers without COPD Smokers with COPD

n=10 n=7

Age (years) 50 (48 - 52) 63 (59 - 65)

Gender (m/f ) 6 / 4 5 / 2

Cigarettes / day 25 (18 - 26) 20 (15 - 23)

Packyears 22 (17 - 26) 40 (33 - 54)

FEV1 post bd (%predicted) 112 (104 - 116) 51 (47 - 64)

FEV1/FVC post bd (%) 83 (79 - 85) 50 (42 - 55)

Sputum*

Cell concentration (106/ml) 2.16 (0.74 – 2.85) 2.50 (0.62 – 5.21)

Neutrophils (%) 56 (48 – 69) 81 (78 – 88)

Eosinophils (%) 0.05 (0.00 – 0.70) 1.60 (0.60 – 3.30)

Macrophages (%) 39 (30 – 49) 13 (6 – 18)

Lymphocytes (%) 1.0 (0.1 – 1.8) 1.1 (0.8 – 2.0)

Bronchial epithelial cells (%) 0.8 (0.1 – 2.1) 0.8 (0.0 – 1.9)

Data are presented as medians with interquartile range or numbers; COPD = chronic obstructive pulmonary disease; FEV

1 = forced expiratory volume in 1 second; FEV

1/FVC = FEV

1 / forced vital capacity; bd = bronchodilator.

* Sputum cell concentrations are presented without squamous cells, bronchial epithelial cell percentages are expressed as percentage of the cell concentration, inflammatory cell percentages are expressed as percentage of only inflammatory cells (excluding bronchial epithelium).

Differential lipid expression in smokers with and without COPDWhen comparing different classes of lipids between smokers with and without COPD, the expression of the sphingolipid class (Figure 1) was significantly higher in smokers with COPD, both in –ESI mode (41.2% versus 28.2%, p=0.004, Figure 2a) and +ESI mode (4.58% versus 2.62%, p=0.004, Figure 2b). Additionally, prenol lipids, the majority of which are tobacco compounds and only detected in +ESI mode, were higher expressed in smokers with COPD than in smokers without COPD. Expression of glycerophospholipids and fatty acids was lower in –ESI mode in smokers with COPD versus smokers without COPD. When assessing expression of the subclasses of sphingolipids, there were no significant differences in expression detected (Supplemental Figure 2).

When analyzing differential expression of individual lipid compounds, 210 lipids were differentially expressed (Supplemental Table 1). A large portion of these lipids were from the sphingolipid pathway. 168 sphingolipids were significantly higher expressed in smokers with COPD (28 ceramides, 11 dihydroceramides, 19 phytoceramides, 36 sphingomyelins and 74 glycosphingolipids (GSLs)). Expression of only 1 neuraminic acid containing GSL was lower in smokers with COPD.

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Figure 2: Relative expression of different lipid classes in –ESI mode (a) and +ESI (b). Data are expressed as mean percentages with SD.

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To investigate whether the differences in lipids found between smokers with and without COPD are still present after smoke cessation we repeated our cross-sectional analyses in a group of 10 COPD patients and 7 subjects without COPD after 2-month smoking cessation. In this analysis, 12 subjects (7 COPD patients and 5 subjects without COPD) overlapped with the earlier cross-sectional study, however the sampling time point differed two months from the previous cross-sectional analysis. After smoking cessation 155 lipids were higher expressed in COPD patients compared to subjects without COPD, 139 being sphingolipids (32 ceramides, 11 dihydroceramides, 18 phytoceramides, 19 sphingomyelins and 59 GSLs) and 16 phosphatidylethanolamines (PE; 6 lysophosphatidylethanolamines (lysoPEs), 10 plasmenyl PEs, Supplemental Table 2).

We observed that the expression of lipids in three sphingolipid subclasses, ceramides (d18:1), dihydroceramides (d18:0) and phytoceramides (t18:0), was dependent on the chain length of the fatty acid attached to the lipid backbone, with higher expression of shorter chain length of fatty acids in smokers with COPD (Figure 3).

Another major lipid class that was significantly higher expressed in smokers with COPD was the class of PEs (n=36), divided by sub-classes lysoPEs (n=14), acyl-PEs (n=2) and plasmalogen PEs (pPE, n=20) (Supplemental Table 1). In contrast to LysoPE species, there were no lysophosphatidylcholine (LysoPC) compounds differently expressed between smokers with and without COPD. This shows specific expression differences between two related subclass lipid species. We therefore determined the ratio of these species calculated against the steadier lysoPC species with identical fatty acid chain length and saturation, showing a significant increase in COPD patients dependent on their fatty acid saturation (Figure 4). In general, higher expression of PEs was only found in compounds containing unsaturated fatty acids, the most being poly-unsaturated, i.e. showing 2 or more double bonds.

Differential lipid expression in smokers without COPD and never-smokersThere were no differences in the expression of lipid classes between smokers without COPD and never-smokers, except for higher expressed prenol lipids in smokers without COPD, which are cigarette smoke compounds (Figure 2).At individual lipid compound only 28 lipids, including 22 sphingolipids, were differentially expressed between smokers without COPD and never-smokers (Supplemental Table 3). Of interest, only two compounds were lower expressed in current-smokers and again these both belong to the neuraminic acid containing glycosphingolipid sub-class. The other 6 lipids that were differentially expressed belonged to the prenol lipids and were almost exclusively found in smokers. These lipids are known tobacco components which include solanesol, its esters, solanesyl palmitate, solanesyl stearate, and its degradation products, bombiprenone, geranylfarnesylacetone and farnesylfarnesylacetone.(20)

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Figure 3: The effect of fatty acid chain length (n) upon differential lipid expression of smokers with and without COPD. (A) Figures Cer(d18:0) refers to dihydroceramides, Figures Cer(d18:1) to ceramides and Figures Cer(t18:0) to phytoceramides. The X-axis represents the fatty acid chain length and the Y-axis represents the difference in expression between smokers with and without COPD. A positive percentage on the Y-axis indicates a higher expression of lipids in sputum of smokers with COPD than in smokers without COPD. Black circles represent the comparison in the cross-sectional study at baseline (20 smokers without COPD, 19 smokers without COPD), grey circles represent the cross-sectional study after 2-month smoking cessation (10 smokers without COPD, 7 smokers with COPD). Solid circles show a significant difference between smokers with COPD versus without COPD (multiple testing corrected p-value ≤0.05); open circles represent no significant difference (multiple testing corrected p-value >0.05). Panel B shows how the backbone of these three sphingolipid subclasses differ at (R).

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*

*

**

* **

*

*

*

*

*

*

Figure 4: The lysoPE/lysoPC abundance ratio (mean+/-SEM) of species with identical fatty acid chain length and saturation in smokers with COPD ( ) and smokers without COPD ( ). LysoPE/lysoPC ratios from species containing fatty acid chains with 2 or more double bonds were significantly different (* p<0.05). Smokers without COPD n=20; smokers with COPD n=10

Effects of smoking cessationWe analyzed induced sputa of 17 individuals before and after 2-month smoking cessation (7 with and 10 without COPD). Analyzed together, 28 lipids were lower expressed after smoking cessation including 3 ceramides, 6 dihydroceramides and 17 GSLs (Supplemental Table 4). This effect was also observed for tobacco related compounds, solanesol and solanesyl palmitate. 12 out of the 28 lipids that were lower expressed upon smoking cessation were higher expressed in smokers without COPD compared with never-smokers.

Correlation between lipid expression and clinical outcome variablesThere were 13 lipids with a ≥2.5 fold change between smokers with and without COPD (4 ceramides, 5 dihydroceramides, 1 sphingomyelin and 3 GSLs). All these lipids were significantly and inversely correlated with FEV

1 % predicted, FEV

1/FVC and RV/TLC %

predicted in smokers with and without COPD (Spearman’s rho between -0,330 and -0.713, Supplemental Table 5). In contrast, only 1 ceramide was significantly associated with CO diffusion (TLCOc/VA %predicted). Lipid expression was also significantly correlated with sputum cell differentials. All 13 lipids positively correlated with the percentage of sputum eosinophils (Spearman’s rho between 0.346 and 0.565) and 11 lipids positively correlated with the percentage of sputum neutrophils (Spearman’s rho between 0.366 and 0.545). Finally, all lipids were positively correlated with the number of packyears smoked (Spearman’s rho between 0.499 and 0.637). Figure 5 shows the correlations for one of these lipids, the ceramide Cer(d18:1/21:0).

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Figure 5: Correlation between clinical outcome variables and ceramide Cer(d18:1/21:0), one of the lipids with a ≥2.5 fold change. FEV

1 = forced expiratory volume in 1 second; RV = residual volume; TLC = total lung capacity;

TLCOc = diffusion capacity for carbon mono-oxide corrected for hemoglobin; VA = alveolar volume. Ceramide expression (X-axis) is shown as normalized peak area.

Discussion This explorative study identified >1500 lipid compounds in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound lipid level, 168 sphingolipids, 36 phosphatidylethanolamine lipids and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared to smokers without COPD. The 13 lipids with a ≥2.5 fold change between smokers with and without COPD showed high correlations with reduced lung function, inflammation in sputum and number of packyears. Two-month smoking cessation reduced expression of 26 sphingolipids in smokers with and without COPD. Smokers without COPD showed higher expression of only 20 (glyco)sphingolipids and 6 tobacco-related compounds compared to never-smokers. Together, our findings suggest that the above described lipids may play a role in the cigarette smoke induced lung disease COPD.

Sphingolipids are lipids of increased importance and can be formed de novo from serine and palmitoyl-CoA (Figure 1). Ceramides form the central hub in the sphingolipid pathway. (6, 7) In COPD, ceramides have previously been implicated in the development of emphysema

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in animal models.(21, 22) Our study confirms these previous observations, since we found significant higher expression of 28 ceramides in sputum from COPD patients, although there was an equal distribution of sphingolipids within the studied sample groups. Oxidative stress and inflammation, which have been put forward as an important component of COPD development and progression, have been shown to induce ceramide generation.(23-25) Ceramides are well known as second messengers involved in apoptosis and may contribute to the development of COPD in various ways.(6, 7, 26) First, they can alter the membrane architecture, influencing the formation of ceramide enriched microdomains or ‘lipid rafts’, which can trap and aggregate receptors and cell-signaling molecules, enabling signal transduction.(27) Second, ceramides directly inhibit pro-growth molecules like Akt and protein kinase C alpha (PKCα) and activate pro-apoptotic molecules, including jun kinases (JNKs), stress-activated protein kinases (SAPKs), kinase suppressor of Ras (KSR), cathepsin D, phosphoprotein phosphatases 1 and 2A (PP1 and PP2A) and phospholipase A

2 (PLA

2).(6, 7, 28-31). Finally, ceramides can alter membrane permeability and induce the

formation of membrane channels, which can lead to release of pro-apoptotic proteins from the mitochondria.(32, 33)

Our study shows that not only ceramides are higher expressed in COPD, but additionally 11 dihydroceramides and 19 phytoceramides. Recent studies suggest that dihydroceramides and phytoceramides are involved in autophagy and may have (anti-)apoptotic effects.(34, 35) Of interest, ceramides, dihydroceramides and phytoceramides with saturated fatty acid side chains showed an increase in COPD that was dependent on fatty acid chain length. Only ceramides with a fatty acid side chain from 14 up to 24 C-atoms were higher expressed in smokers with COPD in our study (Figure 3). Indeed their role in COPD remains unclear, but there are indications that alternate chain length regulates specific effects directed by different enzymes.(36-39) This finding illustrates the power of lipidomics since it allows to interrogate both the behavior of lipid classes and individual lipid species.

Another subclass of sphingolipids that were higher expressed in smokers with COPD are the glycosphingolipids (GSLs). These sphingolipids, usually ceramides modified with one or more sugars, are important components of cell membranes. GSLs play an important role in the formation of glycoprotein micro-domains or `lipid rafts` and have been implicated to play a role in signal transduction, phagocytosis, mast cell degranulation and multidrug resistance.(27, 40-44) To date, no studies investigating the role of GSLs in COPD are available. However, the multidrug resistance pump protein (MRP1) has been shown to be involved in the synthesis of GSLs and we previously showed that single nucleotide polymorphisms in the MRP1 gene are associated with COPD development and severity, strengthening the observed higher expression of GSLs in COPD.(45, 46)

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Phosphatidylethanolamines (PE) belong to another class of lipids that was higher expressed in smokers with COPD, compared to smokers without COPD, especially PE species containing polyunsaturated fatty acid side chains. Sphingolipid turnover through sphingosine-1-phosphate can be responsible for the observed increase in PE levels.(47) LysoPE species are membrane metabolites and most abundant in mitochondria and appear to play an important role in hypoxia and oxidative stress sensing.(48-50) LysoPEs are formed by cleaving of PEs by the enzyme PLA

2, forming lysoPEs and fatty acids, like arachidonic acid

(AA).(51) AA can be further transformed to pro-inflammatory eicosanoids like leukotrienes and prostaglandins. Previous studies have shown that oxidative stress activates cytosolic Phospholipase 2 (PLA

2), leading to the release of AA and increased levels of lysoPEs.(51)

Possibly lysoPEs are only byproducts of AA production. Nevertheless, they do seem to be biologically active and contrary to the pro-inflammatory properties of AA, effects of lysoPEs are shown to be anti-inflammatory.(52) In addition, oxidative stress could directly give rise to chemical degradation of phospholipids to lysophospholipids.(53) This mechanism however, is unlikely to be responsible for the higher expression of lysoPEs, as it can be expected that chemical degradation due to oxidative stress would also affect other lipid classes apart from phosphatidylethanolamines. To date, lysoPEs have not been associated with COPD. This study demonstrates the feasibility to use LC-QTOF-MS(/MS) in induced sputum as a powerful tool to identify hundreds of important lipids and associated lipid-pathways in pulmonary disease. Although over 1500 lipids have been identified, several differentially expressed (sphingo-)lipids in smokers with COPD have not yet been unambiguously determined (data not shown), perhaps excluding important biological signals. Determination of low-abundant lipids, e.g. leukotrienes, remains difficult using this untargeted platform and distinguishing glucose and galactose molecules in GSLs is yet impossible, hampering exact lipid determination. In this study, we used induced sputum as a surrogate of the epithelial lining fluid of the lungs. Sputum induction is a relatively easy, non-invasive procedure and safe to perform, even in COPD patients with exacerbations.(54, 55) However, the use of induced sputum needs some points of attention. First, we normalized the expression of lipids by the total lipid expression in the sample. In this way we could identify differentially expressed lipids without the risk of identifying concentration differences. Second, the sputum sample was centrifuged and only the supernatant was used for analysis. This technique removes cells and large cellular debris and may detect lipids that were either actively excreted by vital cells or released upon destruction of cells.

In this study we used three different cohorts of subjects and we acknowledge that an independent replication study in larger cohorts of well characterized subjects is needed to confirm the findings of this explorative study. On the other hand, all these studies have

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been performed in the same institute, using the same protocols and we were not able to identify cohort effects (data not shown).

In conclusion, lipids from the sphingolipid pathway are higher expressed in smokers with COPD compared to smokers without COPD. Considering their potential biological properties, and the observed correlations with important clinical outcomes of COPD, these lipids may play a role in the pathogenesis of COPD. Lipidome analysis may become an important research tool that can lead to new drug targets and possible new biomarkers in chronic obstructive pulmonary disease.

AcknowledgementsThis research was partially performed within the framework of the Top Institute Pharma project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, with partners University Medical Center Groningen, University Medical Center Utrecht, University Medical Center Maastricht, Nycomed BV, GlaxoSmithKline, Danone, AstraZeneca, and Foundation TI Pharma.

This study was made possible by: Metablys, Research Institute for Chromatography (Kortrijk, Belgium), Royal Netherlands Academy for Arts and Sciences, Lung Foundation Netherlands (NAF 97.74), Stichting Astmabestrijding (SAB 2009/028) and the Agency for Innovation by Science and Technology in Flanders (IWT-Flanders) Belgium.

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21. Petrache I, Medler TR, Richter AT, Kamocki K, Chukwueke U, Zhen L, Gu Y, Adamowicz J, Schweitzer KS, Hubbard WC, Berdyshev EV, Lungarella G, Tuder RM. Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide. Am J Physiol Lung Cell Mol Physiol 2008;295:L44-53.

22. Petrache I, Natarajan V, Zhen L, Medler TR, Richter AT, Cho C, Hubbard WC, Berdyshev EV, Tuder RM. Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice. Nat Med 2005;11:491-498.

23. Levy M, Khan E, Careaga M, Goldkorn T. Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death. Am J Physiol Lung Cell Mol Physiol 2009;297:L125-33.

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24. Chan C, Goldkorn T. Ceramide path in human lung cell death. Am J Respir Cell Mol Biol 2000;22:460-468.

25. Moreno L, Moral-Sanz J, Morales-Cano D, Barreira B, Moreno E, Ferrarini A, Pandolfi R, Ruperez FJ, Cortijo J, Sanchez-Luna M, Villamor E, Perez-Vizcaino F, Cogolludo A. Ceramide mediates acute oxygen sensing in vascular tissues. Antioxid Redox Signal 2013. 20, 1-14

26. Ravid T, Tsaba A, Gee P, Rasooly R, Medina EA, Goldkorn T. Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol 2003;284:L1082-92.

27. Gulbins E, Li PL. Physiological and pathophysiological aspects of ceramide. Am J Physiol Regul Integr Comp Physiol 2006;290:R11-26.

28. Ruvolo PP. Intracellular signal transduction pathways activated by ceramide and its metabolites. Pharmacol Res 2003;47:383-392.

29. Heinrich M, Wickel M, Schneider-Brachert W, Sandberg C, Gahr J, Schwandner R, Weber T, Saftig P, Peters C, Brunner J, Kronke M, Schutze S. Cathepsin D targeted by acid sphingomyelinase-derived ceramide. EMBO J 1999;18:5252-5263.

30. Huwiler A, Johansen B, Skarstad A, Pfeilschifter J. Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release. FASEB J 2001;15:7-9.

31. Zheng W, Kollmeyer J, Symolon H, Momin A, Munter E, Wang E, Kelly S, Allegood JC, Liu Y, Peng Q, Ramaraju H, Sullards MC, Cabot M, Merrill AH,Jr. Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim Biophys Acta 2006;1758:1864-1884.

32. Lindner K, Uhlig U, Uhlig S. Ceramide alters endothelial cell permeability by a nonapoptotic mechanism. Br J Pharmacol 2005;145:132-140.

33. Siskind LJ, Kolesnick RN, Colombini M. Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations. Mitochondrion 2006;6:118-125.

34. Fabrias G, Munoz-Olaya J, Cingolani F, Signorelli P, Casas J, Gagliostro V, Ghidoni R. Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena. Prog Lipid Res 2012;51:82-94.

35. Hwang O, Kim G, Jang YJ, Kim SW, Choi G, Choi HJ, Jeon SY, Lee DG, Lee JD. Synthetic phytoceramides induce apoptosis with higher potency than ceramides. Mol Pharmacol 2001;59:1249-1255.

36. Kroesen BJ, Jacobs S, Pettus BJ, Sietsma H, Kok JW, Hannun YA, de Leij LF. BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome. J Biol Chem 2003;278:14723-14731.

37. Mullen TD, Spassieva S, Jenkins RW, Kitatani K, Bielawski J, Hannun YA, Obeid LM. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism. J Lipid Res 2011;52:68-77.

38. Hartmann D, Lucks J, Fuchs S, Schiffmann S, Schreiber Y, Ferreiros N, Merkens J, Marschalek R, Geisslinger G, Grosch S. Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth. Int J Biochem Cell Biol 2012;44:620-628.

39. Grosch S, Schiffmann S, Geisslinger G. Chain length-specific properties of ceramides. Prog Lipid Res 2012;51:50-62.

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41. Iwabuchi K, Nagaoka I. Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils. Blood 2002;100:1454-1464.

42. Nakayama H, Yoshizaki F, Prinetti A, Sonnino S, Mauri L, Takamori K, Ogawa H, Iwabuchi K. Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms. J Leukoc Biol 2008;83:728-741.

43. Karman J, Tedstone JL, Gumlaw NK, Zhu Y, Yew N, Siegel C, Guo S, Siwkowski A, Ruzek M, Jiang C, Cheng SH. Reducing glycosphingolipid biosynthesis in airway cells partially ameliorates disease manifestations in a mouse model of asthma. Int Immunol 2010;22:593-603.

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Supplemental Information

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Supplemental methods

Induced sputum lipid extractionAll samples were randomized before the lipid extraction. The lipid extraction method was based on the procedure described by Matyash et al.(1) 120 µL of induced lung sputum was transferred to a 2 mL Eppendorf tube and 300 µL of methanol was added. After vortexing for 10 seconds, 1000 µL of MTBE was added after which the sample was incubated for 1 hour at room temperature in a shaker. Subsequently, phase separation was induced by adding 260 µL of water. After 10 minutes incubation at room temperature, the extract was centrifuged for 10 minutes at 1000 x g thereby generating a lower hydrophilic and upper lipophilic phase separated by a protein layer. 1000 µL of the upper phase has been removed and dried in a centrifugal vacuum evaporator (miVac duoconcentrator, Genevac Lim., Ipswich, UK). The dried sample has been reconstituted in 40 µL methyl-tert-butylether/isopropanol 50/50 (v/v). An injection volume of 2 µL and 6 µL of this extract has been used for LC-MS analysis in positive ESI and negative ESI mode, respectively.

Experimental setup, preparation and use of quality control (QC) samples For each experiment, the use of QC samples allowed monitoring the repeatability and stability of the lipid measurement. Three independent analytical experiments have been performed: induced sputum analysis comparing current-smokers with COPD, current-smokers without COPD and never-smokers (53 induced sputum samples, 34 QCs); induced sputum analysis comparing smokers with and without COPD before and after 2-month smoking cessation (34 induced sputum samples, 13 QCs). To prepare QC samples within each experiment, 60 µL aliquots of the study samples were collected in a QC pool. This QC pool was then divided into 120 µL aliquots to obtain representative QC samples. QC samples were prepared simultaneously along with study samples and were analyzed throughout the LC-MS analysis sequences every five study samples. These samples did not contain any biological variability and can thus be considered as technical replicates. The relative standard deviation percent (RSD%) of all significantly up- or downregulated lipids has been calculated to demonstrate the measurement error of the lipidomics analytical platform.

Liquid chromatography-mass spectrometryA 1200 RRLC system (Agilent Technologies, Waldbronn, Germany) was used for RP-LC measurements. Lipid extracts were analyzed on an XBridge BEH C18 Shield column (2.1 x 100 mm; 1.8 µm; Waters, Milford, MA, USA) placed in a Polaratherm 9000 series oven (Selerity Technologies, Salt Lake City, UT, USA) at 80°C. Elution was carried out with a multistep gradient of (A) 20 mM ammonium formate pH 5 and (B) methanol, starting from 50% B

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to 70% B in 5 minutes, followed by a gradient of 70–90% B in 30 minutes. Mobile phase B subsequently reached 100% in 0.1 minutes where it was maintained for an additional 5 minutes. The flow rate was 0.5 mL/min and the injection volume 6 μL for negative ESI and 2 µL for positive ESI measurements. The whole system was allowed to re-equilibrate under starting conditions for 15 min.

High-resolution accurate mass measurements were obtained on an Agilent 6530 Q-TOF mass spectrometer (Agilent Technologies) equipped with a Jetstream ESI source. The instrument was operated in both positive and negative ion electrospray mode. Needle voltage was optimized to +/- 3.5 kV, the drying and sheath gas temperatures were set to 300°C and the drying and sheath gas flow rates were set to 6 and 8 L/min, respectively. Data were collected in centroid mode from m/z 400–1700 in positive ion mode and m/z 200-1700 in negative ion mode at an acquisition rate of 1 spectrum/sec in the extended dynamic range mode (2 GHz), offering an in-spectrum dynamic range of 105 and a resolution of ± 10,000 FWHM in the lipid m/z range. To maintain mass accuracy during the analysis sequence, a reference mass solution was used containing reference ions (922.0097 for positive ESI mode, and 1033.9881 for negative ESI mode). Study samples were analyzed in randomized order in both ionization modes, with QC samples analyzed every 5 study samples. Samples were kept at 4°C in the autosampler tray while waiting for injection.

Tandem mass spectrometry (MS/MS) experiments were performed in the data dependent acquisition mode (DDA). A survey MS scan was alternated with three DDA MS/MS scans resulting in a cycle time of 4 seconds. Singly charged precursor ions were selected based on abundance. After being fragmented twice, a particular m/z value was excluded for 30 s, allowing the MS/MS fragmentation of chromatographically resolved lipid isomers. Subsequent targeted MS/MS or further DDA experiments, thereby adding previously fragmented precursors in an exclusion list, increased the identification rate. The quadrupole was operated at narrow resolution and the collision energy was fixed at either 20 or 35 eV.

Lipid identificationLipid identification was based on accurate mass, MS/MS fragmentation and chromatographic retention time. Lipid identification results from an identification strategy that fully exploits the features of the Q-TOF-MS system. Molecular formulas, based on accurate mass, isotopic abundance, and spacing both in positive and negative ionization modes, are complemented with accurate mass database searching in an in-house build database (populated with LIPID MAPS and HMDB entries and theoretical lipid structures) and MS/MS measurement in both modes. Lipid fragmentation mechanisms/spectra in both positive and negative ionization modes have extensively been reported in literature.(2-11) Each lipid

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class displays characteristic fragmentation spectrum ions in positive and/or negative ESI mode, through neutral loss or the abundance of unique fragment ions. Other parameters such as lipid elution behavior and adduct formation interpretation further assisted in the identification. An in-house accurate mass retention time (AMRT) library was subsequently built with formula, exact mass and retention time of all identified lipids in comma-separated values (.csv) format (compatible with the MassHunter software), providing an automated and targeted data-processing of LC-MS lipid profiles.(2)

Data processing Raw LC-MS data files were processed in an untargeted fashion using the Molecular Feature Extraction (MFE) algorithm incorporated in the MassHunter Qualitative Analysis 5.0 software package (Agilent Technologies). The resulting feature files were subsequently imported in MassProfiler Professional 12.0 (Agilent Technologies) which aligned, visualized and filtered the features. The filtered feature list was exported for recursive peak integration (i.e. targeted feature extraction) using the Find by Ion extraction algorithm in MassHunter Qualitative Analysis 5.0 using predefined mass (25 ppm) and retention time extraction windows (15 sec). The Agile integrator was used, with an absolute peak height cut-off of 1000 counts. After targeted extraction of features, samples were normalized using total lipid abundance, correcting for sputum dilution, and were imported in MassProfiler Professional for statistical analysis.

Supplemental results

Negative ESI Positive ESI

Supplemental Figure 1: Total lipid abundance in induced sputum did not significantly alter between smokers with COPD, smokers without COPD and never-smokers. Box-and-whisker plots represent the minimum and maximum, interquartile range and median total lipid abundance for each sample group. NS = never-smokers (n=14), H_S = smokers without COPD (n=20), COPD = smokers with COPD (n=19), QC = quality control.

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0%

10%

20%

30%

40%

50%

60%

70%

80%

relativ

e qu

antity with

in sam

ple grou

p (%

)

never‐smoker without COPDsmoker without COPDsmoker with COPD

 

 

 

 

 

 

 

 

 

 

 

Supplemental Figure 2: Expression of sphingolipid subclasses in never-smokers, smokers with and without COPD. Sphingolipid subclass expression for each individual sample was calculated by summing up the measured peak areas of sphingolipid species (n = 215). After normalization for sputum dilution, relative quantities for each sphingolipid class were calculated within each sample group. Values are presented as average percentage ± standard deviation. Hex = glucose or galactose; HexNAc = N-acetylglucosamine or N-acetylgalactosamine; never-smokers n= 14, smokers without COPD n=20, smokers with COPD n=19.

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122

Chapter 5

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123

Lipidomic analysis in COPD

Chapter

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.03

SM(d

18:0

/22:

0)C

45H

93N

2O6P

1.76

0.00

611

.45

SM(d

18:0

/24:

0)C

47H

97N

2O6P

1.43

0.02

419

.12

SM(d

18:0

/24:

1)C

47H

95N

2O6P

2.26

0.00

815

.49

SM(d

18:1

/14:

0)C

37H

75N

2O6P

1.84

0.03

222

.78

SM(d

18:1

/15:

0)C

38H

77N

2O6P

1.57

0.02

910

.80

SM(d

18:1

/16:

0)C

39H

79N

2O6P

1.65

0.02

310

.21

SM(d

18:1

/17:

0)C

40H

81N

2O6P

1.74

0.00

910

.97

SM(d

18:1

/18:

0)C

41H

83N

2O6P

1.77

0.00

810

.23

SM(d

18:1

/19:

0)C

42H

85N

2O6P

1.69

0.00

78.

84

124

Chapter 5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Sphi

ngol

ipid

s

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

sphi

ngom

yelin

sph

ytoc

eram

ides

SM(d

18:1

/20:

0)C

43H

87N

2O6P

1.82

0.00

511

.47

SM(d

18:1

/21:

0)C

44H

89N

2O6P

1.67

0.01

010

.06

SM(d

18:1

/22:

0)C

45H

91N

2O6P

1.65

0.01

011

.28

SM(d

18:1

/22:

1)C

45H

89N

2O6P

1.56

0.05

012

.15

SM(d

18:1

/23:

0)C

46H

93N

2O6P

1.64

0.02

413

.72

SM(d

18:1

/24:

0)C

47H

95N

2O6P

1.60

0.02

313

.09

SM(d

18:1

/24:

1)C

47H

93N

2O6P

1.69

0.02

511

.36

SM(d

18:1

/24:

2)C

47H

91N

2O6P

1.93

0.01

512

.42

SM(d

18:1

/26:

1)C

49H

97N

2O6P

1.56

0.01

011

.31

SM(d

18:1

/26:

2)C

49H

95N

2O6P

1.94

0.00

816

.73

SM(t

18:0

/14:

0)C

37H

77N

2O7P

1.47

0.01

518

.00

SM(t

18:0

/16:

0)C

39H

81N

2O7P

2.02

0.00

512

.06

SM(t

18:0

/16:

1)C

39H

79N

2O7P

1.87

0.00

614

.22

SM(t

18:0

/18:

1)C

41H

83N

2O7P

1.94

0.01

015

.33

SM(t

18:0

/22:

0)C

45H

93N

2O7P

1.66

0.00

910

.14

SM(t

18:0

/23:

0)C

46H

95N

2O7P

1.89

0.00

511

.23

SM(t

18:0

/24:

0)C

47H

97N

2O7P

1.91

0.00

514

.78

SM(t

18:0

/24:

1)C

47H

95N

2O7P

1.99

0.00

612

.08

SM(t

18:0

/24:

2)C

47H

93N

2O7P

2.56

0.00

514

.01

SM(t

18:0

/25:

1)C

48H

97N

2O7P

1.72

0.01

014

.88

SM(t

18:0

/26:

1)C

49H

99N

2O7P

1.57

0.00

99.

96

SM(t

18:0

/26:

2)C

49H

97N

2O7P

2.21

0.00

512

.83

125

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Sphi

ngol

ipid

s

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(1

sug

ar)

hexo

syl-d

ihyd

roce

ram

ides

Hex

Cer(d

18:0

/16:

0)C

40H

79N

O8

2.69

0.00

210

.40

hexo

syl-c

eram

ides

Hex

Cer(d

18:1

/16:

0)C

40H

77N

O8

1.91

0.00

19.

79

Hex

Cer(d

18:1

/16:

0(2-

OH

))C

40H

77N

O9

1.96

0.00

19.

49

Hex

Cer(d

18:1

/18:

0)C

42H

81N

O8

1.57

0.03

59.

89

Hex

Cer(d

18:1

/22:

0)C

46H

89N

O8

1.35

0.01

27.

61

Hex

Cer(d

18:1

/23:

0)C

48H

93N

O8

1.26

0.00

59.

12

Hex

Cer(d

18:1

/24:

0(2-

OH

))C

48H

93N

O9

1.53

0.00

510

.84

hexo

syl-p

hyto

cera

mid

esH

exCe

r(t18

:0/1

6:0)

C40

H79

NO

92.

000.

001

7.79

Hex

Cer(t

18:0

/18:

0)C

42H

83N

O9

1.55

0.04

39.

13

Hex

Cer(t

18:0

/20:

0)C

44H

87N

O9

1.70

0.01

910

.85

Hex

Cer(t

18:0

/22:

0)C

46H

91N

O9

1.57

0.00

37.

27

Hex

Cer(t

18:0

/23:

0)C

47H

93N

O9

1.56

0.00

59.

80

Hex

Cer(t

18:0

/24:

0)C

48H

95N

O9

1.44

0.00

76.

64

Hex

Cer(t

18:0

/24:

1)C

48H

93N

O9

1.47

0.01

07.

85

Hex

Cer(t

18:0

/24:

2)C

48H

91N

O9

2.46

0.00

113

.84

glyc

osph

ingo

lipid

s(2

sug

ars)

dihe

xosy

l-cer

amid

esH

ex-H

exCe

r(d18

:1/1

6:0(

2-O

H))

C46

H87

NO

142.

020.

001

10.0

2

Hex

-Hex

Cer(d

18:1

/20:

0(2-

OH

))C

50H

95N

O14

2.21

0.03

338

.76

Hex

-Hex

Cer(d

18:1

/22:

0(2-

OH

))C

52H

99N

O14

2.20

0.00

220

.83

Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))C

54H

103N

O14

1.80

0.00

115

.07

Hex

-Hex

Cer(d

18:1

/26:

0(2-

OH

))C

56H

107N

O14

1.64

0.01

216

.04

dihe

xosy

l-phy

toce

ram

ides

Hex

-Hex

Cer(t

18:0

/16:

0)C

46H

89N

O14

2.17

0.00

19.

98

Hex

-Hex

Cer(t

18:0

/22:

0)C

52H

101N

O14

1.99

0.00

119

.03

Hex

-Hex

Cer(t

18:0

/24:

0)C

54H

105N

O14

1.78

0.00

115

.22

126

Chapter 5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Sphi

ngol

ipid

s

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(2

sug

ars)

dihe

xosy

l-phy

toce

ram

ides

Hex

-Hex

Cer(t

18:0

/24:

1)C

54H

103N

O14

1.53

0.01

110

.19

Hex

-Hex

Cer(t

18:0

/24:

2)C

54H

101N

O14

2.86

0.00

911

.87

glyc

osph

ingo

lipid

s(3

sug

ars)

trih

exos

yl-c

eram

ides

Hex

-Hex

-Hex

Cer(d

18:1

/16:

0)C

52H

97N

O18

2.57

0.00

113

.30

Hex

-Hex

-Hex

Cer(d

18:1

/16:

0(2-

OH

))C

52H

97N

O19

2.51

<0.

001

18.2

4

Hex

-Hex

-Hex

Cer(d

18:1

/17:

0)C

53H

99N

O18

3.50

0.00

116

.48

Hex

-Hex

-Hex

Cer(d

18:1

/18:

0)C

54H

101N

O18

2.34

0.01

126

.88

Hex

-Hex

-Hex

Cer(d

18:1

/18:

0(2-

OH

))C

54H

101N

O19

2.37

0.00

320

.73

Hex

-Hex

-Hex

Cer(d

18:1

/18:

2)C

54H

97N

O18

2.47

<0.

001

18.5

3

Hex

-Hex

-Hex

Cer(d

18:1

/20:

0)C

56H

105N

O18

1.96

0.01

617

.61

Hex

-Hex

-Hex

Cer(d

18:1

/20:

0(2-

OH

))C

56H

105N

O19

3.18

0.00

331

.60

Hex

-Hex

-Hex

Cer(d

18:1

/20:

1)C

56H

103N

O18

2.85

0.03

326

.94

Hex

-Hex

-Hex

Cer(d

18:1

/21:

0)C

57H

107N

O18

2.60

0.03

128

.89

Hex

-Hex

-Hex

Cer(d

18:1

/22:

0)C

58H

109N

O18

1.45

0.04

521

.08

Hex

-Hex

-Hex

Cer(d

18:1

/22:

0(2-

OH

))C

58H

109N

O19

2.48

0.00

327

.34

Hex

-Hex

-Hex

Cer(d

18:1

/22:

2)C

58H

105N

O18

2.88

0.00

933

.49

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0)C

59H

111N

O18

1.48

0.02

918

.28

Hex

-Hex

-Hex

Cer(d

18:1

/23:

1)C

59H

109N

O18

1.97

0.04

018

.30

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0(2-

OH

))C

59H

111N

O19

2.40

0.00

323

.32

Hex

-Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))C

60H

113N

O19

1.87

0.00

622

.17

Hex

-Hex

-Hex

Cer(d

18:1

/24:

1)C

60H

111N

O18

1.55

0.01

212

.74

Hex

-Hex

-Hex

Cer(d

18:1

/24:

2)C

60H

109N

O18

1.74

0.01

620

.72

Hex

-Hex

-Hex

Cer(d

18:1

/25:

0(2-

OH

))C

61H

115N

O19

2.10

0.02

626

.60

Hex

-Hex

-Hex

Cer(d

18:1

/26:

1)C

62H

115N

O18

3.02

0.00

121

.78

127

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Sphi

ngol

ipid

s

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(3

sug

ars)

trih

exos

yl-d

ihyd

roce

ram

ides

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0)C

52H

99N

O18

3.96

0.00

113

.19

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0(2-

OH

))C

52H

99N

O19

3.58

<0.

001

19.5

0

Hex

-Hex

-Hex

Cer(d

18:0

/18:

0)C

54H

103N

O18

4.47

0.00

215

.86

Hex

-Hex

-Hex

Cer(d

18:0

/18:

0(2-

OH

))C

54H

103N

O19

5.28

0.00

129

.29

Hex

-Hex

-Hex

Cer(d

18:0

/20:

0)C

56H

107N

O18

3.61

0.00

220

.88

Hex

-Hex

-Hex

Cer(d

18:0

/22:

0)C

58H

111N

O18

1.96

0.00

419

.07

Hex

-Hex

-Hex

Cer(d

18:0

/22:

0(2-

OH

))C

58H

111N

O19

3.11

0.00

221

.21

Hex

-Hex

-Hex

Cer(d

18:0

/24:

0(2-

OH

))C

60H

115N

O19

2.21

0.02

127

.97

trih

exos

yl-p

hyto

cera

mid

esH

ex-H

ex-H

exCe

r(t18

:0/1

6:0)

C52

H99

NO

192.

740.

001

17.7

8

Hex

-Hex

-Hex

Cer(t

18:0

/16:

0(2-

OH

))C

52H

99N

O20

2.92

0.00

315

.28

Hex

-Hex

-Hex

Cer(t

18:0

/17:

0)C

53H

101N

O19

3.26

0.00

214

.44

Hex

-Hex

-Hex

Cer(t

18:0

/18:

0)C

54H

103N

O19

2.41

0.00

726

.33

Hex

-Hex

-Hex

Cer(t

18:0

/18:

0(2-

OH

))C

54H

103N

O20

1.90

0.03

519

.70

Hex

-Hex

-Hex

Cer(t

18:0

/20:

0)C

56H

107N

O19

2.68

0.01

943

.49

Hex

-Hex

-Hex

Cer(t

18:0

/20:

0(2-

OH

))C

56H

107N

O20

2.31

0.03

325

.83

Hex

-Hex

-Hex

Cer(t

18:0

/22:

0)C

58H

111N

O19

1.74

0.02

421

.84

Hex

-Hex

-Hex

Cer(t

18:0

/22:

1)C

58H

109N

O19

2.17

0.04

847

.33

Hex

-Hex

-Hex

Cer(t

18:0

/22:

2)C

58H

107N

O19

5.15

0.00

216

.38

Hex

-Hex

-Hex

Cer(t

18:0

/24:

1)C

60H

113N

O19

1.64

0.02

618

.31

Hex

-Hex

-Hex

Cer(t

18:0

/24:

2)C

60H

111N

O19

4.50

0.00

324

.85

glyc

osph

ingo

lipid

s(o

ther

)ne

uram

inic

aci

d co

ntai

ning

dih

exos

yl-

cera

mid

esN

euA

c-H

ex-H

exCe

r(d18

:1/2

4:0)

C65

H12

0N2O

210.

620.

022

29.2

8

N-a

cety

lhex

osam

ine-

trih

exos

yl-c

eram

ides

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/1

6:0)

C60

H11

0N2O

231.

910.

013

18.9

8

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/1

8:0)

C62

H11

4N2O

234.

800.

002

26.7

7

128

Chapter 5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Sphi

ngol

ipid

s

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(o

ther

)N

-ace

tylh

exos

amin

e-tr

ihex

osyl

-cer

amid

esH

exN

Ac-

Hex

-Hex

-Hex

Cer(d

18:1

/20:

0)C

64H

118N

2O23

5.09

0.00

237

.29

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

2:0)

C66

H12

2N2O

231.

860.

029

23.1

3

N-a

cety

lhex

osam

ine-

trih

exos

yl-

dihy

droc

eram

ides

H

exN

Ac-

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0(2-

OH

))C

60H

112N

2O24

6.23

0.00

141

.90

N-a

cety

lhex

osam

ine-

trih

exos

yl-

phyt

ocer

amid

esH

exN

Ac-

Hex

-Hex

-Hex

Cer(t

18:0

/16:

1)C

60H

110N

2O24

3.09

0.00

122

.65

Hex

NA

c-H

ex-H

ex-H

exCe

r(t18

:0/1

8:0)

C62

H11

6N2O

243.

280.

007

30.0

2

Hex

NA

c-H

ex-H

ex-H

exCe

r(t18

:0/2

4:0)

C68

H12

8N2O

243.

190.

008

53.7

1

Gly

cero

phos

polip

ids

etha

nola

min

esph

osph

atid

ylet

hano

lam

ines

PE(1

8:0/

20:4

)C

43H

78N

O8P

1.59

0.01

47.

91

PE(3

8:6)

C43

H74

NO

8P1.

550.

013

15.1

7

lyso

phos

phat

idyl

etha

nola

min

esPE

(0:0

/18:

1)C

23H

46N

O7P

1.87

0.04

715

.48

PE(0

:0/1

8:2)

C23

H44

NO

7P2.

450.

030

20.6

7

PE(0

:0/2

0:2)

C25

H48

NO

7P2.

380.

023

19.4

3

PE(2

0:2/

0:0)

C25

H48

NO

7P2.

230.

019

21.8

1

PE(2

0:3/

0:0)

C25

H46

NO

7P2.

290.

009

16.1

7

PE(0

:0/2

0:4)

C25

H44

NO

7P4.

920.

011

24.9

5

PE(2

0:4/

0:0)

C25

H44

NO

7P2.

560.

023

19.2

3

PE(0

:0/2

0:5)

C25

H42

NO

7P6.

300.

016

22.6

6

PE(2

2:4/

0:0)

C27

H48

NO

7P2.

380.

025

18.7

9

PE(0

:0/2

2:4)

C27

H48

NO

7P4.

000.

012

23.2

4

PE(0

:0/2

2:5)

C27

H46

NO

7P5.

190.

010

22.5

5

PE(2

2:5/

0:0)

C27

H46

NO

7P2.

740.

010

13.1

6

129

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Gly

cero

phos

polip

ids

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

etha

nola

min

esly

soph

osph

atid

ylet

hano

lam

ines

PE(0

:0/2

2:6)

C27

H44

NO

7P4.

830.

024

25.2

7

PE(2

2:6/

0:0)

C27

H44

NO

7P2.

520.

030

13.4

5

plas

mal

ogen

pho

spat

idyl

etha

nola

min

esPE

(p16

:0/1

8:1)

C39

H76

NO

7P1.

640.

003

7.07

PE(p

16:0

/20:

4)C

41H

74N

O7P

1.89

0.00

18.

40

PE(p

16:0

/22:

4)C

43H

78N

O7P

2.08

0.00

16.

62

PE(p

16:0

/22:

6)C

43H

74N

O7P

2.44

0.00

111

.20

PE(p

17:0

/22:

6)C

44H

76N

O7P

2.02

0.00

510

.00

PE(p

18:0

/18:

1)C

41H

80N

O7P

1.54

0.04

87.

39

PE(p

18:0

/18:

2)C

41H

78N

O7P

1.64

0.01

29.

19

PE(p

18:0

/20:

4)C

43H

78N

O7P

1.73

0.00

28.

65

PE(p

18:0

/20:

5)C

43H

76N

O7P

1.47

0.00

519

.19

PE(p

18:0

/22:

4)C

45H

82N

O7P

1.71

0.00

56.

40

PE(p

18:0

/22:

6)C

45H

78N

O7P

2.18

0.00

16.

68

PE(p

18:1

/18:

1)C

41H

78N

O7P

1.70

0.04

56.

74

PE(p

20:0

/18:

1)C

43H

84N

O7P

1.91

0.01

88.

14

PEp(

34:2

)C

39H

74N

O7P

1.70

0.00

17.

49

PEp(

38:5

)C

43H

76N

O7P

1.56

0.01

19.

40

PEp(

40:1

)C

45H

88N

O7P

2.38

0.01

49.

80

PEp(

42:3

)C

47H

88N

O7P

1.91

0.02

716

.69

PEp(

42:6

)C

47H

82N

O7P

3.69

0.00

17.

26

PEp(

44:6

)C

49H

86N

O7P

3.93

0.00

112

.21

PEp(

44:7

)C

49H

84N

O7P

3.92

0.00

217

.45

130

Chapter 5

Supp

lem

enta

l Tab

le 1

: Con

tinue

d

Pren

ol li

pids

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

toba

cco

com

poun

dspl

asm

alog

en p

hosp

atid

ylet

hano

lam

ines

sola

neso

lC

45H

74O

3.70

0.02

913

.60

sola

nesy

l pal

mita

teC

61H

104O

24.

610.

050

14.0

4

sola

nesy

l ste

arat

eC

63H

108O

28.

190.

025

18.6

0

bom

bipr

enon

eC

43H

70O

3.10

0.02

512

.54

farn

esyl

farn

esyl

acet

one

C33

H54

O2.

530.

041

10.7

1

Fold

cha

nge

= e

xpre

ssio

n in

smok

ers w

ith C

OPD

/ ex

pres

sion

in sm

oker

s with

out C

OPD

. A fo

ld c

hang

e >

1 is

up-

regu

latio

n in

smok

ers w

ith C

OPD

. A fo

ld c

hang

e be

twee

n 0

and

1 is

dow

n-re

gula

tion

in s

mok

ers

with

CO

PD. T

he L

ipid

Map

s da

taba

se w

as ta

ken

as th

e re

fere

nce

for t

he li

pid

nom

encl

atur

e. L

C-M

S ca

nnot

dis

crim

inat

e be

twee

n ga

lact

ose

and

gluc

ose,

or N

-ace

tylg

luco

sam

ine

and

N-a

cety

lgal

acto

sam

ine.

The

refo

re, l

ipid

nom

encl

atur

e co

ntai

ns h

exos

e (H

ex) i

nste

ad o

f glu

cose

or g

alac

tose

. Rel

ativ

e st

anda

rd d

evia

tion

(RSD

%) h

as b

een

calc

ulat

ed fr

om th

e Q

C sa

mpl

es (n

=34

). A

ll p-

valu

es a

re c

orre

cted

for m

ultip

le te

stin

g w

ith th

e Be

njam

ini H

ochb

erg

Fals

e D

isco

very

Rat

e.

131

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 2

: Diff

eren

tially

regu

late

d lip

ids

afte

r 2-m

onth

sm

okin

g ce

ssat

ion

in s

ubje

cts

with

and

with

out C

OPD

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

sphi

ngol

ipid

sce

ram

ides

Cer(d

18:1

/14:

0)C

32H

63N

O3

1.76

0.04

99.

10

Cer(d

18:1

/15:

0)C

33H

65N

O3

2.36

0.00

78.

12

Cer(d

18:1

/15:

0(2-

OH

))C

33H

65N

O4

1.98

0.03

310

.80

Cer(d

18:1

/16:

0)C

34H

67N

O3

2.29

0.00

76.

46

Cer(d

18:1

/16:

0(2-

OH

))C

34H

67N

O4

2.62

0.00

97.

16

Cer(d

18:1

/17:

0)C

35H

69N

O3

2.89

0.00

75.

78

Cer(d

18:1

/17:

0(2-

OH

))C

35H

69N

O4

2.50

0.01

38.

04

Cer(d

18:1

/17:

1)C

35H

67N

O3

3.38

0.01

124

.89

Cer(d

18:1

/18:

0)C

36H

71N

O3

2.13

0.00

84.

42

Cer(d

18:1

/18:

0(2-

OH

))C

36H

71N

O4

3.27

0.00

915

.69

Cer(d

18:1

/18:

1)C

36H

69N

O3

1.91

0.00

78.

42

Cer(d

18:1

/19:

0)C

37H

73N

O3

2.53

0.00

76.

60

Cer(d

18:1

/20:

0)C

38H

75N

O3

2.47

0.00

96.

63

Cer(d

18:1

/20:

0(2-

OH

))C

38H

75N

O4

1.61

0.03

329

.15

Cer(d

18:1

/20:

1)C

38H

73N

O3

2.66

0.00

710

.54

Cer(d

18:1

/20:

2)C

38H

71N

O3

2.23

0.01

110

.44

Cer(d

18:1

/21:

0)C

39H

77N

O3

2.17

0.00

73.

86

Cer(d

18:1

/21:

0(2-

OH

))C

39H

77N

O4

2.29

0.01

322

.17

Cer(d

18:1

/22:

0)C

40H

79N

O3

2.12

0.00

77.

78

Cer(d

18:1

/22:

0(2-

OH

))C

40H

79N

O4

1.72

0.00

77.

10

Cer(d

18:1

/22:

1)C

40H

77N

O3

2.17

0.01

56.

86

Cer(d

18:1

/22:

2)C

40H

75N

O3

2.33

0.00

78.

59

Cer(d

18:1

/23:

0)C

41H

81N

O3

2.03

0.00

76.

01

Cer(d

18:1

/23:

1)C

41H

79N

O3

2.30

0.00

77.

55

132

Chapter 5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

sphi

ngol

ipid

sce

ram

ides

Cer(d

18:1

/24:

0)C

42H

83N

O3

1.97

0.00

95.

95

Cer(d

18:1

/24:

1)C

42H

81N

O3

2.16

0.00

96.

69

Cer(d

18:1

/24:

2)C

42H

79N

O3

2.49

0.00

76

Cer(d

18:1

/25:

0)C

43H

85N

O3

1.61

0.03

96.

96

Cer(d

18:1

/25:

1)C

43H

83N

O3

2.80

0.00

76.

53

Cer(d

18:1

/25:

2)C

43H

81N

O3

2.17

0.00

78.

79

Cer(d

18:1

/26:

1)C

44H

85N

O3

2.31

0.02

77.

86

Cer(d

18:1

/26:

2)C

44H

83N

O3

2.15

0.00

96.

80

dihy

droc

eram

ides

Cer(d

18:0

/14:

0)C

32H

65N

O3

2.97

0.01

310

.51

Cer(d

18:0

/15:

0)C

33H

67N

O3

3.20

0.00

87.

71

Cer(d

18:0

/16:

0)C

34H

69N

O3

4.50

0.00

77.

27

Cer(d

18:0

/16:

0(2-

OH

))C

34H

69N

O4

2.97

0.00

86.

77

Cer(d

18:0

/17:

0)C

35H

71N

O3

3.48

0.00

79.

02

Cer(d

18:0

/18:

0)C

36H

73N

O3

3.76

0.00

75.

80

Cer(d

18:0

/19:

0)C

37H

75N

O3

3.37

0.00

714

.00

Cer(d

18:0

/20:

0)C

38H

77N

O3

2.96

0.00

75.

79

Cer(d

18:0

/21:

0)C

39H

79N

O3

1.91

0.03

39.

22

Cer(d

18:0

/22:

0)C

40H

81N

O3

2.21

0.01

58.

41

Cer(d

18:0

/23:

0)C

41H

83N

O3

1.96

0.02

75.

18

phyt

ocer

amid

esCe

r(t18

:0/1

5:0)

C33

H67

NO

42.

370.

009

11.7

6

Cer(t

18:0

/16:

0)C

34H

69N

O4

2.87

0.00

710

.43

Cer(t

18:0

/16:

1)C

34H

67N

O4

2.81

0.01

312

.12

Cer(t

18:0

/17:

0)C

35H

71N

O4

2.55

0.00

77.

40

Cer(t

18:0

/18:

0)C

36H

73N

O4

2.47

0.00

95.

35

Cer(t

18:0

/18:

1)C

36H

71N

O4

2.23

0.01

113

.89

133

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

sphi

ngol

ipid

sph

ytoc

eram

ides

Cer(t

18:0

/19:

0)C

37H

75N

O4

2.22

0.00

75.

58

Cer(t

18:0

/20:

0)C

38H

77N

O4

2.04

0.00

98.

33

Cer(t

18:0

/20:

1)C

38H

75N

O4

2.40

0.01

510

.45

Cer(t

18:0

/21:

0)C

39H

79N

O4

2.05

0.00

75.

54

Cer(t

18:0

/22:

0)C

40H

81N

O4

1.74

0.00

73.

23

Cer(t

18:0

/23:

0)C

41H

83N

O4

1.61

0.00

77.

44

Cer(t

18:0

/23:

1)C

41H

81N

O4

2.81

0.00

78.

59

Cer(t

18:0

/24:

0)C

42H

85N

O4

1.53

0.01

85.

44

Cer(t

18:0

/24:

1)C

42H

83N

O4

2.19

0.00

97.

74

Cer(t

18:0

/24:

2)C

42H

81N

O4

3.00

0.00

712

.76

Cer(t

18:0

/25:

1)C

43H

85N

O4

1.50

0.01

126

.48

Cer(t

18:0

/26:

1)C

44H

87N

O4

2.33

0.00

79.

07

sphi

ngom

yelin

sSM

(d18

:0/1

4:0)

C37

H77

N2O

6P2.

800.

033

7.19

SM(d

18:0

/16:

0)C

39H

81N

2O6P

3.50

0.01

88.

54

SM(d

18:1

/14:

0)C

37H

75N

2O6P

2.30

0.04

08.

21

SM(d

18:1

/15:

0)C

38H

77N

2O6P

2.41

0.04

06.

61

SM(d

18:1

/16:

0)C

39H

79N

2O6P

2.64

0.01

87.

58

SM(d

18:1

/17:

0)C

40H

81N

2O6P

2.53

0.05

027

.84

SM(d

18:1

/18:

0)C

41H

83N

2O6P

3.52

0.01

620

.90

SM(d

18:1

/22:

0)C

45H

91N

2O6P

4.50

0.01

524

.38

SM(d

18:1

/23:

0)C

46H

93N

2O6P

2.86

0.01

57.

85

SM(d

18:1

/24:

0)C

47H

95N

2O6P

2.48

0.02

09.

00

SM(d

18:1

/24:

1)C

47H

93N

2O6P

3.24

0.01

528

.02

SM(d

18:1

/24:

2)C

47H

91N

2O6P

3.30

0.02

09.

60

SM(t

18:0

/16:

0)C

39H

81N

2O7P

3.73

0.01

517

.98

134

Chapter 5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

sphi

ngom

yelin

sph

ytoc

eram

ides

SM(t

18:0

/16:

1)C

39H

79N

2O7P

2.53

0.01

612

.39

SM(t

18:0

/18:

0)C

41H

85N

2O7P

2.92

0.02

017

.66

SM(t

18:0

/18:

1)C

41H

83N

2O7P

2.38

0.02

013

.79

SM(t

18:0

/22:

0)C

45H

93N

2O7P

2.76

0.01

510

.45

SM(t

18:0

/24:

1)C

47H

95N

2O7P

3.60

0.01

58.

28

SM(t

18:0

/24:

2)C

47H

93N

2O7P

3.53

0.01

512

.83

glyc

osph

ingo

lipid

s(1

sug

ar)

hexo

syl-c

eram

ides

Hex

Cer(d

18:1

/16:

0(2-

OH

))C

40H

77N

O9

1.89

0.02

78.

25

Hex

Cer(d

18:1

/22:

0)C

46H

89N

O8

1.39

0.02

28.

43

Hex

Cer(d

18:1

/22:

0(2-

OH

))C

46H

89N

O9

2.33

0.00

914

.26

Hex

Cer(d

18:1

/23:

0(2-

OH

))C

47H

91N

O9

1.61

0.01

313

.09

Hex

Cer(d

18:1

/24:

0(2-

OH

))C

48H

93N

O9

1.68

0.02

27.

80

Hex

Cer(t

18:0

/18:

0(2-

OH

))C

42H

83N

O10

2.35

0.01

820

.52

Hex

Cer(t

18:0

/20:

0(2-

OH

))C

44H

87N

O10

5.85

0.02

740

.28

Hex

Cer(t

18:0

/22:

0)C

46H

91N

O9

1.76

0.01

87.

30

Hex

Cer(t

18:0

/23:

0(2-

OH

))C

47H

93N

O10

3.80

0.01

816

.11

Hex

Cer(t

18:0

/24:

2)C

48H

91N

O9

2.60

0.01

57.

87

Hex

Cer(t

18:0

/25:

0)C

49H

97N

O9

1.46

0.03

926

.90

glyc

osph

ingo

lipid

s(2

sug

ars)

dihe

xosy

l-phy

toce

ram

ides

H

ex-H

exCe

r(d18

:1/1

6:0(

2-O

H))

C46

H87

NO

142.

000.

018

8.60

Hex

-Hex

Cer(d

18:1

/17:

0)C

47H

89N

O13

2.77

0.03

911

.94

Hex

-Hex

Cer(d

18:1

/18:

0)C

48H

91N

O13

2.00

0.03

99.

05

Hex

-Hex

Cer(d

18:1

/18:

0(2-

OH

))C

48H

91N

O14

3.48

0.00

817

.50

Hex

-Hex

Cer(d

18:1

/20:

0)C

50H

95N

O13

2.39

0.02

224

.76

Hex

-Hex

Cer(d

18:1

/22:

0(2-

OH

))C

52H

99N

O14

3.03

0.00

712

.77

Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))C

54H

103N

O14

2.43

0.00

79.

36

135

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

glyc

osph

ingo

lipid

s(2

sug

ars)

dihe

xosy

l-phy

toce

ram

ides

Hex

-Hex

Cer(d

18:1

/26:

0(2-

OH

))C

56H

107N

O14

2.28

0.00

810

.20

Hex

-Hex

Cer(t

18:0

/16:

0)C

46H

89N

O14

1.86

0.01

58.

99

Hex

-Hex

Cer(t

18:0

/16:

0(2-

OH

))C

46H

89N

O15

1.93

0.01

38.

93

Hex

-Hex

Cer(t

18:0

/18:

0)C

48H

93N

O14

2.23

0.01

831

.99

Hex

-Hex

Cer(t

18:0

/22:

0)C

52H

101N

O14

2.33

0.00

914

.67

Hex

-Hex

Cer(t

18:0

/24:

0)C

54H

105N

O14

2.34

0.00

733

.01

Hex

-Hex

Cer(t

18:0

/24:

1)C

54H

103N

O14

1.66

0.01

311

.05

Hex

-Hex

Cer(t

18:0

/24:

2)C

54H

101N

O14

2.83

0.02

218

.64

Hex

-Hex

Cer(t

18:0

/26:

0)C

56H

109N

O14

2.08

0.03

913

.60

glyc

osph

ingo

lipid

str

ihex

osyl

-cer

amid

esH

ex-H

ex-H

exCe

r(d18

:1/1

6:0(

2-O

H))

C52

H97

NO

192.

840.

015

15.1

7

Hex

-Hex

-Hex

Cer(d

18:1

/18:

0)C

54H

101N

O18

2.42

0.03

913

.69

Hex

-Hex

-Hex

Cer(d

18:1

/18:

0(2-

OH

))C

54H

101N

O19

4.02

0.01

321

.57

Hex

-Hex

-Hex

Cer(d

18:1

/20:

0(2-

OH

))C

56H

105N

O19

6.63

0.00

926

.02

Hex

-Hex

-Hex

Cer(d

18:1

/22:

0)C

58H

109N

O18

1.75

0.03

916

.75

Hex

-Hex

-Hex

Cer(d

18:1

/22:

0(2-

OH

))C

58H

109N

O19

5.59

0.00

823

.25

Hex

-Hex

-Hex

Cer(d

18:1

/22:

2)C

58H

105N

O18

9.66

0.02

239

.80

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0)C

59H

111N

O18

1.99

0.04

919

.71

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0(2-

OH

))C

59H

111N

O19

5.63

0.01

530

.39

Hex

-Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))C

60H

113N

O19

3.60

0.01

520

.19

Hex

-Hex

-Hex

Cer(d

18:1

/24:

1)C

60H

111N

O18

1.76

0.03

318

.71

Hex

-Hex

-Hex

Cer(d

18:1

/24:

2)C

60H

109N

O18

3.48

0.01

823

.48

Hex

-Hex

-Hex

Cer(d

18:1

/26:

0(2-

OH

))C

62H

117N

O19

3.18

0.03

324

.61

trih

exos

yl-d

ihyd

roce

ram

ides

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0(2-

OH

))C

52H

99N

O19

4.80

0.01

316

.46

Hex

-Hex

-Hex

Cer(d

18:0

/18:

0(2-

OH

))C

54H

103N

O19

15.7

10.

015

44.8

3

Hex

-Hex

-Hex

Cer(d

18:0

/20:

0(2-

OH

))C

56H

107N

O19

18.0

10.

007

25.3

0

136

Chapter 5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

trih

exos

yl-d

ihyd

roce

ram

ides

Hex

-Hex

-Hex

Cer(d

18:0

/22:

0(2-

OH

))C

58H

111N

O19

5.80

0.01

145

.45

Hex

-Hex

-Hex

Cer(d

18:0

/24:

0(2-

OH

))C

60H

115N

O19

2.84

0.04

922

.59

trih

exos

yl-p

hyto

cera

mid

esH

ex-H

ex-H

exCe

r(t18

:0/1

6:0(

2-O

H))

C52

H99

NO

202.

860.

039

18.9

4

Hex

-Hex

-Hex

Cer(t

18:0

/18:

0(2-

OH

))C

54H

103N

O20

2.48

0.04

921

.52

Hex

-Hex

-Hex

Cer(t

18:0

/20:

0(2-

OH

))C

56H

107N

O20

6.31

0.01

825

.22

Hex

-Hex

-Hex

Cer(t

18:0

/22:

0(2-

OH

))C

58H

111N

O20

4.25

0.03

331

.20

Hex

-Hex

-Hex

Cer(t

18:0

/23:

0(2-

OH

))C

59H

113N

O20

2.28

0.04

915

.72

Hex

-Hex

-Hex

Cer(t

18:0

/24:

0(2-

OH

))C

60H

115N

O20

3.84

0.03

928

.22

Hex

-Hex

-Hex

Cer(t

18:0

/24:

1)C

60H

113N

O19

27.5

80.

008

30.6

7

Hex

-Hex

-Hex

Cer(t

18:0

/24:

2)C

60H

111N

O19

3.37

0.01

519

.96

Gly

cosp

hing

olip

ids

(oth

er)

N-a

cety

lhex

osam

ine-

trih

exos

yl-c

eram

ides

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

6:1)

C70

H12

8N2O

2310

.78

0.01

550

.09

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/1

6:0(

2-O

H))

C60

H11

0N2O

242.

440.

033

26.6

0

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

0:0(

2-O

H))

C64

H11

8N2O

242.

900.

027

37.8

3

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

2:0(

2-O

H))

C66

H12

2N2O

248.

220.

022

52.1

0

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

4:0(

2-O

H))

C68

H12

6N2O

2411

.04

0.01

345

.77

N-a

cety

lhex

osam

ine-

trih

exos

yl-

dihy

droc

eram

ides

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:0/1

6:0)

C60

H11

2N2O

237.

880.

027

16.5

8

etha

nola

min

esly

soph

osph

atid

ylet

hano

lam

ines

PE(2

0:2/

0:0)

C25

H48

NO

7P2.

140.

040

22.2

2

PE(0

:0/2

2:4)

C27

H48

NO

7P3.

030.

033

22.8

3

PE(2

2:4/

0:0)

C27

H48

NO

7P3.

320.

016

29.2

1

PE(0

:0/2

2:5)

C27

H46

NO

7P2.

480.

050

13.3

3

PE(2

2:5/

0:0)

C27

H46

NO

7P2.

710.

018

13.2

2

PE(2

2:6/

0:0)

C27

H44

NO

7P2.

550.

020

21.2

8

137

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 2

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

arst

ruct

ure

Fold

cha

nge

p-va

lue

RSD

QC

(%)

plas

mal

ogen

pho

spat

idyl

etha

nola

min

esPE

(p16

:0/1

8:1)

C39

H76

NO

7P2.

570.

015

13.6

7

PE(p

16:0

/20:

4)C

41H

74N

O7P

2.75

0.01

55.

91

PE(p

16:0

/22:

4)C

43H

78N

O7P

3.45

0.01

541

.53

PE(p

16:0

/22:

6)C

43H

74N

O7P

2.56

0.01

53.

45

PE(p

18:0

/18:

1)C

41H

80N

O7P

2.70

0.01

88.

36

PE(p

18:0

/20:

4)C

43H

78N

O7P

2.70

0.02

012

.38

PE(p

18:0

/22:

6)C

45H

78N

O7P

2.22

0.01

67.

87

PE(p

18:1

/18:

1)C

41H

78N

O7P

2.29

0.03

35.

98

PEp(

34:2

)C

39H

74N

O7P

3.26

0.01

58.

19

PEp(

40:4

)C

45H

82N

O7P

3.53

0.01

69.

74

Fold

cha

nge

= e

xpre

ssio

n af

ter s

mok

ing

cess

atio

n in

CO

PD p

atie

nts

/ ex

pres

sion

aft

er s

mok

ing

cess

atio

n in

sub

ject

s w

ithou

t CO

PD. A

fold

cha

nge

>1

is u

p-re

gula

tion

in C

OPD

. A fo

ld c

hang

e be

twee

n 0

and

1 is

dow

n-re

gula

tion

in C

OPD

. The

Lip

idM

aps

data

base

was

tak

en a

s th

e re

fere

nce

for

the

lipid

nom

encl

atur

e. L

C-M

S ca

nnot

dis

crim

inat

e be

twee

n ga

lact

ose

and

gluc

ose,

or

N-a

cety

lglu

cosa

min

e an

d N

-ace

tylg

alac

tosa

min

e. T

here

fore

, lip

id n

omen

clat

ure

cont

ains

hex

ose

(Hex

) ins

tead

of g

luco

se o

r gal

acto

se. R

elat

ive

stan

dard

dev

iatio

n (R

SD%

) has

bee

n ca

lcul

ated

fro

m th

e Q

C s

ampl

es (n

=13

). A

ll p-

valu

es a

re c

orre

cted

for m

ultip

le te

stin

g w

ith th

e Be

njam

ini H

ochb

erg

Fals

e D

isco

very

Rat

e.

138

Chapter 5

Supp

lem

enta

l Tab

le 3

: Diff

eren

tially

exp

ress

ed li

pids

in s

mok

ers

with

out C

OPD

and

nev

er-s

mok

ers

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

cera

mid

esdi

hydr

ocer

amid

esCe

r(d18

:0/1

6:0)

*C

34H

69N

O3

1.71

0.02

19.

57

glyc

osph

ingo

lipid

s di

hexo

syl-c

eram

ides

Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))*C

54H

103N

O14

1.67

0.00

315

.07

trih

exos

yl-c

eram

ides

Hex

-Hex

-Hex

Cer(d

18:1

/16:

0(2-

OH

))C

52H

97N

O19

1.93

0.03

718

.24

Hex

-Hex

-Hex

Cer(d

18:1

/18:

2)C

54H

97N

O18

1.85

0.04

718

.53

Hex

-Hex

-Hex

Cer(d

18:1

/20:

0(2-

OH

))C

56H

105N

O19

2.03

0.02

131

.60

Hex

-Hex

-Hex

Cer(d

18:1

/22:

0(2-

OH

))*C

58H

109N

O19

3.24

0.00

127

.34

Hex

-Hex

-Hex

Cer(d

18:1

/22:

1)C

58H

107N

O18

7.87

0.00

132

.69

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0(2-

OH

))*C

59H

111N

O19

3.54

0.00

123

.32

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0)C

59H

111N

O18

1.89

0.03

418

.28

Hex

-Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))*C

60H

113N

O19

3.58

0.00

122

.17

Hex

-Hex

-Hex

Cer(d

18:1

/25:

0(2-

OH

))C

61H

115N

O19

4.47

0.00

126

.60

Hex

-Hex

-Hex

Cer(d

18:1

/26:

0(2-

OH

))*C

62H

117N

O19

5.35

0.00

133

.31

Hex

-Hex

-Hex

Cer(d

18:1

/26:

0)C

62H

117N

O18

3.51

0.00

123

.51

Hex

-Hex

-Hex

Cer(d

18:1

/26:

1)C

62H

115N

O18

2.95

0.00

221

.78

trih

exos

yl-d

ihyd

roce

ram

ides

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0(2-

OH

))*C

52H

99N

O19

2.86

0.00

819

.50

Hex

-Hex

-Hex

Cer(d

18:0

/22:

0(2-

OH

))*C

58H

111N

O19

3.28

0.00

121

.21

Hex

-Hex

-Hex

Cer(d

18:0

/24:

0(2-

OH

))*C

60H

115N

O19

4.78

0.00

127

.97

Hex

-Hex

-Hex

Cer(d

18:0

/26:

0(2-

OH

))*C

62H

119N

O19

3.25

0.02

933

.78

trih

exos

yl-p

hyto

cera

mid

esH

ex-H

ex-H

exCe

r(t18

:0/2

4:0(

2-O

H))

C60

H11

5NO

202.

400.

022

24.5

8

139

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 3

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(o

ther

)N

-ace

tylh

exos

amin

e-tr

ihex

osyl

-cer

amid

esH

exN

Ac-

Hex

-Hex

-Hex

Cer(t

18:0

/18:

0)C

64H

120N

2O24

3.25

0.00

133

.55

neur

amin

ic a

cid

cont

aini

ng d

ihex

osyl

-ce

ram

ides

Neu

Ac-

Hex

-Hex

Cer(d

18:1

/20:

0)C

61H

112N

2O21

0.15

0.00

130

.85

Neu

Ac-

Hex

-Hex

Cer(d

18:1

/24:

1)C

65H

118N

2O21

0.36

0.00

126

.14

toba

cco

com

poun

dsso

lane

sol*

C45

H74

Ond

<0.

001

13.6

0

sola

nesy

l pal

mita

te*

C61

H10

4O2

nd<

0.00

114

.04

sola

nesy

l ste

arat

eC

63H

108O

2nd

<0.

001

18.6

0

bom

bipr

enon

eC

43H

70O

481.

85<

0.00

112

.54

farn

esyl

farn

esyl

acet

one

C33

H54

Ond

<0.

001

10.7

1

gera

nylfa

rnes

ylac

eton

eC

28H

46O

nd<

0.00

113

.19

Fold

cha

nge

= e

xpre

ssio

n in

sm

oker

s w

ithou

t CO

PD /

exp

ress

ion

in n

ever

sm

oker

s. A

fold

cha

nge

>1

is u

p-re

gula

tion

in s

mok

ers

with

out

COPD

. A fo

ld c

hang

e be

twee

n 0

and

1 is

dow

n-re

gula

tion

in sm

oker

s with

out C

OPD

. nd

- lip

id n

ot d

etec

ted

in n

ever

-sm

oker

s. * T

hese

lipi

ds w

ere

also

diff

eren

tly re

gula

ted

afte

r 2-m

onth

smok

ing

cess

atio

n in

indi

vidu

als w

ith a

nd w

ithou

t CO

PD

(Sup

plem

enta

l tab

le 4

). Th

e Li

pidM

aps

data

base

was

take

n as

the

refe

renc

e fo

r the

lipi

d no

men

clat

ure.

LC

-MS

cann

ot d

iscr

imin

ate

betw

een

gala

ctos

e an

d gl

ucos

e, o

r N-a

cety

lglu

cosa

min

e an

d N

-ace

tylg

alac

tosa

min

e. T

here

fore

, lip

id n

omen

clat

ure

cont

ains

hex

ose

(Hex

) ins

tead

of g

luco

se o

r gal

acto

se. R

elat

ive

stan

dard

dev

iatio

n (R

SD%

) has

bee

n ca

lcul

ated

from

the

QC

sam

ples

(n=

34).

All

p-va

lues

are

cor

rect

ed fo

r mul

tiple

test

ing

with

the

Benj

amin

i Hoc

hber

g Fa

lse

Dis

cove

ry R

ate.

140

Chapter 5

Supp

lem

enta

l Tab

le 4

: Lip

ids

that

are

diff

eren

tly re

gula

ted

afte

r 2-m

onth

sm

okin

g ce

ssat

ion

in in

divi

dual

s w

ith a

nd w

ithou

t CO

PD

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

cera

mid

es c

eram

ides

Cer(d

18:1

/25:

1)C

43H

83N

O3

0.69

0.02

16.

53

Cer(d

18:1

/25:

2)C

43H

81N

O3

0.75

0.04

78.

79

Cer(d

18:1

/26:

2)C

44H

83N

O3

0.76

0.03

86.

80

dihy

droc

eram

ides

Cer(d

18:0

/15:

0)C

33H

67N

O3

0.70

0.04

87.

71

Cer(d

18:0

/16:

0)*

C34

H69

NO

30.

570.

018

7.27

Cer(d

18:0

/17:

0)C

35H

71N

O3

0.56

0.02

99.

02

Cer(d

18:0

/18:

0)C

36H

73N

O3

0.62

0.02

15.

80

Cer(d

18:0

/20:

0)C

38H

77N

O3

0.70

0.02

15.

79

Cer(d

18:0

/22:

0)C

40H

81N

O3

0.76

0.02

98.

41

glyc

osph

ingo

lipid

s di

hexo

syl-c

eram

ides

Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))*C

54H

103N

O14

0.81

0.04

79.

36

trih

exos

yl-c

eram

ides

H

ex-H

ex-H

exCe

r(d18

:1/2

2:0(

2-O

H))*

C58

H10

9NO

190.

490.

018

23.2

5

Hex

-Hex

-Hex

Cer(d

18:1

/23:

0(2-

OH

))*C

59H

111N

O19

0.44

0.02

930

.39

Hex

-Hex

-Hex

Cer(d

18:1

/24:

0(2-

OH

))*C

60H

113N

O19

0.46

0.01

820

.19

Hex

-Hex

-Hex

Cer(d

18:1

/26:

0(2-

OH

))*C

62H

117N

O19

0.49

0.02

524

.61

trih

exos

yl-d

ihyd

roce

ram

ides

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0(2-

OH

))*C

52H

99N

O19

0.52

0.01

816

.46

Hex

-Hex

-Hex

Cer(d

18:0

/18:

0(2-

OH

))C

54H

103N

O19

0.38

0.02

444

.83

Hex

-Hex

-Hex

Cer(d

18:0

/20:

0(2-

OH

))C

56H

107N

O19

0.32

0.04

825

.30

Hex

-Hex

-Hex

Cer(d

18:0

/22:

0(2-

OH

))*C

58H

111N

O19

0.37

0.01

845

.45

Hex

-Hex

-Hex

Cer(d

18:0

/24:

0(2-

OH

))*C

60H

115N

O19

0.40

0.01

822

.59

Hex

-Hex

-Hex

Cer(d

18:0

/26:

0(2-

OH

))*C

62H

119N

O19

0.37

0.03

833

.77

trih

exos

yl-p

hyto

cera

mid

esH

ex-H

ex-H

exCe

r(t18

:0/2

4:1)

C60

H11

3NO

190.

270.

031

30.6

7

Hex

-Hex

-Hex

Cer(t

18:0

/24:

2)C

60H

111N

O19

0.56

0.01

819

.96

141

Lipidomic analysis in COPD

Chapter

5

Supp

lem

enta

l Tab

le 4

: Con

tinue

d

Clas

sSu

bcla

ssLi

pid

Mol

ecul

ar

stru

ctur

eFo

ld c

hang

ep-

valu

eRS

D Q

C (%

)

glyc

osph

ingo

lipid

s(o

ther

)N

-ace

tylh

exos

amin

e-tr

ihex

osyl

-cer

amid

esH

exN

Ac-

Hex

-Hex

-Hex

Cer(d

18:1

/16:

0(2-

OH

))C

68H

126N

2O24

0.61

0.04

826

.60

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

4:0(

2-O

H))

C68

H12

6N2O

240.

270.

018

36.6

0

Hex

NA

c-H

ex-H

ex-H

exCe

r(d18

:1/2

6:1)

C70

H12

8N2O

230.

270.

018

50.0

9

N-a

cety

lhex

osam

ine-

trih

exos

yl-

dihy

droc

eram

ides

H

exN

Ac-

Hex

-Hex

-Hex

Cer(d

18:0

/16:

0)C

60H

112N

2O24

0.26

0.02

516

.58

toba

cco

com

poun

ds

sola

neso

l*C

45H

74O

nd0.

020

10.0

1

sola

nesy

l pal

mita

te*

C61

H10

4O2

nd0.

020

17.6

3

Fold

cha

nge

= e

xpre

ssio

n af

ter

smok

ing

cess

atio

n /

expr

essi

on b

efor

e sm

okin

g ce

ssat

ion.

A f

old

chan

ge >

1 is

up-

regu

latio

n af

ter

smok

ing

cess

atio

n. A

fol

d ch

ange

bet

wee

n 0

and

1 is

do

wn-

regu

latio

n af

ter

smok

ing

cess

atio

n. n

d -

lipid

not

det

ecte

d af

ter

smok

ing

cess

atio

n. *

The

se li

pids

wer

e al

so d

iffer

ently

reg

ulat

ed in

the

com

paris

on b

etw

een

smok

ers

with

out

COPD

an

d ne

ver-

smok

ers

(Sup

plem

enta

l tab

le 3

). Th

e Li

pidM

aps

data

base

was

tak

en a

s th

e re

fere

nce

for

the

lipid

nom

encl

atur

e. L

C-M

S ca

nnot

dis

crim

inat

e be

twee

n ga

lact

ose

and

gluc

ose,

or

N-a

cety

lglu

cosa

min

e an

d N

-ace

tylg

alac

tosa

min

e. T

here

fore

, lip

id n

omen

clat

ure

cont

ains

hex

ose

(Hex

) ins

tead

of g

luco

se o

r gal

acto

se. R

elat

ive

stan

dard

dev

iatio

n (R

SD%

) has

bee

n ca

lcul

ated

fro

m th

e Q

C s

ampl

es (n

=13

). A

ll p-

valu

es a

re c

orre

cted

for m

ultip

le te

stin

g w

ith th

e Be

njam

ini H

ochb

erg

Fals

e D

isco

very

Rat

e.

142

Chapter 5

Supp

lem

enta

l Tab

le 5

: Co

rrel

atio

n be

twee

n lip

id e

xpre

ssio

n an

d cl

inic

al o

utco

me

varia

bles

Pack

year

sFE

V 1 % p

red

FEV 1/F

VCRV

/TLC

% p

red

TLCO

c/VA

% p

red

% N

eutr

ophi

ls%

Eos

inop

hils

Rho

p-va

lue

Rho

p-va

lue

Rho

p-va

lue

Rho

p-va

lue

Rho

p-va

lue

Rho

p-va

lue

Rho

p-va

lue

Cera

mid

esCe

r(d18

:1/2

0:0)

0.59

6<

0.00

1-0

.534

<0.

001

-0.6

35<

0.00

10.

467

0.00

3-0

.217

0.19

60.

276

0.11

40.

418

0.01

4

Cer(d

18:1

/20:

1)0.

573

<0.

001

-0.5

36<

0.00

1-0

.632

<0.

001

0.47

70.

002

-0.3

550.

031

0.23

20.

186

0.40

30.

018

Cer(d

18:1

/21:

0)0.

527

0.00

1-0

.536

<0.

001

-0.5

87<

0.00

10.

572

<0.

001

-0.2

610.

119

0.36

60.

033

0.45

90.

006

Cer(d

18:1

/22:

0)0.

551

<0.

001

-0.5

87<

0.00

1-0

.625

<0.

001

0.53

00.

001

-0.2

730.

102

0.38

00.

027

0.50

20.

002

Dih

ydro

-ce

ram

ides

Cer(d

18:0

/15:

0)0.

552

<0.

001

-0.6

41<

0.00

1-0

.688

<0.

001

0.48

00.

002

-0.1

070.

529

0.39

80.

020

0.36

00.

036

Cer(d

18:0

/16:

0)0.

637

<0.

001

-0.6

82<

0.00

1-0

.707

<0.

001

0.56

6<

0.00

1-0

.254

0.12

90.

516

0.00

20.

424

0.01

2

Cer(d

18:0

/18:

0)

0.61

0<

0.00

1-0

.578

<0.

001

-0.7

13<

0.00

10.

451

0.00

4-0

.164

0.33

10.

428

0.01

20.

346

0.04

5

Cer(d

18:0

/19:

0)

0.54

5<

0.00

1-0

.646

<0.

001

-0.6

75<

0.00

10.

523

0.00

1-0

.106

0.53

20.

475

0.00

50.

450

0.00

8

Cer(d

18:0

/20:

0)

0.49

90.

001

-0.6

17<

0.00

1-0

.687

<0.

001

0.57

0<

0.00

1-0

.156

0.35

80.

545

0.00

10.

441

0.00

9

Sphi

ngo-

mye

lins

SM(t

18:0

/24:

2)0.

631

<0.

001

-0.4

980.

002

-0.5

92<

0.00

10.

330

0.04

6-0

.328

0.05

50.

395

0.02

30.

475

0.00

5

Gly

cosp

hing

o-lip

ids

Hex

-Hex

-Hex

-Ce

r(d18

:1/1

6:0(

2-O

H))

0.52

90.

001

-0.6

41<

0.00

1-0

.551

0.00

10.

555

<0.

001

-0.1

790.

290

0.53

20.

001

0.48

40.

004

Hex

-Hex

-Hex

-Ce

r(d18

:1/1

8:2)

0.52

30.

001

-0.6

39<

0.00

1-0

.546

0.00

10.

558

<0.

001

-0.1

780.

291

0.53

10.

001

0.48

20.

004

Hex

-Hex

-Hex

-Ce

r(d18

:0/1

6:0(

2-O

H))

0.54

9<

0.00

1-0

.538

<0.

001

-0.4

700.

004

0.44

00.

005

-0.0

530.

754

0.44

00.

009

0.56

5<

0.00

1

FEV 1 =

forc

ed e

xpira

tory

vol

ume

in 1

sec

ond;

FVC

= fo

rced

vita

l cap

acity

; RV

= r

esid

ual v

olum

e; T

LC =

tot

al lu

ng c

apac

ity; T

LCO

c =

diff

usio

n ca

paci

ty fo

r ca

rbon

mon

o-ox

ide

corr

ecte

d fo

r he

mog

lobi

n; V

A =

alv

eola

r vol

ume;

pre

d =

pre

dict

ed; R

ho =

Spe

arm

an’s

rank

ed c

orre

latio

n co

effici

ent.

143

Lipidomic analysis in COPD

Chapter

5

References1. Matyash V, Liebisch G, Kurzchalia TV, Shevchenko A, Schwudke D. Lipid extraction by methyl-tert-butyl ether

for high-throughput lipidomics. J Lipid Res 2008;49:1137-1146.

2. Ding J, Sorensen CM, Jaitly N, Jiang H, Orton DJ, Monroe ME, Moore RJ, Smith RD, Metz TO. Application of the accurate mass and time tag approach in studies of the human blood lipidome. J Chromatogr B Analyt Technol Biomed Life Sci 2008;871:243-252.

3. Taguchi R, Houjou T, Nakanishi H, Yamazaki T, Ishida M, Imagawa M, Shimizu T. Focused lipidomics by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2005;823:26-36.

4. Han X, Gross RW. Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom Rev 2005;24:367-412.

5. Cui Z, Thomas MJ. Phospholipid profiling by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877:2709-2715.

6. Ikeda K, Oike Y, Shimizu T, Taguchi R. Global analysis of triacylglycerols including oxidized molecular species by reverse-phase high resolution LC/ESI-QTOF MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877:2639-2647.

7. Hsu FF, Turk J. Structural determination of glycosphingolipids as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisional-activated dissociation on a triple stage quadrupole instrument. J Am Soc Mass Spectrom 2001;12:61-79.

8. Hsu FF, Turk J. Electrospray ionization with low-energy collisionally activated dissociation tandem mass spectrometry of glycerophospholipids: mechanisms of fragmentation and structural characterization. J Chromatogr B Analyt Technol Biomed Life Sci 2009;877:2673-2695.

9. Pulfer M, Murphy RC. Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 2003;22:332-364.

10. Schwudke D, Oegema J, Burton L, Entchev E, Hannich JT, Ejsing CS, Kurzchalia T, Shevchenko A. Lipid profiling by multiple precursor and neutral loss scanning driven by the data-dependent acquisition. Anal Chem 2006;78:585-595.

11. Murphy RC, Axelsen PH. Mass spectrometric analysis of long-chain lipids. Mass Spectrom Rev 2011;30:579-599.

CHAPTER 6

Mitochondrial dysfunction increases

pro-inflammatory cytokine production and

impairs regeneration and glucocorticoid

responsiveness in lung epithelium

Roland F. Hoffmann*, Eef D. Telenga*, Ruben t’Kindt, Harold de Bruin, Antoon J.M. van Oosterhout, Lucie Jorge4, Pat Sandra, Dirkje S. Postma,

Koen Sandra, Nick H.T. ten Hacken*, Irene H. Heijink**both authors contributed equally

Manuscript in preparation

146

Chapter 6

Abstract Rationale: Chronic obstructive pulmonary disease (COPD) is a sustained inflammatory disorder of the lungs with progressive functional decline mainly caused by cigarette smoking. The use of lipidomics, an emerging research field, may provide new insights in the onset and progression of chronic inflammatory diseases like COPD.

Objective: To investigate whether the expression of the (intra)cellular lipidome is affected by long-term cigarette smoke extract (CSE) exposure in the bronchial epithelial cell line BEAS-2B. Additionally, we studied the mRNA expression of key lipid converting enzymes specific by qPCR.

Methods: Cellular changes in lipidome expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) in BEAS-2B cells exposed to 0%, 2,5% and 10% CSE for a period of 6 months.

Measurements and Main Results: We identified >1500 lipid compounds in BEAS-2B cells. Six-month CSE exposure resulted in 13 up-regulated and 13 down-regulated intracellular sphingolipids in BEAS2B cells. Additionally, the mRNA expression of sphingomyelin synthase 2 and fatty acid synthase was significantly reduced upon long-term CSE exposure.

Conclusions: Together, we show an altered lipidome in human bronchial epithelial cells exposed to cigarette smoke for 6 months. Here, mainly lipids from the sphingolipid pathway were affected, providing new insights in our understanding of the role of lipids in cigarette smoke-induced disease.

Key words: Lipidomics, Cigarette Smoke Extract, COPD, Airway Epithelial Cells.

147

Lipidomic analysis in BEAS-2B

Chapter

6

IntroductionLipidome analysis is an emerging technique that unravels the complex composition of the lipidome of the cell. Lipids play an important role in various cellular processes, including metabolism and energy storage, cellular signaling processes, cell survival and proliferation, cell death and of course membrane integrity (1–4). However, the role of the lipidome in the onset or progression of disease is still poorly understood. Several recent studies have linked the disruption of lipid metabolizing enzymes and pathways to neurodegenerative diseases, cancer and inflammatory diseases (3,5–7), indicating that the deregulation of the lipidome may play a role in their pathogenesis. Chronic obstructive pulmonary disease (COPD) is a sustained lung inflammatory disease affecting millions of people worldwide. In the industrialized world, long-term cigarette smoking is the main risk factor for COPD, in combination with a genetic predisposition (8,9). In an explorative study, we have previously shown that lipidome profiles in sputum differ between healthy non-smokers, smokers without COPD and smokers with COPD. Lipid quantity of over 200 individual lipid components, covering four major lipid classes of the sphingolipid pathway, i.e. ceramids, sphingomyelins, glycosphingolipids and ethanolamines, differed between smokers without COPD and smokers with COPD (1). However, in this study we could not specify which cell type contributed to these changes nor could we determine (intra)cellular lipidome changes. As the airway epithelium is the first line of defense against noxious particles such as cigarette smoke, epithelial cells are thought to contribute to the aberrant inflammatory response to cigarette smoking in COPD (8,10–15). The lipidome may play a role in the epithelial pro-inflammatory response to cigarette smoke, as pro-inflammatory cytokines are known to affect the activity of lipid-modifying/metabolizing enzymes, such as phospholipases, sphingomyelinases and sphingosine kinases, leading to changes in lipids that are known to regulate inflammatory signaling (1,6).

We hypothesize that the lipidome may also be affected directly by cigarette smoking. We have previously shown that long-term cigarette exposure of human airway epithelium impairs the structure and function of mitochondria, which are lipid rich organelles important for energy exchange (12). To assess whether cigarette smoking induces alterations in the lipidome, we investigated long-term cigarette smoke exposure in human bronchial epithelial BEAS-2B cells. We studied the effect of 6-month exposure to 2.5% and 10% cigarette smoke extract (CSE) on lipid expression and sphingomyelinase activity in the human bronchial cell line BEAS-2B in vitro. Additionally, gene expression of 6 enzymes that regulate lipid metabolism, Sphingomyelin synthase 1 and 2 (SGMS1 and 2), fatty acid synthase (FASN), sphingomyelin phosphodiesterase 1 (SMPD1), neutral sphingomyelinase activation associated factor (NSMAF) and ceramide kinase (CERK) was quantified by reverse-transcription PCR (qRT-PCR).

148

Chapter 6

Methods and materials

In vitro studyBEAS-2B cells were grown in RPMI 1640 (Lonza, Verviers, Belgium) supplemented with 15% heat-inactivated Fetal Bovine Serum (FBS), penicillin (100 U/ml) (Lonza, Verviers, Belgium) and streptomycin (100 μg/ml) (Lonza, Verviers, Belgium) on collagen-coated plates and 0%, 2.5% and 10% CSE. Cells were grown to ~90 % confluence and passaged twice a week by trypsin. Cigarette smoke extract (CSE) was prepared from filter-less research-grade cigarettes (3R4F, Tobacco Research Institute, University of Kentucky, Lexington, KY) as described previously (16). Smoke from 2 cigarettes was bubbled through 25 ml medium was considered as 100% CSE. CSE was added freshly each time when the cells were passaged.

Airway epithelial (BEAS-2B) cell lipid extraction Once cells were grown to ~ 90% confluence, medium was replaced by serum deprived medium (containing 0 2.5 or 10% CSE) 24 hours before the experiment. Methanol was used to extract the lipids by direct application to the mono-layer cell culture. 200 µL aliquots of methanol extracts were transferred into 500 µL Eppendorf tubes and centrifuged at 15,000 rpm during 10 min. The resulting supernatant (185 µL) was transferred into a vial with insert, dried in centrifugal vacuum evaporator (miVac duoconcentrator, Genevac Lim., Ipswich, UK) and reconstituted in 37 µL chloroform/isopropanol 50/50 (v/v). 10 µL of this extract was injected into the LC-MS system

Lipidomics analysis, experimental setup, preparation and use of quality control (QC) samples For each experiment, the use of QC samples allowed monitoring the repeatability and stability of the lipid measurement. BEAS-2B cell lines were investigated after 6-month exposure to cigarette smoke extract (CSE) (15 cell extracts, 6 QCs). To prepare QC samples within each experiment, 60 µL aliquots of the samples were collected in a QC pool. This QC pool was then divided into 120 µL aliquots to obtain representative QC samples. QC samples were prepared simultaneously along with study samples and were analyzed throughout the LC/MS analysis sequences every five study samples. These samples did not contain any biological variability and can be considered as technical replicates. The relative standard deviation percent (RSD%) of all significantly up- or downregulated lipids has been calculated to demonstrate the measurement error of the lipidomics analytical platform. A more detailed description of the lipidomic analysis is available in the supplement.

149

Lipidomic analysis in BEAS-2B

Chapter

6

Lipid NomenclatureFor this study the International Lipid Classification and Nomenclature Committee (ILCNC) was used, i.e. ‘‘Comprehensive Classification System for Lipids’’ (17–19). Regarding glycosphingolipids, LC-MS is not able to discriminate between galactose and glucose, or N-acetylglucosamine and N-acetylgalactosamine. Therefore, lipid nomenclature of these species contains hexose (Hex) instead of glucose or galactose.

qRT-PCRRNA was extracted from homogenates from the long-term exposed cells using TriReagent (Applied Biosystems/Ambion, Foster City, CA, USA), reverse transcribed to cDNA using iScript cDNA Synthesis Kit (Bio-Rad, Herculus, CA, USA). Sphingomyelin synthase 1 and 2 (SGMS1 and 2), fatty acid synthase (FASN), sphingomyelin phosphodiesterase 1 (SMPD1), neutral sphingomyelinase activation associated factor (NSMAF) and ceramide kinase (CERK) expression was analyzed by real-time PCR using Taqman® (Applied Biosystems, Foster City, CA, USA). Validated probes and housekeeping genes, B2M and PP1A and TaqMan Master Mix were purchased from Applied Biosystems (Foster City, CA, USA).

Statistical analysesDifferences in expression levels were compared with a Mann-Whitney U test for all comparison experiments. Multiple testing correction, according to the false discovery rate, was performed for the results described in table 1 and table 2 (20). Compounds that were significantly differentially expressed (p<0.05) were identified with tandem mass spectrometry (MS/MS).

Results

Long-term exposure of BEAS-2B cells to CSEWe have previously shown that 6-month CSE exposure alters mitochondrial morphology and function in the bronchial epithelial cell line BEAS-2B (22). Therefore, we first tested whether 6-months exposure to 0% (control), 2.5% and 10% CSE also affects the lipidome in BEAS-2B cells. Table 1 shows all lipid compounds that were detected in BEAS-2B cells grown for 6 months without CSE. No significant changes were observed when the cells were grown for 6 months in a low concentration of CSE (2.5%), although a trend towards either up-regulation or down-regulation was visible for 12 sphingolipids (Table 1), including 2 ceramides, 1 dihydroceramide, 3 sphingomyelins, and 6 Glycoshingolipids GSLs (Table 1). The high concentration of CSE (10%) significantly altered the expression of 26 sphingolipids: 4 ceramides, 1 dihydroceramide, 9 sphingomyelins and 12 GSLs (Table 2).

150

Chapter 6

Tabl

e 1:

Diff

eren

tially

regu

late

d lip

ids

afte

r lon

g-te

rm e

xpos

ure

to lo

w C

SE c

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cer

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89.

25

SM(d

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77.

96

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36

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cont

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0.76

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56

Neu

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C65

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500.

098

4.08

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4:1)

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8N2O

210.

740.

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n in

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be G

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n (R

SD%

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n ca

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(n=

6).

151

Lipidomic analysis in BEAS-2B

Chapter

6

Tabl

e 2:

Diff

eren

tially

regu

late

d lip

ids

afte

r lon

g-te

rm e

xpos

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CSE

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cera

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es

cera

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3.52

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814

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Cer(d

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1)C

32H

61N

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0.08

0.02

88.

22

Cer(d

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1)C

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36

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93

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0)C

35H

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0.33

0.02

810

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SM(d

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0)C

37H

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0.66

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89.

25

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1.37

0.02

85.

86

SM(d

18:1

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0)C

40H

81N

2O6P

2.32

0.02

84.

84

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0.37

0.02

87.

96

SM(d

18:1

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1)C

39H

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0.50

0.02

811

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SM(d

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152

Chapter 6

Tabl

e 2:

Con

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0.02

84.

08

Neu

Ac-

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ls. T

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d gl

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r N

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bee

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ples

(n=

6).

153

Lipidomic analysis in BEAS-2B

Chapter

6

Neuraminic acid containing sphingolipids, ceramides and sphingomyelins were all down-regulated, except for 2 specific ceramides and their related sphingomyelins (i.e. d18:1/16:0 and d18:1/17:0 species). In contrast, other glycosphingolipids were significantly up-regulated in cells exposed to 10% CSE (3-9 fold), with the strongest effect on N-acetylhexosamine-trihexosyl-ceramides. Of note, all lipids that tended to increase or decrease in the 2.5% CSE-exposed cells were significantly differentially regulated in 10% CSE-exposed cells in the same direction.

Additionally, we investigated the effect of long-term CSE exposure on sphingomyelinase activity by assessing the ratio of ceramides and sphingomyelin species with identical carbon chain length and saturation (21). Here, a higher ratio indicates that more sphingomyelin is converted to ceramides, reflecting a higher sphingomyelinase activity. The ceramide to sphingomyelin ratio for d18:1/16:0 was increased both in 2.5% and 10% CSE-exposed BEAS-2B cells, while the ratio of d18:1/17:0 and d18:1/18:0 was only significantly increased in BEAS-2B exposed to 10% CSE (Figure 1).  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0

1

2

3

4

5

6

7

8

9

10

Sphing

omye

linase

activ

ity

control

CSE 2.5%

CSE 10%

***

*

Figure 1: Sphingomyelinase activity in BEAS.2B cells (mean+/-SEM). Sphingomyelinase activity was calculated as the ratio between a ceramide and its related sphingomyelin, with identical sphingoid base and fatty acid chain, e.g. Cer(d18:1/16:0) and SM(d18:1/16:0). Sphingomyelinase activity was increased for d18:1/16:0 (2.5% and 10% CSE), d18:1/17:0 (10% CSE) and d18:1/18:0 (10% CSE) in CSE exposed BEAS.2B cells. AUC = area under the curve. * = p<0.05 in comparison with control

154

Chapter 6

qRT-PCR of lipid modifying enzymes in BEAS-2B cells exposed to 0%, 2.5% and 10% CSE for 6 monthsTo uncover which lipid modifying enzymes are affected by cigarette smoke and may be responsible for the observed lipidome alterations, we investigated the transcriptional levels of sphingomyelin synthase 1 and 2 (SGMS1 and 2), fatty acid synthase (FASN), sphingomyelin phosphodiesterase 1 (SMPD1), neutral sphingomyelinase activation associated factor (NSMAF) and ceramide kinase (CERK) in BEAS-2B cells exposed to 0%, 2.5% and 10% CSE for 6 months. SGMS2 transcription was significantly lowered by 2.5% and more strongly by 10% CSE (Figure 2). In contrast, SGMS1 was not significantly altered by 2.5% nor by 10% CSE. Like SGMS2, transcriptional levels of FASN were already lowered by 2.5% CSE exposure and further reduced by 10% CSE. SMPD1 was slightly reduced by the highest concentration of 10% CSE, although this failed to reach statistical significance (p=0.0556). There was no significant difference in transcriptional levels of NSMAF and CERK. These data show that long-term CSE exposure alters the lipid profile in human bronchial epithelial cells and that alterations in expression of FASN and SGMS2, but not SGMS1, may be responsible for the observed changes.

Figure 2: qRT-PCR on long-term cigarette smoke exposed cells, 0% CSE, 2.5% CSE and 10%CSE, show a significant decrease in transcriptional levels of FASN, SGMS2 in 2.5% CSE and 10% CSE when compared to control conditions. A decreasing trend is visible for SMPD1 in cells exposed to 10% CSE when compared to control conditions. No significant changes in transcriptional levels were found for SGMS1, NSMAF and CERK. SGMS = sphingomyelin synthase; FASN = fatty acid synthase; SMPD1 = sphingomyelin phosphodiesterase 1; NSMAF = neutral sphingomyelinase activation associated factor; CERK = ceramide kinase (CERK)

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Chapter

6

DiscussionWe hypothesized that cigarette smoke affects epithelial expression of lipids that are relevant to the pathogenesis of COPD, regulating inflammatory signaling (1,6). In the current study, we show that lipids from the sphingolipid pathway are altered by long-term CSE exposure in human bronchial epithelial cells, which is in line with our previous study showing that the same lipids are down-regulated in sputum of smokers with and without COPD compared to non-smokers (1). This suggests that lipids derived from airway epithelial cells may be responsible for the observed changes in the lipid profile in sputum upon long-term smoking. In our previous study, we found that all lipid components were increased in smokers compared to non-smokers, except for neuraminic acid containing lipids, which was down-regulated. Accordingly, we observed a decrease in three neuraminic acid containing lipid components belonging to the glycosphinogolipids, i.e. sphingolipids, ceramides and sphingomyelins. One identical component, NeuAc-Hex-HexCer(d18:1/24:1), was observed to be elevated in smokers compared to non-smokers. Also, a second identical neuraminic acid containing component, NeuAc-Hex-HexCer(d18:1/24:0), was observed to be increased in COPD patients compared to healthy smokers. On the other hand, we observed an increase in other glycosphinogolipids, especially lipids belonging to N-acetylhexosamine-trihexosyl-ceramides. This is again in line with our previous findings, showing higher levels of the same lipid class in sputum from smokers and especially in COPD (1). Two of the lipid components increased by 10% CSE exposure, HexNAc-Hex-Hex-HexCer(d18:1/16:0) and HexNAc-Hex-Hex-HexCer(d18:1/22:0), were also observed to be increased in sputum from COPD patients compared to healthy smokers. Furthermore, we observed that long-term cigarette smoke exposure significantly reduced the expression of the lipid converting enzymes SGMS2 and FASN in bronchial epithelial cells, which may account for the observed changes in lipid profile. The changes in lipidomic profile and regulating enzymes suggest that cigarette smoke affects the expression of key components regulating lipid expression. Indeed, sphingomyelinase activity, as determined by the ratio of ceramides and sphingomyelin, was increased upon long-term CSE exposure for ceramides and sphingomyelins with a fatty acid chain length of 16 to 18 carbon atoms. SGMS1 and SGMS2 catalyze the conversion of phosphatidylcholine and ceramides to sphingomyelin (SM) and diacylglycerol. This is in line with a recent publication showing that neutral sphingomyelinase 2 (nSMase2) is the sole sphingomyelinase being activated during cigarette smoke-induced oxidative stress and where src acts as a regulator in human airway epithelial cells (23). Thus, the observed reduction in SGMS2 expression upon long-term cigarette smoke exposure may be responsible for the increased sphingomyelinase activity. However, most sphingomyelins, 7 of 9, were down-regulated of which 5 components were identical to the increased sphingomyelins found in COPD patients. These components include the only 2 increased sphingomyelins, SM(d18:1/16:0) and SM(d18:1/17:0), found in 10% CSE exposed BEAS-2B

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cells. The conversion of phosphatidylcholine and ceramides is a process required for cell growth and membrane stability. Sphingomyelin plays a key role in apoptosis regulation (24–27) and alterations in its levels in airway epithelium may thus have consequences for COPD (16,26,27). We also observed that long-term CSE exposure reduces mRNA expression of FASN, a multifunctional enzyme that catalyzes the formation of long-chain fatty acids from one of the most ubiquities lipid component, palmitate. Reduced FASN expression has also been observed under hypoxia induced cell death conditions in hepatocytes (28), whereas cigarette smoke augments hypoxic chemo-sensitivity in humans which may lead to accelerated cell death (29). The observed reduction in FASN expression may also explain the reduction in various long-chain lipid components, as the synthases of these lipids may be hampered in bronchial epithelial cells upon smoke exposure. Thus, our findings on the mRNA expression of FASN and SGM2 are in line with the observed changes in lipid profile, although future studies will be required to confirm that protein levels of these enzymes are also reduced upon long-term CSE exposure. The reduction in neuraminic acid containing lipid components that was observed in vitro as well as in sputum of smokers and COPD patients may have important implications for the development of the disease. Neuraminic acids are terminal monosaccharides modifications of most glycoconjugates and glycosphingolipids. Of these, N-acetylneuraminic acid is involved in a variety of biological interactions including cell adhesion and migration (30), thereby regulating damage and repair processes. Ceramides are known to affect mitochondrial membrane permeability and induce the formation of membrane channels, which in turn, may release pro-apoptotic proteins from the mitochondria (31–33). Thus, the observed cigarette smoke-induced chances may have important implications for mitochondrial function and cell survival, which is in line with our previous findings on the effects of cigarette smoke on mitochondrial structure and function (12). Also, oxidative stress and inflammatory stimuli were shown to induce ceramide generation, which has been linked to the development and progression of COPD disease (26,27,34–36). Two ceramides, Cer(d18:1/16:0) and Cer(d18:1/17:0), were increased in BEAS-2B cells exposed to 10% CSE. Both lipid components were also observed to be elevated in sputum from COPD patients when compared to healthy smokers. The release of ceramides can induce cellular apoptosis and cell death (6,26,27,37). In contrast, 3 other ceramides were actually down regulated (of which 2 of them were actually increased in lung sputum of COPD patients) in BEAS-2B cells exposed to 10% CSE. This might suggest a role for ceramides in possibly a biological signaling response to cigarette smoke induced stress (37,38).In conclusion, our findings show that long-term CSE exposure induces up and down-regulation of lipids belonging to the same class of lipids that were up or down-regulated in induced sputum of smokers with and without COPD. This demonstrates again that LC-QTOF-MS(/MS) LC-MS with MS/MS is also a powerful tool to identify lipids profiles in

157

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Chapter

6

vitro. Lipidome profiling may unravel cigarette-smoke induced changes important for the pathogenesis of COPD.

AcknowledgementsThis research was partially performed within the framework of the Top Institute Pharma project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, with partners University Medical Center Groningen, University Medical Center Utrecht, University Medical Center Maastricht, Nycomed BV, GlaxoSmithKline, Danone, AstraZeneca, and Foundation TI Pharma.

This study was made possible by: Metablys, Research Institute for Chromatography (Kortrijk, Belgium), Royal Netherlands Academy for Arts and Sciences, Lung Foundation Netherlands (NAF 97.74), Stichting Astmabestrijding (SAB 2012/039) and the Agency for Innovation by Science and Technology in Flanders (IWT-Flanders) Belgium.

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25. Grösch S, Schiffmann S, Geisslinger G. Chain length-specific properties of ceramides. Prog Lipid Res 2012;51:50–62.

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26. Levy M, Khan E, Careaga M, Goldkorn T. Neutral sphingomyelinase 2 is activated by cigarette smoke to augment ceramide-induced apoptosis in lung cell death. Am J Physiol Lung Cell Mol Physiol 2009;297:L125–L133.

27. Filosto S, Castillo S, Danielson A, Franzi L, Khan E, Kenyon N et al. Neutral sphingomyelinase 2: a novel target in cigarette smoke-induced apoptosis and lung injury. Am J Respir Cell Mol Biol 2011;44:350–360.

28. Jung SY, Jeon HK, Choi JS, Kim YJ. Reduced expression of FASN through SREBP-1 down-regulation is responsible for hypoxic cell death in HepG2 cells. J Cell Biochem 2012;113:3730–3739.

29. Yamamoto H, Inaba S, Nishiura Y, Kishi F, Kawakami Y. Acute inhalation of cigarette smoke augments hypoxic chemosensitivity in humans. J Appl Physiol 1985;58:717–723.

30. Keppler OT, Horstkorte R, Pawlita M, Schmidt C, Reutter W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications. Glycobiology 2001;11:11R – 18R.

31. Lindner K, Uhlig U, Uhlig S. Ceramide alters endothelial cell permeability by a nonapoptotic mechanism. Br J Pharmacol 2005;145:132–140.

32. Tatsuta T, Scharwey M, Langer T. Mitochondrial lipid trafficking. Trends Cell Biol. 2014;24:44–52.

33. Siskind LJ, Kolesnick RN, Colombini M. Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations. Mitochondrion 2006;6:118–125.

34. Ravid T, Tsaba A, Gee P, Rasooly R, Medina EA, Goldkorn T. Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells. Am J Physiol Lung Cell Mol Physiol 2003;284:L1082–L1092.

35. Moreno L, Moral-Sanz J, Morales-Cano D, Barreira B, Moreno E, Ferrarini A et al. Ceramide mediates acute oxygen sensing in vascular tissues. Antioxid Redox Signal 2014;20:1–14.

36. Chan C, Goldkorn T. Ceramide path in human lung cell death. Am J Respir Cell Mol Biol 2000;22:460–468.

37. Zheng W, Kollmeyer J, Symolon H, Momin A, Munter E, Wang E et al. Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy. Biochim Biophys Acta 2006;1758:1864–1884.

38. Avraham-Davidi I, Grunspan M, Yaniv K. Lipid signaling in the endothelium. Exp Cell Res 2013;:1–8.

CHAPTER 7

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conclusions and future perspectives

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SummaryChronic Obstructive Pulmonary Disease (COPD), a chronic respiratory disease, is one of the leading causes of death today, with a worldwide increase in incidence. It is currently the fourth leading cause of death (97, 98). The majority of COPD patients (excluding alpha1AT deficiency) are characterized by a chronic inflammatory response in the lungs in response to noxious particles and gasses, including cigarette smoke, leading to accelerated lung function decline. Epithelial cells are the first line of defense against inhaled insults such as cigarette smoke. In this thesis we investigated how epithelial cells respond to oxidative stress, specifically with respect to mitochondrial function, and whether mitochondrial dysfunction contributes to the pathogenesis of COPD (5, 6, 15, 72, 80). Because only 10-15% of the smokers eventually develop COPD, we propose that a genetic susceptibility contributes to the onset and progression of the disease (21), leading to overproduction or poor neutralization of ROS. This is supported through GWAS and specific gene association studies (12, 29, 64, 65, 73, 74) as well as familial aggregation studies (52, 61, 75), which may contribute to the onset or progression of the disease. In contrast to asthma, where inhaled glucocorticoids (IGC) are able to suppress inflammatory responses, glucocorticoid (GC) treatment is less beneficial in COPD patients. Reduced sensitivity to IGCs has been clinically associated with neutrophilic airway inflammation. Although neutrophils are considered relatively unresponsive to glucocorticoids, the production of pro-inflammatory cytokines/chemokines involved in neutrophilic infiltration, e.g. CXCL8, by airway epithelium is normally suppressed by corticosteroids (24). However, recent studies have shown that macrophages (37) and bronchial epithelial cells (30) of COPD patients are less responsive to GC, which may thus lead to the development of GC-insensitive airway inflammation. Here, oxidative stress has been implicated as mediator of the observed GC unresponsiveness (30, 44, 69). GCs normally exert a broad spectrum of anti-inflammatory effects after binding to their GC receptor (GRα). After ligand-binding, translocation of the receptor to the nucleus suppresses pro-inflammatory gene transcription through the recruitment of histone deacetylases (HDACs), in particular HDAC2. HDAC2 is able to induce deacetylation of histones containing inflammatory genes, thereby restricting access of the transcriptional machinery and inhibiting transcription (4). It has been shown in COPD macrophages that HDAC2 expression and activity is reduced, and therefore unsuccessful in suppressing the inflammatory response (18, 36, 37). Furthermore, overexpression of HDAC2 in macrophages could overcome GC insensitivity (38). Increased expression of the dominant negative form of the receptor, GRβ, which is seen in steroid resistant neutrophils, may also contribute to GC insensitivity in other cells (81). Furthermore, GCs can directly influence gene transcription and are able to bind to glucocorticoid response elements (GREs) that are present in the promoter of several anti-inflammatory genes (17). Here, posttranslational modification of the GR, by phosphorylation, nitration, acetylation or ubiquitination may

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influence the receptors function and induce GC insensitivity. Cigarette smoke has been shown to induce oxidative stress-dependent activation of p38 MAPK pathway, resulting in GR phosphorylation and inhibiting its translocations to the nucleus (50). Oxidative radicals can either derive from cigarette smoke directly or be generated intracellularly mainly by mitochondria (43). The long term-effects of smoking on mitochondrial function have never been thoroughly investigated in COPD, although some studies that indicate mitochondrial function changes caused by acute cigarette smoke exposure, resulting in OXPHOS and apoptotic abnormalities (77, 84, 85). This thesis aimed to assess the effects of long-term cigarette smoke-induced oxidative stress on GC responsiveness as well as on mitochondrial structure and function in bronchial epithelial cells and the relation between these processes in COPD. Furthermore, we used a novel technique, lipidomics, to monitor cigarette smoke-induced and/or disease related-changes in sputum from COPD patients as well as in long-term cigarette smoke exposed bronchial epithelial cells. To address our hypothesis that cigarette smoke-induced oxidative stress in lung epithelial cells results in mitochondrial defects, in chapter 2 we studied the effect of long-term cigarette smoke exposure on mitochondrial function and morphology in human bronchial epithelial cells. We were especially interested in the long-term effects of CSE, because COPD usually develops after smoking for 20 years or longer. Surprisingly, most in vitro studies rely on relative short cigarette smoke exposure (ranging from 15 min to 48 hours) to understand the acute effects of cigarette smoke on cells that are thought to play key roles in COPD disease. We know from previous studies that long-term cigarette smoke exposure induces changes in the airway epithelial layer, with increased mucus cell hyperplasia and decreased numbers of cilia and ciliated cells (57, 76). In addition to these changes, we hypothesized that long-term cigarette smoke exposure induces mitochondrial dysfunction in airway epithelial cells. In long-term CSE-exposed human bronchial epithelial BEAS-2B cells, we observed increased expression of oxidative stress marker MnSOD and of the inflammatory cytokines CXCL8, IL-1β and IL-6 at mRNA and protein level, which was accompanied by a damaged mitochondrial phenotype including increased fragmentation and branching, and reduced numbers of cristae. All these changes, except for fragmentation, remained present after CSE depletion, suggesting that these are persistent upon smoking cessation. This is in line with the increased mRNA expression levels of OPA1, a critical regulator of cristae formation and fission/fusion, observed in these long-term CSE-exposed cells. We compared the findings in long-term cigarette smoke-exposed BEAS-2B cells with mitochondrial abnormalities in bronchial epithelial cells derived from ex-smoking COPD patients. Here we observed striking similarities, including cristae depletion and increased mitochondrial branching and fragmentation when compared to cells from non-smoking individuals. Also, in bronchial epithelial cells from current smoking COPD patients, we observed higher mRNA expression levels of mitochondrial damage markers PPARGC1α and PINK1 than in cells from

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nonsmoking controls, with an intermediate level of these markers in cells from smoking controls. Both markers are involved in mitochondrial biogenesis and clearance, suggesting an increased mitochondrial turnover due to damage in epithelial cells from COPD patients. Together, our data from this study show that long-term exposure to CSE leads to structural and functional changes in mitochondria that persist upon smoking cessation which could all contribute to (the onset of ) COPD disease by inducing chronic inflammation and hamper tissue repair.

In chapter 3, we investigated the mechanism of cigarette smoke-induced GC unresponsiveness in airway epithelial as well as inflammatory cells. We hypothesized that exposure to cigarette smoke induces GC unresponsiveness by inducing oxidative stress, especially in innate immune cells (e.g. epithelial cells and macrophages) of COPD patients. Previous studies already linked an increased burden of oxidative stress to GC responsiveness in COPD patients (15, 30, 44, 69). We recently observed that oxidative stress reduces GC responsiveness in bronchial epithelial cells and that epithelial cells from COPD patients display reduced GC sensitivity compared to those from non-smokers, with an intermediate effect on control smokers (30). In chapter 3, we confirm that the treatment of epithelial cells with cigarette smoke extract induces oxidative stress, and that this leads to reduced GC responsiveness in bronchial epithelial cells. We next assessed whether GSK3β, a multi-functional redox-sensitive serine/threonine kinase, may be involved in oxidative stress-induced GC unresponsiveness in COPD. Importantly, we found that bronchial epithelial cells and monocytes in lung tissue from smokers and COPD patients display higher levels of phosphorylated GSK3β than in non-smoking controls, a finding that was recapitulated in isolated epithelial cells. We observed that (CSE-induced) oxidative stress is capable of inactivating GSK3β by phosphorylation at serine-9, and that CSE also reduces GC responsiveness in bronchial epithelial cells. We also observed that the pharmacological inhibition of GSK3β leads to reduced GC responsiveness, with a loss of suppressive effects on pro-inflammatory cytokine production. Thus, the increased levels of phopsho-GSK3β in epithelial cells and monocytes may be related to the reduced responsiveness that has been observed in COPD. With respect to the involved mechanism, several studies proposed that oxidative stress-induced histone deacetylase 2 (HDAC2) inactivation in oxidative stress is involved in GC unresponsiveness (4, 5, 17, 53, 93). Although loss of HDAC2 activity appeared, at least in part, responsible for the loss of GC responsiveness upon GSK3β inactivation in monocytes, our data showed that a phospho-p65-dependent mechanism is more likely involved in bronchial epithelial cells (Chapter 2 Supplement). Thus, although the functional output of GSK3β inactivation was similar in inflammatory and structural cells, the downstream mechanisms involved were actually cell-specific.

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GSK3β is involved in the regulation of several mitochondrial processes (13, 51, 59, 79) and in chapter 2 we have shown that cigarette smoking affects mitochondrial function and morphology in lung epithelial cells. This raised the question whether mitochondrial dysfunction in these cells contributes to reduced GC responsiveness as well as chronic airway inflammation and impaired lung tissue repair. In chapter 4 of this thesis, we studied the effect of mitochondrial dysfunction on pro-inflammatory responses, barrier function, wound healing and GC responsiveness in lung epithelial A549 cells depleted of mitochondrial DNA (Rho-0). We have shown that the depletion of mtDNA induces an increase in the baseline production of the pro-inflammatory cytokine IL-6 and chemokines CXCL8 (IL-8), CCL20, CCL3 (MIP1α) and CCL4 (MIP1β). When assessing the suppressive effect of the inhaled glucocorticoid budesonide on the pro-inflammatory response, we observed that the A549-Rho0 cells, depleted of mtDNA, are insensitive to budesonide, in contrast to A549 wild-type cells. In addition, we observed that GCs were unable to significantly improve the barrier function of A549-Rho0 cells, in contrast to A549 wild type cells, while the wound healing response of A549-Rho0 cells was also impaired. Increased lactate levels in A549-Rho0 cells indicated activity of a compensatory secondary metabolism, glycolysis. PI3K, a mediator of the glycolysis, has been linked to GC unresponsiveness in COPD (35, 48, 49). Therefore, we studied the effect of a pharmacological PI3K/Akt inhibitor and observed that this induced a significant decrease in the release of CXCL8. Moreover, in the presence of the Akt inhibitor, the unresponsiveness of A549-Rho0 to budesonide was completely reversed, the suppressive effect on CXCL8 almost returning to the effect in wild type cells. This suggests a role for increased PI3K/Akt signaling in GC unresponsiveness in cells harboring a mitochondrial dysfunction. Together, our data from this chapter demonstrate that mitochondrial dysfunction in epithelial cells may have important consequences for COPD, leading to increased pro-inflammatory activity that cannot be reduced by glucocorticoids and impaired restoration of barrier function upon injury. Thus, the reduced mitochondrial function as observed in epithelial cells from COPD patients (chapter 2) could contribute to the reduced GC responsiveness previously observed in these cells (30).

In chapter 5, we used a novel methodology, lipidomics, to evaluate whether lipid levels in induced sputum of COPD patients may serve as specific markers for COPD. This was especially of interest as changes in lipid metabolism may reflect changes in mitochondrial function. This because the mitochondrial matrix is responsible for fatty acid degradation, a process in which fatty acids are broken down, resulting in release of energy. Induction of sputum by inhaling hypertonic saline has shown to be a useful tool to investigate obstructive airway diseases. Also, in elderly patients with COPD it is considered to be a safe tool for assessing airway inflammation (7, 23), although a disadvantage of induced sputum is that the cellular origin of the lipid compounds cannot be determined. We were able to identify

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more than 1,500 lipid compounds in induced sputum of the participating individuals. Levels of sphingolipids, a major class of lipids, were significantly higher in smokers with COPD than in smokers without COPD. Of these, we have identified more than 200 known single lipid compounds: 168 sphingolipids and 36 phosphatidylethanolamine lipids. In addition, 5 tobacco-related compounds were significantly higher in smokers with COPD compared to smokers without COPD. This latter observation may reflect the differences in pack-years between these groups. In comparison to the never-smoking controls, only 20 (glyco)sphingolipids and 6 tobacco-related compounds were higher expressed in smokers without COPD. After two-months of smoking cessation, we observed a reduction in expression of only 26 sphingolipids in smokers with and without COPD, while the other 175 were still significantly enhanced compared to non-smokers, indicating persistent changes in lipidome profile. In line with these results, several histopathological studies suggest that airway inflammation persists in ex-smoking individuals, although other studies indicate that smoking cessation improves respiratory symptoms and bronchial hyper-responsiveness (90, 91). We cannot exclude that longer periods of smoking cessation will eventually lead to lower expression of additional individual lipid compounds in induced sputum, which requires further investigation. Additionally, we found only one lipid component to be reduced in induced sputum of smokers and COPD patients compared to non-smokers, which was identified as neuraminic acid, a terminal monosaccharides modification of most glycoconjugates and glycosphingolipids. After smoking cessation, neuraminic acid levels were restored, further indicating that its increase was due to cigarette smoking and not a disease-related effect. To conclude, expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD.Many different lung cell types including epithelial cells, macrophages and neutrophils, may be responsible for the observed changes in lipid compounds, either upon active secretion or release of their lipid (membrane) compounds into the lung sputum upon necrotic cell death. The release of lipids may contribute to airway inflammation (86, 92). Free fatty acids can activate TLR-4, thereby inducing similar inflammatory responses as lipopolysaccharides (47). In line with this, we found a correlation with inflammation in sputum and lung function decline for the 13 lipids with the highest increase in smokers with and compared to those without COPD. Furthermore, lipids released from the sphingolipid pathway, such as ceramides, are major players in the induction of apoptosis and cellular senescence, and both cellular responses are increased in the lungs of COPD patients (28, 86). Additionally, cigarette smoke-induced changes in lipid accumulation and/or composition have been reported to be harmful to the cell as they compromise integrity of cell membranes and organelle membranes, including those from mitochondria (47, 92). Thus, the observed changes in lipid profile of smoking COPD patients may be related to the abnormalities in mitochondrial structure in epithelial cells from COPD patients. There are already initial

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studies that use lipidomics and airway epithelial cells for determining airway disease (95).As a follow-up on chapter 5, in chapter 6 we studied whether epithelial cells may be responsible for the observed changes in lipid profile in sputum of smokers, by assessing how long-term cigarette smoke exposure affects the intracellular lipidome expression in human bronchial epithelial cell line BEAS-2B. In this study we showed that lipids from the sphingolipid pathway are altered by long-term CSE exposure in epithelial cells, which is in line with our findings in chapter 5, indicating that the sphingolipids in sputum from smoking individuals may be derived from airway epithelial cells. In further line with our data from chapter 5, we observed significant alterations in 9 sphingomyelins of which 2 components were up-regulated and 7 were down-regulated in long-term CSE-exposed BEAS-2B cells. Additionally, we found significant changes in 5 ceramides, of which 2 were up-regulated and 3 were down-regulated, and we observed and a reduction in 3 neuraminic acid containing lipid components belonging to the class of glycosphinogolipids. Thus, cigarette smoke exposure strongly reduces neuraminic acid containing lipid components both in vivo in sputum and in epithelial cell in vitro. N-acetylneuraminic acid is one of the most important members of this sugar family and is involved in a variety of biological interactions including cell adhesion and migration (42). Moreover, it is an important addition to mucins and has shown to be an antioxidant scavenger (34). Therefore, we expect that alterations in its levels may have implications in cell infiltration and aberrant cellular repair responses of COPD patients. Nine other glycosphinogolipids belonging to the same lipid class showed strong up-regulation upon CSE exposure, especially lipids belonging to N-acetylhexosamine-trihexosyl-ceramides. In line with the observed upregulation of sphingomyelins, sphingomyelinase activity, as determined by the ratio of ceramides and sphingomyelin, showed increased activity in epithelial cells upon long-term exposure to CSE. Furthermore, we observed that CSE exposure lowered the expression of the lipid converting enzymes SGMS2, FASN and SMPD1, which could be responsible for the observed CSE-induced changes in lipid profile in bronchial epithelial cells. We anticipate that especially the CSE-induced changes in sphingolipid compounds may have important consequences, as they regulate various cellular processes like apoptosis, regeneration and senescence (26, 28, 92), all of which are of relevance to COPD.

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General discussionTogether, our study shows that cigarette smoke-induced oxidative stress induces mitochondrial function and morphology changes and that epithelial cells from COPD patients display mitochondrial abnormalities. We hypothesized that oxidative stress results in stronger mitochondrial damage in epithelial cells from COPD patients than control smokers as a consequence of impaired antioxidant responses (15, 22). Normally, cigarette smoking induces an upregulation of antioxidant genes. Increased expression of 16/44 antioxidant-related genes has been observed in airway epithelium from smokers compared to non-smokers (27), while levels of the ROS scavenger Vitamin E were reduced with disease severity in lung tissue of COPD patients (1). Mitochondrial damage may be related to increased pro-inflammatory activity and reduced GC responsiveness in COPD, as depletion of mitochondrial DNA in lung epithelial cells resulted in loss of suppressive effects of glucocorticoids on epithelial pro-inflammatory cytokine production. In addition, mitochondrial dysfunction led to increased glycolysis, which has been associated with increased PI3K activity. PI3K has been implicated in oxidative-stress induced GC unresponsiveness (48, 49). Indeed, we observed that inhibition of PI3K/Akt activity restored the responsiveness of pro-inflammatory epithelial responses to GCs in mtDNA-depleted cells. Of note, PI3K can induce phosphorylation of GSK3β at Ser-9 (94), which results in a reduction of GSK3β activity. We further demonstrated that this inhibitory phosphorylation of GSK3β mediates GC insensitivity in bronchial epithelial cells, and that levels of phosphorylated GSK3β are increased in bronchial epithelial cells of COPD patients. Of interest, it is well known that inhibition of GSK3β activity stimulates glycolytic processes (87), supporting the notion that it may play a role in mitochondrial function. Mitochondria may also be important as sensors of cellular stress by quantitating the local accumulation of specific lipids and glycolipids and initiate stress-induced apoptosis (83). Sphingolipids have been implicated in apoptotic and autophagy processes (46). Accumulation of the sphingolipid ceramide can affect mitochondrial functions either direct or indirectly (82, 83). In early stage apoptosis, a ganglioside (GD3) is actively synthesized and relocates to the mitochondrial membrane initiating apoptosis by opening the transition pore complex (83). Thus, it is tempting to speculate that the cigarette smoke-induced changes in lipid compounds may contribute to the observed mitochondrial abnormalities in COPD and thus the pathogenesis of the disease. We observed higher levels of lipids from the sphingolipid pathway in smokers with COPD compared with smokers without COPD. Specifically, ceramides has been shown to induce alveolar epithelial apoptosis and alveolar enlargement in a mouse model (63). Furthermore, Bowler and co-workers recently reported an association between sphingomyelins and emphysema and between glycosphingolipids and COPD exacerbations (8). Together, we propose that cigarette smoke-induced oxidative stress and changes in lipidome lead to mitochondrial damage and a switch to glycolysis,

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which is accompanied by the activation of PI3K/Akt, with subsequent activation of pro-inflammatory pathways and inactivation of GSK3β, leading to reduced responsiveness to glucocorticoids. In turn, we show that a proper mitochondrial function is important for restoration of the epithelial barrier upon injury, and may thus play a role in the abnormal tissue repair, another important pathological feature of COPD. Excessive ROS production by damaged mitochondria may increase pro-inflammatory signaling (e.g. the PI3K/Akt pathway) and further damage mtDNA components, leading to further impairment of mitochondrial function and enhancement of pro-inflammatory responses (chapter 2, (33)). Mitochondrial impairment in epithelial cells from COPD patients is supported by our finding that the expression of mitochondrial biogenesis gene expression marker PPARGC1α was increased in epithelial cells from COPD patients compared to control smokers. This indicates a necessity for generating new mitochondria in epithelial cells from COPD patients. In contrast to epithelial cells, it was found lowered in skeletal muscle from COPD patients (70). Another mitochondrial damage marker, PINK1, was also significantly increased in epithelial cells from COPD patients compared to non-smokers, although not when compared to control smokers. This is in line with a recent study showing elevated levels of PINK1 in epithelium of COPD patients. Here, authors also showed that PINK1 knock-out mice are protected from cigarette smoke-induced COPD manifestations including airspace enlargement (54).

We have not studied effects of cigarette smoke on cell death in epithelial cells from COPD patients this thesis, but increased epithelial apoptosis has also been observed in COPD (11). GSK3β is able to mediate apoptosis through p53 and BAX activation, triggering the caspase cascade and induce programmed cell death (20, 32, 39, 59). p53 can also influence cell metabolism at specific points, notably through the up-regulation of glutamate synthesis and inhibition of fatty acid synthesis and glycolysis (41, 55, 68). GSK3β, together with the Akt-dependent kinase tau protein kinase 1, is able to interact with pyruvate dehydrogenase (PDH), thereby reducing levels of acetyl-CoA and blocking the TCA cycle, thus impacting on mitochondrial respiration (25, 40). Additionally, GSK3β can negatively regulate several aspects of insulin signaling and thus limit the uptake of glucose (71). Inactivation of GSK3β promotes glucose uptake and together with reduced levels of PDH will generate a metabolic shift towards glycolysis (20, 31). We speculate that especially the effects of GSK3β on the apoptotic response might force the cell to go into necrosis, which may subsequently result in the release various cellular components, including membrane lipids and damage associated danger signals (66) into the environment, contributing to the chronic inflammatory response. An overview of these processes is illustrated in figure 1. Taken together, we expect that more knowledge about mitochondrial function, GSK3β regulation and lipid release will greatly contribute to improve our insight in the inflammatory processes and cellular regeneration

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of COPD disease and might improve the method of treatment and the outcome of this general permanent and progressive disease. This may provide new insights in the role of mitochondria in the development and treatment of Chronic Obstructive Pulmonary Disease.

ATP

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Glycolysis

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NADH

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Healthy individualLung epithelium

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COPD PatientLung epithelium

Increased Mitochondrial and mtDNA Damage Caspase 9 activation

p53BAX BAX

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Figure 1. Model of molecular mitochondrial processes in A) lung epithelial cells from healthy individual and B) lung epithelial cells from COPD patient. A) In epithelial cells from healthy individuals, the majority of ATP is produced by oxidative phosphorylation and an adequate anti-oxidant response (e.g. Vitamin E, Catalase, MnSOD) will neutralize the produced reactive oxygen species. A balanced active and inactive form of GSK3β will ensure limited glycolysis, steroid sensitivity and a proper apoptotic response. B) Hypothetical model of the effects of mitochondrial damage in epithelial cells of COPD patients. Increased ROS production and/or an inadequate anti-oxidant response can trigger an inflammatory response and further mitochondrial damage. Mitochondrial dysfunction can lead to cellular decay, amplify the pro-inflammatory response, and lead to impaired wound healing upon injury. Inactivation of GSK3β reduces steroid responsiveness, and stimulates the glycolysis and can block cells from going into mitochondrial induced apoptosis, potentially inducing necrosis, leading to the release of lipid components.

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Future perspectivesFor a COPD patient, the moments that “take your breath away” are worst in life. COPD is a slow progressive disease and especially the end stage is characterized by very severe airflow limitation and exacerbations that are sometimes life-threatening. A severely impaired quality of life is also common at this stage. Smoking cessation and improvement of physical health until now are considered the only effective interventions inhibiting the further progression of COPD; in particular the further decline in lung function, however, inflicted lung damage cannot be restored. Current treatment of COPD is based on suppressing the inflammatory response in the lung using corticosteroids but this appears to exert little benefit because of an impaired response mechanism. Current research aims to improve the sensitivity of corticosteroids studying the involvement of reactive oxygen species and HDAC2 activity. Studies have shown that oxidative stress induces steroid insensitivity mediated by the activation of Akt and the inhibition of HDAC2 activity (4, 17, 36, 38, 48, 53). Our study supplements these results as we have shown that Akt is indeed involved in GC unresponsiveness in mtDNA-depleted cells, however, we found only partial HDAC2 involvement in steroid insensitivity in human monocytes and no involvement of HDAC2 in bronchial epithelial cells. We did identify a novel player regulating GC response, GSK3β, in both cell types. Pharmacological inhibition of GSK3β led to complete steroid insensitivity in cultured human monocytes and bronchial epithelial cells. Future studies should focus on preventing inactivation/phosphorylation of GSK3β when using corticosteroids to treat COPD patients. Additionally, it would be of interest to study the possible link and role of GSK3β and mitochondrial function. It might be interesting to study the effect of anti-oxidants and/or an Akt inhibitor on the inflammatory response in combination with GC treatment. (Mitochondrial targeted) antioxidants may also prove beneficial in protecting the mitochondria against excessive ROS production. Ahmad et al. have shown that mitochondrial targeted antioxidants greatly improved impaired mitophagy, a phenotype observed in mouse lungs with emphysema as well as in lungs of chronic smokers and patients with COPD (2). Additionally, protection of the mitochondria and stimulating the formation of new mitochondria might be a novel therapeutic strategy for COPD. In this respect, substances like L-carnitine, lipoic acid, and coenzyme Q10 are known to protect mitochondria, while pyrroloquinoline quinone (PQQ) has been shown to stimulate mitochondrial biogenesis by increasing the expression of PPARGC1α (14, 78). We observed that mRNA expression of PPARGC1α is increased in epithelial cells from COPD patients potentially as consequence of an unsuccessful attempt to upregulate mitochondrial biogenesis. Reducing the formation of ROS may also prove beneficial in preventing GSK3β phosphorylation and thus protect mitochondria and promoting the sensitivity to GCs.

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In the future, it will further be of interest to explore abnormalities in autophagy/mitophagy processes in lung epithelial cells of COPD patients. In chapter 2, we imaged epithelial cells of COPD patients and found structural and functional abnormalities in the mitochondria. Normally, when autophagy/mitophagy is hampered, defective mitochondria accumulate. The persistent presence of the defective mitochondria in epithelial cells from COPD patients raised the question whether clearance by mitophagy could be disturbed. A recent study showed that mitophagy-dependent necroptosis contributes to the progression of COPD (54). Also, it has been shown that when autophagy is hampered, this leads to increased release of the pro-inflammatory cytokine IL-1β upon mtDNA exposure. Enhanced levels of IL-1β have been observed in COPD and disturbed autophagy may thus have important consequences for the inflammatory response in COPD (3, 9, 56). mtDNA is also able to induce various inflammatory cytokines next to IL-1β including CLCX8, IL-6 and TNF-α (10, 45, 60, 62, 96). These cytokines can also be released by exposure through cigarette smoke directly or indirectly due to the induction of necrotic cell death leading to the release of DAMPs in the environment. It would be interesting to study whether pro-inflammatory responses of epithelial cells are reduced upon cigarette smoke or DAMP exposure when the autophagy/mitophagy process is blocked. In addition, restoring the neuraminic/sialic acid concentration in respiratory mucus might be a promising therapeutic strategy. The airway epithelium is covered by a renewable layer of gelatinous mucus. Mucus lining the respiratory tract is thought to act as scavenger of free radicals, consisting mostly of water, salt, lipids, ascorbic acid, reduced glutathione (GSH) and mucin (19, 58, 67). Mucins are large neuraminic acid or sialic acid containing glycoproteins present at the interface between epithelium and their extracellular environment. A recent study indicated that sialic acid is the key component in hydroxyl oxidant savaging capacity of mucins (58). Also, sialic acid has been proposed to inhibit viral entry by mimicking sialylated receptors on the cell surface, thus a reduction of sialic acid in the mucus lining could increase the susceptibility for viral infections (16). This may lead to more frequent COPD exacerbations, which are often associated with viral or bacterial infections (88, 89). Neuraminic/Sialic acid containing components were the only components lowered in our lipidomic study (chapter 5). Lowered levels were found in smokers as well as COPD patients, indicating that this was caused by cigarette smoking. This is in line with our prolonged CSE exposed BEAS-2B cell study where neuraminic/sialic acid components were also lowered (chapter 6). This cigarette smoke-induced loss of neuraminic/sialic acid may lead to increased epithelial damage due to impaired scavenging of oxidants. Restoring neuraminic/sialic acid concentration in the airways might protect the respiratory epithelium from further decay.Future research may also focus on the use of lipidomics in recognizing mitochondrial dysfunction in cells or biopsies. We used lipidomics successfully to identify specific lipid components in induced sputum of COPD patients and in lung epithelial cells exposed

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to cigarette smoke. A cell line harboring a mitochondrial dysfunction (Rho-0 or cybrids, a cytoplasmic hybrid formed by the fusion of a whole cell lacking mtDNA with a cytoplast) could serve as a model to generate a specific lipid spectrum specifically for mitochondrial impairment. Mitochondrial fractions can also be isolated from healthy- and affected cells to investigate which lipidomic profile is specific for healthy and dysfunctional mitochondria. These lipidomics spectra can be compared to individual patient samples/spectra for diagnostics use of COPD disease. This would especially be of interest for a disease like COPD because it reduces the invasiveness of current procedures.

Concluding remarksTaken together, our findings suggest an important role for mitochondrial functionality in COPD disease. Mitochondrial function is affected by cigarette smoke-induced oxidative stress, especially in epithelial cells from COPD patients, leading to increased inflammatory signaling, reduced wound healing capabilities and reduced GC responsiveness. These abnormalities observed in mitochondrial function in epithelial cells from COPD patients are accompanied by increased phosphorylation/inactivation of GSK3β. Our data show that both deficiencies lead to GC responsiveness. Glucocorticoids have broad anti-inflammatory effects, but exert little benefit in COPD patients. Understanding the mechanisms of this unresponsiveness may lead to important novel avenues in the treatment of COPD disease. Furthermore, we show that the novel technique lipidomics may be suitable for diagnostic use in induced sputum of COPD patients. This thesis may thus open avenues for further research on the cellular and molecular mechanism underlying the pathophysiology of COPD, and provide novel insights for future therapeutic strategies.

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References1. AH Agler, RG Crystal, JG Mezey J Fuller, C Gao, JG Hansen and PC. Differential Expression of Vitamin E and

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Nederlandse samenvatting

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Nederlandse Samenvatting

Wat is COPDChronische obstructieve longziekte (COPD) is een ziekte die zich voornamelijk in de longen manifesteert

en, vooral bij ouderen, gepaard gaat met een hogere kans op morbiditeit en mortaliteit. De meest

prominente risicofactor van COPD is actief roken of passief meeroken. Daarnaast vormt de inademing

van andere schadelijke deeltjes en gassen zoals fijnstof, brandstoffen, chemicaliën en houtrook een

risico. Slechts 10-15% van de rokers ontwikkelt COPD, wat wijst op een genetische vatbaarheid die

bijdraagt aan de ontwikkeling van COPD. Er zijn ook niet-omgeving gerelateerde risicofactoren voor

het ontwikk e len van COPD zoals genetische afwijkingen, in het bijzonder de alfa1-antitrypsine-

deficiëntie . Het roken van tabak is voor de gezondheidszorg een groot probleem in de westerse

wereld en in de opkomende industrielanden. Schattingen van de `World Health Organization` (WHO)

stellen dat 65 miljoen mensen wereldwijd een matige tot ernstige vorm van COPD hebben, wat leidt

tot hoge zorgkosten. Het is momenteel de vierde belangrijkste doodsoorzaak, en de verwachting is

dat het in 2030 de derde belangrijkste doodsoorzaak wereldwijd zal zijn.

COPD wordt gekenmerkt door chronische ontsteking in de longen die gepaard gaat met toenemende

longschade , onomkeerbare luchtwegobstructie, en versnelde achteruitgang van de longfunctie.

Ingeademde sigarettenrook prikkelt en beschadigt het bekledende luchtwegepitheel, en zorgt ervoor

dat zowel epitheelcellen als macrofagen (beiden behorend tot het aangeboren immuunsysteem)

geactiveerd raken en er een ontstekingsreactie optreedt. De cellen van COPD patiënten reageren

heftiger op de ingeademde rookdeeltjes dan cellen van rokers die geen COPD ontwikkelen, waardoor

er meer on t steking optreedt. Bij COPD patiënten produceren bovengenoemde epitheelcellen en

macrofagen chemische signaalstoffen (cytokines en chemokines, waaronder het zogenaamde

CXCL8/IL-8 ) , die op hun beurt weer andere immuuncellen in de longen en het bloed aantrekken

en activeren, waaronder neutrofielen en cellen van het aangeleerde immuunsysteem, T-cellen. Deze

geactiveerde cellen dragen bij aan het onderhouden van de boven beschreven ontstekingsreactie en

zorgen er voor dat er meer schade optreedt aan het longweefsel (Figuur 1). COPD omvat chronische

bronchitis en longemfyseem. Chronische bronchitis wordt gekenmerkt door een ontstekingsreactie

(‘itis`) in de grote en kleine luchtwegen (de bronchi), die leidt tot verdikking van de luchtwegwand. Bij

patiënten die lijden aan chronische bronchitis toont longfunctie onderzoek een luchtwegobstructie

bij uitademing aan, en de patiënten hebben vaak last van hoesten en slijm opgeven. Longemfyseem

wordt gekenmerkt door een ontstekingsreactie die tot afbraak van eiwitten in de longblaasjes (alveoli)

leidt en uiteindelijk tot verlies van het alveolair weefsel. Hierbij is de rek uit de longen (“slappe long”),

wat leidt t ot verlies van longfunctie, en de patiënt heeft last van kortademigheid bij inspanning.

Deze ziektebeelden kunnen afzonderlijk voorkomen, maar ook naast elkaar aanwezig zijn bij dezelfde

patiënt.

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Figuur 1. Overzicht van een sigarettenrook ontstekingsreactie in de longen. A) beschadiging van de longblaasjes (Emfyseem) en/of B) aantasting van de grote luchtwegen met slijmproductie (Chronische Bronchitis). Sigarettenrook activeert epitheelcellen en Marcofagen in de long die vervolgens chemokines en cytokines uitscheiden zoals, CXCL8, G-CSF, TGFβ, IL-6. Hierdoor worden andere cellen van het immuunsysteem, zoals Neutrofielen (Neu), Natural Killer (NK) cellen en T-cellen, aangetrokken en geactiveerd. Deel van dit figuur was gemodificeerd aan de hand van een figuur getoond op de site van Beth Israël Deaconess Medical Center (http://www.bidmc.org/Centers-and-Departments/Departments/Surgery/Chest-Disease-Center/Programs-and-Services/COPDEmphysemaClinic/DiagnosingCOPD.aspx).

Behandeling van COPDEr is momenteel geen behandeling beschikbaar die COPD kan genezen. De belangrijkste maatregel

is stoppen met roken, waarvan aangetoond is dat het de verslechtering van COPD een halt toe

roept, hoewel het de achteruitgang in longfunctie niet kan terugdraaien. Griepvaccinaties kunnen

virusinfecties en daarmee gepaard gaande longaanvallen (plotselinge verslechtering van het

ziektebeeld) gedeeltelijk voorkomen. Verder kan longrevalidatie de patiënt leren omgaan met

zijn/haar ziekte. Wat betreft de behandeling met geneesmiddelen is deze vooral gericht is op

symptoombestrijding. Voorbeelden hiervan zijn luchtwegverwijders die geïnhaleerd worden zoals

antibiotica, slijmverdunners en glucocorticoïden (GC). GC hebben een sterke ontstekingsremmende

werking en worden daardoor effectief gebruikt bij bijvoorbeeld astma, maar bij COPD patiënten zijn

ze helaas weinig effectief en onderdrukken ze de onderliggende ontstekingsreactie onvoldoende.

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De vraag is waarom deze GC niet effectief zijn bij astma bij COPD. Mogelijk is een van de mogelijke

oorzaken de oxidatieve stress (zie hieronder) die wordt veroorzaakt door het roken bij COPD patiënten.

Roken en oxidatieve stressSigarettenrook veroorzaakt de productie van zogenaamde vrije zuurstofradicalen. Dit zijn

agressieve en uiterst reactieve deeltjes die de cellen in ons lichaam kunnen aanvallen. Als deze vrije

zuurstofradicalen niet op tijd geneutraliseerd worden door antioxidanten, kunnen ze voor het lichaam

belangrijke biologische moleculen beschadigen. Het betreft moleculen zoals vetten en eiwitten, die

ook voorkomen in onze longcellen. De productie van vrije zuurstofradicalen wordt oxidatieve stress

genoemd. Men neemt aan dat de longcellen van COPD patiënten niet de capaciteit hebben om

al deze oxidatieve stress te neutraliseren. Onder meer het eiwit genaamd glucocorticoïdreceptor

α, dat verantwoordelijk is voor een goede werking van GC, kan hierdoor beschadigd raken en

zijn ontstekingsremmende functie verliezen. Ook kunnen vrije radicalen inwerken op andere

belangrijke eiwitten die de ontstekingsremmende activiteit verminderen of kunnen ze deze

eiwitten zelfs `uitzetten`. Van enkele eiwitten (zoals Histone Deacetylase 2, HDAC2, wat een rol

speelt bij de ontstekingsremmende werking van GC) is in de literatuur inderdaad beschreven dat

ze door in-activatie, veroorzaakt door vrije radicalen, de GC-gevoeligheid kunnen verminderen. Of

dit mechanisme ook een rol speelt bij verminderde GC gevoeligheid van een belangrijke aandrijver

van de ontstekingsreactie bij COPD, het longepitheel, is op dit moment onbekend. Het begrijpen

van de specifieke mechanismen van GC ongevoeligheid bij COPD kan leiden tot belangrijke nieuwe

mogelijkheden voor de behandeling van COPD.

MitochondriënMitochondriën zijn organellen in de cel die vaak ‘energie fabrieken` worden genoemd. Ze zijn

samengesteld uit verschillende compartimenten die meerdere gespecialiseerde functies uitoefenen

met betrekking tot de energieproductie in de cel. Mitochondriën zijn ovale of staafvormige

organellen die een dubbele membraanstructuur hebben. Het binnenste membraan wordt lobulair

gevormd waardoor het oppervlak sterk vergroot wordt ten behoeve van de productie van adenosine

trifosfaat (ATP) (Figuur 2). Energie wordt geproduceerd uit een proces dat oxidatieve fosforylering

(OXPHOS) heet. Bij dit proces geven elektronen hun energie af door middel van redoxreacties

die worden uitgewisseld via het binnenmembraan. Vervolgens kunnen de protonenpompen de

productie van energie verzorgen in de vorm van ATP. Naast het produceren van energie zijn de

mitochondriën verantwoordelijk voor het reguleren van geprogrammeerde celdood, calcium opslag,

celgroei (proliferatie), hormoon- en heemsynthese en de overdracht van signalen binnen de cel.

Mitochondriën zijn dus erg belangrijk voor de functie van de cel en het functioneren van het lichaam.

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Figuur 2. Een voorbeeld van een mitochondrion. De zwarte pijlen geven de lobulaire binnenmembraan structuren aan die cristae genoemd worden. Hier wordt energie in de vorm van ATP geproduceerd.

De rol van mitochondriën bij COPDMitochondriën spelen een belangrijke rol in ziekten die geassocieerd worden met oxidatieve stress,

waaronder COPD. Dit komt omdat mitochondriën tijdens de energieproductie een bijproduct in de

vorm van vrije radicalen aanmaken. Deze vrije radicalen zorgen voor oxidatieve stress in de cel, maar

worden normaliter geneutraliseerd door zeer gespecialiseerde enzymen met antioxidant werking,

zoals Superoxide Dismutase, Glutathion en Catalase. Uiteindelijk zetten deze enzymen de agressieve

radicalen om in water (H2O) en zuurstof (O

2). Het lijkt er op dat dit proces bij COPD patiënten niet

meer in balans is, omdat er in verhoogde mate oxidatieve stress aanwezig is. Dit kan komen doordat

de mitochondriën teveel vrije radicalen produceren als het gevolg van het roken (sigarettenrook

bevat ook veel vrije radicalen die vervolgens de mitochondriën), of dat de mitochondriën mogelijk te

weinig (actieve) enzymen hebben die deze radicalen neutraliseren, of een combinatie hiervan. Door

langdurige blootstelling aan oxidatieve stress treedt er in de cel schade op. Deze door oxidatieve

stress geïnduceerde schade is het grootst op de plek waar vrije zuurstofradicalen geproduceerd

worden, en dat is in de mitochondriën zelf. Uiteindelijk leidt dit theoretisch tot een zodanige schade

aan de mitochondriën, dat deze zelf extra vrije radicalen gaan produceren en/of helemaal niet meer

werken. Dit noemen wij een mitochondriale disfunctie.

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In ons onderzoek veronderstellen wij dat de schadelijke effecten van sigarettenrook-geïnduceerde

oxidatieve stress een negatieve invloed hebben op de mitochondriale functie in luchtwegepitheelcellen,

en dat COPD patiënten hiervoor gevoeliger zijn dan rokers die geen COPD ontwikkelen. Ook

vermoeden wij dat een mitochondriale disfunctie bijdraagt aan het ontstaan en verergeren van de

chronische ontsteking bij COPD en dat luchtwegepitheel cellen met slecht werkende mitochondriën

minder goed kunnen herstellen na beschadigende stimuli in het algemeen en roken in het bijzonder.

Bovendien verwachten wij dat oxidatieve stress en mitochondriale disfunctie tot GC-ongevoeligheid

leiden van luchtwegepitheelcellen.

Om aan te tonen of langdurige blootstelling aan sigarettenrook invloed heeft op de werking van

mitochondriën hebben we ons in hoofdstuk 2 gericht op de effecten hiervan in menselijke (humane)

luchtwegepitheelcellen. In het bijzonder hebben we gekeken naar de mitochondriale vorm en functie

in de humane luchtweg/bronchiale epitheelcellijn, welke 6 maanden lang was bloot gesteld aan

sigarettenrookextract. Daarnaast hebben we onderzocht of en hoe de mitochondriale vorm en functie is

veranderd in bronchiale epitheelcellen van COPD patiënten ten opzichte van niet-rokende en rokende

individuen zonder COPD. In zowel de bronchiaal epitheel cellijn als in de luchtwegepitheelcellen van

COPD patiënten, konden we duidelijke afwijkingen in mitochondriale vorm en functie waarnemen.

Zo waren in de langdurig aan sigarettenrookextract blootgestelde luchtwegepitheelcellen meer

vertakte mitochondriën aanwezig en minder cristae zichtbaar in vergelijking tot epitheelcellen die

even lang werden gekweekt, maar niet aan rook werden blootgesteld. Vertakking van mitochondriën

duidt vaak op veroudering van mitochondriën en minder cristae betekent minder oppervlak voor het

produceren van energie (ATP). Soortgelijke afwijkingen vonden we ook in mitochondriën van COPD

patiënten ten opzichte van mitochondriën in gekweekt luchtwegepitheel van gezonde personen

en rokers zonder COPD. In de langdurig aan sigarettenrook blootgestelde luchtwegepitheelcellen

ging dit gepaard met de productie van verschillende ontstekingsbevorderende cytokines zoals

CXCL8, IL-1β en IL-6, die onder meer neutrofielen kunnen aantrekken. Ook zagen we dat belangrijke

mitochondriale stress-markers, zoals PINK1, meer tot expressie kwamen in luchtweg epitheelcellen

van COPD patiënten. Samenvattend bevestigen de bevindingen van dit hoofdstuk de hypothese dat

sigarettenrook schadelijk is voor de mitochondriale functie, dat dit met name in luchtwegepitheel

va COPD patiënten voor mitochondriële afwijkingen zorgt en dat mitochondriale disfunctie gepaard

gaat met de productie van ontstekingsfactoren.

Glycogen Synthese Kinase (GSK3)β is een eiwit dat veelal in het cytosol van de cel is gelokaliseerd

maar daarnaast ook in de mitochondriën voorkomt, waar het een zeer hoge activiteit laat zien en

belangrijke processen reguleert. GSK3β is een eiwit dat reageert op signalering van oxidatieve stress.

Het speelt een rol bij verschillende ontstekingsziekten en in eerder onderzoek T-cellen is gevonden

dat het mogelijk ook een rol speelt bij GC ongevoeligheid. Oxidatieve stress remt de activiteit van

GSK3β en stimuleert de glycolyse, een proces dat energie genereerd uit het omzetten van glucose, ten

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koste van mitochondriale ATP productie via OXPHOS. In hoofdstuk 3 hebben we de mechanismen

voor sigarettenrook geïnduceerde GC-ongevoeligheid cellen van het aangeboren immuunsysteem

van COPD patiënten bestudeerd en gekeken of GSK3β hierbij betrokken is. GSK3β bleek verhoogd

inactief in de macrofagen en epitheelcellen van COPD patiënten vergeleken met gezonde personen

en rokers zonder COPD (zowel in het longweefsel als in gekweekte cellen). In macrofaag- en epitheel-

cellijnen toonden wij aan dat (sigarettenrook geïnduceerde) oxidatieve stress deze inactiviteit konden

veroorzaken. Verder vonden wij dat het (farmacologisch) blokkeren van de werking van GSK3β in

zowel epitheelcellen als macrofagen voor een sterk verminderde gevoeligheid voor GC bleek te

zorgen. Hiermee hebben wij laten zien dat het inactiveren van het eiwit GSK3β door sigarettenrook

in cellen van COPD patiënten mogelijk kan bijdragen aan de ontwikkelingen van GC-ongevoelige

ontsteking bij COPD.

Omdat uit hoofdstuk 2 is gebleken dat er na langdurige blootstelling van epitheel aan sigarettenrook

afwijkingen in de mitochondriën optreden, waren we benieuwd of een mitochondriale disfunctie

ontstekingsreacties en GC ongevoeligheid kan veroorzaken. Daarnaast wilden we bestuderen of

verlies van mitochondriale functie gevolgen heeft voor de herstelreactie van het epitheel als gevolg

van het aanbrengen van schade. We hebben dit in hoofdstuk 4 van dit proefschrift onderzocht

door gebruik te maken van een longepitheel cellijn waarin geen mitochondriaal DNA (mtDNA) meer

aanwezig is, met andere woorden, de mitochondriën zijn disfunctioneel. De vindingen in dit hoofdstuk

laten zien dat een mitochondriale disfunctie een verhoging van meerdere ontstekingsbevorderende

cytokines, zoals CXCL8, CCL20 en IL-6, tot gevolg heeft en dat GC minder effectief in het remmen van

deze factoren in cellen met een mitochondriale disfunctie vergeleken met gezonde cellen. Ook zien

we een minder goed herstel na het aanbrengen van epitheliale schade wanneer de mitochondriën

disfunctioneel zijn. Samenvattend toont dit aan dat goed functionerende mitochondriën belangrijk

zijn bij schadeherstel. Verder kan de mitochondriale disfunctie die we aantroffen in epitheelcellen

van COPD patiënten bijdragen aan de ontstekingsbevorderende activiteit en de verminderde

gevoeligheid voor GC.

COPD wordt gekenmerkt door een chronische ontsteking en lipiden kunnen hieraan mogelijk een

belangrijke bijdrage leveren, omdat lipiden kunnen dienen als signaalmoleculen die een inflammatie

kunnen starten. De mitochondriale afwijking die we in COPD epitheel hebben gevonden, kunnen

ook van invloed zijn op de expressie van lipiden aangezien de mitochondriën de vetstofwisseling

reguleren in de cel. Daarom vroegen wij ons af of er veranderingen optreden in het lipiden profiel van

patiënten met COPD. In hoofdstuk 5 onderzochten we het potentieel om lipidomics, een techniek

die het mogelijk maakt om expressie van specifieke lipiden componenten te herkennen, te gebruiken

om sigarettenrook-geïnduceerde en/of ziekte-gerelateerde veranderingen in het sputum van COPD-

patiënten te herkennen. Het lipidenprofiel van sputum hebben we vergeleken met dat van rokers

zonder COPD en niet-rokers. We hebben meer dan 200 specifieke lipidenmarkers kunnen detecteren

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in sputum van rokende COPD patiënten t.o.v. rokers zonder COPD. Stoppen met roken reduceerde

de expressie van slechts 26 van deze lipiden, wat aangeeft dat de veranderingen in lipidenprofiel van

COPD patiënten grotendeels persistent zijn. 20 (glyco)sphingolipiden componenten waren verhoogd

in rokers zonder COPD in vergelijking met niet-rokers, wat het effect van sigarettenrook weergeeft.

Al deze lipiden behoren tot de sphingolipiden en zouden verantwoordelijk kunnen zijn voor het

induceren van een ontstekingsreactie, aangezien deze groep lipiden sterk wordt geassocieerd met het

veroorzaken van ontsteking. De 13 hoogst tot expressie gebrachte lipiden in rokers met COPD, lieten

een correlatie zien met betrekking tot een minder goede longfunctie. Deze resultaten suggereren

een belangrijke rol voor veranderingen in sphingolipiden expressie bij het ontstaan van COPD. Tevens

zouden sphingolipiden in de toekomst misschien gebruikt kunnen worden als bio-marker voor COPD.

Omdat het nog niet duidelijk is waardoor deze veranderingen in het sputum van COPD patiënten

veroorzaakt werden, en of dit kan komen door de blootstelling van epitheel aan sigarettenrook, hebben

we in hoofdstuk 6 onderzocht of langdurig aan sigarettenrook blootgestelde luchtwegepitheel (zie

hoofdstuk 2) soortgelijke veranderingen laat zien in lipide-profiel. Hier bleek dat dezelfde lipiden van

de sphingolipiden cascade als gevonden in het sputum van COPD patiënten veranderd tot expressie

kwamen in aan sigarettenrook blootgestelde luchtwegepitheel cellen. 13 lipiden waren gestegen in

expressie, maar er waren ook 13 lipiden gedaald in expressie wat mogelijk een rol in lipidensignalering

betekend. Verder was de RNA expressie van `sphingomyelin synthase 2` en `fatty acid synthase`,

enzymen die betrokken zijn bij de expressie van sphingolipiden, flink gereduceerd in de langdurig aan

sigarettenrook blootgestelde longepitheel cellen. Dit betekent dat deze verlaging verantwoordelijk

zou kunnen zijn voor de rook-geïnduceerde verschuiving in lipiden expressie. Deze resultaten geven

duidelijk weer dat het lipiden profiel in epitheelcellen verandert onder invloed van sigarettenrook.

ConclusieSamengevat wijzen onze resultaten er op dat een mitochondriële disfunctie betrokken is bij

de toegenomen ontstekingsbevorderende activiteit, verminderd schade herstel en de GC

ongevoeligheid van het longepitheel bij COPD. De mitochondriale functie wordt beïnvloed door

sigarettenrook geïnduceerde oxidatieve stress en deze waargenomen afwijkingen in mitochondriale

functie in epitheelcellen van COPD patiënten bleken gepaard te gaan met verminderde activiteit

van GSK3β, wat verantwoordelijk kan zijn voor de GC ongevoeligheid. Ook reguleert GSK3β

belangrijke mitochondriale processen waar inactiviteit van GSK3β mogelijk verantwoordelijk is voor

de geobserveerde mitochondriale veranderingen in COPD patiënten. Verder laten we zien dat de

relatief nieuwe techniek, lipidomics, geschikt is voor diagnostische doeleinden in het sputum van

COPD patiënten. Verandering in lipiden profiel kan een verandering in de vetstofwisseling aanduiden

wat een proces is dat door mitochondriën gereguleerd word. Onze resultaten geven aan dat het

van belang is de rol van mitochondriale functie en het eiwit GSK3β bij het ontstaan COPD verder te

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onderzoeken, onder meer door de mitochondriale functie te remmen of de functie van GSK3β uit te

schakelen in proefdiermodellen van COPD. Daarnaast bieden de resultaten nieuwe openingen voor

behandelmogelijkheden gericht op mitochondriale bescherming en reactiveren van GSK3β, wat zou

kunnen leiden tot verhoogde GC gevoeligheid. Verder biedt de lipidomics techniek mogelijkheden

voor betere diagnose van COPD.

APPENDICES

Dankwoord

List of Publications

193

Dankwoord

DankwoordNa een hele tijd gaat het dan toch echt gebeuren, mijn promotietraject afronden. Geduren-de een dergelijke periode werkt men met veel verschillende mensen samen, te veel om op te noemen. Ik vrees dan ook dat ik er wellicht enkele vergeet. Deze mensen wil ik alvast hierbij bedanken.

Meerdere personen hebben een grote bijdrage geleverd aan het tot stand komen van deze thesis. Ik zou deze personen dan ook apart willen noemen.

Irene, ik vind zeker dat ik bij jou moet beginnen. Het was een lange weg, maar uiteindelijk zijn we er dan toch gekomen! Je bent ook nog mijn promotor geworden (in plaats van co). Ik wil je heel hartelijk bedanken voor alles wat je gedaan hebt en voor alle tijd die jij in mijn project hebt gestoken! Onze samenwerking verliep altijd prettig. Dit heeft dan ook geresult-eerd in enkele mooie publicaties en een paar leuke onderzoeksvoorstellen (geaccepteerd of niet). Ik hoop dat we in de toekomst samen kunnen blijven werken!

Antoon, ik wil jou ook graag bedanken. Jij hebt mij de kans gegeven om dit onderzoek te kunnen doen en daardoor de mogelijkheid gegeven om te kunnen promoveren. Wij lagen misschien niet altijd op één lijn, maar ook daar leert men uiteindelijk weer van. Bedankt voor alles en heel veel succes bij GSK.

Nick, ik wil jou, als co-promotor, ook heel erg bedanken. Ik vond het altijd leuk om met je samen te werken, maar ook om te ‘brainstormen’ over onderzoeksvragen. Dit ging altijd bijzonder gemakkelijk en leverde vaak genoeg nieuwe ideeën en energie op om er weer tegenaan te gaan. Ook jij hebt heel veel tijd en energie in het TIP-project gestoken (ook in mijn project), dat uiteindelijk dit alles heeft mogelijk gemaakt. Heel hartelijk bedankt hi-ervoor!

Han, jou wil ik bedanken voor al je hulp met mijn eerste paper van dit boekje. We waren allebei bijzonder gefascineerd door de mitochondriën en met behulp van confocaal en el-ektronen microscopie hebben we hele leuke resultaten behaald. Jouw enthousiasme heeft mij zeker geholpen in moeilijke perioden en uiteindelijk bij het tot stand brengen van een mooie paper. Bedankt hiervoor!

Also, I would like to thank you, Ian, personally, for a great collaboration and your contri-bution in finalizing my PhD. Many thanks! I really appreciate that you could come over to Groningen to be at my defence.

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Dirkje, ook jou zou ik graag willen bedanken. Als wij je nodig waren stond je ook altijd klaar om input te geven en dit heb ik zeer gewaardeerd. Heel erg bedankt hiervoor.

Klary, jouw kennis over mitochondriën heeft mij zeer geholpen en ook geïnspireerd om dit te onderzoeken in COPD.

I would also like to thank my reading committee: Prof. Dr. H.G.D. Leuvenink, Prof. Dr. L. Koen-derman and again Prof. Dr. I.M. Adcock.

Johan, Marnix, Harold en Simone. Jullie hebben allemaal een belangrijk aandeel gehad bij de totstandkoming van dit onderzoek. Hier ben ik jullie heel dankbaar voor. Harold, daar-naast wil ik je ook bedanken dat je mijn paranimf wilt zijn en tevens dank ik je voor de leuke samenwerking. Simone, jij hebt wel een heel groot aandeel gehad en daarom dank ik je extra. Ook met jou heb ik altijd goed kunnen samenwerken. Succes bij ERIBA en met jullie twee koters!

Graag wil ik ook prof. dr. Molema zeer bedanken voor haar motiverende support tijdens mijn promotie-traject. Je was zeker een belangrijke stimulans om door te blijven gaan.

Peter Bron, Ramon Langen en Evert-Jan Uringa, jullie zijn voor mij door de jaren heen belan-grijke rolmodellen geweest in de wetenschap. Jullie zijn alle drie erg kundig in het geduldig overbrengen van kennis en ik ben heel erg blij dat ik daar gebruik van heb kunnen maken. Bedankt hiervoor!

I would also like to thank all the co-authors of this thesis for all their input, effort and contri-bution they made. Eef and Ada, thank you for being great co-first authors :-).

Iedereen van het lab longziekten bedankt! Jan, met name een dankwoord voor jou, aangez-ien jij mij op de kamer tolereerde ;-). Ik heb het altijd zeer leuk gevonden en we konden elkaar goede aanvullingen geven voor onze projecten. Uiteindelijk hebben we hierin een mooi evenwicht in weten te brengen. Dit evenwicht ‘ontspoorde’ soms in een serie ̀ practical jokes` die uiteindelijk, voor zover ik mij kan herinneren, in een glansrijke overwinning voor mij eindigde natuurlijk……. ;-). Dennie, ook jij bedankt voor een leuke tijd bij longziekten en voor het feit dat je bij me op de kamer wilde zitten. Samen kunnen we goed over alles praten en leuke ideeën bedenken. Ook jij bedankt dat je mijn paranimf wil zijn. Marco, jou aanstekelijke enthousiasme hielp mij met mijn verdere onderzoek naar een mitochondriale disfunctie in COPD. Martijn, Dries, Hadi, Soheila, Henk, Sijranke, Neomie, Agnieszka, Benoit, Laura, Maaike (ook bedankt voor je hulp op het eind!), Daan, Rene, Jacobien, Janneke, Uilke, Judith, Theo, Lisette, Delaram, thank you all so much for being great (ex)colleagues! Ook wil ik nog graag het management van de medische biologie, vooral Hans, Annet en Susan,

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Dankwoord

bedanken voor alles. Susan en Ruth, jullie ook bedankt voor een mooie samenwerking bij TIP. Arjan, dank je voor al je inzet tijdens je studieperiode bij ons. Dit heeft toch een leuke paper opgeleverd.

Ook wil ik graag iedereen bedanken van het UMCG Microscopy and Image Center (UMIC) voor een prettige samenwerking. Ben Giepmans, Klaas Sjollema, dank voor jullie bijdrage in confocale microscopie en analyse. Bert, Freark en Ruby, van jullie heb ik veel van elektronen microscopie geleerd. Bedankt hiervoor!

Verder wil ik graag Top Institute Pharma bedanken waar ik aan het project T1-201 “COPD, transition of systemic inflammation into multi-organ pathology”, mocht werken. Daarnaast wil ik ook alle partners bedanken; University Medical Center Groningen, University of Gro-ningen (RUG), GRIAC Research institute Groningen, University Medical Center Utrecht, Uni-versity Medical Center Maastricht, Nycomed BV (now Takeda), GlaxoSmithKline, Danone, AstraZeneca. Tevens wil ik Stichting Astmabestrijding bedanken voor hun bijdrage aan dit onderzoek.

Ik wil ook mijn familie bedanken. Ook jullie hebben bijgedragen aan het tot stand komen van deze thesis. Een hoofdstuk nakijken of op de kinderen passen waardoor ik weer tijd kreeg om me op het boekje te kunnen focussen. Bedankt hiervoor!

En als laatste mijn eigen gezin. Fayenn, ik zou willen dat ik jouw tomeloze energie had, dan was dit boekje waarschijnlijk al lang af geweest :-). Finn, onze nieuwe aanwinst, jij vindt het allemaal wel best zo lachend vanuit je box …;-). En Ingrid ‘last but not least’. De laatste jaren zijn behoorlijk druk geweest, maar op een of andere manier weten we ons er altijd door-heen te slepen. Jij hebt dit ook mogelijk weten te maken en ik wil je daarom zeer bedanken hiervoor. Een PhD succesvol beëindigen doe je niet alleen op het lab, maar ook thuis. Ik ben blij dat ik dat (t)huis met jou deel, ik hou van jou!

Roland Hoffmann

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List of publications

List of Publications Roland F. Hoffmann, Jeroen T.J. Visser, Maikel P. Peppelenbosch. Protection of b-cell

transplants in Type I diabetes by induction of an immunosuppressive environment: The Symbiotic

Defence Cell (SDC) strategy. Bioscience Hypotheses. 2008

G. Jan Zijlstra, Nick H.T. ten Hacken, Roland F. Hoffmann, Antoon J.M. van Oosterhout, Irene H. Heijink. IL-17A induces glucocorticoid insensitivity in human bronchial epithelial cells. ERJ Express. 2011

Adèle T LoTam Loi, Susan JM Hoonhorst, Lorenza Franciosi, Rainer PH Bischoff, Roland F Hoffmann, Antoon JM van Oosterhout, Wim Timens, Dirkje S Postma, Jan-Willem Lammers, Leo Koenderman, Nick HT ten Hacken. Acute and chronic inflammatory responses induced by

smoking in individuals being susceptible and non-susceptible for development of COPD: from

specific disease phenotyping towards novel therapy: a cross-sectional study. BMJ Open. 2012

Hoffmann RF, McLernon S, Feeney A, Hill C, Sleator RD. A single point mutation in the listerial

betL σA-dependent promoter leads to improved osmo- and chill-tolerance and a morphological

shift at elevated osmolarity. Bioengineered. 2013

Irene Heijink, Antoon van Oosterhout, Nathalie Kliphuis, Marnix Jonker, Roland Hoffmann, Eef Telenga, Karin Klooster, Dirk-Jan Slebos, Nick ten Hacken, Dirkje Postma, Maarten van den Berge. Oxidant-induced corticosteroid unresponsiveness in human bronchial epithelial cells. Thorax. 2013

Roland F Hoffmann, Zarrintan S, Brandenburg S.M, Kol A, Jafari S, Dijk F, Kalicharan D, Kelders M, Gosker HR, van der Want J.J, Oosterhout A. J.M, Heijink I.H. Prolonged cigarette smoke

exposure alters mitochondrial structure and function in airway epithelial cells. Respiratory Research. 2013

Eef D. Telenga*, Roland F. Hoffmann*, Ruben t’Kind, Susan J.M. Hoonhorst, Brigitte W.M. Willemse, Antoon J.M. van Oosterhout, Irene Heijink, Maarten van den Berge, Lucie Jorge, Pat Sandra, Dirkje S. Postma, Koen Sandra, Nick H.T. ten Hacken. Untargeted Lipidomic

Analysis in COPD: Uncovering Sphingolipids. American Journal of Respiratory and Critical Care Medicine 2014

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Appendices

M.W. van Gisbergen, A.M. Voets, M.H.W. Starmans, I.F.M. de Coo, R. Yadak , R.F. Hoffmann, P.C. Boutros, H.J.M. Smeets, L. Dubois, P. Lambin. How do changes in the mtDNA and mitochondrial

dysfunction influence cancer and cancer therapy? Challenges, opportunities and models.

Mutation Research – Reviews 2015

Anta Ngkelo*, Roland F. Hoffmann*, Andrew L. Durham, John A. Marwick, Simone M. Brandenburg, Harold G de Bruin, Marnix R. Jonker, Christos Rossios, Eleni Tsitsiou, Gaetano Caramori, Marco Contoli, Paolo Casolari, Francesco Monaco, Filippo Andò, Giuseppe Speciale, lain kilty, Kian Fan Chung, Alberto Papi, Mark A. Lindsay, Nick H.T. ten Hacken, Maarten van den Berge, Wim Timens, Peter J. Barnes, Antoon J. M. van Oosterhout, Ian M. Adcock*, Paul A. Kirkham*, Irene H Heijink*. Glycogen synthase kinase-3β reduces glucocorticoid responsiveness

in COPD by cell-dependent mechanisms. American Journal Physiology-Lung 2015

Roland F. Hoffmann, Yuri M. Moshkin, Stijn Mouton, Nicola A. Grzeschik, Ruby D. Kalicharan, Jeroen Kuipers, Anouk H.G. Wolters, Kazuki Nishida, Aleksander V. Romashchenko, Jan Postberg, Hans Lipps, Eugene Berezikov, Ody C.M. Sibon, Ben N.G. Giepmans and Peter M. Lansdorp. Guanine quadruplex structures localize to heterochromatin. Nucleic Acid Research 2015

Marike W van Gisbergen, An M Voets, Rianne Biemans, Guido RMM Haenen, Marie-José Drittij-Reijnders, Roland F Hoffmann, Irene H Heijink, Hubertus JM Smeets, Kasper MA Rouschop, Ludwig Dubois, Philippe Lambin. Different radiation responses in mtDNA depleted

cells correlate with ROS and GSH levels. Submitted