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Properdin Binds Pseudomonas aeruginosa and is Required for Neutrophil Extracellular Trap Mediated
Activation of Complement Alternative Pathway
by
Joshua Yuen
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Laboratory Medicine & Pathobiology University of Toronto
© Copyright by Joshua Yuen 2013
ii
Properdin Binds Pseudomonas aeruginosa and is Required for
Neutrophil Extracellular Trap Mediated Activation of Complement
Alternative Pathway
Joshua Yuen
Master of Science
Department of Laboratory Medicine & Pathobiology
University of Toronto
2013
Abstract
Neutrophils play an important, yet poorly understood role, in complement mediated pathologies.
Here we identified that neutrophils contain key components from the complement alternative
pathway: properdin (CFP), complement component 3 (C3), complement factor B (CFB), and
complement factor H (CFH). Activation of neutrophils resulted in secretion of these complement
components. When neutrophils are further activated to form neutrophil extracellular traps
(NETs), CFP is deposited onto the surfaces of the NETs. In addition, CFP is able to bind to
Pseudomonas aeruginosa, an opportunistic bacterium which can activate neutrophils to form
NETs. Furthermore, NETs activate complement and increase formation of the terminal
complement complex. The activation of complement on NETs can be initiated through multiple
pathways, however, activation of the alternative pathway is dependent on CFP. This mechanism,
potentially required for effective host defense, may also contribute to complement activation and
disease.
iii
Acknowledgments
I would like to thank my supervisors, Dr. Christoph Licht and Dr. Nades Palaniyar for their
continued support and guidance. Their passion for research was inspiring and helped me become
a better researcher and critical thinker. More importantly, they were determined to guide me
along the way as I developed personal skills that would help me not only succeed academically,
but also succeed in life. For that I will forever be thankful.
To my Master’s advisory committee, Dr. Walter Kahr and Dr. Margaret Rand, you have been
very supportive and encouraging. Thank you.
I would also like to thank my colleagues in both labs for their continuous help and support. It has
been a true privilege to work with such an intelligent group of scientists. They really helped
motivate me to push forward when times were tough. I would especially like to thank David
Douda for all of his help, from optimizing results and experimental procedures to helpful
discussions and technical help. This work has been a collection of our blood, sweat and tears.
I would also like to thank Damien Noon, M.D., Hailu Huang, M.D., Hong Wang, M.D., and Jalil
Nasiri for their technical assistance in collecting peripheral blood.
A special thanks goes out to all the donors who contributed to this research. None of this work
would have been possible without their help.
I would like to thank Dr. Marina Ulanova from the Northern Ontario School of Medicine for
providing the GFP expressing Pseudomonas aeruginosa.
Finally, I would like to thank my parents for their unconditional love and support.
This work was supported by a University of Toronto Fellowship and an Ontario Graduate
Scholarship.
iv
Table of Contents
Acknowledgments .......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ ix
List of Appendices ......................................................................................................................... xi
List of Abbreviations .................................................................................................................... xii
1 Introduction ............................................................................................................................ 1
1.1 The Complement System ....................................................................................................... 1
1.1.1 Classical Pathway ........................................................................................................ 1
1.1.2 Lectin Pathway ............................................................................................................ 2
1.1.3 Alternative Pathway .................................................................................................... 2
1.1.4 Amplification and Terminal Pathway ......................................................................... 4
1.1.5 Other Activation Pathways .......................................................................................... 6
1.2 Complement in Disease ......................................................................................................... 6
1.2.1 Endogenous Activation ............................................................................................... 7
1.2.2 Complement Dysregulation ......................................................................................... 8
1.3 Neutrophils ............................................................................................................................. 9
1.3.1 Neutrophil Activation and Resolution ......................................................................... 9
1.3.2 Neutrophil Extracellular Traps .................................................................................. 12
1.3.3 Neutrophil Extracellular Traps in Disease ................................................................ 15
1.4 Complement and NETs in Disease ...................................................................................... 16
1.5 Complement and Neutrophils .............................................................................................. 22
1.6 Neutrophil Complement Cross-talk .................................................................................... 23
1.7 Rationale .............................................................................................................................. 25
v
1.8 Hypothesis ............................................................................................................................ 27
1.9 Aims ..................................................................................................................................... 27
2 Methods ................................................................................................................................ 28
2.1 Reagents ............................................................................................................................... 28
2.2 Research Ethics Board Approval ......................................................................................... 28
2.3 Neutrophil Isolation ............................................................................................................. 28
2.4 Preparation of Neutrophil Lysates ....................................................................................... 28
2.5 Bacterial Cultures and Strains .............................................................................................. 29
2.5.1 Bacterial Plating ........................................................................................................ 29
2.5.2 Bacterial Growth ....................................................................................................... 29
2.6 Neutrophil Activation .......................................................................................................... 30
2.6.1 Neutrophil Oxidative Burst ....................................................................................... 30
2.6.2 Neutrophil Activation and Secretion ......................................................................... 30
2.6.3 Differential Cell Counts for Nuclear Morphology .................................................... 30
2.6.4 SYTOX Green Plate Reader Assay ........................................................................... 31
2.7 Western Blot ........................................................................................................................ 31
2.7.1 Determining Protein Concentration ........................................................................... 32
2.8 Imaging and Microscopy ..................................................................................................... 32
2.8.1 Acid Washed Cover Slips .......................................................................................... 32
2.8.2 Preparation of Cover Slips for Microscopy ............................................................... 33
2.8.3 Seeding Neutrophils on Coated Cover Slips ............................................................. 33
2.9 Identifying Complement Proteins in Neutrophils ................................................................ 33
2.10 Detection of Complement Proteins in NETs ....................................................................... 34
2.11 Complement Competent Plasma .......................................................................................... 34
2.12 Preparation of Rabbit Erythrocytes ...................................................................................... 35
2.13 Plasma Complement Activity Assay ................................................................................... 35
vi
2.14 Complement Activation and C5b-9 Formation on NETs .................................................... 35
2.15 Inhibiting Properdin Activity in NETs ................................................................................ 36
2.16 Spinning-Disc Confocal Microscopy ................................................................................... 37
2.17 Colocalization Analysis ....................................................................................................... 37
2.18 Statistical Analysis ............................................................................................................... 37
3 Results – Aim 1 .................................................................................................................... 38
3.1 Neutrophils Contain Complement Factors ........................................................................... 38
3.2 Investigating Neutrophil Activation .................................................................................... 41
3.2.1 Neutrophil Activation and Oxidative Burst ............................................................... 41
3.2.2 Complement Protein Localization Upon Activation ................................................. 43
3.2.3 Complement Protein Secretion in Response to Activation ....................................... 45
3.3 Quantification of Nuclear Morphologies During NETosis .................................................. 48
3.4 PMA, but not C5a and fMLP, Induce NET Formation ........................................................ 50
3.5 Neutrophil Extracellular Traps Contain Properdin .............................................................. 52
3.6 NETs Increase Formation of Terminal Complement Complex ........................................... 57
3.7 NETs Activate Complement Alternative Pathway to Form C5b-9 ..................................... 59
3.8 NETs Activate Complement and Activation of Complement AP on NETs is Dependent
on Properdin ......................................................................................................................... 61
4 Results – Aim 2 .................................................................................................................... 64
4.1 Identifying Bacteria That Induce NETosis .......................................................................... 64
4.2 Neutrophil Properdin Binds P. aeruginosa Independent of NETs ...................................... 68
4.3 NETs Increase Formation of C5b-9 in Response to Bacteria .............................................. 70
4.4 NETs Activate Complement Alternative Pathway in Response to Bacteria ....................... 72
4.5 NETs Activate Complement in Response to P. aeruginosa and Complement AP
Activation is Dependent on Properdin ................................................................................. 74
5 Discussion ............................................................................................................................ 76
5.1 Characterizing Interactions of Complement and Neutrophils ............................................. 76
vii
5.2 Properdin is Found on Neutrophil Extracellular Traps ........................................................ 78
5.3 Neutrophil Extracellular Traps Activate Complement ........................................................ 79
5.4 Neutrophils form NETs in Response to Bacteria ................................................................. 80
5.5 NETs Formed in Response to Bacteria Activate Complement ............................................ 80
6 Conclusion and Future Directions ....................................................................................... 84
6.1 Conclusions .......................................................................................................................... 84
6.2 Future Directions ................................................................................................................. 84
7 References ............................................................................................................................ 87
8 Appendix ............................................................................................................................ 103
viii
List of Tables
Table 1. Activators of NETs ......................................................................................................... 14
ix
List of Figures
Figure 1. Complement: Key mediator of innate immunity. ............................................................ 5
Figure 2. Neutrophil activation and regulation. ............................................................................ 11
Figure 3. Complement and NETs lead to endothelial cell damage. .............................................. 17
Figure 4. Complement interaction with NETs. ............................................................................. 26
Figure 5. Neutrophils contain proteins from complement alternative pathway. ........................... 40
Figure 6. Neutrophil stimulation with fMLP and PMA, but not C5a, leads to oxidative burst. ... 42
Figure 7. Complement proteins associate with sialic acid upon activation. ................................. 44
Figure 8. Activated neutrophils secrete complement proteins. ..................................................... 47
Figure 9. Activated neutrophils form neutrophil extracellular traps. ............................................ 49
Figure 10. PMA, but not C5a and fMLP, induces NET formation. .............................................. 51
Figure 11. Neutrophil extracellular traps contain properdin. ........................................................ 53
Figure 12. C3 does not interact with neutrophil extracellular traps. ............................................. 54
Figure 13. CFB does not interact with neutrophil extracellular traps. .......................................... 55
Figure 14. CFH does not interact with neutrophil extracellular traps. .......................................... 56
Figure 15. Neutrophil extracellular traps increase formation of C5b-9. ....................................... 58
Figure 16. NETs activate complement alternative pathway to form C5b-9 ................................. 60
Figure 17. Properdin is required for activation of complement AP on NETs. ............................. 63
Figure 18. Pseudomonas aeruginosa activate neutrophils to form NETs .................................... 66
Figure 19. Neutrophils activated with S. aureus, E. coli, and B. subtilis. .................................... 67
x
Figure 20. Properdin binds P. aeruginosa independent of NETs. ................................................ 69
Figure 21. P. aeruginosa activation of neutrophils increases C5b-9 formation on NETs. ........... 71
Figure 22. NETs activate complement AP to form C5b-9 in presence of P. aeruginosa. ............ 73
Figure 23. NETs activation of complement AP in presence of P. aeruginosa is dependent on
Properdin. ...................................................................................................................................... 75
Figure 24. Proposed model of complement-neutrophil interactions. ............................................ 83
xi
List of Appendices
Appendix A. Optimizing the Protocol for Neutrophil Lysates ................................................... 103
Appendix B. Hemolysis Assay Data ........................................................................................... 106
xii
List of Abbreviations
aHUS atypical Hemolytic Uremic Syndrome
ALT alanine aminotransferase
ACPA anti-citrullinated peptide antibody
ANCA anti- neutrophil cytoplasmic antibody
AP alternative pathway
BCA bicinchoninic acid
C1qR C1q receptor
C3aR C3a receptor
C5aR C5a receptor
CD59 membrane attack complex inhibitor
CFB complement factor B
CFD complement factor D
CFH complement factor H
CFHR1 complement factor H related protein 1
CFI complement factor I
CFP properdin or complement factor P
CGD chronic granulomatous disease
CP classical pathway
CR1 complement receptor 1 (also CD35)
CR3 complement receptor 3 (also Mac-1 or CD11b/CD18)
CR4 complement receptor 4 (CD11c/CD18)
xiii
DAF decay accelerating factor (also CD55)
DAMP damage associated molecular pattern
DPI diphenyleneiodonium
FFP fresh frozen plasma
fMLP n-formyl-methyl-leucyl-phenylalanine
GAGs glycosaminoglycans
G-CSF granulocyte colony stimulating factor
GFP green fluorescent protein
GM-CSF granulocyte-macrophage colony stimulating factor
HUS Hemolytic Uremic Syndrome
HUVEC human umbilical vein endothelial cell
ICAM-1 Intercellular adhesion molecule 1
IL-1β interleukin-1 beta
IL-8 interleukin 8
IL-17 interleukin 17
IL-23 interleukin 23
LPS lipopolysaccharide
LP lectin pathway
MASP MBL associated serine protease
MBL mannose binding lectins
MCP membrane cofactor protein
MOI multiplicity of infection
xiv
MPO myeloperoxidase
NE neutrophil elastase
NETs neutrophil extracellular traps
NOX NADPH oxidase
PAD4 peptidylarginine deiminase 4
PAMP pathogen associated molecular pattern
PKC protein kinase-C
PMA phorbol 12-myristate 13-acetate
PR3 proteinase 3
PSGL-1 p-selectin glycoprotein ligand-1
ROS reactive oxygen species
SLE systemic lupus erythematosus
STX shiga toxin
SVV small vessel vasculitis
TCC terminal complement complex (C5b-9)
TNF-α tumor necrosis factor-α
TLR4 toll-like receptor 4
TSR thrombospondin repeats
1
1 Introduction
1.1 The Complement System
The complement system represents one of the most ancient cornerstones of innate immunity1. It
consists of over 40 soluble and membrane bound proteins2,3
. This highly integrated network,
consisting of many signaling molecules and regulators, acts as an intricate immune surveillance
system to discriminate between healthy host tissue, unwanted cellular debris, apoptotic cells,
necrotic cells and microbial invaders4.
Traditionally, activation of the complement system is achieved through three distinct
initiation pathways: classical (CP), lectin (LP), and alternative (AP). These cascade-like
pathways all culminate to the formation of the convertases, the major enzyme of complement
activation5,6
, which converge to a common effector C3, generating opsonin C3b and
anaphylatoxin C3a. Further activation leads to the formation of C5 convertases which direct the
assembly of the terminal complement complex (C5b-9), a lytic pore which assembles on
membranes to mediate cell destruction5,6
. More recently, a new proteolytic pathway in which
thrombin acts as a C5 convertase, cleaving C5 to the anaphylatoxin C5a, has been described7.
1.1.1 Classical Pathway
The classical pathway is initiated by the calcium dependent C1 complex through its pattern
recognition molecule C1q. Classical pathway is strongly initiated through binding of C1q to IgG
or IgM clusters8. As such, it is often referred to as the antibody-dependent pathway. However,
C1q is also capable of recognizing distinct structures directly on microbial cells9. In addition to
endogenous pattern recognition molecules (immunoglobulins, C-reactive protein), C1q can also
bind to prions, DNA, late apoptotic, and necrotic cells10
. C1q binding leads to conformational
changes in the C1 complex allowing interaction between the C1r and C1s proteases11
. C1r
undergoes autoproteolytic activation cleaving the zymogen of C1s. C1s is a highly specific
protease which cleaves C4 into C4a and C4b, and C2 into C2a and C2b to form the classical
pathway C3 convertase (C4b2b). C4b also opsonizes targets further directing C3 convertase
assmembly on membrane surfaces. This convertase can cleave C3 to initiate amplification and
downstream functions of complement amplification.
2
1.1.2 Lectin Pathway
The lectin pathway is initiated through recognition of carbohydrates on microbial surfaces by
Mannose-binding lectin (MBL) or ficolin. MBL belong to the collectin family of proteins which
contain a carbohydrate recognition domain and a collagen-like domain12
. The carbohydrate
recognition domain of MBL binds to carbohydrates with 3- and 4- hydroxyl groups in the
pyranose ring in a calcium dependent manner13
. This allows for effective recognition of
carbohydrate surfaces (e.g. N-acetyl-glucosamine [GlcNAc]) on pathogens and leaves
mammalian glycoproteins (e.g. galactose and sialic acid), which do not fit this steric
requirement, virtually undetected 14
. Ficolins, like MBL, contain a collagen-like stem structure,
however they also contain a fibrinogen-like domain. Serum ficolin are lectins that have similar
binding specificity as MBL.
MBL-associated serine proteases (MASP) are a serine-protease superfamily (MASP1,
MASP2, sMAP/MAp19, MASP3). Structurally, they are similar to the proteolytic components
(C1r/C1s) of the classical pathway15,16
. Binding of MBL or ficolins to carbohydrates activates
lectin-pathway MASP through mechanisms that are currently unknown17
. MASP2, like C1s,
cleaves C4 and C2 to form the C3 convertase (C3b2b), which is common to the CP C3
convertase16
. In contrast, MASP1 can cleave C2 but is unable to cleave C4, suggesting that it can
enhance the activation of lectin pathway triggered by MBL-MASP2 complexes but not initiate
lectin pathway itself18
.
1.1.3 Alternative Pathway
In contrast to the inducible lectin and classical, the alternative pathway (AP) is constitutively
active and interacts with cell surfaces for constant immune surveillance. Initiation of AP begins
in the fluid phase where C3 is hydrolyzed to C3H2O, exposing new binding sites to allow for rapid
probing of cells in a process commonly referred to as the tick-over pathway19
. Complement
factor B (CFB) protease binds to C3H2O and is cleaved by complement factor D (CFD), in the
presence of Mg2+
, releasing the complement factor B amino terminal fragment (Ba) and
activating the serine protease domain (Bb), to form fluid phase C3 convertase (C3H2OBb)20
.
Fluid phase C3H2OBb cleaves C3 into C3a and C3b. C3b binds to amine and carbohydrate
groups on cell surfaces and engages CFB. Once again, CFB is cleaved by CFD, in the presence
of Mg2+
, to form the AP C3 convertase (C3bBb). The half-life of the C3bBb complex is
3
shortlived (T1/2 ~ 90 sec21,22
) but is stabilized 5 to 10 fold by association with properdin (CFP) to
form a stable C3 convertase (C3bBbP)23
.
On foreign particles, unrestricted activation of AP can lead to rapid amplification of the
complement cascades. This process, however, is immediately regulated on host cells by
regulatory factors. These regulatory factors include complement factor H (CFH), complement
factor I (CFI), and membrane bound: decay accelerating factor (DAF), membrane cofactor
protein (MCP), complement receptor 1 (CR1/CD35) and membrane attack complex inhibitor
(CD59). CFH is the major regulator of the AP24
. CFH regulates AP by either, competitively
binding to C3b preventing the formation of the C3bBb complex, or serving as a cofactor for CFI
mediated cleavage of C3b25,26
. Thus transient formation and regulation of complement AP C3
convertase is in dynamic equilibrium.
In addition to stabilizing the complement AP C3 convertase, CFP also serves as a pattern
recognition molecule to initiate activation of complement AP. Recent studies highlight the
emerging roles for CFP to act as a pattern recognition molecule capable of recognizing pathogen
or damage associated molecular patterns (DAMP/PAMP)27
. Using surface plasmon resonance
methodology, Spitzer et al, demonstrated that CFP binding provides a platform for convertase
assembly28
. CFP has been shown to bind to glycosaminoglycan, Neisseria gonorrhoeae, (LPS)-
defective Escherichia coli, LPS, early/late apoptotic T cells, and necrotic cells via DNA
surfaces28-32
.
Properdin is a 53 kDa protein composed of 7 thrombospondin repeats (TSR)33,34. The TSR
repeats are numbered from 0-6 (TSR 0-6) with TSR 0 starting at the N-terminus35. It is a rod-like
protein, approximately 26 nm in length and 2.6 nm in diameter36. In circulation, CFP is a multimeric
protein that forms dimers, trimers and tetramers in a head to tail fashion33. CFP functions are
dependent on its polymeric nature as the activity of CFP increases with size. The CFP tetramer is
approximately 10 fold more active when compared to the dimer34. The detailed three-dimensional
structure of CFP has not yet been resolved, however a protein surface on TSR 2 and 3 of CFP
containing alternating arginine and tryptophan side chains has been proposed as the site for pattern
recognition37. The distance between the arginines (~9 Å) closely matches the length of a
glycosaminoglycan disaccharide unit. This suggests that interactions between CFP and
glycosaminoglycan are determined through electrostatic interactions33
. The CFP residues that are
proposed to mediate binding to sulfate moieties of glycosaminoglycan may also mediate
4
interactions with other large polyanionic molecules. Because properdin can interact with a
variety of polyanionic macromolecules, any properdin-opsonized target can potentially amplify
complement23
.
1.1.4 Amplification and Terminal Pathway
All surface bound C3 convertase, regardless of origin, induces amplification of AP by cleaving
C3 to deposit C3b in the vicinity of complement activation. In the presence of CFB and CFD,
this allows for an efficient cycle of C3 cleavage and AP convertase assembly. Despite its name,
the alternative pathway contributes to 80-90% of the final C5a generation, regardless of the
initial triggering cascade38
. Amplification of complement by AP causes a rapid increase in C3
cleavage and C3b deposition onto surfaces. Deposition of C3b onto C3 convertases drives the
formation of the C5 convertases (C4b2b3b/C3bBb3b) shifting substrate specificity from C3 to
C539
. C5 is cleaved into C5a, a powerful anaphylatoxin, and C5b, which directs a non enzymatic
assembly of the terminal pathway components C6, C7, C8, and C9 (C5b-9) to form the cell lytic
terminal complement complex (TCC)40
. Thus, activation of complement through its various
initiating pathways can trigger a cascade which leading to the formation of C5b-9 (Figure 1).
5
Figure 1. Complement: Key mediator of innate immunity.
Complement activation can be initiated through three major pathways. The alternative pathway
is constitutively active, while the classical and lectin pathways are inducible. In addition to these
three pathways, serine proteases can directly cleave complement proteins to initiate complement
activity. Complement activation occurs in a sequential manner. All three pathways converge to
form an enzyme known as the C3 convertase. (C3bBb for the alternative pathway and C4bC2b
for the classical and lectin pathway). The C3 convertases cleave C3 to produce anaphylatoxin
C3a and opsonin C3b. Continued activation and deposition of C3b onto cell surfaces leads to the
formation of complement C5 Convertase (C3bBbC3b for the alternative pathway and
C4bC2bC3b for the classical and lectin pathways). C5 convertases cleave C5 into anaphylatoxin
C5a, and generates C5b which initiates the assembly of the terminal complement complex (C5b-
9). Reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Immunology 9,
729-740, 2009.
6
1.1.5 Other Activation Pathways
In addition to the three traditional pathways of complement activation, direct cleavage of C3 or
C5 can also occur through a number of extrinsic proteases. In an in vitro assay, Vogt
demonstrated that leukocyte elastase can cleave C5 to form biologically active C5b-6 like
complex to initiate formation of TCC41
. Using a C3-/-
mice, Huber-Lang et al, demonstrated that
thrombin can cleave C5 to produce biologically active C5a/C5b in the absence of C5
convertase7. Other proteases that have been known to cleave complement C3/C5 include plasmin
and plasma kallikrein2. These extrinsic pathways for complement activation suggest that there is
cross talk between complement, innate immune cells and coagulation.
1.2 Complement in Disease
The primary task of the complement system is to protect the host against invading microbes.
Sensing various danger signals, both host derived and foreign, complement is able to execute its
function in a tightly regulated manner, thus mediating the inflammatory response3. For some
types of danger signals, the complement system serves as an opsonin, tagging molecules for
clearance, where as for others it can mediate a strong inflammatory response42
.
The inflammatory response is a pathophysiological process that engages hundreds of
mediators, cell types, and tissues. Inflammation is often thought of as a response to infection,
however, it can be initiated by any cell stimulus causing injury, including chemical or physical
injury43,44
. The inflammatory process eliminates the initiating factor with minimal destruction to
host tissues. Central to inflammation, complement is intricately involved in regulating this
process4. The mechanisms for complement activation, thus regulation of inflammation, are well
understood in the vasculature. However, soluble components can also infiltrate tissues and
organs to engage exogenous (e.g. infection) and endogenous (e.g. autoimmunity) stimuli that
could cause cell injury45
. Complement activation, under the tight control of inhibitors, can illicit
an appropriate immune response. However, any trigger that tips the delicate balance between
activation and regulation can potentially lead to pathology4,46,47
. There is a paradoxical role for
complement participation in inflammatory disorders. An overly aggressive complement system
results in inflammation-mediated tissue damage, however a deficiency or lack of complement
response leaves the host immune compromised4.
7
The innate immune requirement for complement has been studied for decades but the
appreciation for its link to disease has only emerged in recent years48
. While we recognize that
complement deficiency leads to immune compromised individuals, the focus of our study is on
complement activation and inflammatory damage. Complement activation, through different
intitation pathways, produces pro-inflammatory peptides that mediate disease processes49
.
Deleterious complement activation precipitating into pathology is mediated through two major
mechanisms: Endogenous activation of complement by foreign trigger overwhelms regulatory
machinery to precipitate disease, or insufficient regulation of complement activation via
mutation, decreased expression of regulatory proteins or auto-antibodies leads to increased
complement activation50-52
.
1.2.1 Endogenous Activation
Hemolytic uremic syndrome (HUS) is a kidney disease characterized by microangiopathic
hemolytic anemia, thrombocytopenia and renal impairment. This disease is most commonly
triggered by shiga-like toxin (Stx)-producing E. coli and manifests with diarrhea, often bloody53
.
In North American and Western Europe, 70% of HUS is secondary to infection with E. coli
serotype O157:H753-57
. Retrograde transport of Stx by endothelial cells enzymatically blocks
protein synthesis and inhibits expression of anti apoptotic proteins58,59
. This was traditionally
viewed as the mechanism for mediating cell damage and pathology. However, recent evidence
indicates complement activation also contributes to disease. Stx was demonstrated to activate
complement and bind to the major soluble regulator of complement AP, CFH60
. These data were
further corroborated in a clinical setting as patients with HUS were described to have elevated
levels of complement activation products Bb and SC5b-961
. Furthermore, the use of C5-blocker,
eculizumab, was able to successfully treat a three year old patient suffering from severe Stx
associated HUS62
.
In addition, the circulating levels of CFP in plasma are relatively low (4-6 μg/ml)63
. The
release of CFP from infiltrating neutrophils is a major determinant for complement AP
activation64
. In HUS, neutrophils have been shown to mediate endothelial cell death, and patients
with high peripheral counts of neutrophils have poor prognosis65
. An influx in neutrophils will
lead to localized increase in complement AP activation. Whether the activation of complement
AP by infiltrating neutrophils contributes to pathology has yet to be determined.
8
1.2.2 Complement Dysregulation
Atypical hemolytic uremic syndrom (aHUS) is a rare kidney disease characterized by
microangiopathic hemolytic anemia, thrombocytopenia and renal impairment. This disease
manifests without bloody diarrhea. Genetic analysis has linked aHUS with mutations in genes
encoding for the proteins of complement AP. Gain of function mutations to C366-68
and CFB69,70
and loss of function mutations to CFH71-73
, MCP9,10
, and CFI9,10
have all been described to
contribute to aHUS pathology. Moreover, the presence of anti CFH antibodies may also
precipitate aHUS pathology74,75
. During the onset of disease progression, patients have reduced
levels of circulating C3 and CFB, as well as increased levels of C3 activation fragments.
However, C4 is unaffected, suggesting that this pathology is dependent exclusively on AP76
.
Despite progress in identifying causative factors for aHUS pathogenesis, much of the
disease is enclosed in a shroud of mystery. While genetic mutation has been linked with
pathology, incidence among carriers with predisposing mutations counts for less than 50%76
. In
addition, while aHUS typically presents in childhood, the age of onset varies. Furthermore, the
variable expression of aHUS associated mutations makes it impossible to define a single factor
which may precipitate pathology. Taken together, these findings demonstrate that many other
factors may contribute to complement driven pathologies.
In 2011, a series of reports highlighted the potential or prominence for an infectious
trigger leading to aHUS77-80
. Currently there have been no studies to identify the link between
infection and aHUS pathogenesis. The clinical onset in a majority of familial aHUS cases occurs
subsequent to infection (bacterial or viral)81,82
. Therefore, neutrophils may be considered relevant
as they are the primary sentinel for immune surveillance in the blood stream. Neutrophils have
been shown to be able to directly activate complement. Infiltrating neutrophils can increase local
concentrations of complement CFP, thus activating complement AP83
. Furthermore, proteolytic
enzymes released from activated neutrophils (e.g. elastase) can cleave and activate C3 and C541
.
Neither of these concepts have been investigated within the context of NETs. The propensity for
neutrophil or possibly NETs (Section 1.3.2) to activate complement suggests that they may
trigger clinical manifestations of aHUS, however this has yet to be determined. Thus, we set
forth to investigate the interactions between neutrophils and complement.
9
1.3 Neutrophils
Neutrophil granulocytes, also known as polymorphonuclear leukocytes, are the most abundant
leukocyte in the blood stream. They are highly motile and migrate to sites of infection or
inflammation through chemotaxis. As such, they are commonly referred to as the first line of
defense against infection84
.
They originate in the bone marrow, where under the influence of growth factors and
cytokines, pluripotent hematopoetic cells differentiate to form myeloblasts in a process called
myelopoesis. The cytokine granulocyte colony stimulating factor (G-CSF) is essential for
regulating neutrophil production in response to infection as G-CSF null mice develop chronic
neutropenia85
. The myeloblast progenitors, now committed to become granulocytes, synthesize
proteins and package them into different granules. The formation of granules is a continuous
process in which vesicles bud from the golgi apparatus and fuse forming granular structures.
During the course of maturation from the myeloblast, neutrophils alter their transcription profile
to synthesize various proteins86
. As a result, neutrophils form a continuum of granules with
overlapping contents87
. This is in stark contrast to the traditional method for the classification of
granules into azurophillic (primary), specific (secondary), and gelatinase (tertiary), based on their
protein constituents. While this division is practical, it is now appreciated as largely artificial88-90
.
Unlike the first three classes of granules which are formed from the golgi apparatus,
secretory vesicles are formed during the final stage of neutrophil maturation through endocytosis
in the bone marrow91
. Secretory vesicles are reservoirs for a variety of membrane receptors and
adhesion molecules. They play an important role in mediating the early response for neutrophil
activation. As they are endocytotic in origin, mobilization of secretory vesicles does not result in
the release of proteolytic enzymes, only plasma proteins that form the matrix of secretory
vesicles92
.
1.3.1 Neutrophil Activation and Resolution
Neutrophils are key effectors of innate immune and inflammatory responses93
. As the primary
sentinel for immune surveillance, neutrophils traverse the blood stream continuously and
randomly probing the endothelium of the vessel walls94
. At sites of tissue injury or infection,
stimulants such as bacterial derived lipopolysaccharides (LPS) and N-Formyl-Methyl-Leucyl-
10
Phenylalanine (fMLP), as well as host derived cytokines (e.g. TNF-α, IL-1β, IL-17) activate
endothelial cells to increase the expression of the adhesion molecules P-selectin, E-selectin, and
several members from the integrin superfamily, the ICAMs95
. This process coordinates the
adhesion of neutrophils, which constitutively express P-selectin glycoprotein ligand-1 (PSGL-1)
and L-selectin, to activated endothelium which express E-selectin96
. The selectin mediated
adhesion enables neutrophils to undergo their characteristic rolling, tethering, and
transendothelial cell migration directed by selectins, cytokines and chemokines97
. As neutrophils
travel through the interstitium, they are guided by chemotactic gradients from both host derived
chemokines (e.g. IL-8) and pathogen associated molecular patterns (PAMPs) to sites of infection
and inflammation98
. All the while, these chemoattractants bind to their respective neutrophil
receptors (often G-protein coupled receptors) and initiate the downstream signaling cascade
dominated by the MAPK/ERK pathway in preparation for neutrophil activation99
. Upon arrival
to sites of high chemoattractant concentration, neutrophils coordinate a strict regiment to deal
with potential threats through programmed phagocytosis, degranulation (releasing stored
antimicrobial contents), and NETosis (process of forming Neutrophil Extracellular Traps [NETs
section 1.3.2])100
.
Neutrophil activation is effective when dealing with foreign invaders, however,
deployment of lethal cargo of neutrophils results in collateral damage to host tissue101,102
. Thus,
neutrophils must be swiftly removed from sites of activation before they have detrimental effects
on host tissue. Resolution of inflammation removes apoptotic neutrophils from sites of
inflammation and prevents infiltration of additional cells. This is achieved through two major
mechanisms: phagocytosis and lipid mediators. The phagocytosis of apoptotic neutrophils by
macrophages results in down regulation of IL-23, a cytokine that controls IL-17 production by T-
cells103
. The reduction of IL-17 results in decreased production of G-CSF thus reducing
neutrophil production (Figure 2). In the later stages of the inflammatory response, neutrophils
interact with cells at sites of inflammation to produce anti-inflammatory lipid-mediators (e.g.
lipoxins, resolvins, protectins)104
. These lipid-mediators enhance uptake of apoptotic cells by
macrophages and down regulate neutrophil activation105,106
. The mechanisms for clearance of
NETs are currently unknown, however, DNase has been suggested to play an important role in
NET clearance. DNase impairment has been implicated in NET mediated pathology (see section
on Lupus Nephritis in Section 1.4).
11
Figure 2. Neutrophil activation and regulation.
Neutrophils circulate the blood stream continuously and randomly probing endothelium.
Activation of endothelium results in increased expression of adhesion molecules (P-selectin and
E-Selectin) which capture neutrophils which express PSGL-1 and L-selectin to mediate rolling,
expression of neutrophil integrins and trans-endothelial cell migration. Neutrophils migrate to
sites of infection or inflammation through chemotaxis. Upon arrival to sites of high
chemoattractent concentration, neutrophils respond through generation of cytokines,
phagocytosis, degranulation or NETosis. Resolution of the inflammatory response is achieved
through phagocytosis of apoptotic neutrophils by macrophages. Reprinted from Immunity, 33,
Borregaard, N., Neutrophils, from Marrow to Microbes, 657-670, 2010, with permission from
Elsevier
12
1.3.2 Neutrophil Extracellular Traps
In recent years, a unique form for neutrophil cell death, called NETosis, has been identified
whereby neutrophils actively release decondensed chromatin into extracellular space100,107
. These
structures, termed neutrophil extracellular traps (NETs), contain DNA, histones, granular and
cytoplasmic proteins100
. NETs can ensnare foreign invaders and confer antimicrobial activity by
exposing them to high local concentrations of antimicrobials108
. Mass spectrometry has identified
24 neutrophil proteins associated with NETs109
. This finding reveals that NET proteins are
primarily cationic as they interact with the polyanionic backbone of DNA. Bacteriacidal proteins
include: histones, defensins, elastase and myeloperoxidase, however any pattern recognition
molecule with high affinity for polyanionic compounds can potentially bind to NETs.
The mechanism for NET formation is not completely understood although
decondensation of chromatin is central to NETosis. This can be associated with the
hypercitrullination of histone H3 through peptidylarginine deiminase 4 (PAD4) which converts
arginine to citrulline, thus disrupting the charge interaction between positively charged arginine
and polyanionic DNA110
. PAD4 deficient mice are unable to produce NETs, although this has yet
to be confirmed ex vivo with primary human neutrophils111
. In addition, neutrophil elastase
infiltration from azurophil granules into the nucleus can degrade histones, driving chromatin
decondensation112
.
NET formation is dependent on production of reactive oxygen species (ROS). Hydrogen
peroxide generated by NADPH oxidase (NOX) is further metabolized by MPO. Inhibition of
NADPH oxidase with DPI prevents the formation of NETs. Furthermore, neutrophils from
patients with chronic granulomatous disease (CGD) and MPO deficient neutrophils are unable to
form NETs113,114
. ROS however is not sufficient for NET formation as healthy newborns are
unable to form NETs, despite being capable of full respiratory burst activity115
. Upstream of
NADPH oxidase, the Raf-MEK-ERK axis has been implicated in NET formation. Addition of
U0126, a specific MEK inhibitor, and ERK inhibitory peptide, a specific ERK2 inhibitor,
inhibited the formation of NETs, thus illustrating that the canonical Raf-MEK-ERK pathway is
involved in NETosis116
. In addition, the use of these inhibitors block the production of ROS,
implying that this pathway is upstream of NADPH oxidase116
. Independent of ROS production,
autophagy has also been shown to be required for NET formation. Pharmacological inhibition of
13
PI3K with wortmannin prevents massive vacuolization of neutrophils to form NETs. Inhibition
of either ROS production (pharmacological or genetic) or neutrophil autophagy
(pharmacological) leads to apoptosis117
.
Currently, no surface receptor has been identified to directly induce the formation of
NETs, however, toll-like receptor 4 (TLR4) has been implicated to play an important role118,119
.
NETs can be formed in response to bacteria, protozoa, fungi, virus, host derived factors and
chemical compounds120
. With the exception of S. aureus, all known activators of NETs require
ROS production121
. For a complete summary of all activators of NETs known to date, please
refer to (Table 1).
14
Table 1. Activators of NETs
NETosis Inducer In vitro In vivo Reference(s)
BACTERIA
Eschericia coli 10 MOI; bovine
0.01 MOI; human
-
-
115,118,122
Pseudomonas aeruginosa 0.1-10 MOI; human 1×106 CFU/mouse
123,124
Staphylococcus aureus 0.01-10 MOI; human - 100,121
Shigella flexneri 0.01 MOI; human 2.5-3.0×1010
/rabbit 100
Salmonella enterica 0.01 MOI; human - 100
Group A Streptococcus 0.1 MOI; human 5×107-2×10
8
CFU/mouse
125,126
Streptococcus pneumoniae 0.01 MOI; human 1×107/mouse
127
Mycobacterium tuberculosis 0.1-10 MOI; human - 128
PROTOZOA
Leishmania amazonensis 10 MOI; human - 129
Leishmania donovani 10 MOI; human - 130
Toxoplasma gondii 250 mU/ml; human, mouse
5×107/mouse
131
Eimeria bovis 0.2 (sporozoites) MOI; bovine
- 132
FUNGI
Aspergillus fumigatus 5 (conidia); human - 132
Candida albicans 0.01 MOI; human - 133
Aspergillus nidulans 0.5 (conidia) MOI; human - 113
VIRUS
Human Immunodeficiency Virus (p24 antigen)
1.0-2.4 ng/mL; human - 134
Influenza A virus H1N1 20 MOI; human 100-500 PFU/mouse 135
Influenza A virus H3N2 2 MOI; mouse 2×105 PFU/mouse
136
HOST FACTORS
GM-CSF + C5a 25 ng/ml GM-CSF + 10
-7 M C5a
- 137
15
IL-8 2.5–10 ng/ml; human 138
MIP-2
Singlet oxygen 10 μg/ml Photofrin; human - 139
Platelet activating factor (PAF) 10−10
-10−7
M; human 115
Syncytiotrophoblast microparticles (STBM)
150 μg/ml; human 138
Human β-defensin 1 100 ng/ml; human 140
OTHERS
Glucose oxidase 200-1000 mU/mL; human - 115
Calcium ionophore 5 μg/ml; zebrafish
4 μM; human
- 141,142
Phorbol myristate acetate (PMA) 25 nM - 100 nM; human - 100,117
Bacterial component LPS 100 ng/ml; human 5-25 μg/mouse 100,118,121,124
P. aeruginosa Pyocyanin 10-30 μM; human 143
Adapted and modified from Cheng OZ & Palaniyar N120
.
1.3.3 Neutrophil Extracellular Traps in Disease
Neutrophils are key effectors of innate immune and inflammatory responses. They play a key
role in recognition and elimination of pathogens. The resolution of inflammation is required to
limit the destruction caused by neutrophil activation. It is an active process where apoptotic
neutrophils are removed by macrophages and additional leukocyte infiltration is prevented (See
Neutrophil Activation and Resolution 1.3.1). This process is essential for maintaining tissue
homeostasis144
. If impeded, this process can trigger “non resolving inflammation”, a problematic
condition that contributes to pathology. The participation of neutrophil in mediating acute and
chronic inflammation is well documented145
. However, the involvement of NETs in contributing
to inflammatory damage is a relatively new field. This section will highlight the current opinion
of NETs in the context of disease.
Neutrophil extracellular traps are released in response to various activating stimuli and
effectively ensnare and kill bacteria. They are an effective means for antimicrobial clearance as
bacteria that express DNases as virulence factors are more effective at evading host immune
16
machinery125,127
. This may point to evolutionary pressure to avoid NETs. In addition, restoration
of NADPH oxidase through gene therapy in a patient suffering from CGD cleared persistent
Aspergillus infection and the patients neutrophils demonstrated NET-mediated killing ex vivo113
.
However, NETs do come at a cost. Armed with a hefty arsenal of antimicrobial and
cytotoxic compounds, the increased local concentrations of these compounds acts as a double
edged sword146
. There is no discrimination between host or microbe. The mechanisms for NET
clearance are currently not fully understood, but ineffective clearance can cause deleterious
inflammation on host tissue. NETs have been associated with endothelial and tissue damage as
demonstrated in vitro through propidium iodide staining with human umbilical vein endothelial
cells (HUVEC)118
. The same effect could be observed in vivo in liver microvasculature as NETs
induced a decrease in the number of sinusoids that were perfused and increased serum levels of
alanine aminotransferase (ALT)118
. Furthermore DNA-histone complexes can directly cause
epithelial and endothelial cell death147,148
. In vivo administration of histones resulted in
vacuolated endothelium, intra-alveolar hemmorage, and thrombosis in both micro and macro
vasculature147
. Since their first discovery, NETs have now been implicated in a variety of
inflammatory and autoimmune disease.
1.4 Complement and NETs in Disease
The advent of pharmacological inhibitors and animal models has shed light on our understanding
of the role complement and NETs play in diseases. In this section, we highlight the involvement
of complement and NETs in several inflammatory diseases (Figure 3).
17
Figure 3. Complement and NETs lead to endothelial cell damage. Failure to regulate the inflammatory response can lead to destruction of host tissues. (A)
Overwhelming endogenous activation, or dysregulation of complement activation through
mutation or auto antibodies can lead to a uncontrolled amplification of complement alternative
pathway leading to tissue damage. (B) The cytotoxic DNA-protein complexes in NETs, which
include neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and
proteinase 3 (PR3), can directly cause tissue damage or lead to the presentation of antigens to
form auto antibodies, anti-citrullinated peptide antibody (ACPA) and anti-neutrophil cytoplasmic
antibody (ANCA).
18
Lupus Nephritis
Systemic lupus erythematosus (SLE), or lupus, is a autoimmune disorder that effects multiple
organs with a broad range of clinical manifestations149
. Autoantibody initiated activation of
complement is involved in the pathogenesis of tissue damage in SLE. Evidence for this was first
illustrated when administration of anti-C5 monoclonal antibodies ameliorated the development
of glomerulonephritis in the (NZB × NZW)F1 mouse model of SLE52
. Following this, direct
support for the role of complement AP in disease pathogenesis was discovered as CFB-/-
and
CFD-/-
mice are protected from renal disease in the MRL/lpr mouse model for SLE150,151
. The
level of protection was similar to that found in mice treated with C3 inhibitor Crry152
.
Interestingly, elimination of complement CP activation through C4 blockade in models of SLE
led to an increase in an autoimmune response to auto antigens exacerbating tissue damage153
,
thus highlighting the importance of complement AP in contributing to disease pathology.
The formation of NETs, which contain antimicrobial peptides, DNA and histones has
been associated with the presentation of self-antigens leading to the formation of autoantibodies
in SLE. Post-translational modification of histones within NETs drives the process of auto
immunity as serum autoantibodies from SLE patients recognize many post-translationally
modified histones found in NETs154
.
To further exacerbate the issue, serum from SLE patients cannot remove NETs in a
timely manner, thus allowing for presentation of self-antigens. Impaired NET degradation is a
result of DNase I inhibitors, and anti NET auto-antibodies which prevent DNase I from access to
NETs. Prolonged exposure, of affected skin and kidney from lupus patients, to infiltrating
neutrophil extracellular traps can promote damage to host endothelial cells155
. This correlated
with clinical studies as patients who were unable to degrade NETs have significantly higher anti-
dsDNA and anti nucleus antibody titers, a hallmark test for SLE diagnosis, compared to those
who were able to degrade NETs156
. In addition, serum C1q has been shown to be able to bind
NETs. This suggests that in the presence of impaired NET degradation, NETs activate
complement through CP further amplifying the inflammatory response and exacerbating SLE
pathology157
.
19
Rheumatoid Arthritis
Rheumatoid arthritis is an autoimmune disease characterized by chronic systemic inflammation.
It may affect many tissues and organs, but the principle point of focus is synovial joints158
.
Rheumatoid arthritis has been linked to a single nucleotide polymorphism in the gene for C5
which results in increased concentrations of C5 in serum159
. The implications for AP
involvement in disease progression was illustrated using the K/BxN-derived anti GPI serum
transfer model of Rheumatoid arthritis. In this model, inflammatory joint disease was
ameliorated in CFB-/-
C57BL/6 receiving anti GPI serum but not in C4-/-
mice160
. In the collagen-
induced model of arthritis, CFB-/-
× DBA1/j displayed substantial disease amelioration. These
models again illustrate role complement AP plays disease pathogenisis161
.
In the same K/BxN-murine autoantibody mediated model of arthritis, PAD4 activity is
readily detectable in inflamed synovium of wild type mice. This effect was abrogated when
PAD4 -/-
mice were injected with K/BxN sera to induce disease, suggesting NETs play a
prominent role in disease. Interestingly, PAD4-/-
mice, when compared to PAD4 wt mice, were
not protected from disease pathogenesis. Both mice displayed comparable severity and kinetics
with no statistical significant differences in clinical scores for swelling, joint erosion or
invasion162
. This indicates causes leading to RA disease progression are multi factorial, NETs
may play just one part of many in disease progression.
The presence of auto antibodies mounting immune responses to self antigens is also
prevalent for rheumatoid arthritis patients. Citrullinated proteins are target of anti citrullinated
peptide antibodies (ACPA) in rheumatoid arthritis. Analysis of sera from rheumatoid arthritis
patients revealed that citrullinated H4, from activated neutrophils and NETs, was a target for
ACPA in rheumatoid arthritis163
, suggesting that during NETosis, the presentation of self
antigens can mount for an unwanted immune response.
Asthma
Asthma is a chronic inflammatory disease of the lung airways characterized by recurring
symptoms of airflow obstruction, and bronchospasm. Because complement infiltration out of the
vasculature is poorly understood, asthma is typically associated with hypersensitivity due to IgE
production, cellular immune abnormalities and generation of Th2 cytokines164
. Interestingly, as
20
evidence for the involvement of C3a anaphyltoxin in asthma pathogenesis, guinea pigs with C3a-
receptor (C3aR) defect demonstrate decreased bronchoconstriction165
. Furthermore in a clinical
study, C3a and C5a levels are elevated in bronchoalveolar lavage fluids from asthma patients166
.
In addition, in mouse models of airway hyper-sensitivity, CFB-/-
mice or wild-type mice treated
with monoclonal antibody to complement factor B demonstrated less inflammation and airway
sensitivity to methacholine when compared to wild type controls. C4-/-
mice however were not
protected against injury167
. Therefore, the complement AP, but not classical or lectin pathway,
plays a role in contributing to asthma disease.
Along with chronic inflammation, asthma is also associated with an influx of leukocytes
into the airways. The presence of neutrophilic asthma (e.g. greater proportion of neutrophils than
other leukocytes) dictates greater disease severity and poor prognosis168,169
. In a recent study, out
of 8 potential biomarkers (IL-8, neutrophils, eosinophils, IL-1Rα, IL-1α, IL-5, IL-6, and
RANTES) only neutrophils and IL-8 could be used to distinguish moderate from mild to severe
asthma in patient bronchoalveolar lavage fluid120
. Moreover, a clinical study has identified the
presence of neutrophil extracellular traps in allergic asthmatic airways. In these patients, both IL-
8, neutrophil count and NETs are shown to be increased in asthmatic airways170
. IL-8 has
previously been shown to be a potent activator of NETs138
. These findings suggest that IL-8,
neutrophil count and NETs may contribute to asthma. At this time, however, the mechanism for
NET formation in the asthmatic airway as well as their contribution to disease severity is not
clearly understood.
Vasculitis
Vasculitis is a group of disorders characterized by inflammation of blood vessels leading to
fibrinoid necrosis of vessel walls. Recent studies have shown that complement AP plays an
important role in anti neutrophil cytoplasmic antibody (ANCA) associated form of vasculitis. In
a mouse model for ANCA-vasculitis, anti MPO-IgG or MPO-reacting splenocytes are obtained
from MPO knockout mice immunized with purified mouse MPO. The anti MPO-IgG or MPO-
reacting splenocytes were then transferred into a recipient mouse171
. In this model of ANCA-
associated vasculitis, complement depletion with cobra venom factor completely blocked the
development of glomerulonephritis and vasculitis induced by splenocyte transfer or MPO IgG
injection. Furthermore, using this model for ANCA-vasculitis, C5-/-
-and CFB-/-
were protected
21
from disease induction, however C4-/-
mice developed glomerulonephritis and vasculitis similar
to wild-type172
. This rescue effect was not limited to knockout mice models. Mice treated with
anti-C5 antibody 8 h or one day prior to MPO IgG injection showed no signs of disease
development. In addition, antibody administration one day after MPO-IgG injection resulted in
80% reduction of disease pathogenesis173
.
The presentation of ANCA is strongly linked to small vessel vasculitis (SVV). ANCA has an
activating effect on cytokine-primed neutrophils174
. Activation of TNF-α primed neutrophils with
ANCA leads to the formation of NETs175
. In addition, in situ studies, illustrate that NET and
NET proteins are located in close proximity to neutrophil infiltrates in affected glomeruli and
intersitium of SVV patients175
. Furthermore, NETs contain auto antigens proteinase-3 (PR3) and
myeloperoxidase (MPO) which can activate the inflammatory immune response. The persistent
deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes in SVV patients
highlights the involvement of NETs for vasculitis disease pathogenesis.
Sepsis
Severe sepsis can be detrimental, killing approximately 300,000 to 500,000 North Americans a
year176
. In sepsis, infection with microorganisms can trigger acute inflammatory reactions
associated with cytokine storm and influx of other inflammatory mediators. The result can be
drastic producing hypotension, multi-organ failure and even death. Complement activation in
response to infection is important for immune function, however it must be tightly regulated. In
late stages of sepsis, complement C5a can contribute to organ damage through interaction with
other immune cells or induction of coagulation by C5a mediated expression of tissue factor,
which can lead to thrombocytopenia177
. Often the more profound the thrombocytopenia, the
more severe the sepsis and greater the mortality178
.
Investigations of cross-talk between platelets and neutrophils revealed that TLR4
activated platelets can trigger NETosis118
. LPS or plasma from severely septic patients triggered
platelet TLR4 dependent interaction with neutrophils, leading to the production of NETs. Platelet
derived β-defensin 1, was further demonstrated to be able to activate neutrophils to form
NETs140
. In this setting, the formation of NETs allowed for efficient aid in bacterial trapping
during septic conditions, however, NETs also contributed to tissue damage. Platelet-induced
NETs were associated with hepatotoxicity due to tissue damage118
. This could account for some
22
of the mechanisms contributing to organ failure during sepsis. Furthermore, NETs have also
been demonstrated to display a broad range of procoagulant properties. NETs induce the
formation of a red blood cell-rich thrombi by serving as a scaffold for platelet/RBC adhesion and
aggregation. NETs also serve as a platform binding plasma proteins to promote thrombus
stability179
. In addition, NETs also directly promote thrombus formation as presentation of
nuclear histones promotes thrombin generation through platelet-dependant mechanisms
involving TLR2 and TLR4180
.
1.5 Complement and Neutrophils
The activation of complement leads to the generation of anaphylatoxins C3a and C5a. These
anaphylatoxins initiate a strong inflammatory response and result in leukocyte influx. Generation
of C3a and C5a gradients guide neutrophils to sites of inflammation through chemotaxis181
. In
addition, these anaphylatoxins can serve as ligands binding neutrophil receptors to trigger
neutrophil activation. The activation of neutrophils with C5a usually does not induce NETosis,
however, neutrophils primed with host-derived cytokine, granulocyte-macrophage colony
stimulating factor (GM-CSF), are able to release NETs of mitochondrial origin when activated
with C5a137
. In experimental models, acute inflammatory states in C5-deficient mice result in
reduced neutrophil influx, often associated with attenuated tissue damage. This underlies the
importance of C5aR in mediating the inflammatory response182
.
Neutrophils express both C3a receptor (C3aR) and C5a receptor (C5aR) on their plasma
membranes183,184
. C3aR and C5aR are both classical G protein-coupled receptors. Binding of
ligands to these receptors activates G-protein coupled signaling transduction pathways which
lead to neutrophil chemotaxis and activation. Response to complement anaphylatoxins must be
tightly regulated in situations with sustained complement activation to avoid deleterious effects.
Modulation of neutrophil response to C3a and C5a is achieved through receptor
internalization185
. Binding of C3a and C5a to their respective receptors leads to a ligand-
mediated internalization of the receptor that is modulated by phosphorylation of the C-terminal
domains185,186
. C3a activates the internalization of C3aR and C5a activates the internalization of
C5aR, there is no cross-internalization of receptors185
.
Upon activation, neutrophil granules are mobilized to the surface releasing their cytotoxic
contents at sites of inflammation. The distinction between the different classes of neutrophil
23
granules is not only important in characterizing their constituents, it also reflects on their ability
to mobilize. Granules formed latest having the highest propensity for degranulation, thus
illustrating the neutrophils ability to modulate inflammatory responses187
. The neutrophil
secretory vesicles are mobilized completely by many activating stimuli. They are stores of
adhesion molecules, which upon activation, can mobilize to the surface to initiate leukocyte
adhesion, rolling or transmigration188
. Secretory vesicles also serve as an intracellular reservoir
for complement receptors. There is evidence that secretory vesicles contain C1q Receptor
(C1qR), complement receptor 1 (CR1), complement receptor 3 (CR3), and complement receptor
4 (CR4)189
.
Mobilization of complement receptors to neutrophil surfaces allows for phagocytosis of
complement opsonized particles (e.g. C1q, C3b, or C4b) through IgG independent mechanisms,
thus linking neutrophils to innate immunity190
. Moblization of C1qR mediates enhancement of
C1q opsonized particles for phagocytosis191
. Mobilization of CR1 mediates phagocytosis of
complement opsonized particles. It is also responsible for regulating complement amplification.
In addition, uptake of adenovirus is attributed to CR1, not CR3 or CR4192
. CR3 mobilization
allows for adhesive interaction between the neutrophil complement receptor and its endothelial
cell ligand ICAM-1. This allows for neutrophil adhesion and further activation98
. CR3 is also
responsible for phagocytosis of complement opsonized particles in a urokinase receptor
dependent manner193
. In addition, CR3, demonstrates unique interactions with CFH and CFH
related protein 1 (CFHR1). CFH and CFHR1 binding to neutrophils via CR3 mediates
attachment to Candida albicans and confers antimicrobial resistance194
. CR4 facilitates the
phagocytosis of complement opsonized components190
.
In addition to complement receptors, neutrophils also express complement regulatory
proteins. Neutrophils express DAF, CD59, and MCP on their plasma membrane surfaces.
Expression of complement regulatory proteins can be altered in response to injury195
.
1.6 Neutrophil Complement Cross-talk
Complement activation leads to the formation of powerful anaphylatoxins which can guide
neutrophils to sites of inflammation through chemotaxis. There is, however, a growing body of
evidence to suggest that neutrophils can also initiate complement amplification. Addition of
neutrophil hydrogen peroxide to serum leads to a diminution in serum hemolytic activity as well
24
as reduction in complement C3 and C5. In addition, complement activation products, C3a and
C5a, were produced in serum treated with hydrogen peroxide. This effect could be abolished
with EDTA but not EGTA indicating that neutrophil oxygen radicals can amplify the
complement alternative pathway196
.
In a separate study, purified MPO was able to convert C5 into an activated form which
led to the formation of C5b-9. The mechanism for activation of C5 was dependent on MPO
generation of HOCl from hydrogen peroxidase. This leads to the oxidation, thus activation of C5.
Supernatant from activated neutrophils was also able to activate complement through MPO-
generated hyplocholrite dependent mechanisms197
. Neutrophil elastase was also demonstrated to
be able to cleave C5 to form C5b-941
.
Furthermore, neutrophils are a potent source of properdin, the only positive regulator of
complement AP. CFP is stored in neutrophil secondary granules and is secreted upon
activation198
. Activation of neutrophils can increase local concentrations of CFP, thus amplifying
complement AP. Infiltrating neutophils can often trigger unnecessary inflammatory responses
through propagation of complement AP83
. In addition secreted properdin can bind to activated
neutrophils. This process initiates the formation of complement convertases which ultimately
leads to the formation of C5b-9 on neutrophil surfaces. The result is the production of
anaphylatoxins and release of neutrophil cytokines which further directs infiltration of additional
neutrophils. Thus activated neutrophils amplify complement AP in a positive feedback loop199
.
25
1.7 Rationale
The persistence of both neutrophil NETs and complement amplification in many deleterious
autoimmune or inflammatory diseases suggests that their interactions may exacerbate the
inflammatory immune response. Evidence shows that there is extensive cross-talk between
neutrophil activation and complement activation. However, not much is known within the
context of NETs (e.g. interaction of complement proteins with NETs).
It is interesting to note that DNA exposed on apoptotic and necrotic cells is able to bind
C1q and MBL. Binding of MBL to apoptotic cells enhances phagocytosis by macrophages but
does not activate complement200
. Binding of C1q triggers basal levels of complement activation,
however, the process is often simultaneously inhibited through C4 binding protein (C4BP) and
CFH201
. The binding of these inhibitory proteins not only limits complement activation but
inhibits further release of DNA202
. These inhibitory mechanisms maybe overwhelmed in the
presence of NETS.
NETs have been shown to amplify complement, however the mechanisms for activation
remain elusive. NETs are able to bind C1q which suggest that they may activate complement
through CP157
. NETs, also contain MPO which has been shown to activate complement AP197
. In
addition, neutrophil elastase, a known NET protein, is able to cleave C5 to activate
complement41
. All of these remain viable in explaining the mechanism for neutrophil NET
complement amplification.
In addition, neutrophil CFP has well documented pattern recognition molecule properties.
CFP interacts with a variety of polyanionic macromolecules including DNA expressed on
necrotic or apoptotic cells, however, the interaction with extracellular DNA on NETs has not yet
been investigated. Properdin opsonized targets have potential to amplify complement. In essence,
a complement activating surface is any surface without adequate regulatory protein function to
control the complement AP, or unable to control AP via CFH203
.
Taken together, NET and complement interactions could play a significant role for
antimicrobial activity or in the pathogenesis of many inflammatory and auto immune diseases
(Figure 4). To date, there has been no evidence detailing the mechanisms in which NETs amplify
the complement.
26
Figure 4. Complement interaction with NETs.
Neutrophils are key mediators of innate immune and inflammatory responses. Neutrophil influx
to sites of inflammation can amplify complement activation through release of properdin (CFP).
Neutrophil elastase (NE) and myeloperodiase (MPO) can also contribute to complement
activation. Neutrophils also form NETs to efficiently capture invading microbes, however, the
mechanisms for microbial killing are not fully understood. We propose that neutrophil
extracellular traps can activate complement to enhance microbial clearance. If unregulated, this
process can lead to endothelial cell damage. The dashed line in the center divides the paradoxical
roles of how complement and neutrophil interactions may lead to antimicrobial clearance or
endothelial cell damage.
27
1.8 Hypothesis
Complement factors present in neutrophils participate in antimicrobial function by mounting a
targeted response to infectious agents via NETs by localized complement alternative pathway
activation.
1.9 Aims
Aim 1. To characterize the interaction between the complement system with neutrophils and
neutrophil extracellular traps.
Aim 2. To determine the significance of complement-NET interactions in antimicrobial function.
28
2 Methods
2.1 Reagents
All buffer salts and reagents were obtained from Sigma Aldrich (St. Louis, Mo, USA) unless
otherwise stated.
2.2 Research Ethics Board Approval
Informed and written consent was obtained from the donors and the protocol was approved by
the ethics committee at the Hospital for Sick Children.
2.3 Neutrophil Isolation
Human peripheral whole blood was collected from healthy individuals and aliquoted into BD
vacutainer containing EDTA (1.8 mg/ml). 20 ml of whole blood was layered on top of 20 ml of
Polymorphprep™ (Axis-shield, Oslo, Norway) and separation was achieved by centrifugation at
500 × g for 35 minutes, allowing the rotor to decelerate without brake. The lower band
containing polymorphonuclear cells was harvested and resuspended in an equal volume of a half
strength Hepes-buffered saline (0.425% (w/v) NaCl, 5 mM Hepes-NaOH, pH 7.4) returning the
polymorphonuclear cell suspension to isoosmotic condition. Neutrophils were harvested by
centrifugation at 400 × g for 10 minutes. Removal of residual erythrocyte contamination was
achieved through hypotonic lysis. Polymorphonuclear cell pellets containing erythrocytes were
resuspended in a hypotonic solution (0.2% (w/v) NaCl) for 30 seconds followed by addition of
an equal volume of hypertonic solution (1.6% (w/v), 20 mM Hepes-NaOH, pH 7.4). Following
hypotonic lysis of erythrocytes, the polymorphonuclear cells are washed twice in Hepes-buffered
saline (0.85% (w/v) NaCl, 10 mM Hepes-NaOH, pH 7.4). Cells were harvested and resuspended
in the desired media. Cell counting was performed with a hemocytometer and cells were
resuspended to the desired concentration.
2.4 Preparation of Neutrophil Lysates
Neutrophils were harvested via centrifugation at 1000 × g for 10 minutes. The neutrophil cell
pellet was resuspended in a neutrophil lysis buffer (1% (v/v) Triton X-100, 50 mM Tris, pH 7.4,
10 mM KCl) containing 2 × cOmplete, mini protease inhibitor cocktail (Roche Diagnostics,
Laval, QC, Canada) supplemented with 0.5 mM EDTA, 25 μM leupeptin, 25 μM pepstatin, 25
29
μM aprotinin, 1 mM levamisole, 1 mM Na3VO4, 25 mM NaF, 1 mM PMSF. The sample was
sonicated 3 times for 10 seconds (VWR Sonics model 50D) and incubated for 15 minutes at 4°C.
Neutrophil lysates were centrifuged at 25000 × g for 30 minutes at 4°C and stored at -80°C for
future analysis. Neutrophils were resuspended to a final concentration of 2 × 107 cells/ml.
2.5 Bacterial Cultures and Strains
Bacteria were cultured from glycerol stocks kept at -80°C or from single colonies selected from
LB agar plates. P. aeruginosa mPAO1 (a gift from the lab of Dr. Neil Sweezey, The Hospital for
Sick Children), P. aeruginosa PAKwt (a gift from the lab of Dr. Marina Ulanova, Northern
Ontario School of Medicine), S.aureus RN6390 (a gift from the lab of Dr. John Brumell, The
Hospital for Sick Children), B. subtilis, and E. coli Y1088 (Bacteria from Dr. Nades Palaniyar,
The Hospital for Sick Children) were cultured in LB agar broth. P. aeruginosa PAKgfp (a gift
from the lab of Dr. Marina Ulanova, Northern Ontario School of Medicine) was cultured in LB
agar broth with 30 μg/ml gentamicin (Wisent Bioproducts, Montreal, QC, Canada).
2.5.1 Bacterial Plating
Agar plates were prepared with autoclaved LB-agar. Bacterial cultures were streaked onto agar
plates and incubated overnight at 37°C. PAKgfp was maintained on LB-agar with 30 μg/ml
gentamicin. Agar plates were wrapped in parafilm and stored at 4°C for up to two weeks.
2.5.2 Bacterial Growth
Bacteria cultures were selected from single colonies on LB-agar and grown overnight in sterile
LB-broth. PAKgfp was maintained in LB-broth with 30 μg/ml gentamicin. Prior to neutrophil
actvation, bacterial cultures were diluted by a factor of ten and sub-cultured for 3 hours. Cells
were harvested by centrifugation at 5000 × g for 5 minutes at 4°C and washed three times in 5 ml
of phosphate-buffered saline (PBS, pH 7.4). After the final resuspension, turbidity of the culture
was taken at OD600 and the concentration of the bacteria was approximated using the formula:
Bacteria was resuspended to the appropriate concentration in PBS and used for neutrophil
activation studies at a multiplicity of infection (MOI) of 10 and MOI of 100.
30
2.6 Neutrophil Activation
2.6.1 Neutrophil Oxidative Burst
Purified neutrophils as described in the section Neutrophil Isolation (2.3) were resuspended at 1
× 106
cells/ml in RPMI 1640 (Wisent Bioproducts, Montreal, QC, Canada) supplemented with 10
mM Hepes, pH 7.4. Cells were activated with C5a (1-2 μM), fMLP (1-2 μM), and Phorbol 12-
myristate 13-acetate (PMA, 20 nM) for 30 minutes. Dihydrorhodamine 123 (10 μM) was used
for detection of reactive oxygen species.
Cells were analyzed by flow cytometry using a Gallios Flow Cytometer (Beckman
Coulter, Mississauga, ON, Canada). Cells were gated through forward scatter and side scatter to
determine the neutrophil population. The gated population was further stained for with Hoescht
(1 μg/ml) using filter channel: FL9 (405/450 BP 40) to select for nucleated populations. Reactive
oxygen species was detected using filter channel FL1 (488/429 BP 28.25).
2.6.2 Neutrophil Activation and Secretion
Purified neutrophils as described in the section Neutrophil Isolation (2.3) were resuspended in
RPMI 1640 media (Wisent Bioproducts, Montreal, QC, Canada) supplemented with 10 mM
Hepes, pH 7.4 to a final concentration of 2 × 107 cells/mL. Neutrophils were aliquoted into
Eppendorf tubes and activated with C5a (1 μM), fMLP (1 μM) and PMA (20 nM). Cells were
cultured in a tissue culture incubator (37°C, 5% CO2) for 30 minutes with slight agitation every
10 minutes to prevent cell clumping. Neutrophil stimulation was terminated by removing
neutrophils from tissue culture and incubating at 4°C for 5 minutes. Neutrophils were harvested
via centrifugation at 1000 × g for 10 minutes. The supernatant was carefully collected and
centrifuged at 25000 × g for 10 minutes at 4°C and immediately placed in 2 × neutrophil laemmli
sample buffer. The supernatant was heated at 95°C for 5 minutes and stored at -80°C for future
analysis. Neutrophil lysates were prepared from the remaining cell pellet as described in the
section Preparation of Neutrophil Lysates (2.4)
2.6.3 Differential Cell Counts for Nuclear Morphology
Neutrophils as described in section Seeding Neutrophils on Coated Cover Slips (2.8.3) were
activated with 20 nM PMA and fixed at the following time points (t=0, 30, 60, 120, 180, 240
min) with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA,
31
USA). Cells were stained with DAPI (1 μg/ml) and cover slips were mounted with Dako
Fluorescence Mounting Media (Dako Canada, Burlington, ON, Canada). Images were taken
using a Nikon Eclipse Ti-U Microscope, equipped with QICAM 12 bit Mono Fast 1394, model:
QIC-F-M-12C camera (Qimaging, Surry, BC, Canada) using a 40×/0.60 air immersion objective
operated by NIS-Elements software (Nikon, Mellville, NY, USA). The nuclear morphologies
were categorized as lobular, delobular, decondensed, NETs, and undefined. Differential cell
counts for nuclear morphology was assessed by counting 118 to 220 cells from 5 different fields
of view at various time points. Nuclear morphology types are presented as a percent total
averaged from three independent experiments.
2.6.4 SYTOX Green Plate Reader Assay
Neutrophils (3 × 104 cells) were seeded onto 96 well plates. For inhibition of NETs, neutrophils
were preloaded with 2 μM of NADPH oxidase inhibitor, diphenyleneiodonium (DPI) for one
hour before activation. Neutrophils were activated with PMA (20 nM), C5a (1 μM), and fMLP (1
μM). To study NETs in response to sepsis, Pseudomonas aeruginosa (mPAO1, PAKwt,
PAKgfp), Staphylococcus aureus (RN6390), Escherichia coli (Y1088), and Bascillus subtilis
were used to activate neutrophils at MOIs of 10 and 100.
Cell impermeable DNA dye, SYTOX Green (5 μM), was added to neutrophil suspensions
to detect the presence of extracellular DNA. When neutrophils undergo NETosis, the release of
NETs can be detected by SYTOX green fluorescence intensity. Fluorescence intensity was
measured using the POLARstar Omega microplate reader (BMG Labtech, Ortenberg, Germany)
with excitation/emission (485/520). NET formation was normalized to total DNA in resting
neutrophils permeabilized with 0.5% (w/v) Triton X-100.
2.7 Western Blot
BCA protein determination assay was used prior to preparation of samples for western blot.
Neutrophil lysates were prepared in 2 × neutrophil laemmli buffer (125 mM Tris-HCl, pH 6.8,
6% (w/v) SDS, 8% (v/v) β-mercaptoethanol, 18% (v/v) glycerol, 10 mM EDTA, 25 μM
leupeptin, 25 μM pepstatin, 25 μM aprotinin, 10 mM NaF, 5 mM Na3VO4, 1mM levamisole,
0.05% (w/v) bromphenol blue) and heated at 95°C for 5 minutes with shaking. For non reducing
samples, neutrophil lysates were prepared in 2 × neutrophil laemmli sample buffer in the absence
32
of β-mercaptoethanol. 50 μg of protein from neutrophil lysates were loaded and separated by
10% SDS-polyacrylamide gel electrophoresis running at 200 V for 45 minutes. Protein gels were
transferred onto nitrocellulose membrane for 70 minutes at 100 V. Membranes were blocked
with 5% (w/v) skim-milk in Tris-Buffered-Saline, pH 7.6, + 0.05% (v/v) Tween-20 (TBST) for
one hour at room temperature. For identification of complement proteins, membranes were
incubated with goat polyclonal antibody to complement proteins (1:1000 dilution; Complement
Technology, Tyler, TX, USA) in 5% (w/v) skim-milk in TBST overnight at 4°C with rocking.
For loading controls, membranes were incubated with mouse monoclonal antibody to β-actin (β-
actin, BA3R, 1:10000 dilution; Thermo Fisher Scientific, Rockford, IL, USA) in 5% (w/v) skim-
milk in TBST for 1 hour at room temperature with shaking. Membranes were washed 3 times,
with TBST for 5 minutes, and incubated with secondary antibody in 5% (w/v) skim-milk in
TBST at room temperature for 1 hour with shaking. Membranes were washed 5 times with TBST
for 5 minutes. Proteins were detected using Western Lighting™ Plus-ECL, Enhanced
(PerkinElmer, Waltham, MA, USA) and developed on radiographic film on a Kodak X-Omat
2000a processor.
2.7.1 Determining Protein Concentration
A bicinchoninic acid (BCA) assay was performed on neutrophil lysates to determine total protein
concentration. BCA protein assay kit was used according to manufacturer protocol without
deviation (Thermo Fisher Scientific, Rockford, IL, USA). 50 μg of protein was loaded onto SDS-
PAGE for western blot.
2.8 Imaging and Microscopy
2.8.1 Acid Washed Cover Slips
Coverslips were acid washed prior to use for microscopy to remove manufacturing residue, oils
and other contaminants such as lipopolysacchardie (LPS) to prevent non specific activation of
neutrophils. 12-mm round glass coverslips (Fisher Scientific) were submerged in nitric acid for
48 hour. Coverslips were washed with deionized tissue-culture-grade water for 15-20 exchanges
or until wash exchanges reach neutral pH, followed by two exchanges of 95% (v/v) ethanol to
remove residual water. Cover slips were stored in 70% (v/v) ethanol for future use.
33
2.8.2 Preparation of Cover Slips for Microscopy
Acid washed cover slips were removed from ethanol storage and excess ethanol was drained
using Kimwipes. Cover slips were flame dried and placed into a 6-well tissue culture plate.
Three cover slips were placed in each well. Cover slips were coated with 75 μl of 0.001% Poly-
L-Lysine for 60 minutes at room temperature. Cover slips were washed three times with PBS (~3
mL). Poly-L-lysine coated cover slips were used in all experiments prepared for analysis with
micrsocopy.
2.8.3 Seeding Neutrophils on Coated Cover Slips
Purified neutrophils as described in Neutrophil Isolation (2.3) were resuspended in RPMI 1640
media (Wisent Bioproducts, Montreal, QC, Canada) supplemented with 10 mM Hepes, pH 7.4,
to a concentration of 1 × 106 cells/ml. 1 ml of the neutrophil suspension was placed in tissue
culture dish containing the coated cover slips (2.8.3). Neutrophils adhere to cover slips after 1
hour incubation (37°C, 5% CO2).
2.9 Identifying Complement Proteins in Neutrophils
Neutrophils were prepared for microscopy as described in Seeding Neutrophils on Coated Cover
Slips (2.8.3) and activated with PMA (20 nM) for 30 minutes (37°C, 5% CO2). No exogenous
plasma or serum was added to the culturing media. Samples were fixed with 4% (w/v)
paraformaldehyde (Electron Microscopy Sciences, Fort Washington, PA) in PBS. Samples were
stained with 5 μg/mL Wheat Germ Agglutinin, Alexa Fluor® 555 conjugate (Invitrogen, Eugene,
OR, USA) for 10 min and washed in PBS. Samples were permeabilized with 0.2% (v/v) Triton
X-100 for 20 minutes, and washed in PBS. Samples were blocked in 3% (v/v) gelatin from cold
water fish skin (v/v), and stained with anti complement antibodies: rabbit polyclonal antibody to
properdin (SC-68366, 1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit
polyclonal antibody to CFB (SC-67151, 1:50 dilution; Santa Cruz Biotechnology, Dallas, TX,
USA), rabbit polyclonal antibody to complement C3 (ab97462, 1:100 dilution; Abcam,
Cambridge, MA, USA), and goat polyclonal antibody to CFH (AF4779, 1:50; R&D Systems,
Minneapolis, MN, USA) for 1h. Samples were washed in PBS and incubated with respective
secondary donkey-anti primary antibodies with Alexa Fluor® 488 conjugate (Invitrogen,
Eugene, OR, USA). Cell nuclei were stained by DAPI (1 μg/ml). Samples were washed in PBS
34
and cover slips were mounted with Dako Fluorescence Mounting Media (Dako Canada,
Burlington, ON, Canada) for analysis with spinning disc confocal microscopy.
2.10 Detection of Complement Proteins in NETs
Neutrophils were prepared for microscopy as described in Seeding Neutrophils on Coated Cover
Slips (2.8.3) and activated with PMA (20 nM) or P. aeruginosa PAKgfp (MOI of 10) for 240
minutes (37°C, 5% CO2). No exogenous plasma or serum is added to the culturing media. The
samples were fixed with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, Fort
Washington, PA) in PBS. Samples were blocked with 3% (v/v) gelatin from cold water fish skin
for 1h followed by incubation with anti-complement antibodies: rabbit polyclonal antibody to
properdin (SC-68366, 1:50 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit
polyclonal antibody to CFB (SC-67151, 1:50 dilution; Santa Cruz Biotechnology, Dallas, TX,
USA), rabbit polyclonal antibody to complement C3 (ab97462, 1:100 dilution; Abcam,
Cambridge, MA, USA), and goat polyclonal antibody to CFH (AF4779, 1:50; R&D Systems,
Minneapolis, MN, USA) for 1h. Neutrophils activated with PMA were co-stained with mouse
monoclonal antibody to neutrophil myeloperoxidase (ab25989, 1:500 dilution; Abcam,
Cambridge, MA, USA). Samples were washed in PBS and incubated with secondary donkey
anti-primary antibodies conjugated with Alexa Fluor ®555 (Invitrogen, Eugene, OR, USA).
PAKgfp signal was enhanced with mouse monoclonal antibody to GFP (A11120, 1:100 dilution;
Invitrogen, Eugene, OR, USA) primary for 1h, washed in PBS, followed by incubation with
donkey anti-mouse conjugated with Alexa Fluor ® 488 (Invitrogen, Eugene, OR, USA). NETs
were detected by DAPI (1 μg/ml) staining. Samples were washed in PBS and cover slips were
mounted with Dako Fluorescence Mounting Media (Dako Canada, Burlington, ON, Canada) for
analysis with spinning disc confocal microscopy.
2.11 Complement Competent Plasma
Human peripheral whole blood was collected in 10 μg/ml Refludan® (Bayer Healthcare, Wayne,
NJ, USA) and centrifuged at 1500 × g for 15 minutes. The platelet poor plasma (PPP) was
collected and sterile filtered through 0.2 μm syringe filters. PPP samples were stored at -80°C for
future use. Refludan ® is the pharmacological trade name for lepuridin (recombinant hirudin
derived from yeast cells). It prevents coagulation through inhibition of thrombin. It is
35
advantageous when collecting complement competent plasma as it does not chelate Ca2+
or
Mg2+
, thus allowing for complement activity.
2.12 Preparation of Rabbit Erythrocytes
Rabbit erythrocytes (Complement Technologies, Tyler, TX, USA) were aliquoted into
Eppendorf tubes and harvested through centrifugation at 6000 × g for 60 seconds. Rabbit
erythrocytes were washed through continuous cycles of centrifugation and resuspension in
complement alternative pathway buffer (AP buffer), containing 20 mM Hepes, pH 7.4, 144 mM
NaCl, 7 mM MgCl2, and 10 mM EGTA, until supernatant is clear. Cells are resuspended to a
final concentration of 5 × 108 erythocytes/ml.
2.13 Plasma Complement Activity Assay
To inhibit complement AP, mouse monoclonal antibody to properdin (Anti Factor P#1, A233;
Quidel Corporation, San Diego, CA, USA) was serially diluted in 40% (v/v) Refludan® (Bayer
Healthcare, Wayne, NJ, USA) fresh frozen PPP/AP buffer. 50 μl of the 40% (v/v) plasma/AP
buffer, containing inhibitory antibody, was added to 50 μl of freshly prepared rabbit erythrocytes
(5 × 108 erythrocytes/ml) in AP buffer to give a final concentration of 20% (v/v) plasma/AP
buffer. Plasma and rabbit erythrocytes were incubated for 15 minutes at 37°C. Residual
erythrocytes were removed through centrifugation at 6000 × g for 60 seconds. 100 ul of
supernatant from each reaction was added to a 96 well tissue culture dish. Free hemoglobin was
measured in supernatants with end point absorption at 405 nm on a POLARstar Omega
microplate reader (BMG Labtech, Ortenberg, Germany). Samples were normalized to total
hemolysis of 2.5 × 107 erythocytes (50 μl) of rabbit erythrocytes in 100 μl of distilled water.
2.14 Complement Activation and C5b-9 Formation on NETs
Neutrophils were prepared for microscopy as described in Seeding Neutrophils on Coated Cover
Slips (2.8.3) with slight deviation. A single cover slip was placed in a 12 well tissue culture plate
and 500 μl of the neutrophil suspension (1 × 106 neutrophils/ml) in RPMI 1640 (Wisent
Bioproducts, Montreal, QC, Canada) supplemented with 10 mM Hepes, pH 7.4 was placed in
each well. Neutrophil samples were preloaded with DNase I (50 μg/ml) to digest all extracellular
DNA including NETs and activated with PMA (20 nM) or P. aeruginosa PAKgfp (MOI of 10)
for 240 minutes (37°C, 5% CO2).
36
After 240 minutes of activation, tissue culture plates were centrifuged at 200 × g for 5
minutes 4°C. The media was carefully removed and 500 μl of 20% (v/v) Refludan® (Bayer
Healthcare, Wayne, NJ, USA) fresh frozen PPP prepared in RPMI 1640 media or AP buffer was
added to the 12 well tissue culture plate. For experiments with AP buffer, a buffer exchange was
performed with 3 washes with PBS, followed by one wash with AP buffer, to remove
phosphates, prior to the addition of 20% (v/v) Refludan® (Bayer Healthcare, Wayne, NJ, USA)
fresh frozen PPP in AP buffer. Samples were incubated for 15 minutes (37°C, 5% CO2) and
fixed with 4% (w/v) paraformaldehyde (Electron Micrscopy Sciences, Fort Washington, PA) in
PBS.
Samples were blocked with 3% (v/v) gelatin from cold water fish skin for 1h and
incubated with mouse monoclonal antibody to C5b-9 (DIA 011-01, 1:200 dilution; Antibody
Shop, Gentofte, Denmark) for 1h. Samples were washed in PBS and incubated with donkey anti-
mouse secondary antibody conjugated with Alexa Fluor ® 555 (Invitrogen, Eugene, OR, USA).
PAKgfp was enhanced with Alexa Fluor ® 488 conjugated rabbit polyclonal antibody to GFP
(A21311, 1:400 dilution; Invitrogen, Eugene, OR, USA). NETs were detected by DAPI (1
μg/ml) staining. Samples were washed in PBS and cover slips were mounted with Dako
Fluorescence Mounting Media (Dako Canada, Burlington, ON, Canada) for analysis with
spinning disc confocal microscopy.
2.15 Inhibiting Properdin Activity in NETs
Purified neutrophils were prepared for addition of 20% (v/v) Refludan® (Bayer Healthcare,
Wayne, NJ, USA) fresh frozen PPP in RPMI 1640 media or AP buffer as described in
Complement Activation and C5b-9 Formation on NETs (2.14). For inhibition of properdin
activity, mouse monoclonal antibody to properdin (Anti Factor P#1, A233; Quidel Corporation,
San Diego, CA, USA) was added to 20% plasma at a concentration previously determined using
the procedure outlined in Plasma Complement Activity Assay (2.13). Samples were incubated
for 15 minutes (37°C, 5% CO2) and fixed with 4% (w/v) paraformaldehyde (Electron Micrscopy
Sciences, Fort Washington, PA) in PBS.
Samples were blocked with 3% (v/v) gelatin from cold water fish skin for 1h and
incubated with rabbit polyclonal antibody to C5b-9 (ab55811, 1:1000 dilution; Abcam,
Cambridge, MA, USA). Samples were washed in PBS and incubated with donkey anti-rabbit
37
secondary antibody conjugated with Alexa Fluor ® 555 (Invitrogen, Eugene, OR, USA). NETs
were detected by DAPI (1 μg/ml) staining. Samples were washed in PBS and cover slips were
mounted with Dako Fluorescence Mounting Media (Dako Canada, Burlington, ON, Canada) for
analysis with spinning disc confocal microscopy.
2.16 Spinning-Disc Confocal Microscopy
Images were taken on an Olympus IX81 inverted fluorescence microscope using a 60×/1.35 oil
immersion objective equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and
Yokogawa CSU X1 spinning disc confocal scan head (with Spectral Aurora Borealis upgrade).
The unit is equipped with 4 separate diode-pumped solid state laser lines (Spectral Applied
Research, 405 nm, 491 nm, 561 nm, 642 nm) with emission filters: 447 nm ± 60, 525 nm ± 50,
593 nm ± 40, 620 nm ± 60, 676 ± 29 and 700 nm ± 75, and 1.5X magnification lens (Spectral
Applied Research). Confocal images were taken with an Improvision Piezo Focus Drive. Z-
stacks were taken at 0.25 μm. Images taken using the spinning disc confocal microscope was
deconvolved by iterative restoration using Volocity Software (PerkinElmer, Waltham, MA,
USA) with confidence limit set to 95% and iteration limit set to 20.
2.17 Colocalization Analysis
Colocalization analysis was performed using Volocity Software (PerkinElmer, Waltham, MA,
USA). Thresholds were selected to the background and thresholded Pearson’s correlation was
recorded as measure of colocalization.
2.18 Statistical Analysis
Student t-test or one-way/two-way ANOVA with Tukey’s multiple comparison test was used for
statistical comparison as needed. A p value was set at 0.05, 0.01 or 0.001 for statistical
significance. All statistical analysis was performed using GraphPad Prism(GraphPad Software,
La Jolla, CA, USA) statistical analysis software (Version 6.0).
38
3 Results – Aim 1
To characterize the interaction between the complement system with neutrophils and neutrophil
extracellular traps.
3.1 Neutrophils Contain Complement Factors
The first aim of our study was to characterize the interaction between complement proteins and
neutrophils. Infiltrating neutrophils are often associated with amplification of complement
response leading to inflammatory damage83
. However, the mechanisms in which neutrophils
amplify complement activation are not fully understood. Existing literature suggests that
neutrophils are key mediators of complement AP because they are a potent source of
properdin198
. In addition, early reports have shown that neutrophils may also contain complement
factor B, complement components C3, C6 and C7204
, however these early reports have not
gathered much attention. There is currently a large gap in knowledge surrounding complement
proteins and neutrophils.
To consolidate the data from existing literature, we performed a series of western blots to
identify complement proteins that are contained in human neutrophils. Human neutrophils were
isolated from healthy donors using polymorph prep. Neutrophil lysates were prepared at a final
concentration of 2 × 107 neutrophils/ml. Total protein concentration was determined using a
BCA assay, and 50 μg of protein was loaded onto SDS-PAGE for western blot.
Consistent with the literature, western blot analysis revealed that neutrophils express
properdin (CFP) (Figure 5A), complement component C3 (C3) (Figure 5B), and complement
factor B (CFB) (Figure 5C). In addition, we were able to demonstrate that neutrophils contain
complement factor H (CFH) (Figure 5D) the major soluble regulator of complement AP. To our
knowledge, this is the first report of neutrophils as a source of CFH. All complement antibodies
were tested on diluted human plasma which is an abundant source for complement proteins. The
antibodies for CFP, C3, CFB and CFH all yielded a strong specific band in human plasma
diluted 1:50 (plasma:PBS) after 5 seconds of exposure on radiographic film. This suggests
neutrophils contain a very high concentration of CFP, as we were able to observe a distinct band
on radiographic film after 5 seconds of exposure. The concentrations for C3, CFB and CFH were
much lower as distinct bands only visible on radiographic film after 5 minutes of exposure. In
39
addition, using western blot analysis, we screened for complement components C1q, C2, C4, C5,
C6, C7, C8, C9, CFD, and CFI, however we were unable to detect the presence of these proteins
(Data not shown).
The presence of CFP, C3, CFB and CFH in resting neutrophils is consistent with the observation
that neutrophils are key mediators of complement alternative pathway. CFP, C3, and CFB are all
major components required to form the complement AP C3 convertase. In the presence of
circulating CFD (1-9 μg/ml), neutrophils express all the necessary constituents to activate
complement AP205
. The presence of CFH in neutrophils suggests that neutrophils may undergo
some form of self regulation of complement activity. This further strengthens the notion that
neutrophils are key mediators of complement AP.
40
Figure 5. Neutrophils contain proteins from complement alternative pathway.
Representative western blot for detection of complement proteins contained in resting
neutrophils. 50 μg of protein from neutrophil lysates was used for detection of complement
protein. (A) CFP, 10% (w/v) non reducing SDS-PAGE, 5 sec exposure on radiographic film. (B)
C3, 10% reducing SDS-PAGE, 5 min exposure on radiographic film. (C) CFB, 10% reducing
SDS-PAGE, 5 min exposure on radiographic film. (D) CFH, 10% (w/v) non-reducing SDS-
PAGE, 5 min exposure on radiographic film.
41
3.2 Investigating Neutrophil Activation
Neutrophils are dynamic cells which traverse through the blood stream continuously and
randomly probing endothelium of vessel walls. Upon activation, neutrophils rapidly degranulate
to modify expression of adhesion molecules on the plasma membrane and secrete proteins into
the extracellular environment. To further characterize the interaction between complement and
neutrophils, we examined the localization and protein secretion for complement proteins we
previously identified (Section 3.1) in response to various activating stimuli. From a panel of
activating agonists, we selected two potent mediators of the inflammatory response. We
activated neutrophils with complement anaphylatoxin C5a, and formylated peptide fMLP. In
addition, we also activated neutrophils with PMA, a potent protein kinase-C (PKC) activator.
3.2.1 Neutrophil Activation and Oxidative Burst
Neutrophil activation leads to an NADPH-mediated formation of reactive oxygen species (ROS)
known as an oxidative burst. This process is essential for immune function. We investigated the
effect of C5a, fMLP and PMA on neutrophil ROS production. Neutrophils were analyzed by
flow cytometry using a Gallios Flow Cytometer (Beckman Coulter) and dihydrorhodamine 123
was used for detection of ROS.
We were unable to detect the presence of ROS in neutrophils stimulated with
complement anaphylatoxin C5a (Figure 6A). Stimulation with fMLP resulted in a 1.5 fold
increase in ROS production (Figure 6B), and stimulation with PMA resulted in approximately a
9 fold increase in ROS production (Figure 6C). These data suggest that activation of formylated
peptide receptor or PKC, respective targets for ligands fMLP and PMA, leads to neutrophil
oxidative burst, while activation of C5aR does not lead to oxidative burst. In this way,
neutrophils can modulate their response to activating stimuli. Recruitment of neutrophils to sites
of inflammation with anaphylatoxin C5a does not lead to neutrophil ROS production and
oxidative stress, while PKC activation, or stimulation of neutrophils with fMLP, can lead to ROS
production and oxidative stress.
42
Figure 6. Neutrophil stimulation with fMLP and PMA, but not C5a, leads to oxidative
burst.
Neutrophils (1 × 106 cells/ml) were activated at 37°C for 30 min in the presence of
dihydrorhodamine 123 (10 μM) Cells were analyzed by flow cytometry on a Gallios Flow
Cytometer (Beckman Coulter). (A) Activation with C5a does not lead to oxidative burst.
Activation with (B) fMLP and (C) PMA leads to oxidative burst. Representative histogram is
shown. Results are given as median fluorescence intensity (MFI) from three independent
experiments. Student t-test, *P < 0.05, **p < 0.01, n=3.
43
3.2.2 Complement Protein Localization Upon Activation
Neutrophil activation is a dynamic process where mobilization of neutrophil granules results in
exocytosis of proteins into the extracellular environment and modification of adhesion molecules
expressed on the plasma membrane. Upregulation of adhesion molecules enable neutrophils to
undergo their characteristic rolling, tethering, and transendothelial cell migration. In addition,
modulation of surface receptors allows for interaction with various ligands.
Neutrophil properdin has been shown to bind neutrophil surfaces and propagate
complement AP in a positive feedback loop for complement and neutrophil activation199
. In
addition, CFH can act as an adhesion protein and bind to integrin receptor CR3
(CD11b/CD18)206
. This suggests that activated neutrophils should bind complement proteins to
their surfaces.
To determine localization of the proteins previously identified (3.1), we utilized spinning-
disc immunofluoresecence confocal microscopy to analyze the cellular distribution of CFP (sc-
68366; Santa Cruz Biotechnology), C3 (ab97462; Abcam), CFB (sc-67151; Santa Cruz
Biotechnology), and CFH (AF4779; R&D Systems) upon activation with PMA (Figure 7). We
observed that upon neutrophil activation (Figure 7BDFH), complement proteins associate with
sialic acid surfaces, as indicated by an increase in colocalization between the complement
proteins and neutrophil sialic acids stained with wheat germ agglutinin.
During neutrophil activation, endogenous sialidases and sialyltransferases modulate the
expression of sialic acid on neutrophils and endothelial cells207
. Furthermore, neutrophil sialidase
activity on endothelial cell surfaces increases neutrophil adhesion and migration across
endothelium208
. Currently, it is unknown why complement proteins associate with sialic acid
upon activation, however this interaction suggests that activated neutrophils can potentially
mediate complement AP by modulating expression of sialic acids.
44
Figure 7. Complement proteins associate with sialic acid upon activation.
Isolated neutrophils were left (ACEG) untreated or (BDFH) activated with PMA (20 nM) for 30
min. Cells were permeabilzed 0.2% (v/v) triton X-100 for 20 minutes. Samples were prepared
for immunostaining for detection of (AB) Properdin (sc-68366; Santa Cruz Biotechnology);
(CD) C3 (ab97462; Abcam); (EF) CFB (sc-67151; Santa Cruz Biotechnology), and (FG) CFH
(AF4779; R&D Systems). Neutrophil sialic acid was stained with Wheat Germ Agglutinin 568
(Invitrogen). Neutrophil nucleus was stained with DAPI. Z-stack images shown are
representative of images from three independent experiments taken with a 60×/1.35 oil
immersion objective. (ABGH) Scale bar, 3.60 μm. (CDEF) Scale Bar, 3.70 μm.
45
3.2.3 Complement Protein Secretion in Response to Activation
Activation of neutrophils results in secretion of proteins stored in granules. Isolated neutrophils
(2 × 107
cells/ml) were activated with C5a (1 μM), fMLP (1 μM), and PMA (20 nM) to
determine if complement proteins are secreted. Neutrophil cell fractions were harvested through
centrifugation at 1000 × g and supernatants were collected for secreted proteins. Total protein
concentration was determined using a BCA assay, and 50 μg of protein was loaded onto SDS-
PAGE for western blot.
Activation of neutrophils with C5a, fMLP, and PMA led to secretion of complement
proteins (Figure 8). The absence of structural protein β-actin in the supernatant fractions
indicates that proteins collected are representative of secreted proteins. Activation of neutrophils
did not result in cell lysis which would release β-actin into the solution. CFP secretion from
activated neutrophils varied with response to different activating stimuli (Figure 8A). The
incremental increase in CFP secretion corresponds with the ROS production from the activating
stimuli (Figure 6). Activation of neutrophils, with PMA, resulted in the greatest concentration of
CFP secretion. Activation, with fMLP, resulted in lower concentrations of CFP secretion when
compared to PMA, and activation with C5a resulted in the least amount of CFP secretion. In
addition, CFP is retained in neutrophils even after secretion as indicated by the band found in the
lysate fraction of the western blot. Likewise, CFB also displayed a varying response of protein
secretion with an incremental increase corresponding to the ROS production of the activating
stimuli Figure 6. Activation with C5a and fMLP resulted in lower levels of CFB secretion, and
activation with PMA resulted in greater amounts of CFB secretion. We also noticed that only full
length CFB (90 kDa) is secreted into the supernatant while cleaved fragment CFB Bb (60 kDa) is
retained within the neutrophil (Figure 8C). C3 (Figure 8B) and CFH (Figure 8D) were
completely secreted into the supernatant upon activation with C5a, fMLP and PMA.
The varying secretion profiles for the complement proteins suggests that these proteins
are stored in different granules. The high propensity for mobilization of CFH and C3 suggest that
these proteins are likely to be stored in secretory granules92
. Neutrophil properdin is stored in
secondary granules198
, which have a much lower propensity for mobilization as demonstrated by
the varied response to activating stimuli209
. The secretion profile for CFB is similar to CFP
suggesting that they may be stored in the same granule. The varied secretion of complement
46
proteins in response to activation suggests a role for neutrophils in modulating complement
activation. Early activation and secretion of CFH and C3 can serve to regulate complement
activity and modulate complement response, where as activation and mobilization of CFP and
CFB can greatly amplify the complement AP.
47
Figure 8. Activated neutrophils secrete complement proteins.
Neutrophils were activated with different activating agents for 30 minutes. Cells were harvested
through centrifugation. Neutrophil lysates were prepared from the harvested cell pellet at a
concentration of 2 × 107 cells/ml. 50 μg of protein of lysate and equal volumes of supernatant, 50
μl, was loaded for detection of complement proteins. (A) CFP, 10% (w/v) non reducing SDS-
PAGE, 5 sec exposure on radiographic film. (B) C3, 10% (w/v) reducing SDS-PAGE, 5 min
exposure on radiographic film. (C) CFB, 10% (w/v) reducing SDS-PAGE, 5 min exposure on
radiographic film. (D) CFH, 10% (w/v) non-reducing SDS-PAGE, 5 min exposure on
radiographic film.
48
3.3 Quantification of Nuclear Morphologies During NETosis
Activated neutrophils can generate NETs consisting of DNA and antimicrobial peptides to
effectively ensnare and kill bacteria 100
. To validate our method of producing NETs, we activated
neutrophils with PMA (20 nM) and fixed them with paraformaldehyde (4% w/v) at various time
points (0, 30, 60, 120, 180, 240 min). Cells were stained with DAPI and analyzed with
fluorescence microscopy (Figure 9) .
When neutrophils are activated to form NETs, four distinct nuclear morphologies can be
identified (Figure 9A). Resting neutrophils are multilobed (multilobed nuclei), however as the
genomic DNA begins to decondense in preparation for NETs, the nucleus begins to lose its
multilobed structure and appears to be delobulated (delobulated nuclei). As the nuclear envelope
disintegrates to allow mixing of DNA with neutrophil granule proteins, the nucleus begins to
look diffuse (decondensed nuclei). Finally, neutrophil DNA along with its antimicrobial peptides
are released to form NETs (Neutrophil Extracellular Traps).
Upon identifying four distinct nuclear morphologies, we examined the population of each
morphology during the course of NET formation along various time points. In our experiment,
the earliest time for detection of NETs was at 120 minutes, however the percentage difference
between NETs and other nuclear morphologies was not statistically significant. At 120 minutes,
the predominant nuclear morphology was the delobulated nucleus (p<0.0001) (Figure 9B). At
180, NETs were the predominant nuclear morphology (p<0.0001) (Figure 9B). The percentage
of nucleus found in the decondensed stage remains very low throughout neutrophil activation.
This suggests that this morphology is short lived and as the nuclear envelope disappears, nuclear
DNA rapidly mixes with cytosolic and granular proteins to form NETs. The differential cell
counts for nuclear morphology validates that our procedure for inducing NETosis in neutrophils
isolated from human peripheral whole blood is consistent with the methods described in
literature100,116
.
49
Figure 9. Activated neutrophils form neutrophil extracellular traps.
Neutrophils were activated with PMA (20 nM) to induce the formation of NETs. Samples were
fixed with 4% (w/v) paraformaldehyde and stained with DAPI for microscopy. (A) Four distinct
nuclear morphologies (lobulated, delobulated, decondensed, NETs) can be identified during
NETosis. (B) Percentage difference for each nuclear morphology at various time points during
neutrophil activation. Percentage difference for nuclear morphologies was identified through
manual counting of at least 118 to 220 cells from 5 different focal planes at 40 × magnification.
Data are presented as mean ± SEM from 3 independent experiments. Statistical significance is
shown only if percentage of nuclear morphology is significant compared to all other
morphologies at the same time point. 2 way ANOVA with Tukey’s multiple comparison test, *p
<0.05, #p<0.001,
$p<0.0001.
50
3.4 PMA, but not C5a and fMLP, Induce NET Formation
Neutrophils are recruited to sites of tissue (e.g. endothelium) injury and/or infection by
cytokines (e.g. IL-1β and TNF-α), growth factors (e.g. G-CSF), anaphylatoxins (e.g. C5a) and
other cell damage signals (e.g. fMLP). Upon arrival to sites of high chemoattractant
concentration, neutrophils coordinate a strict regiment to deal with potential threats through
programmed phagocytosis, degranulation, and NETosis.
To determine whether neutrophil activation with complement anaphylatoxin C5a, or
pathogen/damage signal fMLP, can lead to the formation of neutrophil extracellular traps, we
utilized a SYTOX green plate reader assay. SYTOX green is a cell impermeable DNA dye that is
excitable at 488 nm. It exhibits a 500-fold fluorescence enhancement upon binding to nucleic
acids. Using this assay, we were able to detect the presence of extracellular DNA as fluorescence
emission at 520 nm. The fluorescence was then used to quantify the formation of NETs.
Neutrophils (3 × 104) in RPMI media were seeded onto 96 well plates in the presence of
SYTOX green (5μM). Neutrophils were activated with C5a (1 μM, 2 μM), fMLP (1 μM, 2 μM)
and PMA (20 nm) and fluorescence (485/520) was taken at various time points to monitor the
formation of neutrophil extracellular traps. Our data indicates that neither C5a (Figure 10A), nor
fMLP (Figure 10B), were was able to induce the formation of NETs. Only activation with PMA
(Figure 10C) led to the formation of NETs.
The mechanism for NETosis is heavily dependent on the formation of ROS210
. Our
earlier data demonstrate that stimulation of neutrophils with C5a did not lead to an oxidative
burst and activation with fMLP only led to a 1.5 fold increase in ROS (Figure 6). In contrast,
stimulation of neutrophils with PMA leads to a 9 fold increase in production of ROS (Figure 6).
Thus, our data indicate that neutrophil response to C5a and fMLP does not lead to the formation
of NETs. This may be due to insufficient production of oxygen radicals.
51
Figure 10. PMA, but not C5a and fMLP, induces NET formation.
Neutrophils (3 × 104) were seeded onto a 96 well tissue culture dish in the presence of SYTOX
green (5μM). NET formation was inhibited with addition of NOX inhibitor DPI (2μM). (A) C5a
(1μM or 2μM) activation of neutrophils did not lead to the formation of NETs. Data are
presented as mean ± SEM from 3 to 7 individual experiments. (B) fMLP (1μM or 2μM)
activation of neutrophils did not lead to the formation of NETs. Data are presented as mean ±
SEM from 3 to 7 individual experiments. (C) PMA (20 nM) activation of neutrophils led to the
formation of NETs. Data are presented as mean ± SEM from 7 individual experiments.
Fluorescence emission of SYTOX green was normalized to total DNA of resting neutrophils
permeabilized with 0.5% triton X-100. Two-way ANOVA with Tukey’s multiple comparison
test, $P < 0.0001.
52
3.5 Neutrophil Extracellular Traps Contain Properdin
Although not exclusive, the association of proteins to NETs can be associated with charge
interactions133
. Complement regulatory proteins, CFP and CFH, have been shown to associate
with polyanionic compounds23,211
. Moreover, our previous data suggest that C3 and CFB
associate with sialic acid upon neutrophil activation (Figure 7). Taken together, these data
strongly suggest that complement proteins may associate with polyanionic structures such as
neutrophil extracellular traps.
To determine if the complement AP proteins contained in neutrophils are present in
NETs, we isolated neutrophils from human peripheral whole blood and prepared them for
spinning-disc immunofluorescence confocal microscopy. Neutrophils were seeded onto 12 mm
coverslips, coated with poly-L-lysine, and activated with PMA (20 nM). After 240 minutes of
activation, neutrophils were fixed with 4% (w/v) paraformaldehyde. Cells were not
permeabilized because we were only interested in extracellular structures. Samples were stained
for CFP (sc-68366; Santa Cruz Biotechnology) (Figure 11), C3 (ab97462; Abcam) (Figure 12),
CFB (sc-57151; Santa Cruz Biotechnology) (Figure 13), and CFH (AF4779; R&D Systems)
(Figure 14). Samples were co stained with myeloperoxidase (ab25989; Abcam), a protein which
is known to be found in NETs. Our data indicates that CFP (Figure 11) can be found in NETs, as
it follows same pattern of distribution as neutrophil MPO and extracellular DNA stained with
DAPI. C3 (Figure 12), CFB (Figure 13) and CFH (Figure 14) were not found on NETs. From
these data, we can conclude that neutrophil properdin is associated with neutrophil extracellular
traps.
However, with these current data, we cannot rule out the possibility that other
complement proteins may also be present in NETs. We previously identified the concentration of
CFP to be much higher in neutrophils than the concentration of C3, CFB and CFH (Figure 5).
The concentration of these proteins may be below the limit of detection for immunofluorescence
microscopy. This is further complicated because attempts to detect these proteins through
increased exposure of fluorophore to the microscope laser or increasing detection sensitivity of
the camera can lead to autofluorescence in neutrophils 212-214
. Other more sensitive techniques
(e.g. ELISA) maybe required to definitively determine whether neutrophil complement proteins
are found in NETs. Our current data only demonstrate that CFP is associated with NETs.
53
Figure 11. Neutrophil extracellular traps contain properdin.
Neutrophil extracellular traps are prepared for spinning-disc immunofluorescence microscopy.
Properdin is found in secondary granules in resting neutrophils. In order to detect only properdin
on NETs, samples were not permeabilized. (A) Properdin was detected using secondary donkey
anti-rabbit Alexa Fluor ® 555 antibody. (B) Myeloperoxidase was detected using secondary
donkey anti-mouse Alexa Fluor ® 488 antibody. (C) Neutrophil extracellular traps were detected
with DAPI. (D) CFP and MPO colocalize to neutrophil extracellular traps. Z-stack images shown
are representative of images from five independent experiments taken with a 60×/1.35 oil
immersion objective. Scale bar, 9.00 μm.
54
Figure 12. C3 does not interact with neutrophil extracellular traps.
Neutrophil extracellular traps are prepared for spinning-disc immunofluorescence microscopy.
Samples are not permeabilized in order to detect protein associated with extracellular DNA. (A)
C3 was detected using secondary donkey anti-rabbit Alexa Fluor ® 555 antibody. (B)
Myeloperoxidase was detected using secondary donkey anti-mouse Alexa Fluor ® 488 antibody.
(C) Neutrophil extracellular traps were detected with DAPI. (D) C3 immunostain does not
associate with MPO or extracellular DNA. Z-stack images shown are representative of images
from five independent experiments taken with a 60×/1.35 oil immersion objective. Scale bar,
9.00μm
55
Figure 13. CFB does not interact with neutrophil extracellular traps.
Neutrophil extracellular traps are prepared for spinning-disc immunofluorescence microscopy.
Samples were not permeabilized in order to detect protein associated with extracellular DNA.
(A) CFB was detected using secondary donkey anti-rabbit Alexa Fluor ® 555 antibody. (B)
Myeloperoxidase was detected using secondary donkey anti-mouse Alexa Fluor ® 488 antibody.
(C) Neutrophil extracellular traps were detected with DAPI. (D) CFB does not appear to
associate with MPO or extracellular DNA. Z-stack images shown are representative of images
from five independent experiments taken with a 60×/1.35 oil immersion objective. Scale bar,
9.00μm
56
Figure 14. CFH does not interact with neutrophil extracellular traps.
Neutrophil extracellular traps are prepared for spinning-disc immunofluorescence microscopy.
Samples were not permeabilized in order to detect protein associated with extracellular DNA.
(A) CFH was detected using secondary donkey anti-goat Alexa Fluor ® 555 antibody. (B)
Myeloperoxidase was detected using secondary donkey anti-mouse Alexa Fluor ® 488 antibody.
(C) Neutrophil extracellular traps were detected with DAPI. (D) CFH immunostain does not
associate with MPO or extracellular DNA. Z-stack images shown are representative of images
from five independent experiments taken with a 60×/1.35 oil immersion objective. Scale bar,
9.00μm
57
3.6 NETs Increase Formation of Terminal Complement Complex
The presence of properdin on neutrophil extracellular traps suggests that NETs may activate
complement through the complement alternative pathway23
. Although there are several pathways
for complement initiation and activation, all pathways converge to the formation of the terminal
complement complex (C5b-9). Thus, complement activation can readily be detected through
formation of C5b-9.
To determine the effect of NETs on complement activation, neutrophils were isolated and
activated with PMA (20 nM) for 240 minutes in RPMI1640 supplemented with 10 mM Hepes.
After activation, neutrophils were centrifuged at 200 × g for 5 minutes and RPMI media was
replaced with 20% (v/v) sterile filtered autologous FFP in RPMI media. Complement competent
plasma was prepared with Refludan ®, a potent thrombin inhibitor to prevent coagulation. Since,
complement CP is dependent on Ca2+
and complement AP is dependent on Mg2+
, the use of
chelating agents as anticoagulants, such as EDTA, will inhibit complement activation and
therefore cannot be used to prepare complement competent plasma. After addition of 20% (v/v)
autologous FFP:RPMI, neutrophils were incubated at 37°C, 5% (v/v) CO2 for 15 minutes to
allow for the formation of C5b-9 and fixed with 4% (w/v) paraformaldehyde for spinning-disc
immunofluorescence confocal microscopy. In the rabbit hemolysis assay (Appendix B),
incubation of rabbit erythrocytes with 20% complement competent plasma leads to 67 to 100%
hemolysis (Table B2). Thus, complement activation is a rapid process and incubation of samples
with complement competent plasma for 15 minutes is sufficient for complement activation and
formation of C5b-9.
Our data indicate that addition of plasma did not initiate the formation of C5b-9 in non
stimulated neutrophils (Figure 15ADG). In the presence of NETs, the addition of complement
competent plasma led to the formation of C5b-9 (Figure 15BEH). This effect was abolished
when neutrophils were activated in the presence of DNase I (50 μg/ml) before the addition of
complement competent plasma (Figure 15CFI). Together, these data indicate that NETs can
activate complement pathways which result in the formation of C5b-9.
58
Figure 15. Neutrophil extracellular traps increase formation of C5b-9. Neutrophils were left untreated (ADG) activated with 20 nM PMA (BEH) and treated with
DNase I to disseminate the formation of NETs (CFI). After induction of NETs, 20% (v/v) sterile
filtered autologous FFP was added to samples. (A-C) NETs were visualized with DAPI, (D-F)
C5b-9 was detected using mouse monoclonal antibody to C5b-9 (DIA 011-01; Antibody shop).
Representative image from three independent experiments taken with 60×/1.35 oil immersion
objective is shown. Data are presented in extended focus. Scale bar, 9.00μm.
59
3.7 NETs Activate Complement Alternative Pathway to Form C5b-9
The presence of NETs is able to initiate complement activation to form C5b-9 (Figure 15).
However, the presence of complement AP proteins identified in neutrophils (Figure 5) strongly
suggests that complement amplification may be through complement AP.
In order to determine if NETs can activate specifically the complement AP, we utilized a
complement AP buffer, containing 20 mM Hepes, pH 7.4, 144 mM NaCl, 7 mM MgCl2, and 10
mM EGTA. EGTA is a chelating agent with low affinity for Mg2+
making it selective for Ca2+
.
The presence of EGTA inhibits complement CP, while the addition of Mg2+
enables the
activation of complement AP.
Neutrophils were activated with PMA (20 nM) for 240 minutes in RPMI1640
supplemented with 10 mM Hepes. After activation, neutrophils were centrifuged at 200 × g for 5
minutes and a buffer exchange was performed through three washes with PBS followed by one
wash with AP buffer. 20% (v/v) sterile filtered autologous FFP in AP buffer was added to each
sample and neutrophils were incubated at 37°C, 5% CO2 for 15 minutes to allow for the
formation of C5b-9. Samples were fixed with 4% (w/v) paraformaldehyde and prepared for
spinning-disc immunofluorescence confocal microscopy.
Our data indicate that addition of plasma in AP buffer did not initiate the formation of
C5b-9 in non stimulated neutrophils (Figure 16ADG). In the presence of NETs, the addition of
20% (v/v) plasma in AP buffer led to the formation of C5b-9 (Figure 16BEH). The pattern of
C5b-9 formation detected in AP buffer (Figure 16) varies with the pattern detected in RPMI
media (Figure 15). Complement activation is dependent on the presence of divalent cations Ca2+
and Mg2+
. RPMI media (Wisent Bioproducts, Montreal, QC, Canada) contains 400 μM of Ca2+
and Mg2+
. In contrast, AP buffer contains 7 mM of Mg2+
. In a buffer designed for complement
activation, we see a greater formation of C5b-9. This effect was abolished when neutrophils were
activated in the presence of DNase I to digest extracellular DNA (50 μg/ml) before the addition
of plasma in AP buffer (Figure 16CFI). Together, these data indicate that NETs can activate
complement through the alternative pathway to form C5b-9.
60
Figure 16. NETs activate complement alternative pathway to form C5b-9 Neutrophils were left untreated (ADG) activated with 20 nM PMA (BEH) and treated with
DNase I to disseminate the formation of NETs (CFI). A buffer exchange was performed with
three washes of PBS and one wash of AP buffer, followed by addition of 20% (v/v) autologous
plasma in AP buffer. (A-C) NETs were visualized with DAPI, (D-F) C5b-9 was detected using
mouse monoclonal antibody to C5b-9 (DIA 011-01; Antibody shop). Image was taken with
60×/1.35 oil immersion objective. Data are presented in extended focus. Scale bar, 9.00μm.
61
3.8 NETs Activate Complement and Activation of Complement AP on NETs is Dependent on Properdin
Properdin is the major positive regulator of complement AP. Blocking of properdin can inhibit
the amplification of complement AP without affecting other modes of complement
amplification215
. To investigate if other modes of complement activation are activated in NETs,
we utilized a mouse monoclonal antibody to properdin (A233; Quidel Corporation) that is
capable of blocking its activity215
. We performed a serial dilution of the mouse monoclonal
antibody, and complement activity was determined through rabbit erythrocyte hemolysis assay.
Rabbit erythrocytes do not contain sialic acid therefore are unable to bind CFH. In addition, they
lack the membrane bound regulators for complement AP, thus the constitutively active
complement AP is able to propagate on rabbit erythrocyte surface to form C5b-9 leading to cell
lysis. Complement activity is determined through measuring free hemoglobin in supernatants
with end point absorption at 405 nm.
The concentration of antibody required to inhibit the activation complement AP in 20%
(v/v) plasma varied from donor to donor. In three independent donors, we determined the
effective range varied from 2-8 μg/ml. The data presented correspond to 20% (v/v) plasma that
showed effective inhibition of complement AP from 4μg/ml of blocking antibody (Figure 17A).
To examine whether NETs activate other modes of complement amplification, neutrophils were
activated with PMA (20 nM) for 240 minutes to induce the formation of NETs. After induction
of NETosis, a buffer exchange is performed and neutrophil media is replaced with 20% (v/v)
autologous FFP in AP buffer or 20% (v/v) plasma in RPMI1650 supplemented with 10 mM
Hepes. Both these buffers are incubated in the presence or absence of 4 μg/ml of the blocking
antibody to CFP.
Our data reveal that NETs are able to propagate the assembly of C5b-9 when
supplemented with 20% (v/v) autologous FFP in AP buffer (Figure 17B). However inhibition of
CFP is able to stop the complement activation and C5b-9 formation (Figure 17C). NETs
supplemented with 20% (v/v) autologous FFP in RPMI are also able to propagate complement
activation to form C5b-9 (Figure 17D), however addition of mAb to CFP in RPMI does not
inhibit C5b-9 formation (Figure 17E).
62
Together, these data indicate that activation of complement AP on NETs is dependent on
properdin. However, inhibition of properdin had no effect on complement activation in RPMI
media, thus revealing that NETs activate complement through other modes of complement
activation in addition to complement AP.
63
Figure 17. Properdin is required for activation of complement AP on NETs. Mouse monoclonal antibody to properdin inhibits the activation of complement alternative
pathway on NETs. (A) Serial dilution is performed to determine the concentration of mouse
mAb to CFP required to inhibit complement AP as determined by rabbit erythrocyte hemolysis.
Neutrophils are activated with PMA (20 nM) to induce the formation of NETs. A buffer
exchange was performed followed by addition of (B) 20% (v/v) plasma:AP buffer – mAb CFP,
(C) 20% (v/v) plasma:AP buffer + mAb CFP, (D) 20% (v/v) plasma:RPMI1640 + 10 mM hepes
– mAb CFP, (E) 20% (v/v) plasma:RPMI1640 + 10 mM Hepes + mAb CFP. Image was taken
with 60×/1.35 oil immersion objective. Data are presented in extended focus. Scale bar, 9.00μm.
64
4 Results – Aim 2
To determine the significance of complement-NET interactions in antimicrobial function.
4.1 Identifying Bacteria That Induce NETosis
In order to determine the significance of neutrophil-complement interactions with regards to
antimicrobial function, we set out to identify bacteria, which can activate neutrophils to induce
the formation of neutrophil extracellular traps. Using the SYTOX green plate reader assay, we
activated neutrophils with a panel of different bacteria to examine their roles in activating
NETosis.
From our selected panel of bacteria, we identified that activation of neutrophils with
Pseudomonas aeruginosa (Figure 18) and Staphylococcus aureus (Figure 19A) led to the
formation of NETs. We had access to three different strains of P. aeruginosa. All three strains
mPAO1 (Figure 18A), PAKwt (Figure 18B), and PAKgfp (Figure 18C) led to the formation of
NETs and displayed similar kinetics to PMA activated NETs. Moreover, NET formation is
concentration dependent with regards to the number of bacteria used to activate neutrophils.
With all three strains, activation with a multiplicity of infection of 100 (MOI of 100) led to an
increase in NETs when compared with activation with a multiplicity of infection of 10 (MOI of
10).
Activation of neutrophils with S. aureus (RN6390), led formation of neutrophil
extracellular traps (Figure 19A). The formation of NETs in response to S. aureus was also
concentration dependent. Activation of neutrophils with MOI of 100 led to an increase in NET
production when compared to activation with MOI of 10. Activation of neutrophils with S.
aureus at MOI 100 led to a robust NET formation with SYTOX fluorescence being detected at
levels greater than total neutrophil DNA. Moreover, S.aureus activation using a MOI of 100
could not be completely inhibited with NOX inhibitor DPI, suggesting that the formation of
NETs in response to S. aureus has both ROS dependent and independent mechanisms.
Previous reports demonstrated that activation of neutrophils with bacterial
lipopolysaccharide (LPS) can lead to the formation of NETs100
. Interestingly, our data showed
that activation of neutrophils with gram-negative Escherichia coli (Y1088) at MOI 10 does lead
to the formation of NETs. The neutrophil response to E. coli is concentration dependent as
65
activation with a MOI of 100 led to the formation of NETs. There is a delayed onset for the
release of extracellular traps when neutrophils are activated with E. coli at a MOI of 100,
however there is a rapid increase of extracellular DNA at 180 minutes (Figure 19B).
Activation of neutrophils with Gram-positive Bacillus subtilis was also concentration
dependent. Activation with B. subtilis at a MOI of 10 did not lead to the formation of NETs.
Activation with B. subtilis at MOI 100 led to the formation of neutrophil extracellular traps
(Figure 19C).
Together, these data indicate that there are other factors outside of Gram-classification
which can influence neutrophils to form NETs in response to bacteria. LPS alone may not be
sufficient to induce the formation of NETs, as Gram-negative E. coli are poor inducers of
NETosis. In addition, bacterial LPS may not be required to induce formation of NETs, as Gram-
positive S. aureus is a strong inducer of NETosis. The neutrophil response to different virulent
factors may also play a role in triggering NETosis, for example, pyocyanin is a virulent factor
associated with P. aeruginosa and has been demonstrated to activate NET formation in an
NADPH dependent manner143
. Other virulent factors specific for activating NETosis have yet to
be identified. It may be possible that more virulent strains of otherwise harmless bacteria (e.g. E.
coli O157:H7) may possess virulent factors which can potentially trigger NETosis. Thus, our
current findings can only be applied to these specific strains of bacteria tested.
66
Figure 18. Pseudomonas aeruginosa activate neutrophils to form NETs
Neutrophils (3 × 104) in RPMI were seeded onto a 96 well tissue culture dish in the presence of
SYTOX green (5μM). NET formation was inhibited with addition of NOX2 inhibitor DPI
(2μM). Neutrophils were activated with different strains of Pseudomonas aeruginosa.(A) P.
aeruginosa, mPAO1. Data are presented as mean ± SEM from 5 to 7 individual experiments. (B)
P. aeruginosa, PAKwt. Data are presented as mean ± SEM from 5 to 7 individual experiments.
(C) P. aeruginosa, PAKgfp. Data are presented as mean ± SEM from 4 to 7 individual
experiments. Fluorescence emission of SYTOX green was normalized to total DNA of resting
neutrophils permeabilized with 0.5% triton X-100. Statistical significance compared to SYTOX
fluorescence from resting neutrophils. 1denotes statistical significance between different
multiplicities of infection. 2 way ANOVA with Tukey’s multiple comparison test, *p <0.05,
**p<0.01, #p<0.001,
$p<0.0001.
67
Figure 19. Neutrophils activated with S. aureus, E. coli, and B. subtilis.
Neutrophils (3 × 104) in RPMI were seeded onto a 96 well tissue culture dish in the presence of
SYTOX green (5μM). NOX2 dependent NETosis was inhibited with DPI (2μM). (A) S. aureus,
RN6390. Data are presented as mean ± SEM from 4 to 7 individual experiments. (B) E. coli,
Y1088. Data are presented as mean ± SEM from 5 to 7 individual experiments. (C) B. subtilis,
unknown strain. Data are presented as mean ± SEM from 4 to 7 individual experiments.
Fluorescence emission of SYTOX green was normalized to total DNA of resting neutrophils
permeabilized with 0.5% triton X-100. Statistical significance compared to SYTOX fluorescence
from resting neutrophils. 1 denotes statistical significance between different multiplicities of
infection. 2 way ANOVA with Tukey’s multiple comparison test, *p <0.05, **p<0.01, #p<0.001,
$p<0.0001.
68
4.2 Neutrophil Properdin Binds P. aeruginosa Independent of NETs
In the previous chapter, we demonstrated that neutrophil properdin can be found on NETs
(Section 3.5). To determine if neutrophils can target CFP onto bacteria through NETs, we
utilized a transgenic strain of pseudomonas, transformed using a mini-Tn7 transposon delivery
system. This chromosomally labeled P. aeruginosa (PAKgfp) expresses gfp under a
constitutively active promoter 216
.
Our data indicate that neutrophil properdin binds to Pseudomonas aeruginosa PAKgfp
(Figure 20). However, this was independent of neutrophil NET formation. Activation of
neutrophils with PAKgfp for 30 minutes (Figure 20ADGJ) led to CFP deposition onto bacteria.
Neutrophil activation at 30 minutes shows secretion of CFP in response to formylated peptides,
however, no NETs are formed at this time (Figure 8). Activation of neutrophils to induce the
formation of NETs also led to deposition of CFP onto bacteria (Figure 20BEHK). Addition of
DNase I to disseminate NETs did not result in decreased deposition of CFP to PAKgfp (Figure
20CFIL).
Statistical analysis of thresholded Pearson’s correlation between neutrophil properdin and
PAKgfp confirmed that properdin binds bacteria independent of NETs. Colocalization data were
obtained from 40 individually selected bacteria from 3 focal planes and average thresholded
pearson’s correlation from 3 independent experiments ± SEM was calculated. The colocalization
of neutrophil CFP to PAKgfp after 30 minutes of activation was 0.801 ± 0.038, the
colocalization of neutrophil CFP to PAKgfp in the presence of NETs was 0.740 ± 0.007, and
colocalization of neutrophil CFP to PAKgfp in the presence of DNase I was 0.779 ± 0.014.
Statistical analysis using 2 way ANOVA with Tukey’s multiple comparison test reported no
statistical significance between these values. Therefore, while neutrophil CFP is capable of
binding to P. aeruginosa, the formation of NETs does not actively target P. aeruginosa to
deposit CFP onto bacteria surfaces.
69
Figure 20. Properdin binds P. aeruginosa independent of NETs.
Neutrophils were activated with PAKgfp for 30 min (ADGJ), 240 min (BEHK) and 240 min
with DNase I (CFIL). Neutrophil properdin (D-F) binds to PAKgfp (G-I). Merged images (J-L)
with Pearson’s thresholded correlation ± SEM from three independent experiments. Z-stack
images shown are representative of images from three independent experiments taken with a
60×/1.35 oil immersion objective. Scale bar, 9.00μm.
70
4.3 NETs Increase Formation of C5b-9 in Response to Bacteria
In the previous chapter (Section 3.6) we demonstrated that NETs increase complement activation
and lead to the formation of the membrane attack complex. Although CFP was not targeted to P.
aeruginosa via NETs, the activation of complement on bacteria trapped by NETs can still be an
effective means of microbial clearance.
Neutrophils were activated with PAKgfp in the absence or presence of DNase I for 240
minutes. After activation, neutrophils were supplemented with 20% sterile autologous FFP and
incubated for an additional 15 minutes at 37°C, 5% CO2 to allow for the assembly of C5b-9.
Samples were fixed with 4% (w/v) paraformaldehyde for spinning-disc immunofluorescence
confocal microscopy.
We demonstrated that supplementing neutrophils with complement competent plasma,
did not initiate to the formation of C5b-9 in resting neutrophils that were not activated with P.
aeruginosa PAKgfp (Figure 21ADGJ). In the presence of NETs, the addition of complement
competent plasma led to the formation of C5b-9 (Figure 21BEHK). The formation of C5b-9 was
abolished when neutrophils were activated in the presence of DNase I (50 μg/ml), to disseminate
extracellular DNA, before the addition of complement competent plasma (Figure 21CFIL).
Together, these data indicate that NETs enhance complement amplification on trapped bacteria.
71
Figure 21. P. aeruginosa activation of neutrophils increases C5b-9 formation on NETs.
Neutrophils untreated (ADGJ), activated with PAKgfp (BEHK) in the presence of DNase I
(CFIL). Addition of 20% (v/v) autologous FFP to NETs (A-C) leads to increased deposition of
C5b-9 (D-F) onto PAKgfp (G-I) activated neutrophils. Representative image from three
independent experiments taken with 60×/1.35 oil immersion objective is shown. Data are
presented in extended focus. Scale bar, 9.00 μm.
72
4.4 NETs Activate Complement Alternative Pathway in Response to Bacteria
Complement activation can be initiated through several pathways, all of which converge to form
the terminal complement complex (C5b-9). NETs, which ensnare bacteria, are able to initiate the
complement response which results in the formation of C5b-9 (Figure 21). The abundance of
CFP in neutrophils suggests that they may be key mediators of the complement AP. The
dependency of the different complement pathways on different cations allows us to carefully
dissect which pathways of complement are activated to form C5b-9. Complement CP is
dependent on Ca2+
while complement AP is dependent on Mg2+
.
To determine if NETs can activate complement AP in response to P. aeruginosa, we
activated neutrophils with PAKgfp at a MOI of 10 to induce NETosis and performed a buffer
exchange. 20% (v/v) sterile filtered autologous FFP in AP buffer was added to each sample and
neutrophils were incubated at 37°C, 5% CO2 for 15 minutes to allow for the formation of C5b-9.
Samples were fixed with 4% (w/v) paraformaldehyde and prepared for spinning-disc
immunofluorescence confocal microscopy.
Our data indicate that addition of plasma in AP buffer does not initiate the formation of
C5b-9 in non stimulated neutrophils (Figure 22ADGJ). In the presence of NETs, the addition of
20% (v/v) plasma in AP buffer lead to the formation of C5b-9 (Figure 22BEHK). The ability of
NETs to propagate the formation of C5b-9 was abolished when neutrophils were activated in the
presence DNase I (50 μg/ml) before the addition of plasma in AP buffer (Figure 22CFIL).
Together, these data indicate that NETs formed in response to bacteria are effective at activating
complement AP. This suggests that NETs enhance complement amplification on ensnared
bacteria.
73
Figure 22. NETs activate complement AP to form C5b-9 in presence of P. aeruginosa.
Neutrophils were left untreated (ADGJ), activated with PAKgfp (BEHK) and treated with DNase
I (CFIL). A buffer exchanged was performed, followed by addition of 20% (v/v) autologous
plasma in AP buffer. NETs were visualized with (A-C) DAPI, and immunostained for (D-F)
C5b-9, and (G-I) GFP. Image was taken with 60×/1.35 oil immersion objective. Data are
presented in extended focus. Scale bar, 9.00 μm.
74
4.5 NETs Activate Complement in Response to P. aeruginosa and Complement AP Activation is Dependent on Properdin
Neutrophil extracellular traps formed in response to PMA are able to activate multiple
complement pathways (Section 3.8). As a mechanism for antimicrobial activity, NETs are very
potent at ensnaring invading bacteria. The ability to initiate multiple pathways of complement
activation on bacteria trapped in NETs could be a potent mechanism for microbial clearance.
To examine whether NETs can activate other pathways of complement activation in
response to P. aeruginosa, we utilized a mouse monoclonal antibody to properdin (A233; Quidel
Corporation) that is capable of blocking its activity215
. We used concentrations of this antibody
previously established to be effective in blocking complement AP activity (Figure 17A).
Neutrophils were activated with PAKgfp at a MOI of 10 for 240 min to induce the formation of
NETs. Following activation, a buffer exchange was performed and neutrophil media was
replaced with 20% (v/v) autologous FFP in AP buffer or 20% (v/v) plasma in RPMI1650
supplemented with 10 mM Hepes. Both these buffers are incubated in the presence or absence of
the mouse monoclonal antibody to properdin (A233; Quidel Corporation).
Neutrophils supplemented with 20% (v/v) autologous FFP in AP buffer are able to
propagate the assembly of C5b-9 in response to PAKgfp (Figure 23A). However, the formation
of C5b-9 in AP buffer was inhibited by the addition of the mouse monoclonal antibody to CFP
(Figure 23B). This suggests that complement AP activation on NETs in response to PAKgfp is
dependent on CFP. The addition of 20% (v/v) autologous FFP in RPMI to neutrophils was also
able to propagate the assembly of C5b-9 (Figure 23C), however the addition of mouse
monoclonal antibody to properdin did not block the assembly of C5b-9 (Figure 23D). This
suggests that in addition to complement AP, NETs are able to initiate the assembly of C5b-9
through other pathways of complement activation.
Together, these data indicate that activation of complement AP on NETs in response to P.
aeruginosa is dependent on properdin. However, inhibition of CFP had no effect on complement
activation in RPMI media, revealing that, in addition to complement AP, NETs activate
complement through other pathways in response to P. aeruginosa. The ability for NETs to
activate complement on bacteria may contribute to antimicrobial clearance.
75
Figure 23. NETs activation of complement AP in presence of P. aeruginosa is dependent on
Properdin.
Addition of mAb to properdin stops activation of complement AP on NETs. Neutrophils are
activated with PAKgfp at a MOI of 10. A buffer exchange was performed followed by addition
of (A-B) 20% (v/v) plasma:AP buffer, or (C-D) RPMI1640 + 10 mM Hepes; (AC) without mAb
to properdin, or (BD) with addition of mAb to Properdin. Images were taken with 60×/1.35 oil
immersion objective. Data are presented in extended focus. Scale bar, 9.00 μm.
76
5 Discussion
Failure to regulate the immune response can often lead to deleterious inflammatory damage
associated with neutrophil influx and complement activation. While evidence highlights
extensive cross-talk between neutrophil and complement activation, little is known with regards
to NETs. The goal of this work was to characterize the interactions between the complement
system and NETs in order to shed light on how the link between neutrophils and innate immunity
can contribute to disease pathology. For this we conducted ex vivo studies using neutrophils
purified from human periphal whole blood. We were able to demonstrate that, neutrophils, in
response to certain stimuli, can activate to form NETs which are able to propagate complement
activation to form C5b-9. The activation of complement AP on NETs is dependent on CFP,
however NETs are also able to initiate other pathways of complement activation that are
independent of CFP to form C5b-9. This suggests that NETs are able to amplify complement
activation through multiple pathways.
5.1 Characterizing Interactions of Complement and Neutrophils
We first identified complement proteins that are expressed in purified neutrophils. We found that
under resting conditions, neutrophils contain CFP, C3, CFB and CFH (Figure 5). Neutrophils are a
potent source of CFP in circulation. They synthesize CFP and store the protein in secondary granules
for release upon activation198. As a result, CFP was the most abundant protein of the four
complement proteins we identified in neutrophils. Consistent with literature, we also found that
neutrophils express C3 and CFB, although these proteins were found at much lower
concentrations198,217. CFP, C3 and CFB all play an important role in amplifying complement AP.
Certainly, in the presence of circulating CFD (1-8 μg/ml), neutrophils express all the necessary
constituents to activate complement through the AP205. In addition, we were able to detect expression
of CFH, the major soluble regulator of complement AP, in resting neutrophils. To our knowledge this
is the first time anyone has shown expression of CFH in neutrophil. The role neutrophil CFH plays in
the immune response is currently unknown. It may act as an adhesion molecule for phagocytosis, as
binding of CFH to CR3 mediates attachment to Candida albicans to enhance neutrophil
antimicrobial activity194. The identification of these proteins in neutrophils, which are intricately
involved in complement AP activation and regulation, suggest that they play a prominent role in
activating complement AP.
77
In order to gain a better understanding of how these complement proteins interact with
neutrophils, we decided to investigate the neutrophil response to various activating stimuli. We
selected complement C5a, a powerful anaphylatoxin, fMLP, which can serve as both a PAMP
and a DAMP, and PMA, a potent PKC activator. Neutrophil activation is associated with an
oxidative burst to produce ROS. Using flow cytometry, we measured neutrophil ROS production
using DHR123. Our findings indicate that activation with fMLP leads to mild oxidative burst and
activation with PMA leads to a dramatic production of ROS, however activation of neutrophil
with C5a is not sufficient to induce respiratory burst (Figure 6). This is consistent with the
literature as C5a has a priming effect on neutrophils. Activation with C5a alone is not sufficient to
induce oxidative burst as measured with DHR123218. However, priming of neutrophils enhances its
ability to produce reactive oxygen metabolites in response to activating stimuli219,220. Therefore, in
the presence of complement amplification and c5a anaphylatoxin production, neutrophils maybe
primed to more readily form ROS and undergo NETosis.
Another hallmark of neutrophil activation is granule mobilization and degranulation. To
further characterize neutrophil complement interactions in response to activation, we looked for
secretion of the previously identified complement proteins. We found that CFP and CFB secretion
varied with response to different activating stimuli, while CFH and C3 were completely secreted into
the supernatant (Figure 8). This sheds light on the granular localization of complement proteins
within neutrophils. As a reservoir for surface receptors, secretory vesicles have a low threshold for
mobilization and are completely mobilized to the surface upon activation in response to chemokines
(e.g. fMLP)92. The complete mobilization of CFH and C3 in response to all three of the activating
stimuli suggest that they are found in neutrophil secretory granules. This class of granule is relatively
new and a complete characterization of protein contents in the secretory vesicle has not yet been
performed95. Tertiary granules have a higher threshold for mobilization (20% mobilized by
fMLP)209,221, secondary granules have an even higher threshold for mobilization and primary
granules are often only mobilized partially88,209. CFP is a neutrophil protein known to be found in
secondary granules198. As a result, it is only partially mobilized in response to activation with C5a
and fMLP. The localization of CFB in neutrophils is currently unknown, however the varied
response of CFB secretion suggests that it is in one of these 3 other classes of granules that are
formed earlier during neutrophil development. The varying thresholds for granule mobilization
serves as a way for regulating neutrophil activation. In this way, mobilization of secretory vesicles
can decorate neutrophil plasma membrane with receptors vital for immune response without release
78
of harmful granule proteins95. Secretion of C3 and CFH at a much lower threshold suggest roles for
neutrophil in regulating complement response and opsonization without formation of convertases and
C5b-9. Secretion of CFB and CFP from neutrophils represents an efficient means for complement AP
amplification.
5.2 Properdin is Found on Neutrophil Extracellular Traps
To further characterize the interaction between complement system in neutrophils, we looked at
NETs. Using a SYTOX green plate reader assay, we found that neither C5a or fMLP alone was
sufficient to induce the process of NETosis (Figure 10). This indicates that complement
activation to produce anaphylatoxins or formylated peptides alone are not sufficient to trigger
neutrophils to form NETs. C5a however may play an important role in neutrophil priming to
enhance the process of NETosis. Consistent with literature, activation of neutrophils with PMA
triggered a robust oxidative burst (Figure 6) and formation of NETs (Figure 10)100.
Using PMA as a model for NETosis, we set forth to characterize the interaction of CFP, C3,
CFB and CFH with NETs. In 2009, mass spectrometry analysis identified 24 proteins to be
associated with NETs109. The complement proteins we identified were not present in this study.
However, complement proteins are often over looked in mass spectrometry analysis as they are often
associated with plasma proteins. Our findings indicate that CFP can be found on NETs (Figure 11).
C3 (Figure 12), CFB (Figure 13) and CFH (Figure 14), were not identified on NETs. However, our
findings suggest that the concentration of CFP in neutrophils is much greater than C3, CFB or CFH
(Figure 5). The concentration of these proteins dispersed in NETs may be below the limit of
detection for immunofluorescence microscopy. Attempts to detect these proteins by increasing
exposure of the fluorophore to the microscope laser or increasing detection sensitivity of the camera
can lead to autofluorescence in neutrophils 212-214
. Thus at this time, we are unable to conclude that
these proteins are not associated with NETs.
The mechanism for deposition of CFP onto NETs may in part be due to its pattern
recognition molecule properties. As a known pattern recognition molecule, CFP has been reported to
be able to bind to necrotic cells via surface DNA31. In circulation, CFP is a multimeric protein that
forms dimers, trimers and tetramers in a head to tail fashion33. The monomer is a 53 kDa protein
composed exclusively of 7 thrombospondin repeats (TSR)33,34. TSRs contain two arginine residues
which are thought to mediate binding to negatively charged sulfate moieties on glycosaminoglycans
(GAGs)23. While mechanism for CFP binding to DNA is currently unknown, like GAG, DNA is a
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large polyanionic compound. The properdin residues proposed to bind sulfate moieties of GAGs may
also be responsible for mediating properdin deposition onto NETs23. However, as the mechanisms for
NETosis are still not fully understood, we cannot the rule out the process of mechanical mixing in the
neutrophil as granular proteins rupture and the nuclear envelope decondenses prior the release of
NETs.
5.3 Neutrophil Extracellular Traps Activate Complement
CFP deposition onto NETs implied that NETs may amplify complement AP through CFP
dependent mechanisms. In addition, NETs are void of CFH, therefore they act as an activating
surface because they do not contain the proteins necessary for regulating complement AP203
. We
were able to successfully show the activation of complement on NETs through spinning disc
immunofluorescence confocal microscopy. Looking downstream to the final component of
activation, we were able to detect the formation of C5b-9 in the presence of NETs (Figure 15).
This process was dependent on NETs as activation of neutrophils in the presence of DNase I
completely abolished C5b-9 formation. To our knowledge, this is the first time anyone has shown a
direct link between NETs and complement activation leading to C5b-9. These data suggest that NETs
and complement interactions play a significant role in contributing to tissue damage during a
deleterious inflammatory response.
Mechanistically, we determined that complement activation on NETs is achieved through
multiple pathways. CFP is necessary for activation of complement AP on NETs as inhibition of CFP
is sufficient to stop the assembly of C5b-9 in 20% (v/v) plasma in AP buffer (Figure 17). The
inhibition of CFP in 20% (v/v) plasma in RPMI buffer was not sufficient in stopping the assembly of
C5b-9. Without chelation of Ca2+ or Mg2+ in RPMI buffer, all potential modes of complement
activation can be initiated. No current studies have demonstrated that MBL binding to NETs can
initiate complement LP, however the deposition of C1q on NETs has been shown to activate
complement CP157. In addition, neutrophil MPO and elastase have been associated with complement
activation. MPO production of HOCl oxidizes C5 and activates it to form C5b-9197. Neutrophil
elastase cleaves C5 to produce a functionally active C5b6 which can initiate the assembly of C5b-941.
Therefore, it may be possible for NETs to activate complement through non canonical pathways.
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5.4 Neutrophils form NETs in Response to Bacteria
Much of the previous work was conducted through activation with PMA. While these data
demonstrate clearly, the amplification of complement through NETs, little is known with regards
to how this interaction contributes to antimicrobial function. Using the SYTOX green plate
reader assay, we probed for the ability of neutrophils to form NETs in response to infection with
Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Bacillus subtilis
Consistent with literature120
, we were able to demonstrate that P. aeruginosa (Figure 18), and S.
aureus (Figure 19) were able to induce the formation of NETs. We also report that E. coli and B.
subtilis are poor inducers of NETosis. Activation of neutrophils at a multiplicity of 10 was
insufficient to induce NETosis, only activation of neutrophils with E. coli and B. subtilis at MOI
100 was able to induce NETosis (Figure 19).
We postulate that this may be indicative of the virulence of the pathogenic bacteria.
Pseudomonas aeruginosa and Staphyloccocus aureus are both opportunistic pathogens. P.
aeruginosa and S. aureus are opportunistic pathogens that often establish themselves in
immunocompromised individuals (e.g. patients with cystic fibrosis)222
. P. aeruginosa is a major
cause of infections worldwide (10% of all infections in EU hospitals) and is a series threat to
public health223
. S. aureus is a common cause for sepsis with a significant associated rate of
mortality224
. Neutrophils may respond to these virulent strains of bacteria with an “exaggerated”
response by forming NETs. Whether the formation of NETs in response to virulent strains of
bacteria attenuates or exacerbates disease prognosis has yet to be determined. In contrast, E. coli
and B. subtilis are considered non pathogenic bacteria which are often found in the human
gastrointenstinal tract. However, virulent strains of these bacteria do exist. E. coli O157:H7, for
example, is a virulent strain that causes hemolytic uremic syndrome (HUS), a disease
characterized by bloody diarrhea, microangiopathic hemolytic anemia, thrombocytopenia, and
renal impairment53
. Currently, no research has been conducted with regards to neutrophil
induction of NETosis with E. coli O157:H7. In the future, it may be worthy to investigate
whether virulent strains of different strains of bacteria are able to induce the formation of NETs.
5.5 NETs Formed in Response to Bacteria Activate Complement
To further explore the interaction between complement and NETs, and understanding how they
may facilitate an antimicrobial response, we looked at the interaction between NETS and P.
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aeruginosa. We chose to focus on gram-negative P. aeruginosa because the pattern recognition
molecule properties of CFP have been demonstrated to be able to bind bacterial LPS30
. To study
NET, CFP, P. aeruginosa interactions, we utilized a strain of P. aeruginosa PAKgfp that
expresses green fluorescent protein (GFP) through a constitutively active promoter (a gift from
the lab of Dr. Marina Ulanova, Northern Ontario School of Medicine)216
. While we were able
demonstrate the deposition of CFP onto pseudomonas (Figure 20), our data indicate that this
phenomenon occurs independent of NETs. Thus, neutrophil CFP found on NETs is not actively
targeted to bacterial surfaces to mount an antimicrobial response.
While NETs do not actively deposit CFP onto bacteria, our data clearly illustrate the
bacterial trapping capabilities of NETs (Figure 20). PAKgfp trapped in NETs (Figure 20BEHK)
can be found at a much higher concentration compared to neutrophils that are activated with
PAKgfp in the presence of DNase I to disseminate NETs (Figure 20CFIL). We previously
demonstrate that NETs, formed in response to PMA, can act as an activating surface to propagate
the formation of C5b-9 (Figure 15). Although, NETs do not actively target CFP onto bacteria,
bacterial trapping and complement amplification can work together to mount an effective
immune response. We then asked whether NETs formed in response to infection can initiate a
complement response. Our data demonstrate that NETs formed in response to PAKgfp can
initiate complement activation leading to the formation of C5b-9 (Figure 21). This process was
dependent on utilizing NETs as a complement activating surface, because activating neutrophils
in the presence of DNase I was able to abolish the formation of complement C5b-9. To our
knowledge, this is the first demonstration linking NETs to bacterial trapping and complement
amplification. These data suggest that in response to infection, neutrophils may form NETS to
facilitate an effective immune response. However, this also suggests that if this process
overwhelms regulatory mechanisms, infections can trigger an unwanted deleterious
inflammatory pathology.
Mechanistically, NETs that are formed in response to P. aeruginosa activate complement
in the same manner as NETs formed in response to PMA. The activation of complement AP on
NETs formed in response to bacteria is dependent on CFP as inhibition of CFP with mAb is
sufficient to stop the assembly of C5b-9 in 20% (v/v) plasma in AP buffer (Figure 23B). The
addition of mAb to CFP to 20% (v/v) plasma in RPMI was not sufficient in stopping the formation of
C5b-9 on NETs formed in response to bacteria (Figure 23D). Thus, in addition to complement AP,
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other pathways of complement activation are at work on NETs. Therefore, in addition to containing
many antimicrobial peptides, the activation of complement on NETs may be an effective method for
antimicrobial clearance.
Integrating our findings from the interaction between complement and neutrophils, we
propose a model where infectious triggers can initiate a neutrophil response for mobilization and
activation (Figure 24). Neutrophil activation prompts the secretion of complement proteins contained
in neutrophils to modulate and activate the complement response. In addition, the formation of NETs
is effective in capturing bacteria and initiates complement amplification. Overall, the interactions
between complement and neutrophils may work together to mount an effective immune response.
83
Figure 24. Proposed model of complement-neutrophil interactions. The activation of neutrophils leads to the secretion of complement proteins. Secretion of CFP is
capable of binding to invading microbes. Further activation of neutrophils will lead to the
formation of NETs. CFP is able to bind to both bacteria and NETs, however whether CFP
enhances the ability for NETs to capture bacteria has yet to be determined. NETs initiate the
amplification of multiple complement pathways which leads to the formation of C5b-9. The
activation of complement AP initiated by NETs is dependent on CFP.
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6 Conclusion and Future Directions
6.1 Conclusions
From the work presented here, we conclude that neutrophils contain key proteins of the
complement alternative pathway. This further strengthens the notion that neutrophils are key
mediators of inflammatory and immune responses. Neutrophil activation coordinates a strict
regiment to deal with potential threats (e.g. infection or tissue damage) through programmed
phagocytosis, degranulation and NETosis. We show that in response to activating stimuli (e.g.
C5a, fMLP, and PMA) complement proteins contained in neutrophils are secreted. Also, secreted
properdin is effective in recognizing and binding to P. aeruginosa. In addition, neutrophil
properdin was demonstrated to be deposited on NETs. The addition of complement competent
plasma to NETs revealed that they are able to initiate and activate complement. Inhibition of
properdin in AP buffer was sufficient in stopping the formation of C5b-9, however it could not
stop the formation of C5b-9 in RPMI buffer. Thus, we demonstrate that while NETs are able to
initiate complement AP, they also activate complement through other initiating pathways.
Neutrophils are a major determinant of local complement AP activity as neutrophil
release of CFP is able to dramatically increase local concentrations64
. Therefore we proposed that
NETs may also lead to activation of complement AP. We successfully demonstrated that NETs
can activate complement, however, a caveat to our current finding is that we were not be able to
demonstrate that this property is dependent on neutrophil properdin. Circulating CFP is found at
relatively low concentrations in plasma (4-6 μg/ml), however this concentration is still sufficient
in stabilizing the complement AP C3 convertase to amplify this constitutively active pathway63
.
Further research needs to be conducted to elucidate the mechanisms by which NETs activate
complement. In addition, while our ex vivo model successfully demonstrates that NETs are able
to activate complement, further research needs to be conducted to investigate whether this
interaction can contribute to anti microbial activity or endothelial cell damage.
6.2 Future Directions
Our work demonstrates the potential for NETs to interact with complement to mediate an
inflammatory response. There are still many questions which remain to be addressed to bridge
85
the gap between this ex vivo model and understanding how this interaction can contribute to
microbial clearance or pathology in an in vivo setting.
We demonstrated that NETs are formed in response to PMA, P. aeruginosa, and S.
aureus. The activation of neutrophils with C5a did not induce the formation of NETs, however, a
study has demonstrated that C5a primed neutrophils enhances neutrophil oxidative burst in
response to ANCA220
. Since the formation of NETs is largely dependent on production of ROS,
it is important to address whether C5a primed neutrophils are more susceptible to NETosis, thus
further bridging the gap between complement and neutrophils.
Our analysis using spinning-disc confocal microscopy reveals that CFP is found on
NETs, however we were unable to identify the presence of the other complement proteins
contained in neutrophils on the NETs. As we alluded to earlier, at this time, we can not conclude
that these proteins are not found in NETs as the low concentration of these proteins may be
below the detection limits for immunofluorescence microscopy. In order to address this question,
an optimized technique for excising proteins from NETs needs to be established and more
sensitive methods (e.g. ELISA or Mass-spectrometry) need to be utilized to definitively answer
this question.
In our hypothesis, we proposed that neutrophils mount a targeted antimicrobial response
through NETs to initiate complement. We anticipated that NETs would enhance the deposition
of CFP onto bacteria. Our finding that NETs do not enhance the deposition of CFP onto P.
aeruginosa was an interesting revelation. However, the ability of CFP to act as a pattern
recognition molecule allows it to bind various ligands. In addition, CFP exists as a multimer
allowing it to interact with multiple ligands. CFP has been shown to bind to both LPS and DNA.
Therefore, it is likely that the deposition of CFP onto P. aeruginosa may enhance the ability of
NETs to trap bacteria. Further studies need to be conducted to address this.
In order to look for the activation of complement on NETs, we probed for the assembly
of C5b-9. Our data clearly demonstrate that NETs can activate complement, and that
complement AP activation on NETs is dependent on CFP. However, the formation of C5b-9 on
NETs is somewhat puzzling. At this moment, there does not appear to be a discernible pattern for
C5b-9 deposition. C5b-9 does not appear to show preference for NET DNA, nor does it show
preference to PAKgfp. However, C5b-9 does appear to be localized to various clusters scattered
86
throughout the NET. This suggests that there is an activating factor which directs the formation
of C5b-9 on NETs. Further studies need to be conducted to identify where C5b-9 is being
assembled on NETs.
We also need to address the caveat in our current finding. While we show that NETs are
complement activating, we have not shown that this process is dependent on neutrophil CFP. To
fully understand the mechanism on how NETs can activate the different complement initiating
pathways, we need to utilize the full arsenal of inhibitory antibodies against complement proteins
and utilize complement-depleted plasma, which is commercially available . However, addressing
this question will be technically challenging, because complement depleted plasmas are often
also complement in active. We may also utilize properdin knockout mouse models to address
this caveat, however the murine complement system is different from the human complement
system. Complement deficient mice do not display the same pathological symptoms or
phenotypes as their human counterparts.
Our ex vivo model successfully demonstrated that NETs are able to activate complement
activity. However, whether this process mounts an antimicrobial response or contributes to cell
damage remains to be addressed. To address whether complement and NETs work together to
mount an antimicrobial response, bacteria colony counts need to be performed in the presence or
absence of NETs and/or complement competent/inactive plasma. The introduction of endothelial
cells to our ex vivo model (e.g. HUVEC, BOEC)225
will be required to address whether
complement-NET interactions can lead to endothelial cell damage.
Neutrophils have often been shown to play an important, but poorly understood role in
complement mediated pathologies. Here we show that NETs are able to initiate complement
activity. However, in patients who have deficiencies in complement regulatory proteins, this
localized activation of complement may overwhelm the delicate balance of complement
activation and regulation to precipitate into pathology. It would be worth investigating if there is
increased complement activity on NETs for patients who have known mutations in complement
regulatory proteins. This work may answer many of the mysteries behind complement mutations
and pathology.
87
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8 Appendix
Appendix A. Optimizing the Protocol for Neutrophil Lysates
When I first embarked on this project, one of the greatest challenges was to prepare a neutrophil
lysate. The trouble with preparing a neutrophil lysate arises from the neutrophil granules which
are very abundant in proteases. In addition, neutrophil myeloperoxidase is able to produce
hypochlorous acid, thus creating an environment that is unfavorable for recovering proteins.
In the literature, neutrophil research is often conducted with microscopy or flow
cytometry. For molecular work, neutrophil proteins are usually collected using a lysis buffer,
however the contents of the lysis buffer are often not disclosed. My first attempts to prepare a
neutrophil lysate using a lysis buffer containing 1% (v/v) Triton X-100 supplemented with 1 ×
cOmplete, mini protease inhibitor cocktail (Roche Diagnostics, Laval, QC, Canada) were
unsuccessful. In addition, heat treatment to prevent protein degradation was also unsuccessful226
.
While I was able to detect CFP in lysates, I was not able to detect loading controls such as β-
actin or GAPDH through western blot.
Moving forward, with advice from Dr. Lisa Robinson (The Hospital for Sick Children) I
was able to successfully prepare my first neutrophil lysate using a lysis buffer containing 1%
(v/v) Triton X-100, 45mM Tris-HCL pH 7.4, 120 mM NaCl, 1mM PMSF, 1mM Na3VO4, 2mM
PMSF and 2 × cOmplete, mini protease inhibitor cocktail. Neutrophils were lysed on ice for 10
minutes followed by sonication. To evaluate the efficiency of the different methods for preparing
neutrophil lysates, samples were separated with 10% (w/v) SDS-PAGE and proteins were
stained with Coomassie Brilliant Blue (Figure A1).
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Figure A1. Evaluating different methods for preparing neutrophil lysates.
Coomassie Blue staining was used to determine the most effective method for preparing
neutrophil lysates. Neutrophils were isolated from peripheral whole blood and lysates were
prepared using (A) 1% (v/v) Triton X-100 + 1 × cOmplete, mini protease inhibitor cocktail. (B)
1% (v/v) Triton X-100 + 1 × cOmplete, mini protease inhibitor cocktail heat treated at 95°C. (C)
1% (v/v) Triton X-100, 45mM Tris-HCL pH 7.4, 120 mM NaCl, 1mM PMSF, 1mM Na3VO4,
2mM PMSF and 2 × cOmplete, mini protease inhibitor cocktail. Samples were separated with
10% (w/v) SDS-PAGE.
105
To further validate that this method for preparing neutrophil lysates was effective, I
utilized immunoblot analysis to detect common proteins that are used as loading controls.
Neutrophil lysates were separated with 10% (w/v) SDS-PAGE, transferred onto nitrocellulose
membrane. Western blot analysis was able to successfully detect myosin, β-actin, and GAPDH
in neutrophil lysates. In addition, we were able to identify the large membrane receptor CR1 in
neutrophil lysates (Figure A2).
Figure A2. Detection of proteins from neutrophil lysates. Neutrophil lysates were prepared and separated on 10% (w/v) SDS-PAGE. (A) Detection of
common loading controls, Myosin, β-actin, and GAPDH. (B) Detection of known neutrophil
membrane protein complement receptor 1.
The protocol for the preparation of neutrophil lysates has since evolved to what is now described
in this thesis (section 2.4) after continued consultation with literature and discussions with
experts in neutrophil biology at the international symposium on The Neutrophil in Immunity
(Quebec City, QC, Canada).
106
Appendix B. Hemolysis Assay Data
A rabbit erythrocyte hemolysis assay was used to determine complement activity. A serial
dilution of the monoclonal antibody to CFP was performed in 20% (v/v) plasma in AP buffer to
determine the effective concentration of antibody required to inhibit complement AP on rabbit
erythrocytes. The effective concentration of antibody required to inhibit complement AP on
rabbit erythrocytes was dependent on donor plasma and varied from 2-8 μg/ml. The hemolysis
assay was performed on three separate dates and raw data for hemolysis determined through end
point reading at 405 nm can be found in (Table B1). The values are normalized to total
hemolysis of 2.5 × 107 rabbit erythocytes (50 μl) in distilled water (Table B2). The plasma from
June 19, 2013 displayed decreased complement activity after blockade with 2 μg/ml of
monoclonal antibody to CFP. Plasma from June 20, 2013 and July 4, 2013 showed decreased
complement activity after 4 μg/ml of monoclonal antibody to CFP (Figure B1). The data and
figures presented in this thesis corresponds neutrophils and plasma collected from the donor on
July 4, 2013 where 4 μg/ml of monoclonal antibody to CFP could effectively stop complement
AP activity (Figure B1C).
Table B1. Hemolysis absorption at 405 nm. Raw data as determined through endpoint reading at 405 nm. Controls A: AP Buffer, B: Rabbit
erythrocyte in AP buffer, C: Total hemolysis, D: 20% (v/v) plasma in AP buffer
Controls mouse monoclonal anti Factor P (μg/ml)
Date: A B C D 8 4 2 1 0.5 0.25 0.125
June 19, 2013 0.039 0.336 2.324 1.574 N/A 0.735 0.805 1.209 1.403 1.324 1.369
June 20, 2013 0.036 0.142 2.545 2.034 0.586 0.839 1.73 1.875 1.831 2.031 2.306
July 4, 2013 0.043 0.143 2.912 3.136 0.438 0.572 2.769 2.797 2.82 3.5 2.716
Table B2. Hemolysis absorption normalized to total hemolysis. Hemolysis data normalized to total hemolysis of rabbit erythrocytes resuspended in distilled
water. Controls A: AP Buffer, B: Rabbit erythrocyte in AP buffer, C: Total hemolysis, D: 20%
(v/v) plasma in AP buffer
Controls mouse monoclonal anti Factor P (μg/ml)
Date: A B C D 8 4 2 1 0.5 0.25 0.125
June 19, 2013 0.017 0.145 1 0.677 N/A 0.316 0.346 0.520 0.604 0.570 0.589
June 20, 2013 0.014 0.056 1 0.799 0.252 0.330 0.680 0.737 0.719 0.798 0.906
July 4, 2013 0.015 0.049 1 1.077 0.172 0.196 0.951 0.961 0.968 1.202 0.933
107
Figure B1. Complement activity assay determined by rabbit erythrocyte hemolysis.
Activity of complement AP was determined using rabbit erythrocyte hemolysis. A serial dilution
was performed to determine the concentration of mouse monoclonal antibody to CFP required to
inhibit complement AP. Three independent experiments from dates (A) June 19, 2013 (B) June
20, 2013 (C) July 4, 2013.
