Author's Accepted Manuscript

Oxidative stress-dependent activation of theeIF2α-ATF4 UPR branch by skin sensitizerDNFB modulates dendritic-like cell matura-tion and inflammatory status in a biphasicmanner

Andreia Luís, João Demétrio Martins, Ana Silva,Isabel Ferreira, Maria Teresa Cruz, BrunoMiguel Neves

PII: S0891-5849(14)00427-4DOI: http://dx.doi.org/10.1016/j.freeradbiomed.2014.09.008Reference: FRB12145

To appear in: Free Radical Biology and Medicine

Cite this article as: Andreia Luís, João Demétrio Martins, Ana Silva, IsabelFerreira, Maria Teresa Cruz, Bruno Miguel Neves, Oxidative stress-dependentactivation of the eIF2α-ATF4 UPR branch by skin sensitizer DNFB modulatesdendritic-like cell maturation and inflammatory status in a biphasic manner,Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freerad-biomed.2014.09.008

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Title

Oxidative stress-dependent activation of the eIF2�-ATF4 UPR branch by skin

sensitizer DNFB modulates dendritic-like cell maturation and inflammatory status

in a biphasic manner

Authors:

Andreia Luís*, João Demétrio Martins

†,‡, Ana Silva

†, Isabel Ferreira

†, Maria Teresa

Cruz†‡

, Bruno Miguel Neves*,†,1

Affiliations

*Department of Chemistry, Mass Spectrometry Centre, QOPNA, University of Aveiro,

Campus Universitário de Santiago, 3810-193 Aveiro - Portugal

† Centre for Neuroscience and Cell Biology, University of Coimbra 3004-517, Coimbra

– Portugal

‡Faculty of Pharmacy, University of Coimbra 3004-517, Coimbra – Portugal

Corresponding author:

1Bruno Miguel Neves: Department of Chemistry, Mass Spectrometry Center, QOPNA,

University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro – Portugal

(E-mail: neves_bruno@sapo.pt; Telephone: +351-964182278

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Abstract

The pathogenesis of allergic contact dermatitis, the most common manifestation of

immunotoxicity in humans, is intimately connected to hapten-induced maturation of

dendritic cells (DC). The molecular mechanisms driving this maturational program are

not completely known, however initial danger signals such as the generation of reactive

oxygen species (ROS) were shown to play a critical role. Recent evidence linking ROS

production, endoplasmic reticulum (ER) stress and the pathogenesis of several

inflammatory diseases lead us to analyze, in the present work, the ability of the skin

sensitizer 1-Fluoro-2, 4-dinitrobenzene (DNFB) to evoke ER stress in DC-like THP-1

cells and the concomitant consequences to their immunobiology. We found that DNFB

triggers a ROS-dependent activation of the PERK-eIF2�-ATF4 unfolded protein

response (UPR) branch conferring cytoprotection and modulating the

maturation/proinflammatory cell status in a biphasic manner. Early DNFB induction of

ATF4 positively modulates autophagy related genes MAP1LC3B and ATG3 and

stabilizes the transcription factor Nrf2 causing a strong induction of HMOX1

detoxifying gene. Moreover, we observed that in a first phase, DNFB-induced ATF4

up-regulates IL8 mRNA levels while blocking CD86, IL1B, IL12B and CXL10

transcription. Later, following ATF4 decay, HMOX1 and IL8 transcription drastically

decrease and CD86, IL1B and Il12B are up-regulated. Overall, our results evidence a

connection between sensitizer-induced redox imbalance and the establishment of ER

stress in DC-like cells and provide new insights into the role of UPR effectors such as

ATF4 to the complex DC maturational program.

Keywords: Allergic contact dermatitis, ROS, ER stress, dendritic cell maturation,

ATF4, autophagy.

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Introduction

Allergic contact dermatitis (ACD) is a T-cell-mediated type IV hypersensitivity reaction

caused by skin exposure to low molecular weight reactive chemicals (haptens). It is the

most common manifestation of immunotoxicity in humans with a prevalence reaching

20% in developed countries [1, 2]. The pathophysiology of ACD encompasses two

distinct stages: 1) a sensitization phase following the first contact with the hapten and 2)

an elicitation phase triggered by subsequent exposures. During the sensitization phase,

chemicals react with cellular and soluble proteins forming immunogenic hapten-protein

complexes. Antigen presenting cells in the skin, such as Langerhans cells and dermal

dendritic cells (DC), capture these haptenized-proteins, mature and migrate to draining

lymph nodes where they prime naïve T-cells. T-cells become activated and expand into

allergen-specific effector T-cells that disseminate systemically and elicit a strong

inflammatory reaction upon later contact with the same chemical [3]. Despite intense

investigation, the molecular mechanisms by which skin sensitizers trigger and shape DC

maturation are not completely known. Recent evidence suggest that haptens activate

innate inflammatory pathways common to anti-infectious responses [4-6], a sterile

inflammatory process termed xenoinflammation [7]. In this process, early danger

signals such as ATP/ADP [8, 9], hyaluronic acid fragments [10] and reactive oxygen

species (ROS) [11-13] activate intracellular signal transduction pathways that ultimately

are responsible for the DC maturation program (reviewed in [14]). Although sensitizer-

evoked oxidative and electrophilic stresses are well characterized modulators of DC

immunobiology, data regarding the possible contribution of endoplasmic reticulum

(ER) stress is still lacking. In eukaryote cells, ER plays crucial roles in multiple cellular

processes including lipid biosynthesis, intracellular calcium homeostasis and protein

folding and assembly. As consequence of insults such as oxidative stress, iron

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imbalance, leaking of Ca2+

, protein overload, and hypoxia, unfolded/misfolded proteins

accumulate in the ER lumen triggering ER stress [15]. This leads to the activation of an

integrated program known as the unfolded protein response (UPR), that in mammals

comprises 3 parallel branches: inositol-requiring kinase 1 (IRE1), protein kinase-like

ER kinase (PERK) and activating transcription factor (ATF6) pathways. UPR primarily

drives the reestablishment of ER homeostasis by slowing down translational processes,

chaperoning misfolded proteins and by promoting cytoprotective mechanisms such as

ER-associated protein degradation (ERAD) and autophagy. However, if unresolved, ER

stress results in inflammation within affected tissues and can ultimately lead to

apoptotic cell death [16].

Intracellular redox imbalance and ER stress are closely linked events. Oxidative stress

affects ER functions through perturbation of protein processing/transport and by

disturbing Ca2+

homeostasis and perturbations in protein folding increase mitochondrial

ROS production causing alterations in cellular redox status [17-19]. Since ROS are

common early danger signals in ACD and a major cause of ER stress, it is plausible that

sensitizer-induced redox imbalance may evoke ER stress in DC with consequences to

their maturational program. Although data regarding the role of ER stress and UPR

effectors on DCs immunobiology is scarce, recent studies have unravelled important

relations between them. ER stress-induced transcription factor C/EBP homologous

protein (CHOP) was shown to synergize with TLR signalling for the expression of IL-

23 cytokine in monocyte-derived dendritic cells [20]. It was also demonstrated that ER

stress and its effector XBP-1 contribute to dendritic cell maturation [21] and that certain

types of ER stress prime DC to respond to innate immune stimuli by activating

interferon regulatory factor 3 (IRF3) with a consequent increase in type I interferons

expression [22].

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Therefore, in the present study, we sought to investigate the ability of the skin sensitizer

1-Fluoro-2, 4-dinitrobenzene (DNFB) to evoke ER stress in a DC-like cell model and to

assess the relevance of mounted UPR to DC immunobiology. We found that DNFB

triggers the PERK-eIF2�-ATF4 branch of the UPR in a ROS-dependent way and that

this axis mainly drives cytoprotective effects. Early DNFB-induced ATF4 up-regulates

autophagy related genes such as MAP1LC3B and ATG3 and cooperates with

transcription factor Nrf2 to strongly induce HMOX1 detoxifying gene. We also

observed that transiently expression of ATF4 contributes to the modulation of

maturation/proinflammatory DC-like cell status by initially limiting IL1B, IL12B, CD86

and CXCL10 gene transcription while exacerbating IL8 production. Following ATF4

decay, IL8 mRNA levels dramatically fall and IL1B, IL12B and CD86 are strongly

induced. It is, to our knowledge, the first report showing the involvement of ER stress

and its UPR effectors in the skin sensitizer-induced maturation/activation of dendritic-

like cells.

Materials and Methods

Materials

RPMI 1640 medium, penicillin, streptomycin, the anti-tubulin antibody, acridine orange

(AO) and N-Acetyl cysteine (NAC) were obtained from Sigma Chemical Co. (St. Louis,

MO, USA). Fetal Bovine Serum (FBS), TRIzol, CellRox Green Reagent, DAPI and

Alexa Fluor® 555 Phalloidin were from Invitrogen (Paisley, UK). The protease and

phosphatase inhibitor cocktails were obtained from Roche (Mannheim, Germany).

Pharmacological inhibitors SB203580, U0126, SP600125, Sal003 and GSK2606414

were from Calbiochem (San Diego, California, USA). Antibodies against phospho-

p44/p42 MAPK (ERK1/ERK2), phospho-p38 MAPK, phospho-SAPK/JNK, phospho-

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eIF2�, CHOP, p62, LC3B and total p38 MAPK were from Cell Signaling Technologies

(Danvers, MA, USA). The anti-JNK1 and anti-ERK antibodies were from Millipore

(Bedford, MA, USA), anti-XBP1-s, anti-GRP78, Alexa-Fluor®-488 anti-CD86, Alexa

Fluor®-488 Mouse IgG2b, � Isotype control�antibodies and recombinant human IL-4

and GM-CSF were from Biolegend (San Diego, CA, USA). Anti-Nrf2 antibody was

from Santa Cruz Biotechnology (Dallas, TX, USA) and anti-ATF6 was from Novus

Biologicals (Cambridge, UK). The alkaline phosphatase-linked secondary antibodies

the enhanced chemifluorescence (ECF) reagent and Ficoll-Paque Plus were obtained

from GE Healthcare (Chalfont St. Giles, UK), and the polyvinylidene difluoride

(PVDF) membranes were from Millipore Corporation (Bedford, MA, USA). iScript

Select cDNA Synthesis Kit and SYBR Green master mix were from BioRad (Hercules,

CA, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA) or

from Merck (Darmstadt, Germany).

THP-1 Cell culture

THP-1 human monocytic cell line (ATCC TIB-202, American Tissue Culture

Collection, Manassas, VA, USA) was cultured and maintained at a cell density between

0.2x106 and 0.8x10

6 cells/mL in RPMI 1640 medium supplemented with 10% of heat-

inactivated FBS, 25 mM glucose, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL

penicillin and 100 �g/mL streptomycin (complete RPMI). Cells were sub-cultured every

2–3 days and kept in culture for a maximum of 2 months.

Differentiation of monocyte-derived DC (MoDC)

Peripheral blood mononuclear cells were obtained from fresh EDTA-treated blood of

healthy human donors (after their informed consent), by density gradient centrifugation

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with Ficoll-Paque Plus. Briefly, blood was diluted with equal parts of PBS and 20 ml

samples were seeded in 50 ml Falcon tubes containing 15 ml of Ficoll-Paque Plus. The

tubes were centrifuged at 450g, for 30 min at 20 �C and the mononuclear fraction

collected and washed twice with PBS. After counting, peripheral blood mononuclear

cells (PBMC) were resuspended in the appropriate volume of PBS supplemented with

0.5% FBS and 2mM EDTA (MACS buffer) and monocytes isolated with human

Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) according to

manufacturer instructions. Briefly, T cells, NK cells, B cells, dendritic cells and

basophils, were indirectly magnetically labeled using a cocktail of biotin-conjugated

antibodies against CD3, CD7, CD16, CD19, CD56, CD123 and Glycophorin A, and

highly pure unlabeled monocytes collected by negative selection. Monocytes were then

cultured for 7 days, in complete RPMI-1640 medium supplemented with 500 U/ml IL-4

and 800 U/ml GM-CSF. One half of the culture medium was replaced on day 3 and day

5, by fresh medium containing the same cytokines concentrations. At day 7, the

phenotype of these cells is consistent with an immature DC population: HLA-DR+,

CD1a+, CD83

−, CD80

low and CD86

low [23].

Cell viability assay

Cell viability was assessed by resazurin assay [24]. Briefly, 0.2x106 cells/well in a 96

well plate were exposed to different DNFB concentrations (2, 4, 8, 10 and 15 µM), for

24h. Three hours before the end of exposure, resazurin solution was added to each well

to a final concentration of 50 µM. Absorbance was then read at 570 and 600 nm in a

standard spectrophotometer MultiSkan Go (Thermo Fisher Scientific, Waltham, MA,

USA). Since a certain level of cytotoxicity is required for effective DC activation [25]

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the concentration of DNFB that induced up to 20% cytotoxicity was determined and

used hereinafter in all experiment unless stated otherwise (Supplementary Figure 1).

Total and nuclear cell lysates preparation

To obtain total cell lysates for Western blot analysis, THP-1 cells were plated at 2.4x106

cells/well in 6-well microplates in a final volume of 3 ml. The cells were then incubated

with 8 µM DNFB for the indicated time periods. As a positive control, parallel assays

were performed by exposing cells to Tunicamycin (5 µg/ml), a known inducer of ER

stress. In some experiments cells were treated with different concentrations of DNFB (4

µM, 8 µM, 16 µM and 24 µM) for 1 or 8 h. To address the possible crosstalk between

the UPR response and the DNFB-induced MAPK kinases activation, cells were pre-

treated for 1 h with NAC (5 mM) or with the pharmacological inhibitors SB203580 (10

µM), U0126 (10 µM), SP600125 (20 µM), Sal003 (20 µM) and GSK2606414 (1 µM)

and then stimulated with 8 µM DNFB for the indicated time periods. At the end of

exposure, cells were washed in 1 ml ice-cold PBS and harvested in RIPA lysis buffer

(50 mM Tris–HCl (pH 8.0), 1% Nonidet P-40, 150 mM NaCl, 0.5% sodium

deoxycholate, 0.1% SDS, 2 mM EDTA and 1 mM DTT) freshly supplemented with

protease and phosphatase inhibitor cocktails. The nuclei and the insoluble cell debris

were removed by centrifugation at 4ºC, at 12,000 g for 10 min. The post-nuclear

extracts were collected and used as total cell lysates.

For nuclear cells extracts, 9 x106cell were used for each indicated condition and extracts

obtained with the Nuclear Extract Kit (Actif Motif, Carlsbad, CA, USA) according to

manufacturer´s instructions. Protein concentration was determined using the

bicinchoninic acid method and the cell lysates denatured at 95ºC, for 5 min, in sample

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buffer (0.125 mM Tris pH 6.8; 2%, w/v SDS; 100 mM DTT; 10% glycerol and

bromophenol blue) being then stored at -20ºC for posterior use.

Western blot analysis

Briefly, 30 µg of protein were separated by electrophoresis on a 12% (v/v) SDS-

polyacrylamide gel, transferred to polyvinylidene fluoride (PVDF) membranes and

blocked with 5% (w/v) fat-free dry milk in Tris-buffered saline, containing 0.1% (v/v)

Tween-20 (TBS-T), for 1 h at room temperature. Blots were then incubated overnight at

4ºC with the primary antibodies diluted (1:1000) in 1% (w/v) fat-free dry milk TBS-T.

The membranes were washed with TBS-T and incubated, for 1 h at room temperature,

with alkaline phosphatase-conjugated anti-rabbit (1:20,000) or anti-mouse (1:20,000)

antibodies. The immune complexes were detected by membrane exposure to the ECF

reagent for 5 minutes, followed by scanning for blue excited fluorescence on the

Typhoon imager (GE Heatlcare, Chalfont St. Giles, UK). The generated signals were

analysed using Total Lab 2009 software (TotalLab Ltd, Durham, USA). To demonstrate

equivalent protein loading, membranes were stripped and reprobed with antibodies to

total ERK1/2, SAPK/JNK, p38 MAPK, or with an anti-tubulin antibody. Briefly,

membranes were washed twice (15 min each) in TBS-T with strong agitation and once

for 5 min in MilliQ H2O followed by 5 min in 0.2M NaOH. The membranes where then

washed for 5 min in MilliQ H2O and blocked for 1h at room temperature in 5% (w/v)

fat-free dry milk in TBS-T. At the end of blocking process membranes were incubated

with primary antibodies and finally, blots were developed with alkaline phosphatase-

conjugated secondary antibodies and visualized by enhanced chemifluorescence as

described above.

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Analysis of gene expression by qPCR

After cell treatment for the indicated times, total RNA was isolated with TRIzol reagent

according to the manufacturer’s instructions. The RNA concentration and possible

contamination were assessed by OD260 measurement using a NanoDrop

spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and samples stored in

RNA Storage Solution (Ambion, Foster City, CA, USA) at -80°C until use. Briefly, 1

µg of total RNA was reverse-transcribed using the iScript Select cDNA Synthesis Kit

and real-time quantitative PCR (qPCR) reactions were performed using SYBR Green on

a Bio-Rad MyCycler iQ5 as previously described [26]. After amplification, the

fluorescence threshold was calculated using the iCycle iQ System software and the

results normalized using HPRT1 as reference gene. This gene was experimentally

determined with Genex software (MultiD Analyses AB, Göteberg, Sweden) as the most

stable for the treatment conditions used. The final comparison of transcript ratios

between samples was calculated by relative quantification method corrected for specific

primer efficiencies [27]. Primer sequences (Supplementary Table 1) were designed

using Beacon Designer software version 7.7 (Premier Biosoft International, Palo Alto,

CA, USA) and thoroughly tested.

ROS production assay

To visualize DNFB-induced ROS formation, 1x105

THP-1 cells were plated in Poly-L-

Lysine coated µ-Chamber slides (IBIDI GmbH, Germany) and, after an overnight

stabilizing period, loaded with 5 �M Cell ROX® Green Reagent for 30 min. The cells

were then washed three times and treated with 8 µM DNFB during 30 min following a

new washing step with PBS and fixation during 15 min with 4% paraformaldehyde.

Cell permeabilization was achieved by treatment with 0.2% (v/v) Triton-X-100, 200

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mM Glycine in PBS for 10 min at room temperature and F-actin stained by adding

Alexa Fluor® 555 Phalloidin diluted 1:100 in PBS during 30 min. After washed twice

with PBS, cells were finally exposed to DAPI nuclear probe (100 nM) for 2 min and the

slides analysed with a fluorescent microscope (Nikon Corporation, Japan) at 630X

magnification. Images were captured with a DS-Fi2 High-definition digital camera and

analysed in NIS-Elements Imaging Software (Nikon Corporation, Japan)

Determination of lysosomal membrane destabilization

The effects of DNFB on lysosome functional state and lysosomal-cytosolic pH gradient

were analysed using acridine orange (AO). AO is a lysosomotropic metachromatic

fluorochrome that once excited with blue light, emits red fluorescence when highly

concentrated at acidic pH (intact lysosomes) and green fluorescence at low

concentrations (in the cytosol and the nucleus) [28]. For the assay, 1x105

THP-1 cells

were treated with 8 µM DNFB during 4, 8 or 24 h, washed with PBS and resuspended

in medium containing 5 µg/ml AO. Following 20 min incubation at 37ºC, cells where

washed twice with PBS and analysed by fluorescence microscopy.

Flow Cytometry analysis

For the analysis of surface expression of the costimulatory marker CD86, THP1 cells

were plated at 1 × 106 /well in 12-wells microplates and then treated with 8 µM DNFB,

20 µM Sal003 or Sal003 + DNFB for 24 h. Cells were then washed in PBS and

incubated for 30 minutes at 4ºC with 1:20 Alexa-Fluor 488® conjugated CD86 antibody

or respective isotype control (Mouse IgG2b, k). After two washing steps with PBS/2%

FBS, cells were analyzed by flow cytometry in a FACSCalibur (BD Biosciences)

equipped with CellQuest Pro software.

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Statistical analysis

Results are presented as mean ± the standard deviation (SD) of the indicated number of

experiments. Comparisons between two groups were made by the two-sided unpaired

Student’s t test and multiple group comparisons by One-Way ANOVA analysis, with a

Bonferroni´s Multiple Comparison post-test. Statistical analysis was performed using

GraphPad Prism, version 5.02 (GraphPad Software, San Diego, CA, USA).

Significance levels are as follows: *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001

Results

DNFB induces a rapid activation of the eIF2�-ATF4 UPR branch and a

posttranslational modification of the ER major chaperone GRP78

To test our hypothesis that skin sensitizers such as DNFB may induce ER stress in DC

affecting their immunobiology, we started to address its effects on the levels of key

proteins from the three UPR branches. Parallel experiments were performed with

Tunicamycin, a well-known ER stress inducer. We observed that, in contrast to

Tunicamycin, DNFB does not trigger a complete canonical ER stress response (Figure

1A and 1B). While Tunicamycin clearly activates the three branches of UPR, IRE-

XBP1s, PERK-eIF2� and ATF6 (Figure 1B), DNFB mainly affects the PERK-eIF2�-

ATF4 branch, causing a strong and time sustained phosphorylation of eIF2� and an

early increase of ATF4 protein levels (Figure 1A). Of note is also the fact that DNFB,

although it just slightly up-regulates the levels of GRP78, causes the appearance of a

low molecular weight immunoreactive protein form in a time dependent manner (Figure

1A). This suggests that DNFB may induce a posttranslational modification in GRP78

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with possible consequences to its functions. Several studies report GRP78 post-

translational modifications such as phosphorylation and ADP ribosylation, being the

existence of these modified forms correlated with the physiological activity of the ER

[29, 30]. Since immunoreactive form detected in DNFB-treated cells presents a low

molecular weight we hypothesize that DNFB-induced ROS formation may be causing

the oxidative cleavage of GRP78 in a process similar to the recently described HSP90

oxidative cleavage [31].

Results from gene expression study corroborate those from protein analysis as we

observed that DNFB modestly induce common ER stress controlled transcripts. Among

the studied genes, the most robustly up regulated were those encoding for the

detoxifying proteins selenoprotein S (SELS) and Homocysteine-responsive endoplasmic

reticulum-resident ubiquitin-like domain member 1 protein (HERP), with maximal

increases observed at 3h post treatment (3.9 and 6.7 fold change relatively to control for

SELS and HERP, respectively) (Figure 1E). Regarding CHOP and GRP78, despite

statistically significant, the observed increases at early time points are very modest (5.1

and 2.4, respectively) and the levels rapidly decrease to values similar to untreated cells

(Figure 1E). This observation is in accordance with the results from Western Blot

analysis where CHOP protein remained undetectable upon DNFB exposure and levels

of unmodified GRP78 were just slightly up-regulated. In contrast, treatment of THP-1

cells with Tunicamycin caused a significant and sustained increase of all analysed

genes, being CHOP and HERP the most robustly up regulated (Figure 1F).

Effects of DNFB over eIF2�, GRP78 and ATF4 are ROS-dependent

The production of ROS following DCs exposure to skin sensitizers is assumed to be an

early danger signal that triggers intracellular signalling cascades that ultimately

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modulate cell maturation [13]. Considering the hypothesis that DNFB may trigger ER

stress through a ROS dependent mechanism we have analysed DNFB ability to

effectively induce oxidative stress in THP-1 cells. For this purpose, the levels of

oxidative stress were measured by fluorescence microscopy with the ROS sensitive

probe Cell ROX® Green Reagent. Upon oxidation, this probe binds to DNA with a

strong fluorescence increase, being its signal primarily localized in the nucleus and

mitochondria. We observed that DNFB causes a strong and rapid ROS accumulation in

THP-1 cells (Figure 2A), confirming that this event may represent an early danger

signal during the sensitization phase of ACD. Next, in order to evaluate the relationship

between DNFB-induced redox imbalance and the observed effects on eIF2�, GRP78

and ATF4, cells were pre-treated with the antioxidant N-acetylcysteine (NAC) and these

proteins levels determined by Western blotting. The observed DNFB effects were dose-

dependent and cell pre-treatment with NAC significantly mitigates the DNFB-induced

phosphorylation of eIF2�, the expression of ATF4 and the low molecular weight

immunoreactive GRP78 form (Figure 2B). This indicates that the observed activation of

eIF2�-ATF4 UPR branch is, at least in part, a consequence of the redox imbalance

caused by DNFB.

In order to conclude about the occurrence of this events in a more accurate dendritic cell

model we performed several experiments in primary DC differentiated from human

peripheral blood monocytes. As can be seen in Figure 3, the response pattern of

primary DC to DNFB was similar to the one obtained for THP-1 cells: DNFB rapidly

induces the phosphorylation of eIF2a and a concomitant increase of ATF4 protein levels

and this effect is almost completely reverted by pre-treatment with NAC.

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DNFB-activated MAPK and the elicited-UPR effectors are not interrelated

The activation of MAPKs in DC, particularly p38 MAPK, is a common feature of skin

sensitizers [14]. Therefore, we first assessed the effects of DNFB on the levels of

phosphorylated p38, ERK1/2 and JNK1/2 in a 24 hours’ time-course experiment. As

can be seen in Figure 4A, exposure to DNFB caused a rapid and significant activation

of p38 MAPK and JNK signalling pathways. These two kinases presented however a

distinct behaviour over time: while JNK1/2 remained highly phosphorylated during the

24h of the assay, p38 presented maximal activation at 1h post cell treatment with

phosphorylation progressively decaying over time (Figure 4A). The obtained results

suggest a constitutive activation of ERK1/2 in THP-1 that is not significantly increased

when cells were exposed to DNFB.

We further investigated the existence of a possible crosstalk between the DNFB-

activated MAPKs and the observed eIF2�-ATF4 axis activation. For this purpose THP-

1 cells were pre-treated with the antioxidant NAC or with pharmacological inhibitors of

the different pathways as described in Material and Methods section. As shown in

Figure 4B, pre-treatment of cells with NAC markedly reduces the DNFB-induced

phosphorylation of JNK, p38 and eIF2�. These results highlight ROS as key early

danger signals that are central for the activation of signalling cascades in skin

sensitizers-exposed DC. Treatment with the PERK inhibitor GSK2606414 resulted in a

significant decrease in eIF2� phosphorylated levels, indicating that activation of the

eIF2�-ATF4 branch is at least in part a direct consequence of DNFB-induced ER stress.

Additionally, Sal003, an analogue of the eIF2�-specific phosphatase inhibitor Salubrinal

with higher solubility, significantly increased the eIF2� phosphorylation induced by

DNFB. Neither GSK2606414 nor Sal003 had significant effects on the levels of p-

JNK1/2, p-38 and p-ERK, indicating that DNFB-evoked ER stress does not influence

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MAPKs activation (Figure 4B). One the other hand, inhibition of MAPKs pathways

does not significantly affect eIF2� phosphorylation triggered by DNFB. Overall, these

results suggest that DNFB-induced MAPKs and eIF2�-ATF4 axis activation are both

ROS-dependent but not interrelated with one another and probably trigger

complementary phenotypic and functional programs in dendritic cells.

ATF4 cooperates in the DNFB-induced transcription of autophagy-related genes

MAP1LC3B and ATG3

The eIF2�-ATF4 pathway was recently shown to regulate crucial aspects of the stress-

induced autophagic process [32]. Therefore, we sought to investigate whether DNFB

triggers autophagy and whether ATF4 is involved in the process. For this purpose we

analysed the levels of microtubule-associated protein light chain 3 (LC3) and p62. As

can be observed in Figure 5A, DNFB significantly induced the levels of LC3B-II in a

concentration-dependent way, and this effect was completely abrogated by cell pre-

treatment with NAC. Since the amount of LC3B-II at a certain time point does not

specifically indicates activation of autophagy, and may evenly be caused by a blockade

of autophagic flux [33], we also analysed the levels of p62. p62 is a protein that binds to

LC3, serving as a selective substrate of autophagy. Therefore, in situations where

autophagy is activated, p62 levels are expected to decrease. We found that DNFB-

treated cells do not present decreased levels of p62 and rather a slight concentration-

dependent increase was observed (Figure 5A). These results suggested that DNFB

could be blocking the autophagic flux in THP-1 cells. To explore this hypothesis we

performed an LC3B-II turnover assay and assessed the effects of DNFB over lysosomal

membrane integrity. In the LC3 turnover assay, degradation of LC3-II inside the

autolysosome was estimated by comparing LC3-II levels of DNFB-treated cells in the

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presence or absence of the lysosomal inhibitor chloroquine (Figure 5B). LC3B-II levels

were significantly higher when cells were simultaneously exposed to DNFB and

chloroquine than when they were treated with each chemical individually. This

indicated that even though DNFB may be blocking autophagic flux it is also inducing

the autophagic machinery, or at least the expression of LC3. Moreover, we found that

the observed DNFB-induced blockade of autophagic flux was in part due to a

destabilization of lysosomal membranes. As shown in Figure 5C, at early time points

(4h), DNFB causes a partial lysosomal membrane rupture that results in acridine orange

diffusion from the lysosomes to the cytosol. This is observed as a decrease in

punctuated red fluorescence (lysosomes) with a concomitant green fluorescence

increase in the cytosol and nucleus (Figure 5C). This effect appears to be reversible

since cells regain normal lysosomal function 24h post DNFB exposure. Once again, the

destabilization caused by DNFB was almost completely abolished when cells were pre-

treated with NAC, indicating a direct role for redox imbalance (Figure 5C).

Next we sought to investigate the role of eIF2�-ATF4 axis on DNFB-induced

autophagy. As initial experimental approach we tried to block PERK-dependent eIF2�

phosphorylation by treatment with the PERK inhibitor GSK2606414, however

paradoxal effects were observed: for short time periods (1h) as shown in Figure 4B we

observed the expected decrease in p-eIF2� and concomitantly in ATF4. However for

long exposure times (>4h), GSK2606414 caused a paradoxal increase in ATF4 levels

(data not shown). We speculate that prolonged inhibition of PERK may be rendering

eIF2� more susceptible to phosphorylation by its other kinases (HRI, PKR and GCN2).

As alternative approach we treated THP-1 cells with Sal003, a specific inhibitor of

eIF2� dephosphorylation that has as main result the preferential translation of proteins

with open reading frames (uORFs) in there 5´UTR mRNA sequence, particularly ATF4.

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This gave us a similar outcome of ATF4 overexpression. We checked in these

conditions the resultant effects on the levels of LC3B-II and on the transcription of

several autophagy related genes. We observed that ATF4 was significantly unregulated

at 1h post DNFB exposure but decreased to barely detectable levels after 8h (Figure

5D). This decay was completely prevented in cells simultaneously exposed to Sal003.

Moreover, stabilizing DNFB-induced ATF4 expression with Sal003 resulted in a

significant increase in LC3B-II protein levels (DNFB vs Sal003+DNFB; p<0.001),

indicating that this transcription factor may play a role on DNFB-induced autophagic

process (Figure 5D). However, although cell exposure to Sal003 alone also resulted in

ATF4 up-regulation, this was not followed by an LC3B-II increase, who may indicate

that ATF4 cooperates with other DNFB-induced transcription factors to increase LC3

expression. These results were corroborated by gene expression analysis. As shown in

Figure 5E, DNFB significantly induced the transcription of MAP1LC3B gene

(6.17±0.33) and concomitant treatment with Sal003 resulted in an even higher induction

(9.55±1.25) (DNFB vs Sal003+DNFB; p<0.05). ATG3 gene transcription followed a

similar behaviour although the differences between DNFB and Sal003+DNFB were not

statistically significant. As observed at the protein level for LC3B-II and p62, Sal003

doesn’t have significant effects on the transcription of these genes.

The transient activation of eIF2�-ATF4 axis by DNFB modulates DC-like cell

maturation and inflammatory status in a biphasic manner.

Finally, we addressed the relevance of eIF2�-ATF4 axis on the DNFB-induced THP-1

maturation/inflammatory status. The mRNA levels of the pro-inflammatory cytokines

IL-1� and IL-12p40, the chemokines IL-8 and CXCL10, the co-stimulatory molecule

CD86 and of the detoxifying protein HMOX-1 were evaluated by qPCR at 6 and 24h

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post DNFB exposure. In some experiments, DNFB-induced ATF4 expression was

stabilized over 24h by cell treatment with Sal003. Two distinct transcription patterns for

the genes analysed were observed upon DNFB treatment. The detoxifying protein

HMOX-1 and the pro-inflammatory cytokine/chemokine IL-8 were rapidly and robustly

induced after 6h, 2037±301 and 98±35 fold changes, respectively, but drastically

decrease to 16±6 and 25±7 after 24h (Figure 6A). Exposing cells simultaneously to

DNFB and Sal003 resulted in a significant inhibition of this decay, indicating a clear

role for ATF4 on the positive transcriptional control of HMOX1 and IL8 genes. By

contrast, CD86, IL-1� and IL-12p40 were only significantly up-regulated after 24h and

these increases were robustly blocked by ATF4 stabilization (Figure 6A). The

transcription of CXCL10 gene was, in turn, found to be significantly reduced by DNFB

at both time points and, contrarily to the other genes analysed, cell treatment with

Sal003 alone also caused a significant modulation. Finally, flow cytometry experiments

were performed to analyse the effects of DNFB-induced ATF4 stabilization on CD86

surface expression. In agreement to results from qPCR experiments, treatment of cells

with DNFB caused and increase in CD86 positive population, being this increase

strongly impaired by pre-treatment with Sal003 (Figure 6B).

As the positive transcriptional control played by ATF4 appeared to result from its

cooperation with other transcription factors, we addressed whether it interacts with Nrf2

and NF-�B. As shown in Figure 7, the nuclear levels of ATF4 and Nrf2 have a similar

kinetic: following DNFB exposure they are both rapidly translocated to the nucleus and

then suffer a rapid decay over time. Strikingly, we found that blocking ATF4 decay by

cell treatment with Sal003 resulted in nuclear stabilization of DNFB-induced Nrf2

levels. This indicates that ATF4 cooperates with and stabilizes Nrf2, thus modulating

the transcription of ARE regulated genes. This explains the observed positive effect of

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ATF4 stabilization on the transcription of HMOX1 (Figure 6A), as this gene is known to

be highly dependent on the Keap1-Nrf2-ARE pathway. Regarding the NF-kB

transcription factor, we did not observe any significant nuclear translocation of the p65

subunit following DNFB exposure either with or without ATF4 stabilization by Sal003

(Figure 7).

Overall, these results evidence that, in a first phase, the early DNFB-induced ATF4

cooperates with other transcription factors, namely Nrf2, to up-regulate the transcription

of HMOX1 and IL8 while blocking the transcription of CD86, IL1B, IL12B and CXL10.

In a second phase, following ATF4 decay (Figures 5D and 7), HMOX1 and IL8 gene

transcription drastically decrease and CD86 and IL1B are up-regulated.

Discussion

The chemical-induced activation/maturation of DC is a keystone in the pathogenesis of

allergic contact dermatitis. Mature DC migrate to lymph nodes and effectively present

processed antigens to naïve T-cells leading to their polarization in effector and memory

T-cell populations. The sensitizer-induced DC maturation process is not completely

disclosed, however it was shown to be strongly dependent on initial danger signals such

as imbalance of cell redox status and release of intracellular ATP [8, 13, 34]. Reactive

oxygen species deplete thiol groups and cause the activation of intracellular signalling

pathways such as p38 and JNK MAPK, which ultimately drive the phenotypical and

functional changes that characterize DC maturation. In recent years, several studies

have shown a close link between ROS production, endoplasmic reticulum stress and the

pathogenesis of several chronic inflammatory, neurodegenerative, cardiovascular and

autoimmune diseases [17]. Despite intense research activity regarding ER stress and its

consequences, the information concerning its effects on DC immunobiology remains

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scarce. Therefore, in this work, we evaluated the ability of the extreme skin sensitizer

DNFB to induce ER stress in DC-like THP-1 cells, as well as the relevance of the

evoked UPR to cell detoxifying and maturation/inflammatory status. We were able to

demonstrate that DNFB triggers, in a ROS-dependent way, the PERK-eIF2�-ATF4

branch of the UPR. This axis mainly drives an early cytoprotective program while

transiently blocking THP-1 cell maturation.

In accordance to previous works, we observed a rapid and strong increase in ROS

production following DNFB exposure [11]. This redox imbalance was shown to cause

the observed UPR activation given that p-eIF2� and ATF4 protein levels were markedly

reduced in cells pre-treated with the antioxidant NAC. Therefore, our observations

support growing evidence of a close interplay between oxidative stress and endoplasmic

reticulum dysfunction. Accumulation of ROS causes leak of Ca2+

from the ER lumen by

mechanisms that may involve oxidation of critical thiol groups in the ryanodine receptor

causing its inactivation [35, 36]. In turn, increases in cytosolic Ca2+

stimulate

mitochondrial electron-transport chain activity, leading to generation of more ROS.

This vicious cycle culminates in a massive depletion of Ca2+

stores who causes ER

dysfunction and misfolded protein accumulation. Recently, Malhotra and collaborators

showed that the relationship between oxidative stress and ER stress is bidirectional and

that the accumulation of unfolded proteins in the ER lumen is per se sufficient to trigger

ROS production [37].

In the present study, we found that DNFB mainly activates the PERK-eIF2�-ATF4

branch of the UPR. In mammals, in addition to PERK, eIF2� may be phosphorylated by

PKR, a sensor of viral RNA, by general control non depressible 2 (GCN2), a sensor of

amino acid starvation, and by heme-regulated inhibitor (HRI) in heme depletion

conditions [38]. Although we cannot discard the involvement of these kinases in the

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observed eIF2� phosphorylation, our results point to a major contribution of the ER

stress related kinase PERK since p-eIF2� levels were significantly reduced by cell pre-

treatment with GSK2606414. GSK2606414 is a new potent and selective PERK

inhibitor that has been shown to completely block PERK-dependent eIF2�

phosphorylation in vitro and in vivo [39]. Physiologically, the major consequence of

eIF2� phosphorylation is a rapid attenuation of mRNA translation in order to prevent

the influx of newly synthesized polypeptides into the stressed ER [38]. Besides this

general protein synthesis inhibition, specific proteins such as ATF4 are up-regulated

[40]. In our study, to address the contribution of ATF4 to DC immunobiology we

treated cells with Sal003, a selective inhibitor of p-eIF2� phosphatases. Although this

may cause the translation of other proteins with uORFs such as ATF5, CEBPA and

CEBPB the main consequence is the selective translation of ATF4. Hence, although

very unlikely, we admit that Sal003 may be exerting non-ATF4-dependent effects. The

transcription factor ATF4 controls the expression of genes involved in oxidative stress

detoxification, amino acid synthesis, differentiation, metastasis and angiogenesis [41-

45]. Among ATF4 downstream targets genes, CHOP is one of the most studied, which

serve to amplify the restructuring of the transcriptome to manage stress or to direct cell

fate toward apoptosis. In the present work, although increased levels of ATF4 were

detected in DNFB-treated cells, we did not observe a concomitant up-regulation of

CHOP. Accordingly, Harding and co-workers have shown that in Perk-/- fibroblasts,

forced ATF4 overexpression in absence of an additional stress stimulus was not

sufficient for CHOP induction [45]. Therefore, our findings reinforce the idea that

additional stress signals other than those transmitted by the PERK-eIF2�-ATF4 branch

are required for CHOP induction in response to ER stress. Another possible explanation

for the lack of CHOP expression despite the observed ATF4 increase is the repressor

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effect of DNFB-induced Nrf2 over the CHOP promoter. Several works report that the

activation of Nrf2 pathway negatively correlates with CHOP expression, and recently it

was demonstrated that Nrf2 affects CHOP transcription by precluding the binding of

transcription factors such ATF4 to the CHOP promoter [46, 47].

Given that ER stress effector proteins such as IRE1�, PERK and eIF2� were shown to

crosstalk with MAPKs, particularly JNK and p38 [48-50], we analysed the existence of

a possible link between DNFB-induced eIF2� phosphorylation and MAPKs activation.

In accordance to previous reports we observed that DNFB rapidly triggers p38 and JNK

signalling pathways and that this activation is dependent on sensitizer-evoked oxidative

stress [11, 13]. In our experimental conditions we were unable to found a significant

relation between eIF2�-ATF4 axis and MAPKs activation although we do not discard a

possible interaction in prolonged time exposures.

The UPR may be primarily viewed as a cellular response to restore ER homeostasis

through promotion of cytoprotective mechanisms such as ERAD and autophagy [51,

52]. Recently, a connection between ROS production, activation of PERK-eIF2�-ATF4

pathway and autophagy was also established [53]. In the referred work, extracellular

matrix detachment of mammary epithelial cells resulted in ROS-dependent activation of

canonical PERK-eIF2�-ATF4 pathway which in turn induced the autophagy regulators

ATG6 and ATG8. In agreement with these findings, we observed that DNFB-induced

ROS-dependent activation of the eIF2�-ATF4 axis positively modulates the autophagy

machinery in THP-1 cells. We showed an ATF4-dependent transcriptional up-

regulation of the autophagy related genes MAP1LC3B and ATG3. Supporting our

results, B´Chir and collaborators elegantly demonstrated that the eIF2a-ATF4 pathway

fine-tunes the autophagy gene transcription program in response to ER stress [32].

These authors identified three classes of autophagy-related genes according to their

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dependence on ATF4 and CHOP transcription factors: genes such as MAP1LC3B,

ATG3 and ATG12 are only dependent on ATF4; ATG5 and ATG10 are only dependent

on CHOP; and p62 and ATG7 are dependent both on ATF4 and CHOP. Given that in

our experimental model CHOP is not induced, this may explain why we did not observe

significant alterations in p62 transcription.

Finally we addressed whether the DNFB-induced eIF2�-ATF4 axis influenced DC

activation/ maturation status and found that ATF4 modulates, in a time-dependent

manner, the maturation and pro-inflammatory profile of THP-1 cells. We demonstrated

that DNFB-induced ATF4 co-operates with other transcription factors to positively

regulate the transcription of IL8 and HMOX1 while blocking the transcription of CD86,

IL1B and CXL10 genes. As ATF4 protein half-life is approximately 1h [54], the levels

induced by DNFB rapidly decay, resulting in an early transcription of IL8 and HMOX1

followed by a late up-regulation of CD86, IL1B and IL12B. Thus, our results indicate

that ATF4, when induced in DC, restrains the maturational signals conferred by other

signalling pathways. Since DNFB induced in our model, a time sustained activation of

JNK, the late CD86, IL1B, IL12B up-regulation may result from the activity of

transcription factors under its control such as c-Jun, Elk-1 and ATF-2. Accordingly,

several studies showed that JNK activation by skin sensitizers is directly involved in the

up regulation of cytokines and co-stimulatory molecules such as IL-6, IL-12, CD86 and

CD83, [55, 56]. The positive and negative transcriptional effects of ATF4 are well

documented in literature (review in [57]) and our results are in agreement with recent

findings where ATF4 was shown to modulate the TLR4-triggered cytokine production

in THP-1cells [58]. In the referred study, knockdown of ATF4 resulted in increased

expression of IL1-�, CXCL10 and MIF while CCL5, IL-8, IL-6 and IFN-� were down-

regulated. Authors additionally showed that the ATF4 positive transcriptional regulation

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over the studied cytokines was in part due to heterodimers formation with c-Jun [58].

To date, JNK is the only kinase known to phosphorylate and activate c-Jun [59]. As

DNFB strongly activates JNK, we can hypothesize that, in our experimental model,

ATF4 may similarly be forming heterodimers with c-Jun with consequent positive

regulation of IL8 gene. Besides heterodimerization with C/EBPs and AP-1 family

proteins [60], ATF4 can also transactivate gene expression by interacting with other

binding partners such as p300 [61], Satb2 [62], CEP290 [63] and Nrf2 [64]. Under

sensitizer-evoked oxidative and electrophilic stresses, Nrf2 has been shown to

translocate to the nucleus, bind to antioxidant response elements (ARE) and activate the

transcription of detoxifying enzymes such as HMOX-1 and NQO1 [65]. Accordingly,

we found that DNFB induces a rapid activation and nuclear translocation of Nrf2 with a

kinetic very similar to that observed for ATF4. Moreover, we demonstrated that

sustainment of DNFB-induced ATF4 results in stabilization of nuclear levels of Nrf2,

explaining the continued HMOX1 gene transcription observed in Sal003+DNFB treated

cells. In agreement to this role for PERK-eIF2�-ATF4 axis in ROS detoxification,

Rouschop and collaborators recently demonstrated that PERK-eIF2�-ATF4 signaling

induces uptake of cysteine and glutathione synthesis, conferring protection against ROS

produced during hypoxia [66]. Finally, several early studies showed that Nrf2 may also

be a direct substrate for the PERK kinase activity [46], which could equally explain our

observations.

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Conclusions

Overall, we show that the skin sensitizer DNFB triggers a ROS-dependent activation of

PERK-eIF2�-ATF4 UPR branch in DC-like cells. We demonstrate that the eIF2�-ATF4

axis drives early cytoprotective effects by up-regulating the autophagy related genes

MAP1LC3B and ATG3 and that ATF4 cooperates with Nrf2 to strongly induce HMOX1

detoxifying gene expression. Additionally, we show that transiently DNFB-induced up-

regulation of ATF4 modulates THP-1 cell maturation through negative transcriptional

regulation of CD86, IL1B, IL12B and CXL10 and through induction of IL8 transcription.

Our results evidence for the first time a connection between sensitizer-induced redox

imbalance and the establishment of ER stress in DC-like cells and provide new insights

about the role of UPR effectors to the complex DC maturational program.

Acknowledgments

We would like to thank Fundação para a Ciência e a Tecnologia (FCT, Portugal), the

European Union, QREN, FEDER, COMPETE, for funding the Organic Chemistry

Research Unit (QOPNA) (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-

FEDER-037296) and Centro de Neurociências e Biologia Celular (CNC) (PEst-C/SAU/

LA0001/2013-2014 and PTDC/SAU-OSM/099762/2008). João Demétrio Martins had a

FCT grant number SFRH/BD/73065/2010.

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Supplementary Data

Supplementary Table 1: Primer sequences for targeted cDNAs

Supplementary Figure 1: Effect of DNFB on THP-1 cell viability

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Figure legends

Figure 1: DNFB treatment evokes several ER stress effectors in THP-1 cells. (A

and B) THP-1 total protein extracts were prepared after the indicated time exposure to 8

µM DNFB (A) or 5 µg/ml Tunicamycin (B) and protein levels of GRP78, CHOP, p-

eIF2�, ATF4 and XBP-1s were assessed by Western blotting. (C and D) The graphs

represent the quantitative values as mean ± S.D. of optical densities relatively to control

for five independent experiments. (E and F) RNA was extracted after 3h, 6h and 24h

THP-1 cell stimulation with DNFB (C) or Tunicamycin (D). The relative expression of

indicated genes was assessed by qPCR and normalized using HPRT1 as reference gene.

Each value represents the mean ± S.D. of three to five independent experiments.

(*p<0.05; **p<0.01; ***p<0.001: control vs treatment)

Figure 2: DNFB activation of eIF2�-ATF4 axis and effects over GRP78 are ROS-

dependent. (A) THP1 cells were loaded with the fluorogenic probe Cell ROX® Green

Reagent for 30 min and then treated with 8 µM DNFB during additional 30 min. In

some experiments 5 mM NAC was added 1h prior to DNFB exposure. ROS formation

was visualized by increased green fluorescence; F actin cytoskeleton fibers were stained

with Alexa Fluor® 555 Phalloidin (red) and DAPI was used to label the nuclei (blue).

Images representative of different fields were acquired with a DS-Fi2 high-definition

digital camera coupled to a Nikon fluorescent microscope (magnification 630x; scale

bar = 10µm). (B) Cells were exposed to 4, 8 or 16 µM of DNFB and where indicated,

cells simultaneously treated with 5 mM NAC. After indicated times, proteins were

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extracted and the levels of GRP78, ATF4 and p-eIF2� analysed by Western blotting.

Results are expressed as % of intensity relatively to control. Each value represents the

mean ± S.D. from 3 independent experiments (*p<0.05; **p<0.01; ***p<0.001: control

vs treatment; #p<0.05: DNFB vs NAC + DNFB)

Figure 3: Primary dendritic cells show a DNFB-induced ROS-dependent activation

of eIF2�-ATF4 axis similar to the observed for THP-1 cells

Primary dendritic cells were obtained by culturing human peripheral blood monocytes

in GM-CSF and IL-4-supplemented culture medium, for 7 days, as described in

“Material and Methods”. Cells (1x106) were then exposed to 8µM DNFB during 1h and

where indicated, simultaneously treated with 5 mM NAC. Proteins were extracted and

the levels of ATF4 and p-eIF2� analysed by Western blotting.

Figure 4: DNFB-activated MAPK and eIF2�-ATF4 axis are not interrelated. (A)

Proteins were extracted after the indicated time exposure of THP-1 cells to 8 µM DNFB

and 30 µg of protein were separated on a 12% SDS-polyacrylamide gel. Phosphorylated

levels of JNK1/2, p38 and ERK1/2 were assessed by Western blotting. (B) THP1 cells

were pre-treated for 1h with the indicated inhibitors and then stimulated with 8 µM

DNFB. Phosphorylated levels of JNK1/2, p38, ERK and eIF2� were assessed by

Western blotting. Results were expressed as % of intensity relatively to control. Each

value represents the mean ± S.D. from 3 independent experiments (****p<0.0001:

control vs treatment; ##

p<0.01; ####

p<0.0001: DNFB vs inhibitor + DNFB).

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Figure 5: DNFB transiently destabilizes lysosomal membranes and DNFB-induced

ATF4 positively modulates the transcription of the autophagy-related genes

MAP1LC3B and ATG3. (A) THP-1 cells were exposed to the indicated concentrations

of DNFB for 8h. In some experiments 5 mM NAC was added prior to DNFB exposure.

Western blotting was performed to evaluate the levels of LC3B-II and p62 proteins.

Results were expressed as % of intensity relatively to control. The bars represent the

mean ± S.D. from 3 independent experiments (*p<0.05; **p<0.01; ***p<0.001;

****p<0.0001: control vs treatment; ###

p<0.001: DNFB vs NAC + DNFB). (B) Cells

were treated during 8h with 8 µM DNFB, 50 µM chloroquine or with DNFB and

chloroquine simultaneously. Western blotting was performed to assess the levels of

LC3B-II. Results are expressed as % of intensity relatively to control. Each value

represents the mean ± S.D. from 3 independent experiments. (*p<0.05; **p<0.01;

***p<0.001; ****p<0.0001: control vs treatment; ###

p<0.001: DNFB vs chloroquine +

DNFB). (C) THP-1 cells were treated with 8 µM DNFB during 4, 8 or 24 h, washed

with PBS and resuspended in medium containing 5 µg/ml acridine orange. In some

experiments 5 mM NAC was added prior DNFB exposure. Following 20 min

incubation cells where washed and analysed by fluorescence microscopy (magnification

630x; scale bar = 10µm). (D) THP-1 cells were exposed to DNFB, Sal003 or Sal003 +

DNFB during the indicated time periods and ATF4 and LC3B-II protein analysed by

Western blotting. Results are expressed as % of intensity relatively to control. Each

value represents the mean ± S.D. from 3 to 5 independent experiments. (*p<0.05;

**p<0.01; ***p<0.001; ****p<0.0001: control vs treatment; ##

p<0.01; ###

p<0.001:

DNFB 1h vs DNFB 8h; §§

p<0.01: DNFB 8h vs Sal003 + DNFB 8h). (E) Cells were

treated with DNFB, Sal003 or Sal003+DNFB during 6h and RNA extracted for qPCR

analysis. The relative expression of the indicated genes was normalized using HPRT1 as

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reference gene. Each value represents the mean ± S.D. from three to five independent

experiments. (*p<0.05; **p<0.01; ***p<0.001: control vs treatment; #p<0.05: DNFB vs

Sal003 + DNFB)

Figure 6: ATF4 positively modulates HMOX1 and IL-8 while limiting CD86, IL1B,

IL12B and CXL10 transcription. (A) THP-1 cells were exposed to DNFB, Sal003, or

Sal003 + DNFB for the indicated times and RNA extracted for qPCR analysis. The

relative expression of the indicated genes was normalized using HPRT1 as reference

gene. Each value represents the mean ± S.D. from three to five independent

experiments. (*p<0.05; **p<0.01; ***p<0.001: control vs treatment; ##

p<0.01;

###p<0.001;

####p<0.0001: DNFB 6h vs DNFB 24h;

§p<0.05;

§§p<0.01;

§§§p<0.001:

DNFB 24h vs Sal003 + DNFB 24h).

Figure 7: ATF4 stabilizes the DNFB-induced nuclear levels of Nrf2.

Nuclear extracts were prepared and the protein levels of ATF4, Nrf2 and p65 analyzed

by Western blotting. Results are expressed as % of intensity relatively to control. Each

value represents the mean ± S.D. from 3 independent experiments. (*p<0.05; **p<0.01;

***p<0.001: control vs treatment; #p<0.05: DNFB 8h vs Sal003 + DNFB 8h)

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Highlights

- Skin sensitizer DNFB triggers a ROS-dependent activation of ER stress in DCs

- The PERK-eIF2�-ATF4 branch of UPR is the main pathway affected by DNFB

- ATF4 positively modulates autophagy related genes MAP1LC3B and ATG3

- ATF4 up-regulates IL8 while blocking CD86, IL1B, IL12B and CXL10

- The eIF2�-ATF4 axis confers cytoprotection and modulates DC maturation status

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