Recapitulates Some Features of Psoriasis Α , and TNF- Α Oncostatin M, IL-1 Synergistic Action of IL-17A, IL-22, Skin Inflammat

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Skin Inflammation Induced by the Synergistic Action of IL-17A, IL-22, Oncostatin M, IL-1α, and TNF-α Recapitulates Some Features of Psoriasis This information is current as of September 27, 2021. Karline Guilloteau, Isabelle Paris, Nathalie Pedretti, Katia Boniface, Franck Juchaux, Vincent Huguier, Gerard Guillet, François-Xavier Bernard, Jean-Claude Lecron and Franck Morel

J Immunol 2010; 184:5263-5270; Prepublished online 24 Downloaded from March 2010; doi: 10.4049/jimmunol.0902464 http://www.jimmunol.org/content/184/9/5263 http://www.jimmunol.org/

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Skin Inﬂammation Induced by the Synergistic Action of IL-17A, IL-22, Oncostatin M, IL-1a, and TNF-a Recapitulates Some Features of Psoriasis

Karline Guilloteau,*,† Isabelle Paris,*,‡ Nathalie Pedretti,*,† Katia Boniface,*,1 Franck Juchaux,*,† Vincent Huguier,x Gerard Guillet,{ Franc¸ois-Xavier Bernard,*,† Jean-Claude Lecron,*,‡ and Franck Morel*

Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screen- ing of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-a, and IL-1a as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production Downloaded from of CXCL8 and b-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of http://www.jimmunol.org/ BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-a, and IL-1a on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production. The Journal of Immunology, 2010, 184: 5263–5270.

eratinocytes play an important role in the regulation of keratinocyte (NHEK) cultures with those of human biopsies from skin inflammation, responding to environmental and inflammatory cutaneous diseases, especially psoriasis, revealed by guest on September 27, 2021 K immune cells stimuli. Over the past few years, an in- a number of common features. Indeed, IL-1a, IL-22, TNF-a, and creasing number of reports has demonstrated that keratinocytes are oncostatin M (OSM) induce a “psoriasiform” profile on NHEKs direct targets for a specific set of cytokines, leading to the regu- in vitro (1–4, 7). These in vitro studies identified cytokines able to lation of their biological properties, such as secretion of cytokines, induce specific expression patterns related to innate immune re- chemokines, and antimicrobial peptides, their differentiation and sponse, such as IL-1a, TNF-a, or IL-17A, or related to the ker- migration capacities (1–6). The comparison of the biological atinocyte differentiation program, such as IL-22 or OSM. Some of profile induced in vitro by cytokines on normal human epidermal these cytokines, or related ones, such as IL-1a, IL-22, or IL-23, can induce skin inflammation in animal models (7–10). Moderate to full redundancy within each cytokine family has also been *Laboratoire Inflammation, Tissus Epithe´liaux et Cytokines, Unite´s Propres de described for IL-1, IL-6, IL-10, IL-17, and TNF families (11–17). Recherche, Equipe d’Accueil, Poˆle Biologie Sante´ and ‡Laboratoire Proteínes et Inflammation, Centre Hospitalier Universitaire de Poitiers, Poˆle Biologie Sante´, It appears that single cytokine stimulation generates a rather Universite´ de Poitiers; xService de Chirurgie Plastique, and {Service de Dermato- limited effect on keratinocytes, namely, a limited number and/or † logie, Centre Hospitalier Universitaire de Poitiers, Poitiers; and BIOalternatives, a limited modulated expression of targeted genes, reflecting only Gençay, France partial features with psoriasis. Recent data showing that immune 1Current address: Department of Immunology, Schering-Plough Biopharma, Palo Alto, CA. cells infiltrating psoriatic skin secreted large amounts of several Received for publication July 29, 2009. Accepted for publication February 25, 2010. inflammatory cytokines, including IL-22, IL-17A, and IL-23, led to classify psoriasis as the paradigm of an inflammatory disease This work was supported by grants from a clinical research program from Poitiers University Hospital, La Ligue Contre le Cancer, Association Nationale de Recherche involving the proinflammatory Th17 subset, challenging the Th1 et de la Technologie, and from le Conseil Re´gional de la Re´gion Poitou-Charentes. K.G. theory previously admitted (4, 18–21). We previously reported was supported by Convention Industrielle de Formation par la Recherche financing. that supernatants from T cells isolated from psoriatic skin were Address correspondence and reprint requests to Dr. Franck Morel and Dr. Jean- able to induce a “psoriasis-like” phenotype in NHEKs (18, 21), Claude Lecron, Laboratoire Inflammation, Tissus Epithe´liaux et Cytokines, UPRES EA 4331, Poˆle Biologie Sante´, 40 Avenue du Recteur Pineau, Universite´ de Poitiers, which would result from synergistic effects among the cytokines 86022 Poitiers Cedex, France. E-mail addresses: [email protected] and jean- produced by these T cells. For example combination of IL-17A [email protected] and IFN-g or IL-17A and TNF-a results in a synergistic effect on The online version of this article contains supplemental material. CXCL8 production (22, 23). IL-17A and IL-22 synergize in the Abbreviations used in this paper: BD, b-defensin; NHEK, normal human epidermal ker- upregulation of b-defensin 2 (BD2) and S100A9 production (24). atinocyte; NI, noninjected; OSM, oncostatin M; QRT-PCR, quantitative real-time PCR. Our goal was to identify an optimal and relevant cytokine com- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 bination able to synergize to generate in vitro an inflammatory www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902464 5264 CYTOKINE-INDUCED KERATINOCYTE INFLAMMATION keratinocyte model recapitulating some features of lesional pso- was taken after 24 h of treatment at the site of injection and immediately riatic skin. frozen in liquid nitrogen for RNA quantification. Culture supernatants were collected for cytokine ELISA determination. Materials and Methods Macroarrays analysis Skin samples The comparison of the effects of 36 different cytokines on the expression of 154 genes of potential interest for skin physiology was performed using The use of skin samples for this study was approved by the Ethical home-made cDNA macroarrays analysis (Supplemental Table I). After Committee of the Poitiers Hospital. After informed consent, lesional skin cytokine stimulation, total RNA was extracted using TRIzol Reagent biopsies were obtained from eight different patients with moderate-to- (Invitrogen Life Technologies, Cergy Pontoise, France) and conventional severe plaque psoriasis (mean age = 47 y; skin involvement 30–90% of body 33P-cDNA target synthesis and hybridization were performed (4). Genes . surface area) that did not receive any therapy for 4 wk. Normal skin were considered regulated if expression levels differed .3-fold relative to biopsies were obtained from surgical samples of healthy breast skin. untreated control and 3-fold relative to mean background noise. Cell cultures, cytokines, and reagents Real-time PCR analysis NHEK were obtained as previously described, from surgical samples of NHEK and skin total RNAwere isolatedand reverse transcribedas previously healthy breast skin (1). NHEK were cultured to 80% of confluence and then described (1). Quantitative real-time PCR (QRT-PCR) was carried using starved for 24 h in Keratinocyte serum-free medium without addition of the LightCycler-FastStart DNA MasterPlus SYBR Green I kit on LightCycler growth factors before stimulation. Cells were stimulated with or without 480 (Roche Diagnostics, Meylan, France). The reaction components were 10 ng/ml recombinant IL-17A, OSM, TNF-a, IL-22, and IL-1a alone or in 13 DNA Master Mix, and 0.5 mM HPLC purified sense and antisense oli- combination (R&D Systems Europe, Lille, France) during 24 h for mRNA gonucleotides purchased from Eurogentec (Eurogentec France, Angers, quantification or 48–96 h for protein quantification. France), designed using Primer3 software. Samples were normalized to Downloaded from In vivo murine skin inflammation. Outbred OF1 mice were purchased from three independent control housekeeping genes (G3PDH, RPL13A, or ACTB Charles River Laboratories (Chatillon, France). Ear intradermally injections for human samples and G3PDH, HMBS, or b2-microglobulin for mouse samples) and reported according to the ΔΔCT method as RNA fold increase: were realized at day 0 under brief isoflurane (Forene, Abott France, Rungis, ΔΔCT ΔCT sample 2 ΔCT reference France) gas anesthesia. The 250 ng carrier-free IL-17A, OSM, TNF-a, IL-22, 2 =2 . For comparison of normal skin and pso- and IL-1a (R&D Systems Europe) or PBS or 10 mg LPS (Sigma-Aldrich, riatic skin the REST 2008 software was used (25). Saint Quentin Fallavier, France) were injected in a total volume of 20 ml. After Cytokine measurement by ELISA. Levels of BD2, BD3, and CXCL8 were 24 or 48 h, ears were collected and frozen immediately in liquid nitrogen for determined using Human ELISA development kit (PeproTech, Neuilly sur H&E staining, immunohistochemistry analysis, or RNA quantification. Seine, France), and CXCL1 and CXCL5 with DuoSet reagents (R&D Sys- http://www.jimmunol.org/ Ex vivo human skin culture. Pieces (2 3 2 cm) of healthy breast skin were tems Europe). washed in PBS and intradermally injected with 10 ng each cytokines (IL- Western blotting analysis 17A, OSM, TNF-a, IL-22, and IL-1a) or with PBS 0.1% BSA in a total volume of 50 ml. Each sample was placed individually in a 6-well plate After 96 h of stimulation, NHEK lysis was performed as previously described and incubated up to 72 h at 37˚C, 5% CO2, in SkinEthic maintenance (1). After separation by SDS-PAGE, proteins were transferred to nitrocellulose medium (SkinEthic Laboratories, Nice, France). Punch biopsy of 4 mm membranes (Amersham Pharmacia Biotech, Orsay, France) by electroblotting. by guest on September 27, 2021

FIGURE 1. Cytokine-stimulated keratinocyte transcriptional proﬁle. Com- parison of the effects of 36 cytokines on the overall expression of a panel of 154 genes of potential interest for skin physiology was performed using home- made cDNA macroarrays analysis. NHEK were cultured with 10 ng/ml of each cytokine for 24 h. Total RNA was extracted and conventional 33P-cDNA target synthesis and hybridization were performed. For each cytokine, the relative expression of each gene in the stimulated culture is plotted versus that of the control culture. The Journal of Immunology 5265

S100A7 was detected with mouse anti-human S100A7 mAb (Imgenex, San Diego, CA) and sheep anti-mouse IgG peroxidase-conjugated polyclonal Ab (Amersham Biosciences, Orsay, France). Bound Ab were detected by chem- iluminescence (ECL Hyperfilm and ECL Plus Reagen, Amersham Bio- sciences). Chemotaxis assay. Chemotaxis assay was performed using 24-well transwell inserts (transparent polyethylene terephthalate membrane, 3-mm pore; Becton Dickinson Biosciences, Le Pont de Claix, France). Human neutrophils obtained from peripheral blood of healthy volunteers were labeled with 5 mM Calcein AM (Molecular Probes, Invitrogen Life Technologies). NHEK culture supernatants were incubated or not with anti-CXCL8 (10 mg /ml), anti-CXCL1/2/3, and/or anti-CXCL5 mAb (25 mg/ml) (all from R&D Systems) for 30 min at 37˚C. The 400 ml NHEK supernatants were added to the lower chamber of a Transwell plate and 200 ml calcein- labeled neutrophils were added to the upper chamber. After incubation 2 h at 37˚C, 5% CO2, the number of migrating cells in the lower chamber was determined by measuring calcein fluorescence. Results are expressed as percentage of migrating neutrophils per well. Assay for antibacterial activity. Supernatants from NHEK treated or not for 96 h with cytokines were 50-fold concentrated by centrifugation using Amicon Ultra 3000 Da (Millipore, Saint-Quentin en-Yveline, France) and dialyzed against sodium phosphate buffer (10 mM, pH 7.4). Escherichia coli (ATCC 29325) was grown to exponential phase, bacterial concentra- Downloaded from 5 tion was adjusted to 2.10 bacteria/ml in 10 mM sodium phosphate buffer FIGURE 2. BD2 and CXCL8 production by keratinocyte after stimu- and mixed (ratio 2:1) with concentrated NHEK supernatants or with hu- lation by IL-1a, IL-17A, IL-22, OSM, and TNF-a or by cytokines of the man recombinant BD2 (PeproTech). After incubation at 37˚C for 1 h, serial same family. NHEK were cultured with (A) 10 ng/ml IL-1a, IL-17A, IL- dilutions of bacterial suspensions were plated onto Brain Heart Infusion agar plates and cultured for 24 h at 37˚C for determination of bacterial 22, OSM, and TNF-a alone or in combination (M5) or (B) after sub- CFU. Results are expressed as percentage of CFU in control condition with stitution by cytokines of the same family. After 96 h, BD2 and CXCL8 the mean and SEM of three independents experiments. secreted in culture supernatants were measured by ELISA (seven in- http://www.jimmunol.org/ Histological studies and immunohistochemistry for Gr-1. The 6-mm sec- dependent experiments). Statistical comparisons were performed using tions of mouse ear were fixed in 10% formalin in PBS. Ear thickness was either Mann-Whitney U test or Kruskal-Wallis ANOVA and Dunn’s test measured after H&E coloration at three different points in the injection for multiple comparisons. pp , 0.05; ppp , 0.01; pppp , 0.001. area. SD from the mean is shown for three separate experiments. Sections of mouse ear were stained for granulocytes by using rat IgG2b we identified major contributors of keratinocyte inflammation and anti-mouseLy-6GmAb(Gr-1,BectonDickinsonBiosciences)orwithisotype control (IgG2b, Caltag, Invitrogen Life Technologies) associated with analyzed cytokine redundancy. IL-17A and TNF-a were more crit- a donkey anti-rat IgG Alexa Fluor 488-conjugated secondary Ab (Invitrogen ical to the activity of the M5 combination than IL-1a,OSM,orIL-22 Life Technologies). Confocal microscopy was carried out on a Olympus on BD2 and CXCL8 production (Fig. 2B). Substitution by cytokines FV1000 confocal. Pictures are representative of three experiments. of the same family demonstrated a total redundancy between IL-1a by guest on September 27, 2021 Statistical analysis and IL-1b, strong redundancy between TNF-a and TNF-b, limited redundancy between IL-17A and IL-17F, IL-22, and IL-24 and weak Statistical analysis of significance was calculated using either Mann- redundancy between OSM and IL-6 (Fig. 2B). Whitney U test or Kruskal Wallis one-way ANOVA by ranks with a Dunn’s test. The p values # 0.05 were considered as significant, and all data are represented as mean and SEM. Comparison study used the Spearman rank correlation test.

Results Identification of cytokines able to modify keratinocyte transcriptional profile To identify major skin inflammation inducers we screened the activity of 36 different cytokines previously described for their effect in the skin or for their involvement during regulation of the immune/inflammatory response. We compared their effects on the expression of 154 genes of potential interest for skin physiology (Supplemental Table I). Among the cytokines able to modify the expression of at least five genes (with at least a 3-fold increase or decrease), we identified IL-22, IL-24, IL-6, OSM, IL-1a, IL-1b, TNF-a, and IL-17A (Fig. 1, Supplemental Table II). IL-1a, IL-17A, IL-22, OSM, and TNF-a strongly induce BD2 and CXCL8 production by keratinocyte We further selected the most potent cytokine from each family, based FIGURE 3. Expression of antimicrobial peptides and chemokines by on the number of genes regulated and on the fold increase or decrease human skin explants after cytokine injections. Human skin explants were injected with 10 ng IL-1a, IL-17A, IL-22, OSM, and TNF-a (M5), with gene expression (Supplemental Table II), namely, IL-1a,IL-17A, PBS or noninjected (NI) and cultured for 24, 48, and 72 h (four independent IL-22, OSM, and TNF-a (named M5 combination) and showed their experiments). A, After 24 h treatment, QRT-PCR analysis was carried out strong synergistic activity on the production of BD2 (161900 pg/ml for S100A7, CXCL8 and BD2, normalized using housekeeping genes and versus 85 pg/ml for control culture) and CXCL8 (41800 pg/ml versus expressed as the fold increase above normal skin. B, BD2 secreted in culture 80 pg/ml) (Fig. 2A). By successive removal of each cytokine of the supernatants was measured by ELISA. Statistical comparisons were per- M5 combination and replacement by a cytokine of the same family, formed using Mann-Whitney U test. pp , 0.05; ppp , 0.01. 5266 CYTOKINE-INDUCED KERATINOCYTE INFLAMMATION

Ex vivo and invivo proinflammatory activities of the cytokine mix transcription of the antimicrobial peptide encoding genes S100A9 To evaluate the proinflammatory activities of the M5 cytokine and BD3 was strongly induced by the M5 combination (Fig. 4B). combination in the skin, we setup human ex vivo and murine in vivo Modification of keratinocyte transcriptional profile induced by skin challenges. Human normal skin explants were injected with the proinflammatory cytokine combination M5 cytokine mix and cultured for 24 h before S100A7, CXCL8, and To further characterize the activity of the cytokine mix on innate BD2 mRNA quantification. A strong increase of S100A7, CXCL8, immunity, the antimicrobial peptides and chemokines transcrip- and BD2 gene transcription was observed after injection of the M5 tional profile was determined by QRT-PCR analysis. The tran- combination when compared with the saline-injected or noninjected scription of 9 chemokines and 12 antimicrobial peptides encoding skin explants (Fig. 3). BD2 quantification by ELISA in the skin genes was upregulated in response to the M5 combination (Fig. 5). explants culture supernatants confirmed the 10- to 100-fold in- Among these 21 genes, expression of 19 was upregulated by IL-1a, creased in BD2 production over a 3-d culture period (Fig. 3). 16 by IL-17A, 20 by TNF-a, 14 by IL-22, and 13 by OSM (Fig. 5). To assess in vivo the effect of the proinflammatory mixture, the A strong transcriptional synergy with the M5 cytokine combination M5 cytokine combination was injected intradermally in mouse ears was observed for BD2, BD3, LL37, RNASE7, PI3, S100A7, in parallel to LPS that was used as a positive control. After 48 h, S100A7A, S100A12, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, redness and swelling were observed in M5-injected ears as well as and CXCL8, whereas only an additive effect of M5 was observed a 2-fold increase in ear thickness in comparison with saline-injected for S100A8, S100A9, CCL5, CCL20, WFDC5, and WFDC12. ears (Fig. 4A,4B). Interestingly, histological analysis revealed an Finally, the activity of the M5 combination on CCL27 gene tran- important inflammatory cellular infiltrate in ears injected with M5, scription seems to be due to the redundant activity of IL-1a and Downloaded from comparable to the one obtained in LPS-injected ears. Immunohis- TNF-a (Fig. 5). tological analysis with anti-Gr1 mAb revealed abundant granulocytes in ear tissue from LPS- or M5-injected mice compared with Production of CXCL chemokines and chemotactic activity saline-injected mice (Fig. 4A). This infiltrate was predominantly We further examined the production of CXCL1, CXCL5, and present in the dermis and associated with an increase in CXCL1, CXCL8 proteins by ELISA. As shown in Fig. 6A, untreated NHEK CXCL3,andtoalesserextentCXCL2gene transcription. In addition, secreted low levels of CXCL1, CXCL5, and CXCL8. IL-1a was http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Cytokine-induced skin ear inflammation. Ears from outbred OF1 mice (n =3 for each group) were injected intradermally with 250ngIL-1a, IL-17A, IL-22, OSM, and TNF-a, with PBS or with 10 mg LPS. A, On day 2, ears were collected for staining with H&E (original magnification 3100) and immunodetection of neutrophils using anti-Gr-1 mAb (original magnification 3200). B, On day 1, QRT-PCR analysis was carried out on RNA isolated from treated ears, normalized using housekeeping genes, and expressed as the fold increase above untreated skin. Ear thickness at day 2 was measured. Statistical comparisons were performed using Kruskal-Wallis ANOVA and Dunn’s test for multiple comparisons. pp , 0.05; ppp , 0.01; pppp , 0.001. The Journal of Immunology 5267 Downloaded from http://www.jimmunol.org/

FIGURE 5. Antimicrobial peptides and chemokines gene expression by cytokine-stimulated keratinocytes. NHEK were cultured in the presence or by guest on September 27, 2021 absence of 10 ng/ml IL-1a, IL-17A, IL-22, OSM, and TNF-a alone or in FIGURE 6. Neutrophil chemotactic activity and antimicrobial activity of combination (M5) for 24 h. QRT-PCR analysis was carried out on total cytokine-stimulated keratinocytes. A, Keratinocytes were treated with RNA from six independent NHEK cultures. mRNA expression levels are 10 ng/ml cytokines alone or in combination (M5). After 96 h, chemokines normalized using housekeeping genes and expressed as the fold increase secreted in culture supernatants were measured by ELISA and culture su- above unstimulated cultures. pernatants were tested for chemotactic activity on human neutrophils, with or without blocking mAbs against CXCL1/2/3 (a1/2/3), CXCL5 (a5), CXCL8 (a8), or the combination of these three mAbs (3a). B, Keratinocytes the most effective, followed by IL-17A or TNF-a, and finally IL- were treated with 10 ng/ml cytokines alone or in combination (M5). After 22 or OSM with a discrete effect on chemokine production. 96 h, BD2 and BD3 secreted in culture supernatants were measured by However, in combination, IL-17A, OSM, TNF-a, IL-22, and IL- ELISA and S100A7 in cell lysates detected by Western blot. Antimicrobial 1a synergistically induced a massive secretion of CXCL1, activity of the keratinocyte culture supernatants (control or M5) against CXCL5, and CXCL8 with induction factors of 190, 80, and 650, E. coli was analyzed using a CFU assay. A total of 5 mg/ml recombinant respectively, when compared with controls (Fig. 6A). BD2 was used as positive control. Statistical comparisons were performed using either Mann-Whitney U test or Kruskal-Wallis ANOVA and Dunn’s We next analyzed the chemotactic activity of supernatants from test for multiple comparisons. pp , 0.05; ppp , 0.01; pppp , 0.001. control or stimulated NHEK. Supernatants from M5-stimulated NHEK had a significantly increased chemotactic activity for neutrophils compared with supernatants from unstimulated cul- production (Fig. 6B), in agreement with previously observed in- tures (Fig. 6A). Neutrophil chemotactic activity was significantly creased gene transcription. reduced when neutralizing the activities of CXCL1/2/3 or CXCL5, Expression of S100A7 in NHEK was evaluated by Western and totally blocked when inhibiting CXCL8 (Fig. 6A). blotting analysis. S100A7 was barely detected in unstimulated NHEK (Fig. 6B). Each of the five cytokines alone induced S100A7 Antimicrobial peptides production and activity production, IL-1a being the most potent inducer, and a stronger Because we showed that antimicrobial peptide gene expression was expression was observed when the M5 combination was used. strongly induced after NHEK stimulation by the M5 combination, We next evaluated the antimicrobial activity of M5-stimulated we further quantified BD2 and BD3 protein by ELISA in NHEK NHEK culture supernatants against E. coli, and showed that they supernatants. As shown in Fig. 6B, BD2 was significantly induced exhibited strong antibacterial activity compared with the un- by the five cytokines alone, among them IL-1a and IL-17A being stimulated NHEK supernatants. A similar activity for recombinant the most potent inducers. A strong synergy was seen with the M5 BD2 was observed (Fig. 6B). combination with an induction factor of 24,000 when compared with unstimulated keratinocytes. A significant increase of BD3 Transcriptional profile of lesional psoriatic human skin production was seen for IL-1a, IL-22, or OSM stimulation. Again, To evaluate the pathophysiological relevance of the inflammatory the M5 combination resulted in a strong synergistic effect on BD3 phenotype observed in vitro when stimulating NHEK with the M5 5268 CYTOKINE-INDUCED KERATINOCYTE INFLAMMATION combination, we quantified the gene expression of several proinflammatory cytokines and their potential targets, in particular chemokines and antimicrobial peptides, in normal skin and psoriatic skin. We were able to detect overexpression of IL-23, IL-17A, IL-22, and OSM in psoriatic skin as compared with normal skin, as well as a small increased expression of IL-1b, whereas IL-1a and TNF-a expression were not different. Among the 12 antimicrobial peptides analyzed, the transcription of 10 of them was higher in psoriatic skin compared with normal skin, with .100-fold increases for BD2, S100A7A, S100A12, PI3, S100A7, S100A9, and S100A8 (Table I). Expression of CXCL8, CXCL1, CXCL6, CCL20, CXCL5, and CCL5 encoding genes was also strongly FIGURE 7. Transcriptional profiles of in vitro inflammatory keratino- increased in psoriatic skin compared with normal skin, but CCL27 cytes and psoriatic skin. QRT-PCR analysis was carried out on NHEK expression was lower (Table I). In summary, among the 21 genes cultures stimulated or not with M5, control skin, and psoriatic skin. The overexpressed in our in vitro model, the expression of 18 was ratio of relative expression of M5 versus unstimulated cultures (n = 6) and higher in psoriatic skin compared with normal skin, and a corre- the ratio of relative expression of psoriatic versus normal skin (n = 8) were lation was found between these two sets of data (Fig. 7). compared. Comparison study was performed using the Spearman rank correlation test (r = 0.58; p = 0.0063).

Discussion Downloaded from To identify major skin inﬂammation inducers we conﬁrmed that IL- and IL-24 (11, 12), TNF-a and TNF-b (14), or IL-17A and IL-17F 22, IL-20, IL-6, OSM, IL-1a, IL-1b, TNF-a, and IL-17A represent (13, 17), the former being always the most active. We extended the potent keratinocytes modulators, and demonstrated a powerful characterization of this synergic effect using QRT-PCR analysis, synergy between IL-17A, IL-22, OSM, TNF-a, and IL-1a on the and observed increased expression of 12 antimicrobial peptides expression of CXCL8 and BD2. We showed a total redundancy and 9 chemokines in M5-stimulated NHEK. IL-1a, IL-17A, and

between IL-1a and IL-1b, strong redundancy between TNF-a and TNF-a were the most potent inducers; however, the addition of http://www.jimmunol.org/ TNF-b, limited redundancy between IL-17A and IL-17F, IL-22 IL-22 and OSM increased the activity of the cytokine mixture. and IL-24, and weak redundancy between OSM and IL-6. A such Interestingly, the presence of NFkB, STAT3, and MAPK signaling partial redundancy has been described on keratinocytes for IL-22 cytokines seems crucial for this synergy, that could be reinforced by C/EBP transcription factors activation and/or mRNA stabili- zation induced by IL-17A (26). Antimicrobial peptides expressed Table I. Transcriptional profile of psoriatic skin versus normal skin in this in vitro NHEK model, in particular BD2, S100A7, LL37, and RNAse7, are crucial for the skin innate defense as direct Ratio Gene Psoriasis/Controla p Valueb bacterial killing compounds (27–30). Because antimicrobial peptides can demonstrate synergy with each other (31), the antimi- by guest on September 27, 2021 Cytokine crobial peptides induced in our system should generate a broad IL-17A 24 ,0.001 IL-23 16 ,0.001 spectrum protection against Gram-positive and Gram-negative IL-22 16 0.004 bacteria, fungi, and viruses. Host-defense function may be com- OSM 5 0.003 pleted by chemokines, which have also direct antimicrobial ac- IL-1b 2.4 0.131 tivity, in particular CCL20, CXCL1, CXCL2, and CXCL3, also TNF-a 0.8 0.342 IL-1a 0.8 0.537 induced in our model (32). Alternatively some antimicrobial Chemokine peptides have developed the capacity to play important roles in CXCL8 38 0.002 adaptive immune responses against microbial invasion. Indeed CXCL1 38 0.001 BD2 may recruit dendritic cells and T cells to the site of microbial CXCL6 26 0.001 invasion through interaction with CCR6 (33). More recently CCL20 19 0.001 CXCL5 9 0.003 S100A7 was identified as chemotactic for neutrophils, monocytes, CCL5 3.2 0.022 and lymphocytes, by interacting with the receptor for advanced CXCL3 2.4 0.433 glycation end products (34). In addition, a role of S100A8 and CXCL2 2.3 0.487 S100A9 in the release of neutrophils from the bone marrow, and CCL27 0.03 0.001 Antimicrobial peptide subsequent neutrophil chemotaxis and adhesion could be involved BD2 5962 ,0.001 in the recruitment of this inflammatory infiltrates (35, 36). How- S100A7A 1804 ,0.001 ever, the strongest synergy of the M5 combination targeted, mainly, S100A12 792 0.001 neutrophils attracting CXCL chemokines. Experiments using , PI3 934 0.001 blocking mAbs demonstrated that the neutrophil chemotactic ac- S100A7 224 0.001 S100A9 305 0.001 tivity was mainly due to CXCL8 production. The M5 combination- S100A8 287 0.001 induced CCL20 expression, predominantly due to activity of IL-1a, LL37 16 0.001 TNF-a, and IL-17A, may also contribute to the recruitment of WFDC12 5 0.004 CCR6-expressing Th17 cells, and generate a positive feedback loop RNase7 3 0.007 BD3 1.6 0.170 as described previously (37). WFDC5 0.7 0.313 The combined activity of IL-1a, IL-17A, IL-22, OSM, and TNF-a led to high levels of host defense proteins expression, QRT-PCR analysis was carried out on total RNA from eight psoriatic skins and eight normal skins. Relative expression levels are normalized using housekeeping generating an in vitro NHEK model of skin inflammation. The genes and expressed as the ratio between psoriatic skin and normal skin. pathophysiological relevance of this model was assessed by aRelative gene expression ratio calculated using REST 2008 software. bThe p values for the pairwise fixed reallocation randomization test were calcu- comparison of its transcriptional profile with that of psoriatic skin. lated using REST 2008 software. We confirmed increased expression of IL-17A, IL-22, IL-23, and The Journal of Immunology 5269

OSM in psoriatic skin. We detected similar TNF-a mRNA levels tured keratinocytes: dominance of innate immune responses in psoriasis. Am. J. Pathol. 171: 32–42. in lesional psoriatic skin compared with normal skin, but TNF-a 6. Nograles, K. E., L. C. Zaba, E. Guttman-Yassky, J. Fuentes-Duculan, M. Sua´rez- expression has been described to be regulated posttranscription- Farinãs, I. Cardinale, A. Khatcherian, J. Gonzalez, K. C. Pierson, T. R. White, et ally (38). A correlation was found between in vitro and in vivo al. 2008. Th17 cytokines interleukin (IL)-17 and IL-22 modulate distinct inflammatory and keratinocyte-response pathways. Br. J. Dermatol. 159: 1092– transcriptional profiles, demonstrating that a part of the charac- 1102. teristic “signature” for psoriasis was obtained in the present 7. Groves, R. W., H. Mizutani, J. D. Kieffer, and T. S. Kupper. 1995. Inflammatory in vitro model of keratinocyte inflammation (39). 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compared with atopic dermatitis. J. Invest. Dermatol. 125: 1163–1173. 3194. http://www.jimmunol.org/ by guest on September 27, 2021 Supplemental Table I: Macroarray composition

Family Gene Innate immunity : DEFB1, DEFB3, DEFB2, S100A7, S100A8, S100A9, S100A12, SLPI, PI3, TLR1, TLR2, TLR4, CXCL2, CXCL5, CXCL6, CXCL8 Cytokines, growth factors & receptors : BMP1, CTGF, EGF, EGFR, FGF2, FGF7, FGFR2, GHR, GHSR, GMCSF, HBEGF, IFNGR1, IFNGR2, IGF1, IGF1R, IL10RA, IL10RB, IL1A, IL1B, IL1R1, IL1R2, IL1RA, IL20RA, IL20RB, IL22RA1, IL23R, IL27RA, IL31RA, IL4R, IL6R, IL6ST, IL8RB, KLF10, LIFR, OSMR, PDGFA, PIAS3, PLAB, STAT3, TGFB1, TNF, VEGFA Epidermal structure & differentiation : AQP3, BGN, BPAG1, CALML3, CALML5, CASP14, CD44, CDH1, CDH3, CDSN, CHP, CLDN1, COL17A1, COL4A2, COL7A1, CRABP2, CTNNA1, DCN, DSC1, DSG1, DSP, EPPK1, EVPL, FABP5, FLG, FN1, FTH1, HAS3, HAX1, ID1, ITGA2, ITGB1, ITGB6, IVL, KI67, KLK5, KLK6, KLK7, KRT1, KRT10, KRT13, KRT14, KRT16, KRT18, KRT19, KRT2E, KRT4, KRT5, KRT6A, KRT6B, LAMC2, LAMR1, LCE3D, LGALS7, LOR, MMP1, MMP11, MMP14, MMP2, MMP3, MMP9, NICE1, PLAU, PSORS1, PXN, SBS, SCEL, LCE2B, SERPINB2, SERPINE1, SFN, SPARC, SPRR1A, SPRR1B, SPRR2A, SYND1, TGM1, TGM3, THH, TIMP1, TIMP3, TMSB10, TNC, P53, TP73L, ZYX Stress : CAT, HMOX1, HSP27, HSPA1A, HSPCA, MT1E, MT1H, SOD1, SOD2, TXN

Supplemental Table II: Cytokine-stimulated keratinocyte transcriptional profile

Cytokine Family Cytokine Fold expression above control Number of modified gene expression HBD2 HBD3 S100A7 S100A8 S100A9 S100A12 PI3 CXCL8 KRT10 IL-1 IL-1α 304 - 6 7 7 5 4 25 - 7 IL-1β 304 - 5 5 6 4 4 26 - 7 IL-6 IL-11, IL-27, IL-31, LIF, CNTF, ------0 CT-1, CLC, NP IL-6 5 3 5 3 4 - - - - 5 OSM 7 5 9 5 5 - - - 0.2 6 IL-10 IL-10, IL-19, IL-26, IL-28, IL-29 ------0 IL-20 3 - 4 ------2 IL-22 11 5 10 4 4 - - 3 0.3 7 IL-24 8 3 6 3 3 - - - - 5 IL-17 IL-17A 310 3 11 6 7 7 6 4 - 8 IFN IFNα - 4 ------1 IFNγ ------3 0.3 2 TNF TNFα 125 3 4 4 4 4 4 20 0.2 9 Miscellaneaous IL-2, IL-4, IL-8, IL-12, IL-13, IL-15, ------0 IL-18, IL-23, TSLP

NHEK were cultured with or without 10 ng/ml of each cytokine for 24 h. Total RNA was extracted and conventional 33P-cDNA target synthesis and hybridization were performed on home-made cDNA macroarrays. Signal intensities are quantified and the results are expressed as the fold increase above unstimulated control culture.

- : expression not modified compared to control (<3 fold increase or decrease)