Limited Presence of IL-22 Binding Protein, a Natural IL-22 Inhibitor, Strengthens Psoriatic Skin Inflammation

This information is current as Jérôme C. Martin, Kerstin Wolk, Gaëlle Bériou, Ahmed of September 25, 2021. Abidi, Ellen Witte-Händel, Cédric Louvet, Georgios Kokolakis, Lucile Drujont, Laure Dumoutier, Jean-Christophe Renauld, Robert Sabat and Régis Josien J Immunol 2017; 198:3671-3678; Prepublished online 29

March 2017; Downloaded from doi: 10.4049/jimmunol.1700021 http://www.jimmunol.org/content/198/9/3671

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Limited Presence of IL-22 Binding Protein, a Natural IL-22 Inhibitor, Strengthens Psoriatic Skin Inﬂammation

Je´roˆme C. Martin,*,†,‡,x,1 Kerstin Wolk,{,‖,#,1 Gae¨lle Be´riou,*,† Ahmed Abidi,*,†,** Ellen Witte-Ha¨ndel,{,‖ Ce´dric Louvet,*,† Georgios Kokolakis,{ Lucile Drujont,*,† Laure Dumoutier,††,‡‡ Jean-Christophe Renauld,††,‡‡ Robert Sabat,{,‖,xx,2 and Re´gis Josien*,†,‡,x,2

Psoriasis is a chronic inﬂammatory disease resulting from dysregulated immune activation associated with a large local secretion of cytokines. Among them, IL-22 largely contributes to epithelial remodeling and inﬂammation through inhibiting the terminal differentiation of keratinocytes and inducing antimicrobial peptides and selected chemokines. The activity of IL-22 is regulated by

IL-22 binding protein (IL-22BP); however, the expression and role of IL-22BP in psoriatic skin has remained unknown so far. Downloaded from Here we showed that nonaffected skin of psoriasis patients displayed lower expression of IL-22BP than skin of healthy controls. Furthermore, the strong IL-22 increase in lesional psoriatic skin was accompanied by a moderate induction of IL-22BP. To investigate the role of IL-22BP in controlling IL-22 during skin inflammation, we used imiquimod-induced skin disease in rodents and showed that rats with genetic IL-22BP deficiency (Il22ra22/2) displayed exacerbated disease that associated with enhanced expression of IL-22–inducible antimicrobial peptides. We further recapitulated these findings in mice injected with an anti–IL-

22BP neutralizing Ab. Hypothesizing that the IL-22/IL-22BP expression ratio reﬂects the level of bioactive IL-22 in psoriasis skin, http://www.jimmunol.org/ we found positive correlations with the expression of IL-22–inducible molecules (IL-20, IL-24, IL-36g, CXCL1, and BD2) in keratinocytes. Finally, we observed that serum IL-22/IL-22BP protein ratio strongly correlated with psoriasis severity. In conclusion, we propose that although IL-22BP can control deleterious actions of IL-22 in the skin, its limited production prevents a sufﬁcient neutralization of IL-22 and contributes to the development and maintenance of epidermal alterations in psoriasis. The Journal of Immunology, 2017, 198: 3671–3678.

soriasis is a chronic inflammatory skin disease that affects cruitment of further immune cells into the skin to create a self- ∼2% of the Caucasian population (1). Typical macro- sustained inflammatory milieu. Moreover, the high keratinocyte P scopic skin alterations present as sharply demarcated, red, production of antimicrobial peptides prevents infections of the by guest on September 25, 2021 and slightly raised lesions with silver-whitish scales. It is gener- highly disturbed psoriatic epidermis. Among a range of immune ally acknowledged that these skin changes result from chronic mediators present in the psoriatic skin, an essential role for IL-23 dysregulated activation of the cutaneous immune system that—by and its downstream cytokine IL-17A in the maintenance of pso- secreted cytokines—alters the biology of local tissue cells (2, 3). riatic lesions has been recently confirmed by the dramatic clinical Epidermal keratinocytes respond by hyperproliferation and im- success of their therapeutic blockade in these patients (4–7). pairment of their terminal differentiation, leading to epidermal Another major IL-23 downstream cytokine is the IL-10 family thickening, hypogranularity, and hyperkeratosis. At the same time, member IL-22. IL-22 is also present in large quantities in psoriatic these cells secrete high amounts of chemokines enabling the re- lesions (8). Main producers of IL-22 include different CD4+ T cell

*Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Europeén des Sciences de la Transplantation et de l’Immunologie (IHU-Cesti) Proj- Universite´ de Nantes, 44093 Nantes Cedex 1, France; †Institut de Transplantation ect. The IHU-Cesti Project is also supported by Nantes Me´tropole and the Pays de la Urologie Ne´phrologie, Centre Hospitalier Universitaire Nantes, 44093 Nantes Cedex Loire region. J.C.M. was supported by a grant from Centre Hospitalier Universitaire 1, France; ‡Faculte´ de Me´decine, Universite´ de Nantes, 44093 Nantes Cedex 1, (CHU) Nantes (Appel d’Offre Interne 2013 RC14_0042); J.C.M. also received sup- France; xLaboratoire d’Immunologie, Centre Hospitalier Universitaire Nantes, port from CHU Nantes through Anneé Supple´mentaire d’Internat. G.B. was sup- 44093 Nantes Cedex 1, France; {Psoriasis Research and Treatment Center, ported by the Pays de la Loire Region through the IMmunoBIOthe´rapies et Dermatology/Medical Immunology, University Hospital Charite´, D-10117 Berlin, Cellules Dendritiques Network. A.A. was supported by a French-Tunisian UTIQUE Germany; ‖Interdisciplinary Group of Molecular Immunopathology, University grant from the 2015 Hubert Curien program. K.W. and R.S. were supported by Grant Hospital Charite´, D-10117 Berlin, Germany; #Berlin-Brandenburg Center for Re- 01ZX1312A from the German Federal Ministry of Education and Research. generative Therapies, University Hospital Charite´, 13353 Berlin, Germany; Address correspondence and reprint requests to Dr. Je´roˆme C. Martin at the current **Faculte´ des Sciences Mathe´matiques, Physiques et Naturelles, Universite´ de address: Department of Oncological Science, Icahn School of Medicine at Mount Tunis El Manar, 2092 Tunis, Tunisia ††Ludwig Institute for Cancer Research, Sinai, 1470 Madison Avenue, New York, NY 10029, Prof. Re´gis Josien, INSERM B-1200 Brussels, Belgium; ‡‡Institut de Duve, Universite´ Catholique de Louvain, xx UMR1064-ITUN, CHU Nantes Hoˆtel Dieu, Universite´ de Nantes, 30 Boulevard Jean B-1200 Brussels, Belgium; and Research Center Immunosciences, University Monnet, 44093 Nantes Cedex 1, France, or Dr. Robert Sabat, Interdisciplinary Group Hospital Charite´, D-10117 Berlin, Germany of Molecular Immunopathology, Dermatology/Medical Immunology, University 1J.C.M. and K.W. contributed equally to this work and are cofirst authors. Hospital Charite´, Charite´platz 1, D-10117 Berlin, Germany. E-mail addresses: [email protected] (J.C.M.), [email protected] (R.J.), or robert. 2R.S. and R.J. contributed equally to this work and are colast authors. [email protected] (R.S.) ORCIDs: 0000-0002-7689-4130 (K.W.); 0000-0002-8042-7885 (G.K.); 0000-0003- Abbreviations used in this article: IL-22BP, IL-22 binding protein; IMID, immune- 1736-2131 (J.-C.R.); 0000-0001-7900-7413 (R.J.). mediated inflammatory disease; PASI, psoriasis area and severity index; qRT-PCR, Received for publication January 5, 2017. Accepted for publication March 1, 2017. quantitative RT-PCR.

This work was supported by the National Research Agency via Investment into the Ó Future Program Grant ANR-10-IBHU-005 for the Institut-Hospitalo Universitaire–Centre Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700021 3672 IL-22BP LIMITS IL-22–DEPENDENT INFLAMMATION IN PSORIASIS populations and group 3 innate lymphoid cells (9). Because the 23 reagent (rodent samples) (both from Applied Biosystems) and the expression of the membrane IL-22R is restricted to epithelial/ StepOne Plus device (Applied Biosystems). Primers and double-labeled epithelioid cells (8), IL-22 assumes major cross-talk functions be- fluorescent probes were either self-designed (human IL-20, IL-22, IL-24, IL-22BP, and BD2) (26) or purchased from Applied Biosystems (all others). tween immune and epithelial cells, especially at body barriers (10, Expressions were normalized to HPRT using the 2-DD cycle threshold 11). By inducing antimicrobial peptides and proinflammatory che- method, and results were expressed in arbitrary units. mokines, as well as by inhibiting the terminal differentiation of ELISA keratinocytes, IL-22 has been shown to largely contribute to inflammation and epithelial remodeling of the psoriatic skin (12–16). Detection of blood serum levels of IL-22 and IL-22BP was performed using Interestingly, the IL-22 activity is regulated by IL-22 binding protein ELISA kits from Bio-Techne (Quantikine system) and Biozol (Cloud- (IL-22BP). IL-22BP is a soluble single chain receptor encoded by Clone), respectively. the gene IL22RA2 that specifically prevents the binding of IL-22 to Imiquimod-induced psoriasis-like skin inflammation IL-22R (17–20). Recently, we and others showed that conventional Psoriasis-like skin inflammation was induced in the right ear of Il22ra2+/+ dendritic cell–derived IL-22BP exerts important inhibitory functions and Il22ra22/2 rats, and in the back skin of C57BL/6J mice as previously that impair the IL-22–mediated protection during acute colitis in described (27). Briefly, topical application of Aldara cream (3M Pharma- rodents, although preventing potentially dangerous long-lasting ceuticals) was performed daily for 5 d. Skin thickness was measured daily proliferative effects on intestinal epithelial cells (21–23). Whether with a Digimatic Caliper and the percentage of skin thickness increase IL-22BP is expressed and plays a role in psoriatic skin inflammation relative to day 0 was calculated every day. Animals were sacrificed at day 5 for qRT-PCR and histopathological analyses on skin tissues. Skin sections is, however, unknown so far. Our study answers these questions in a were stained with H&E. translational approach using skin and blood samples from healthy Downloaded from control donors and psoriasis patients, in vitro experiments with hu- In vivo neutralization of IL-22BP man keratinocytes and reconstituted epidermis, and genetic deletion Anti–IL-22BP neutralizing Ab (28) or control isotype was injected i.p. at a and pharmacological blockade approaches in rodents. dose of 10 mg/kg at days 21, 0, 2, and 4 in Aldara-treated mice. Statistical analysis Materials and Methods Statistical analysis was performed with GraphPad Prism Software http://www.jimmunol.org/ Patients (GraphPad Software, San Diego, CA) or IBM SPSS Statistics 23.0 software For analyses of skin expression, biopsies were obtained from adult healthy (IBM, New York). Mean comparisons of unpaired samples were performed participants (22–63 y old [mean 6 SD: 44.0 6 10.9], 28.6% female), using the Mann–Whitney U test. The Wilcoxon matched-pairs signed-rank patients with plaque psoriasis (25–67 y old [mean 6 SD: 46.4 6 12.8], test (two-tailed) was used for paired samples. Correlations were calculated , 22.7% female, 68.2% moderate to severe disease [psoriasis area and se- using the Spearman rank correlation test. The p values 0.05 were con- verity index (PASI) $10]), and patients with atopic dermatitis (22–40 y old sidered statistically significant. [mean 6 SD: 30.0 6 7.7], 50.0% female, 100% moderate to severe disease). For analyses of blood mediators, blood samples were obtained from Results adult control participants (26–57 y old [mean 6 SD: 38.3 6 9.3], 72.0% female) and patients with plaque psoriasis (19–64 y old [mean 6 SD: 41.2 6 Psoriatic skin shows relative IL-22BP deficiency 13.7], 61.1% female, 27.8% moderate to severe disease). The skin and To assess whether IL-22BP could exert a regulatory role in psoriasis, by guest on September 25, 2021 blood samples were approved by the clinical institutional review board of we first analyzed IL22RA2 expression in the skin from psoriasis pa- the Charite´ University Medicine, Berlin. Written consent was obtained from all participants. The study was conducted according to the principles tients as well as from atopic dermatitis patients and healthy donors as of the Declaration of Helsinki. comparison groups. Confirming our previous works (17, 20), constitutive expression of IL-22BP was found in healthy donors’ skin. Epidermis model IL-22BP was also detected in the nonaffected skin of psoriasis patients. Underdeveloped EpiDerm-201 human epidermis models (MatTek) were However, although IL-22BP expression in atopic dermatitis appeared cultured in inserts at the air–liquid interface as described previously (24) rather increased as compared with healthy donors, levels in the and stimulated or not (control) by supplementing the culture medium with nonaffected skin of psoriasis patients were in fact lower (Fig. 1A). We either 20 ng/ml IL-22, 10 ng/ml IL-17A, a mixture of both, or 20 ng/ml IL-24 (all from R&D Systems, Wiesbaden-Nordenstadt, Germany). After 72 h, next analyzed IL-22 and IL-22BP expression in paired biopsies from samples were taken for quantitative RT-PCR (qRT-PCR) analysis. nonaffected, perilesional, and lesional skin of psoriasis patients. In line with previous studies including ours (8, 15, 29), IL-22 expression Animals was almost absent in nonlesional skin but showed a strong upregu- Il22ra22/2 and Il22ra2+/+ control littermate rats were generated on the lation in perilesional (∼1200-fold) and lesional (2500-fold) skins Sprague-Dawley background using zinc-finger nucleases (Sigma-Aldrich, (Fig. 1B). Importantly, in contrast to IL-22, the increase of IL-22BP St. Louis, MO) at our local Rats Transgenesis Platform facility IBISA- expression was minimal in perilesional and lesional psoriatic skin CNRS as described previously (22). C57BL/6J mice were purchased from ∼ Centre d’Elevage Janvier (Le Genest-Saint-Isle, France). ( 2-fold induction) (Fig. 1B). Thus, the very small IL-22/IL-22BP All animals were kept under specific pathogen-free conditions. All ratio in nonlesional skin (0.01) was largely increased in perilesional animal studies were conducted in accordance with the EU Directive 2010/ and lesional psoriatic skin to ∼3 and 7, respectively. 63/EU for animal experiments, and the guidelines of the French Agriculture Taken together, these data suggest that constitutive low levels of Ministry. These studies were approved by the Veterinary Departmental Services committee (E.44011). IL-22BP in nonlesional psoriatic skin and their limited upregulation in lesional skin could enable broadly unregulated IL-22 to be qRT-PCR analysis effective and therefore might contribute to the development and Snap frozen skin samples or epidermis models were mechanically ho- maintenance of epidermal alterations in these patients. mogenized (25). Total cellular RNA was isolated using Invisorb RNA kit II IL-22BP deficiency exacerbates imiquimod-induced skin (Invitek/Stratec Molecular) (human samples) or TRIzol reagent (Invi- trogen) (rodent samples) according to the manufacturers’ instructions. inflammation in the rat Reverse transcription was performed using murine moloney leukemia virus In order to demonstrate that IL-22BP deficiency indeed strengthens reverse transcriptase (Invitrogen) following the manufacturer’s instructions. Quantitative PCR on reverse transcribed mRNA was performed us- IL-22–mediated pathogenicity in vivo during skin inflammation, ing the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific/ we first took advantage of IL-22BP–deficient rats we generated Fermentas) (human samples) or the TaqMan Fast Advanced Master Mix recently (22) and characterized the impact of IL-22BP deficiency The Journal of Immunology 3673

FIGURE 1. Psoriatic skin shows a relative deficiency in IL-22BP expression. (A) Skin from healthy control donors (n = 11) and nonlesional skin from psoriasis patients (n = 9) and atopic dermatitis patients (n = 4) was analyzed for IL-22BP (IL22RA2) expression by qRT-PCR. Expression was normalized to HPRT transcripts. Mean 6 SEM data are shown; data were analysed using Mann–Whitney U test. (B) Paired nonlesional, perilesional, and lesional skin Downloaded from from psoriasis patients (n = 6–7) was analyzed for IL-22 and IL-22BP expression as in (A). Mean 6 SEM data are shown. Data were analysed using Wilcoxon matched-pairs signed-rank test. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not significant. on the severity of skin lesions in the model of skin inflammation critically depends on IL-23 and downstream mediators IL-17 and induced by imiquimod (30). Following daily topical application, IL-22 (30–32). As expected, imiquimod application on the right TLR7 activation by imiquimod and inflammasome activation ear of Il22ra2+/+ rats led to skin erythema and thickening within 5 d http://www.jimmunol.org/ by the vehicle lead to an acute skin inflammatory process that (Fig. 2A, 2B). Histological analyses confirmed the induction of by guest on September 25, 2021

FIGURE 2. IL-22BP deficiency exacerbates imiquimod-induced psoriasis-like skin disease. Psoriasis-like skin disease was induced in the right ear of Il22ra2+/+ and Il22ra22/2 rats (n = 12 each). (A) Representative pictures of skin lesions at days 0 and 5. (B) Percentage of skin thickness increase (mean 6 SEM). (C) Representative hematoxylin–eosin–saffron staining of ear skin at days 0 and 5. Original magnification 3100 (D) Kinetics of IL-22 and IL-22BP (Il22ra2) expression in imiquimod-treated skin were assessed by qRT-PCR (n = 3 for each time point). (E) IL-17A, IFN-g, TNF-a, and IL-22 expression were analyzed by qRT-PCR at day 5 of imiquimod application. Expression was normalized to HPRT transcripts. Each symbol (square or triangle) corresponds to one rat. (F) Lipocalin-2 (Lcn2) and b-defensin-2 (Defb4) expression were analyzed by qRT-PCR at day 5 of imiquimod application. Expression was normalized to HPRT transcripts. Each symbol (square or triangle) corresponds to one rat. *p , 0.05, **p , 0.01, ***p , 0.001, Mann–Whitney U test. 3674 IL-22BP LIMITS IL-22–DEPENDENT INFLAMMATION IN PSORIASIS acanthosis, parakeratosis, Munro’s microabscesses, and dermal Collectively, data obtained from rodent experiments thus argued immune infiltration (Fig. 2C). Importantly, skin alterations were in favor of a protective role of IL-22BP during skin inflammation characterized by a strong induction of IL-22 and a moderate in- through its ability to neutralize IL-22 pathogenic actions on crease of IL-22BP, somehow mimicking the regulation of these two keratinocytes. molecules in human psoriatic skin (Fig. 2D). Confirming our ex- IL-22/IL-22BP ratio correlates with the expression of IL-22 pectation that IL-22BP should negatively regulate IL-22 pathoge- target molecules in psoriasis nicity during skin inflammation, clinical and histological skin alterations were severely worsened in Il22ra22/2 versus Il22ra2+/+ To extend our findings in rodents and provide evidence that littermate controls (Fig. 2A–C). In agreement with their more severe IL-22BP controls IL-22 in human psoriatic skin, we hypothesized phenotype, enhanced expression of inflammatory cytokines, in- that the IL-22/IL-22BP ratio should reflect the level of free bio- cluding IL-17A, TNF-a,andIFN-g, was detected in lesional skin of active IL-22 (i.e., not bound to IL-22BP) and should thus correlate Il22ra22/2 rats (Fig. 2E). Moreover, concordant with exacerbated with the expression of several molecules induced by IL-22 in the actions of IL-22 in the absence of IL-22BP, b-defensin-2 (BD2) and lesional psoriatic skin. As demonstrated in Fig. 4A, IL-22/IL-22BP lipocalin-2 (LCN2), two antimicrobial peptides known to be induced ratios effectively positively correlated with IL-20 and IL-36g, two by IL-22 in rodent epithelial cells (33, 34), showed significantly cytokines coinduced in keratinocytes by IL-22 (35, 36) and higher expression in the lesional skin of Il22ra22/2 versus Il22ra2+/+ playing a key role in psoriatic skin alterations (37, 38). Positive littermate controls (Fig. 2F). Of note, there was no significant dif- correlations were also measured between IL-22/IL-22BP ratios ference in the expression of these inflammatory cytokines and an- and the neutrophil-recruiting chemokine CXCL1, as well as the 2 2 timicrobial peptides in the skin of Il22ra2 / rat versus Il22ra2+/+ antimicrobial peptide BD2 (Fig. 4A), both genes also known to be Downloaded from littermate controls at steady state (data not shown). strongly induced in keratinocytes by IL-22 (8, 14). As expected, the expression of CXCL1 and BD2 also positively correlated with IL-22BP blockade exacerbates imiquimod-induced skin that of IL-17A, although without reaching statistical significance inflammation in the mouse for BD2 (p = 0.057) (Fig. 4B). This clearly matched the known To further demonstrate the protective role of IL-22BP against IL-22 in facts about IL-17A and IL-22 synergistic actions in inducing these

skin inflammation and avoid any potential bias that might arise from two molecules in keratinocytes (39). Finally, we observed a pos- http://www.jimmunol.org/ studies in genetically engineered animals, we sought to recapitulate the itive correlation between the IL-22/IL-22BP ratio and IL-24 data obtained with IL-22BP–deficient rats through a pharmacological (Fig. 4A), another member of the IL-10 family (9) known to blockade of endogenous IL-22BP during imiquimod-induced skin participate in psoriasis skin lesions (40–42), but whose relation- disease in wild-type animals. As no neutralizing Ab against rat ship with IL-22 has remained poorly established so far. IL-22BP is currently available but does exist against mouse IL-22BP (28), we launched a new set of experiments in wild-type mice treated IL-22 induces IL-24 expression in keratinocytes with imiquimod and injected i.p. with anti–IL-22BP or isotypic To get more insight into the relationship between IL-22 and IL-24, control Ab (Fig. 3A). Back skin lesions, including erythema, thick- we moved to in vitro experiments using reconstituted human ening, and scaling, were dramatically worsened in mice treated with epidermis. IL-24 has been shown to be produced by keratinocytes by guest on September 25, 2021 the anti–IL-22BP Ab as compared with those injected with the control (41), and we observed that its expression was in fact directly in- Ab (Fig. 3B, 3C), as confirmed by histological analyses (Fig. 3D). duced by IL-22 stimulation (Fig. 5A). Interestingly, a similar

FIGURE 3. IL-22BP blockade exacerbates imiquimod-induced psoriasis-like skin disease. Psoriasis-like skin disease was induced in the back skin of C57BL/6J mice treated with an anti–IL-22BP neutralizing Ab (n = 12) or isotype control Ab (n = 6). (A) Experimental protocol. (B) Representative pictures of skin lesions at days 0 and 5. (C) Percentage of skin thickness increase (mean 6 SEM). (D) Representative hematoxylin-eosin-saffron staining of back skin lesions at day 5. Original magniﬁcation 3200. *p , 0.05, **p , 0.01, Mann–Whitney U test. The Journal of Immunology 3675 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. IL-22/IL-22BP expression ratio correlates with expressions of psoriasis-relevant molecules in lesional psoriatic skin. Lesional skin from psoriasis patients (n = 17–21) was analyzed by qRT-PCR. Expression was normalized to HPRT transcripts. The ratio of IL-22 and IL-22BP (IL22RA2) expression data (A) as well as IL-17 expression (B) was correlated with expressions of other psoriasis-relevant molecules. Spearman correlation coefficients and respective p values are indicated. effect was observed after IL-17A stimulation, and the presence of IL-22 were significantly elevated in psoriasis patients (Fig. 6A). We both cytokines in the culture medium further increased the level of also found a slight but significant elevation of IL-22BP serum induction (Fig. 5B). This suggested that, as for BD2 and CXCL1 concentration in psoriasis patients as compared with healthy con- (39, 43), synergistic actions existed between IL-22 and IL-17A in trols (Fig. 6A). These data thus reflected the extent of the expres- regards to IL-24 induction in keratinocytes. Importantly, further sional increase of IL-22 and IL-22BP in psoriatic lesions (Fig. 1A). characterization of the IL-24 action showed its autocrine action to Moreover, the IL-22/IL-22BP serum protein ratio showed excellent inhibit the expression of genes, including CALML5, involved in positive correlation with disease severity, as assessed by the PASI, the terminal differentiation of keratinocytes (Fig. 5C). but not with disease duration (Fig. 6B). These data thus further Together, these data indicate that, as previously described for substantiated a pathophysiological role for IL-22BP in controlling IL-20 (35, 44), IL-22 together with IL-17, induces the expression IL-22–dependent skin inflammation in psoriasis by suggesting that of IL-24, which, by inhibiting keratinocytes differentiation, sup- the higher the level of free IL-22, the more severe psoriasis is. ports the installation of a self-sustained amplification loop responsible for skin alterations in psoriasis. Discussion Psoriasis is a common chronic skin disease whose pathogenesis is Ratio of IL-22 and IL-22BP blood protein levels correlates now admitted to largely consist of deregulated responses of the with psoriasis disease severity immune system promoting the production of high amounts of In the last part of our study we quantified IL-22 and IL-22BP protein cytokines that are directly responsible for skin alterations (2). Due concentrations in sera from psoriasis patients and healthy controls. to the role of the immune system, the high prevalence of the Confirming previous reports including ours (13, 29, 45), levels of disease, and the easily visible and quantifiable skin lesions, 3676 IL-22BP LIMITS IL-22–DEPENDENT INFLAMMATION IN PSORIASIS

FIGURE 5. IL-24 is a target molecule of IL-22 and alters the keratinocyte terminal differentiation. (A and B) Human reconstituted epidermis models were cultured in inserts at the air–liquid interface during 72 h in culture medium supplemented or not (control) with IL-22, IL-17, or both. IL-24 expression was analyzed by qRT-PCR. Expression was normalized to HPRT transcripts. (A) Mean 6 SEM data are shown (n = 8). (B) Percentage of expression relative to control is shown as mean 6 SEM (n = 3). (C) Human reconstituted epidermis models were cultured as in (A) and (B) in the absence (control) or presence of IL-24. CALML5 expression was analyzed by qRT-PCR. Expression was normalized to HPRT transcripts. Mean 6 SEM data are shown (n = 6). *p , 0.05, **p , 0.01, Wilcoxon matched-pairs signed-rank test. Downloaded from psoriasis can even be seen as an easy-to-access model of immune- IL-22BP expression we measured in nonlesional skin of psoriasis mediated inflammatory diseases (IMIDs). This is especially true patients, as compared with healthy donors and atopic dermatitis for IMIDs, which, like psoriasis, depend on the IL-23/IL-17/IL-22 patients, may predispose to emergence and maintenance of pso- pathway, including ankylosing spondylitis or inflammatory bowel riatic lesions. The cellular and molecular mechanisms of this disease for instance (46). Concordantly, the efficacy of several decrease nevertheless still remain to be understood. When pre-

novel immune therapies targeting components of this pathway paring this manuscript, a report appeared in which IL-22BP ex- http://www.jimmunol.org/ have often been successfully tried in psoriasis, firstly, as illus- pression was analyzed specifically in epidermis and dermis of skin trated, with anti–IL-17 or anti–IL-23 Abs (4–7). Beyond this, biopsies from psoriasis patients (47). In contrast to our study, the however, there is currently very limited knowledge about the in authors did not observe any difference in both compartments be- situ regulation of the activity of these cytokines in psoriasis. tween healthy donors and nonlesional skin of psoriasis patients This question is particularly relevant for IL-22, which is part of (47). The reason for this discrepancy is unclear but might be due the rare cytokines that possess a specific endogenous, secreted to the long-lasting and stressing procedure the authors used in inhibitor that is IL-22BP (17–20). In a previous work, we showed their study to separate the epidermis from the dermis, which may that IL-22BP impaired the protective functions of IL-22 on the gut have altered mRNA expressions and masked any possible preex- epithelium during dextran sulfate sodium–induced colitis, and isting differences. by guest on September 25, 2021 suggested it may thus have a pathogenic role in inflammatory To demonstrate that IL-22BP is able to dampen the extent and bowel disease (22). In this study, we provide evidence suggesting severity of IL-22–mediated skin inflammation in psoriasis, we that insufficient production of IL-22BP in the psoriatic skin con- chose to use the highly IL-22–dependent model of skin inflam- tributes to the development and maintenance of epidermal alter- mation induced by the topical application of imiquimod (30, 32) ations. We indeed showed that, concomitant to the strong through two complementary approaches. We first showed that, in induction of IL-22 in the inflamed psoriatic skin, the IL-22BP comparison with littermate controls, a constitutive deficiency of increase was very moderate. Based on our results obtained in IL-22BP in Il22ra22/2 rats was associated with increased severity animal models, it is tempting to speculate that lower levels of of the disease because of unleashed actions of IL-22, as further

FIGURE 6. The ratio of blood IL-22 and IL-22BP correlates with psoriasis disease severity. (A) Levels of IL-22 and IL-22BP were analyzed in the blood of 20 healthy control participants and 18 psoriasis patients by ELISA. Mean 6 SEM data are shown. *p , 0.05 (Mann–Whitney U test). (B) Levels of IL-22 and IL-22BP were analyzed in the blood of 18 psoriasis patients by ELISA. Molar ratios of IL-22 and IL-22BP molecules were calculated and tested for correlation with PASI and disease duration by the Spearman rank correlation test. Spearman correlation coefficients and p values are indicated. The Journal of Immunology 3677 confirmed by enhanced expression of known IL-22–inducible Acknowledgments genes in the lesional skin. These data confirm a recently accepted We are grateful to La Plate-Forme MicroPICell (Structure Fe´de´rative de publication showing the same model of skin inflammation in Recherche François Bonamy, Nantes University, INSERM) for help with 2 2 Il22ra2 / mice (47). Importantly, we further corroborated these immunohistology experiments. data by performing for the first time, to our knowledge, a pharmacological blockade of endogenous IL-22BP through injecting a Disclosures neutralizing Ab (28) to imiquimod-treated mice and thus reca- The authors have no financial conflicts of interest. pitulating our observations made in IL-22BP–deficient rats. Re- sults from these two strategies thus allowed us to formally confirm that IL-22BP is able to play protective functions during skin in- References 1. Boehncke, W.-H., and M. P. Scho¨n. 2015. Psoriasis. Lancet 386: 983–994. flammation by actively inhibiting the extent of IL-22 action. 2. Lowes, M. A., M. Sua´rez-Farinãs, and J. G. Krueger. 2014. Immunology of Moreover, this data obtained with an IL-22BP neutralizing Ab is psoriasis. Annu. Rev. Immunol. 32: 227–255. of particular relevance because it highlights the possibility to 3. Sabat, R., S. Philipp, C. Ho¨flich, S. Kreutzer, E. Wallace, K. 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