Epidermis model
qRT-PCR analysis
Imiquimod-induced psoriasis-like skin inflammation
In vivo neutralization of IL-22BP
Psoriatic skin shows relative IL-22BP deficiency
IL-22BP deficiency exacerbates imiquimod-induced skin inflammation in the rat
IL-22BP blockade exacerbates imiquimod-induced skin inflammation in the mouse
IL-22/IL-22BP ratio correlates with the expression of IL-22 target molecules in psoriasis
IL-22 induces IL-24 expression in keratinocytes
Ratio of IL-22 and IL-22BP blood protein levels correlates with psoriasis disease severity
Limited Presence of IL-22 Binding Protein, a Natural IL-22 Inhibitor, Strengthens Psoriatic Skin Inflammation
Jérôme C. Martin;
Jérôme C. Martin
*Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes, 44093 Nantes Cedex 1, France;
†Institut de Transplantation Urologie Néphrologie, Centre Hospitalier Universitaire Nantes, 44093 Nantes Cedex 1, France;
‡Faculté de Médecine, Université de Nantes, 44093 Nantes Cedex 1, France;
§Laboratoire d'Immunologie, Centre Hospitalier Universitaire Nantes, 44093 Nantes Cedex 1, France;
Address correspondence and reprint requests to Dr. Jérôme C. Martin at the current address: Department of Oncological Science, Icahn School of Medicine at Mount Sinai, 1470 Madison Avenue, New York, NY 10029, Prof. Régis Josien, INSERM UMR1064-ITUN, CHU Nantes Hôtel Dieu, Université de Nantes, 30 Boulevard Jean Monnet, 44093 Nantes Cedex 1, France, or Dr. Robert Sabat, Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charité, Charitéplatz 1, D-10117 Berlin, Germany. E-mail addresses: jerome.martin@mssm.edu (J.C.M.), regis.josien@univ-nantes.fr (R.J.), or robert.sabat@charite.de (R.S.)
Kerstin Wolk
¶Psoriasis Research and Treatment Center, Dermatology/Medical Immunology, University Hospital Charité, D-10117 Berlin, Germany;
‖Interdisciplinary Group of Molecular Immunopathology, University Hospital Charité, D-10117 Berlin, Germany;
#Berlin-Brandenburg Center for Regenerative Therapies, University Hospital Charité, 13353 Berlin, Germany;
Gaëlle Bériou;
Gaëlle Bériou
Ahmed Abidi;
Ahmed Abidi
**Faculté des Sciences Mathématiques, Physiques et Naturelles, Université de Tunis El Manar, 2092 Tunis, Tunisia
Ellen Witte-Händel;
Ellen Witte-Händel
Cédric Louvet;
Cédric Louvet
Georgios Kokolakis
Lucile Drujont;
Lucile Drujont
Laure Dumoutier;
Laure Dumoutier
††Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium;
‡‡Institut de Duve, Université Catholique de Louvain, B-1200 Brussels, Belgium; and
Jean-Christophe Renauld
Robert Sabat;
Robert Sabat
§§Research Center Immunosciences, University Hospital Charité, D-10117 Berlin, Germany
Régis Josien
J.C.M. and K.W. contributed equally to this work and are cofirst authors.
R.S. and R.J. contributed equally to this work and are colast authors.
Received: January 05 2017
Accepted: March 01 2017
Published: May 01 2017
J Immunol (2017) 198 (9): 3671–3678.
Jérôme C. Martin, Kerstin Wolk, Gaëlle Bériou, Ahmed Abidi, Ellen Witte-Händel, Cédric Louvet, Georgios Kokolakis, Lucile Drujont, Laure Dumoutier, Jean-Christophe Renauld, Robert Sabat, Régis Josien; Limited Presence of IL-22 Binding Protein, a Natural IL-22 Inhibitor, Strengthens Psoriatic Skin Inflammation. J Immunol 1 May 2017; 198 (9): 3671–3678. https://doi.org/10.4049/jimmunol.1700021
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. 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 (Il22ra2−/−) 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, we found positive correlations with the expression of IL-22–inducible molecules (IL-20, IL-24, IL-36γ, 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.
Psoriasis is a chronic inflammatory skin disease that affects ∼2% of the Caucasian population (1). Typical macroscopic skin alterations present as sharply demarcated, red, and slightly raised lesions with silver-whitish scales. It is generally acknowledged that these skin changes result from chronic dysregulated activation of the cutaneous immune system that—by secreted cytokines—alters the biology of local tissue cells (2, 3). Epidermal keratinocytes respond by hyperproliferation and impairment of their terminal differentiation, leading to epidermal thickening, hypogranularity, and hyperkeratosis. At the same time, these cells secrete high amounts of chemokines enabling the recruitment of further immune cells into the skin to create a self-sustained inflammatory milieu. Moreover, the high keratinocyte production of antimicrobial peptides prevents infections of the highly disturbed psoriatic epidermis. Among a range of immune mediators present in the psoriatic skin, an essential role for IL-23 and its downstream cytokine IL-17A in the maintenance of psoriatic lesions has been recently confirmed by the dramatic clinical success of their therapeutic blockade in these patients (4–7).
Another major IL-23 downstream cytokine is the IL-10 family member IL-22. IL-22 is also present in large quantities in psoriatic lesions (8). Main producers of IL-22 include different CD4+ T cell populations and group 3 innate lymphoid cells (9). Because the expression of the membrane IL-22R is restricted to epithelial/epithelioid cells (8), IL-22 assumes major cross-talk functions between immune and epithelial cells, especially at body barriers (10, 11). By inducing antimicrobial peptides and proinflammatory chemokines, as well as by inhibiting the terminal differentiation of keratinocytes, IL-22 has been shown to largely contribute to inflammation and epithelial remodeling of the psoriatic skin (12–16). Interestingly, the IL-22 activity is regulated by IL-22 binding protein (IL-22BP). IL-22BP is a soluble single chain receptor encoded by the gene IL22RA2 that specifically prevents the binding of IL-22 to IL-22R (17–20). Recently, we and others showed that conventional dendritic cell–derived IL-22BP exerts important inhibitory functions that impair the IL-22–mediated protection during acute colitis in rodents, although preventing potentially dangerous long-lasting proliferative effects on intestinal epithelial cells (21–23). Whether IL-22BP is expressed and plays a role in psoriatic skin inflammation is, however, unknown so far. Our study answers these questions in a translational approach using skin and blood samples from healthy control donors and psoriasis patients, in vitro experiments with human keratinocytes and reconstituted epidermis, and genetic deletion and pharmacological blockade approaches in rodents.
For analyses of skin expression, biopsies were obtained from adult healthy participants (22–63 y old [mean ± SD: 44.0 ± 10.9], 28.6% female), patients with plaque psoriasis (25–67 y old [mean ± SD: 46.4 ± 12.8], 22.7% female, 68.2% moderate to severe disease [psoriasis area and severity index (PASI) ≥10]), and patients with atopic dermatitis (22–40 y old [mean ± SD: 30.0 ± 7.7], 50.0% female, 100% moderate to severe disease). For analyses of blood mediators, blood samples were obtained from adult control participants (26–57 y old [mean ± SD: 38.3 ± 9.3], 72.0% female) and patients with plaque psoriasis (19–64 y old [mean ± SD: 41.2 ± 13.7], 61.1% female, 27.8% moderate to severe disease). The skin and blood samples were approved by the clinical institutional review board of the Charité University Medicine, Berlin. Written consent was obtained from all participants. The study was conducted according to the principles of the Declaration of Helsinki.
Underdeveloped EpiDerm-201 human epidermis models (MatTek) were cultured in inserts at the air–liquid interface as described previously (24) and stimulated or not (control) by supplementing the culture medium with 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, samples were taken for quantitative RT-PCR (qRT-PCR) analysis.
Il22ra2−/− and Il22ra2+/+ control littermate rats were generated on the Sprague-Dawley background using zinc-finger nucleases (Sigma-Aldrich, St. Louis, MO) at our local Rats Transgenesis Platform facility IBISA-CNRS as described previously (22). C57BL/6J mice were purchased from Centre d'Elevage Janvier (Le Genest-Saint-Isle, France).
All animals were kept under specific pathogen-free conditions. All animal studies were conducted in accordance with the EU Directive 2010/63/EU for animal experiments, and the guidelines of the French Agriculture Ministry. These studies were approved by the Veterinary Departmental Services committee (E.44011).
Snap frozen skin samples or epidermis models were mechanically homogenized (25). Total cellular RNA was isolated using Invisorb RNA kit II (Invitek/Stratec Molecular) (human samples) or TRIzol reagent (Invitrogen) (rodent samples) according to the manufacturers' instructions. Reverse transcription was performed using murine moloney leukemia virus reverse transcriptase (Invitrogen) following the manufacturer's instructions. Quantitative PCR on reverse transcribed mRNA was performed using the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific/Fermentas) (human samples) or the TaqMan Fast Advanced Master Mix 2× reagent (rodent samples) (both from Applied Biosystems) and the StepOne Plus device (Applied Biosystems). Primers and double-labeled 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). Expressions were normalized to HPRT using the 2-ΔΔ cycle threshold method, and results were expressed in arbitrary units.
Detection of blood serum levels of IL-22 and IL-22BP was performed using ELISA kits from Bio-Techne (Quantikine system) and Biozol (Cloud-Clone), respectively.
Psoriasis-like skin inflammation was induced in the right ear of Il22ra2+/+ and Il22ra2−/− rats, and in the back skin of C57BL/6J mice as previously described (27). Briefly, topical application of Aldara cream (3M Pharmaceuticals) was performed daily for 5 d. Skin thickness was measured daily with a Digimatic Caliper and the percentage of skin thickness increase 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 were stained with H&E.
Anti–IL-22BP neutralizing Ab (28) or control isotype was injected i.p. at a dose of 10 mg/kg at days −1, 0, 2, and 4 in Aldara-treated mice.
Statistical analysis was performed with GraphPad Prism Software (GraphPad Software, San Diego, CA) or IBM SPSS Statistics 23.0 software (IBM, New York). Mean comparisons of unpaired samples were performed using the Mann–Whitney U test. The Wilcoxon matched-pairs signed-rank test (two-tailed) was used for paired samples. Correlations were calculated using the Spearman rank correlation test. The p values <0.05 were considered statistically significant.
To assess whether IL-22BP could exert a regulatory role in psoriasis, we first analyzed IL22RA2 expression in the skin from psoriasis patients as well as from atopic dermatitis patients and healthy donors as comparison groups. Confirming our previous works (17, 20), constitutive expression of IL-22BP was found in healthy donors' skin. IL-22BP was also detected in the nonaffected skin of psoriasis patients. However, although IL-22BP expression in atopic dermatitis appeared rather increased as compared with healthy donors, levels in the nonaffected skin of psoriasis patients were in fact lower (Fig. 1A). We next analyzed IL-22 and IL-22BP expression in paired biopsies from nonaffected, perilesional, and lesional skin of psoriasis patients. In line with previous studies including ours (8, 15, 29), IL-22 expression was almost absent in nonlesional skin but showed a strong upregulation in perilesional (∼1200-fold) and lesional (2500-fold) skins (Fig. 1B). Importantly, in contrast to IL-22, the increase of IL-22BP expression was minimal in perilesional and lesional psoriatic skin (∼2-fold induction) (Fig. 1B). Thus, the very small IL-22/IL-22BP ratio in nonlesional skin (0.01) was largely increased in perilesional and lesional psoriatic skin to ∼3 and 7, respectively.
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 ± SEM data are shown; data were analysed using Mann–Whitney U test. (B) Paired nonlesional, perilesional, and lesional skin from psoriasis patients (n = 6–7) was analyzed for IL-22 and IL-22BP expression as in (A). Mean ± 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.
Taken together, these data suggest that constitutive low levels of IL-22BP in nonlesional psoriatic skin and their limited upregulation in lesional skin could enable broadly unregulated IL-22 to be effective and therefore might contribute to the development and maintenance of epidermal alterations in these patients.
In order to demonstrate that IL-22BP deficiency indeed strengthens IL-22–mediated pathogenicity in vivo during skin inflammation, we first took advantage of IL-22BP–deficient rats we generated recently (22) and characterized the impact of IL-22BP deficiency on the severity of skin lesions in the model of skin inflammation induced by imiquimod (30). Following daily topical application, TLR7 activation by imiquimod and inflammasome activation by the vehicle lead to an acute skin inflammatory process that critically depends on IL-23 and downstream mediators IL-17 and IL-22 (30–32). As expected, imiquimod application on the right ear of Il22ra2+/+ rats led to skin erythema and thickening within 5 d (Fig. 2A, 2B). Histological analyses confirmed the induction of acanthosis, parakeratosis, Munro's microabscesses, and dermal immune infiltration (Fig. 2C). Importantly, skin alterations were characterized by a strong induction of IL-22 and a moderate increase of IL-22BP, somehow mimicking the regulation of these two molecules in human psoriatic skin (Fig. 2D). Confirming our expectation that IL-22BP should negatively regulate IL-22 pathogenicity during skin inflammation, clinical and histological skin alterations were severely worsened in Il22ra2−/− versus Il22ra2+/+ littermate controls (Fig. 2A–C). In agreement with their more severe phenotype, enhanced expression of inflammatory cytokines, including IL-17A, TNF-α, and IFN-γ, was detected in lesional skin of Il22ra2−/− rats (Fig. 2E). Moreover, concordant with exacerbated actions of IL-22 in the absence of IL-22BP, β-defensin-2 (BD2) and lipocalin-2 (LCN2), two antimicrobial peptides known to be induced by IL-22 in rodent epithelial cells (33, 34), showed significantly higher expression in the lesional skin of Il22ra2−/− versus Il22ra2+/+ littermate controls (Fig. 2F). Of note, there was no significant difference in the expression of these inflammatory cytokines and antimicrobial peptides in the skin of Il22ra2−/− rat versus Il22ra2+/+ littermate controls at steady state (data not shown).
IL-22BP deficiency exacerbates imiquimod-induced psoriasis-like skin disease. Psoriasis-like skin disease was induced in the right ear of Il22ra2+/+ and Il22ra2−/− rats (n = 12 each). (A) Representative pictures of skin lesions at days 0 and 5. (B) Percentage of skin thickness increase (mean ± SEM). (C) Representative hematoxylin–eosin–saffron staining of ear skin at days 0 and 5. Original magnification ×100 (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-γ, TNF-α, 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 β-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.
To further demonstrate the protective role of IL-22BP against IL-22 in skin inflammation and avoid any potential bias that might arise from studies in genetically engineered animals, we sought to recapitulate the data obtained with IL-22BP–deficient rats through a pharmacological blockade of endogenous IL-22BP during imiquimod-induced skin disease in wild-type animals. As no neutralizing Ab against rat 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 with imiquimod and injected i.p. with anti–IL-22BP or isotypic control Ab (Fig. 3A). Back skin lesions, including erythema, thickening, and scaling, were dramatically worsened in mice treated with the anti–IL-22BP Ab as compared with those injected with the control Ab (Fig. 3B, 3C), as confirmed by histological analyses (Fig. 3D).
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 ± SEM). (D) Representative hematoxylin-eosin-saffron staining of back skin lesions at day 5. Original magnification ×200. *p < 0.05, **p < 0.01, Mann–Whitney U test.
Collectively, data obtained from rodent experiments thus argued in favor of a protective role of IL-22BP during skin inflammation through its ability to neutralize IL-22 pathogenic actions on keratinocytes.
To extend our findings in rodents and provide evidence that IL-22BP controls IL-22 in human psoriatic skin, we hypothesized that the IL-22/IL-22BP ratio should reflect the level of free bioactive IL-22 (i.e., not bound to IL-22BP) and should thus correlate with the expression of several molecules induced by IL-22 in the lesional psoriatic skin. As demonstrated in Fig. 4A, IL-22/IL-22BP ratios effectively positively correlated with IL-20 and IL-36γ, two cytokines coinduced in keratinocytes by IL-22 (35, 36) and playing a key role in psoriatic skin alterations (37, 38). Positive correlations were also measured between IL-22/IL-22BP ratios and the neutrophil-recruiting chemokine CXCL1, as well as the antimicrobial peptide BD2 (Fig. 4A), both genes also known to be strongly induced in keratinocytes by IL-22 (8, 14). As expected, the expression of CXCL1 and BD2 also positively correlated with that of IL-17A, although without reaching statistical significance for BD2 (p = 0.057) (Fig. 4B). This clearly matched the known facts about IL-17A and IL-22 synergistic actions in inducing these two molecules in keratinocytes (39). Finally, we observed a positive correlation between the IL-22/IL-22BP ratio and IL-24 (Fig. 4A), another member of the IL-10 family (9) known to participate in psoriasis skin lesions (40–42), but whose relationship with IL-22 has remained poorly established so far.
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.
To get more insight into the relationship between IL-22 and IL-24, we moved to in vitro experiments using reconstituted human epidermis. IL-24 has been shown to be produced by keratinocytes (41), and we observed that its expression was in fact directly induced by IL-22 stimulation (Fig. 5A). Interestingly, a similar effect was observed after IL-17A stimulation, and the presence of both cytokines in the culture medium further increased the level of induction (Fig. 5B). This suggested that, as for BD2 and CXCL1 (39, 43), synergistic actions existed between IL-22 and IL-17A in regards to IL-24 induction in keratinocytes. Importantly, further characterization of the IL-24 action showed its autocrine action to inhibit the expression of genes, including CALML5, involved in the terminal differentiation of keratinocytes (Fig. 5C).
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 ± SEM data are shown (n = 8). (B) Percentage of expression relative to control is shown as mean ± 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 ± SEM data are shown (n = 6). *p < 0.05, **p < 0.01, Wilcoxon matched-pairs signed-rank test.
Together, these data indicate that, as previously described for IL-20 (35, 44), IL-22 together with IL-17, induces the expression of IL-24, which, by inhibiting keratinocytes differentiation, supports the installation of a self-sustained amplification loop responsible for skin alterations in psoriasis.
In the last part of our study we quantified IL-22 and IL-22BP protein concentrations in sera from psoriasis patients and healthy controls. Confirming previous reports including ours (13, 29, 45), levels of IL-22 were significantly elevated in psoriasis patients (Fig. 6A). We also found a slight but significant elevation of IL-22BP serum concentration in psoriasis patients as compared with healthy controls (Fig. 6A). These data thus reflected the extent of the expressional increase of IL-22 and IL-22BP in psoriatic lesions (Fig. 1A). Moreover, the IL-22/IL-22BP serum protein ratio showed excellent positive correlation with disease severity, as assessed by the PASI, but not with disease duration (Fig. 6B). These data thus further substantiated a pathophysiological role for IL-22BP in controlling IL-22–dependent skin inflammation in psoriasis by suggesting that the higher the level of free IL-22, the more severe psoriasis is.
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 ± 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.
Psoriasis is a common chronic skin disease whose pathogenesis is now admitted to largely consist of deregulated responses of the immune system promoting the production of high amounts of cytokines that are directly responsible for skin alterations (2). Due to the role of the immune system, the high prevalence of the disease, and the easily visible and quantifiable skin lesions, psoriasis can even be seen as an easy-to-access model of immune-mediated inflammatory diseases (IMIDs). This is especially true for IMIDs, which, like psoriasis, depend on the IL-23/IL-17/IL-22 pathway, including ankylosing spondylitis or inflammatory bowel disease for instance (46). Concordantly, the efficacy of several novel immune therapies targeting components of this pathway have often been successfully tried in psoriasis, firstly, as illustrated, with anti–IL-17 or anti–IL-23 Abs (4–7). Beyond this, however, there is currently very limited knowledge about the in situ regulation of the activity of these cytokines in psoriasis.
This question is particularly relevant for IL-22, which is part of the rare cytokines that possess a specific endogenous, secreted inhibitor that is IL-22BP (17–20). In a previous work, we showed that IL-22BP impaired the protective functions of IL-22 on the gut epithelium during dextran sulfate sodium–induced colitis, and suggested it may thus have a pathogenic role in inflammatory bowel disease (22). In this study, we provide evidence suggesting that insufficient production of IL-22BP in the psoriatic skin contributes to the development and maintenance of epidermal alterations. We indeed showed that, concomitant to the strong induction of IL-22 in the inflamed psoriatic skin, the IL-22BP increase was very moderate. Based on our results obtained in animal models, it is tempting to speculate that lower levels of IL-22BP expression we measured in nonlesional skin of psoriasis patients, as compared with healthy donors and atopic dermatitis patients, may predispose to emergence and maintenance of psoriatic lesions. The cellular and molecular mechanisms of this decrease nevertheless still remain to be understood. When preparing this manuscript, a report appeared in which IL-22BP expression was analyzed specifically in epidermis and dermis of skin biopsies from psoriasis patients (47). In contrast to our study, the authors did not observe any difference in both compartments between healthy donors and nonlesional skin of psoriasis patients (47). The reason for this discrepancy is unclear but might be due to the long-lasting and stressing procedure the authors used in their study to separate the epidermis from the dermis, which may have altered mRNA expressions and masked any possible preexisting differences.
To demonstrate that IL-22BP is able to dampen the extent and severity of IL-22–mediated skin inflammation in psoriasis, we chose to use the highly IL-22–dependent model of skin inflammation induced by the topical application of imiquimod (30, 32) through two complementary approaches. We first showed that, in comparison with littermate controls, a constitutive deficiency of IL-22BP in Il22ra2−/− rats was associated with increased severity of the disease because of unleashed actions of IL-22, as further confirmed by enhanced expression of known IL-22–inducible genes in the lesional skin. These data confirm a recently accepted publication showing the same model of skin inflammation in Il22ra2−/− mice (47). Importantly, we further corroborated these data by performing for the first time, to our knowledge, a pharmacological blockade of endogenous IL-22BP through injecting a neutralizing Ab (28) to imiquimod-treated mice and thus recapitulating our observations made in IL-22BP–deficient rats. Results from these two strategies thus allowed us to formally confirm that IL-22BP is able to play protective functions during skin inflammation by actively inhibiting the extent of IL-22 action. Moreover, this data obtained with an IL-22BP neutralizing Ab is of particular relevance because it highlights the possibility to target IL-22BP in conditions where the IL-22BP–mediated control of IL-22 is expected to be detrimental, like inflammatory bowel disease, as we previously suggested (22). In this context, it is important to note that not only the structure of respective genes, mRNA transcripts, and proteins (48), but also the IL-22–neutralizing capacity are virtually identical between human and rodent IL-22BP (23).
No correlation existed between IL-22BP and IL-22 expression in the lesional psoriatic skin (data not shown), suggesting that different inducer signals are involved in their respective upregulation. This different regulation of IL-22 and IL-22BP indicates that accurate interpretation of high levels of IL-22 production in the psoriatic skin must take concomitant levels of IL-22BP into account. In this sense, we observed a close positive association between the IL-22/IL-22BP ratio and the expression of several IL-22–inducible molecules known for their pathogenic roles in psoriasis (i.e., IL-20, IL-36γ, and CXCL1). Furthermore, the IL-22/IL-22BP ratio also positively correlated with IL-24, another member of the IL-20 subfamily of cytokines sharing pathogenic functions with IL-22 in psoriasis (9, 40–42). As previously suggested by in vitro experiments in cultured keratinocytes transfected with IL-22R cDNA (44), we confirmed IL-24 as an additional target gene of IL-22 in psoriasis. Interestingly, similar to other genes including those encoding CXCL1 and BD2 (39), IL-24 expression was further increased when IL-17A was added to IL-22 in the culture medium. This suggested that a number of pathogenic effects attributed to either IL-22 or IL-17A in the psoriatic skin are in fact dependent on synergic actions of both cytokines on keratinocytes, and are thus subjected to IL-22BP negative regulation. For psoriasis pathogenesis, this also indicates the existence of a self-sustained amplification loop in which the T cell–derived cytokines not only directly induce the visible epidermal alterations in psoriasis but also provoke the expression of keratinocyte mediators further worsening these alterations.
Finally, we substantiated the importance of the protective functions assumed by IL-22BP through addressing its serum levels in relation to those of IL-22 in psoriasis patients. We indeed showed that the IL-22/IL-22BP serum protein ratio strongly correlated with the disease severity as assessed by the PASI, which suggests that the higher the control of IL-22 by IL-22BP, the less severe psoriasis is. In future prospective studies it will be interesting to address whether the IL-22/IL-22BP blood protein ratio may predict therapy success (e.g., anti–IL-17 or anti-p40 therapy), proofing the value of this ratio as a potential new biomarker in psoriasis.
In conclusion, we showed that IL-22BP critically controls deleterious actions of IL-22 during skin inflammation in rodents and suggest it is insufficiently present to limit the extent of IL-22 pathogenicity in human psoriasis. Together with our recent report on the role of IL-22BP during inflammatory bowel diseases, our results identify IL-22BP as a critical regulator of IL-22 actions on epithelial cells during IMIDs.
We are grateful to La Plate-Forme MicroPICell (Structure Fédérative de Recherche François Bonamy, Nantes University, INSERM) for help with immunohistology experiments.
This work was supported by the National Research Agency via Investment into the Future Program Grant ANR-10-IBHU-005 for the Institut-Hospitalo Universitaire–Centre Européen des Sciences de la Transplantation et de l'Immunologie (IHU-Cesti) Project. The IHU-Cesti Project is also supported by Nantes Métropole and the Pays de la Loire region. J.C.M. was supported by a grant from Centre Hospitalier Universitaire (CHU) Nantes (Appel d'Offre Interne 2013 RC14_0042); J.C.M. also received support from CHU Nantes through Année Supplémentaire d'Internat. G.B. was supported by the Pays de la Loire Region through the IMmunoBIOthérapies et Cellules Dendritiques Network. A.A. was supported by a French-Tunisian UTIQUE grant from the 2015 Hubert Curien program. K.W. and R.S. were supported by Grant 01ZX1312A from the German Federal Ministry of Education and Research.
Abbreviations used in this article:
IL-22BP
IL-22 binding protein
IMID
immune-mediated inflammatory disease
psoriasis area and severity index
qRT-PCR
quantitative RT-PCR.
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ERASURE Study Group
FIXTURE Study Group
Secukinumab in plaque psoriasis--results of two phase 3 trials.
Strober
Menter
Weglowska
Toth
Kerdel
Phase 3 studies comparing brodalumab with ustekinumab in psoriasis.
IL-22 increases the innate immunity of tissues.
Rutz
The IL-20 subfamily of cytokines--from host defence to tissue homeostasis.
Nat. Rev. Immunol.
Therapeutic opportunities of the IL-22-IL-22R1 system.
Nat. Rev. Drug Discov.
Dudakov
J. A.
Hanash
M. R. M.
Interleukin-22: immunobiology and pathology.
F.-X.
Lecron
J.-C.
IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.
W.-D.
IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis.
Eur. J. Immunol.
Haugen
H. S.
Waggie
Vom Baur
IL-22 and IL-20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-gamma are not.
J. Mol. Med. (Berl.)
G. K.
Ainali
Semenova
Hundhausen
Barinaga
A. E.
M. M.
Integrative biology approach identifies cytokine targeting strategies for psoriasis.
Sci. Transl. Med.
223ra22
H.-L.
Liang
Napierata
Senices
Dunussi-Joannopoulos
IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation.
J. Clin. Invest.
Colau
Renauld
Cloning and characterization of IL-22 binding protein, a natural antagonist of IL-10-related T cell-derived inducible factor/IL-22.
Kotenko
S. V.
Izotova
L. S.
Mirochnitchenko
O. V.
Esterova
Dickensheets
R. P.
Pestka
Identification, cloning, and characterization of a novel soluble receptor that binds IL-22 and neutralizes its activity.
Presnell
Parrish-Novak
Kindsvogel
Jaspers
A soluble class II cytokine receptor, IL-22RA2, is a naturally occurring IL-22 antagonist.
B. H.
Schoenemeyer
A novel, soluble homologue of the human IL-10 receptor with preferential expression in placenta.
Genes Immun.
J. C. J.
Bériou
Heslan
Utriainen
Aumeunier
C. L.
Mowat
Cerovic
S. A.
Interleukin-22 binding protein (IL-22BP) is constitutively expressed by a subset of conventional dendritic cells and is strongly induced by retinoic acid.
Mucosal Immunol.
Bossard
Abidi
Ménoret
Thinard
Anegon
IL-22BP is produced by eosinophils in human gut and blocks IL-22 protective actions during colitis.
Gagliani
Zenewicz
F. J.
Bosurgi
Hedl
Jr.,
A. J.
IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine.
Gellrich
Gulati
Humme
Gonsior
M. E.
Deficient cutaneous antibacterial competence in cutaneous T-cell lymphomas: role of Th2-mediated biased Th17 function.
Clin. Cancer Res.
Raftery
Kokolakis
Schönrich
Prösch
IL-29 is produced by T(H)17 cells and mediates the cutaneous antiviral competence in psoriasis.
204ra129
Hoeflich
Schneider-Burrus
Roewert
H. J.
Deficiency of IL-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa.
Drujont
Lancien
Bouchet-Delbos
RORγt+ cells selectively express redundant cation channels linked to the Golgi apparatus.
Sci. Rep.
Vanhaudenarde
Induction of autoantibodies against mouse soluble proteins after immunization with living cells presenting the autoantigen at the cell surface in fusion with a human type 2 transmembrane protein.
J. Immunol. Methods
Guignouard
Pedretti
Delwail
Dagregorio
Yssel
A role for T cell-derived interleukin 22 in psoriatic skin inflammation.
Clin. Exp. Immunol.
F. O.
TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
van der Fits
Mourits
Voerman
J. S. A.
A.-M.
Prens
Lubberts
Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
Van Belle
de Heusch
Hendrickx
Warnier
Fouser
IL-22 is required for imiquimod-induced psoriasiform skin inflammation in mice.
Zelante
Iannitti
Pieraccini
Zecchi
D'Angelo
Massi-Benedetti
Fallarino
Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22.
Aujla
Y. R.
D. J.
Pociask
Reinhart
T. A.
McAllister
Edeal
Gaus
IL-22 mediates mucosal host defense against gram-negative bacterial pneumonia.
Nat. Med.
Schulze-Tanzil
The Th17 cytokine IL-22 induces IL-20 production in keratinocytes: a novel immunological cascade with potential relevance in psoriasis.
H. E.
O'Toole
Nickerson-Nutter
Inter-regulation of Th17 cytokines and the IL-36 cytokines in vitro and in vivo: implications in psoriasis pathogenesis.
J. Invest. Dermatol.
W. F.
Brender
Carollo
Haldeman
Interleukin 20: discovery, receptor identification, and role in epidermal function.
Rosenwald
Kisielow
Psoriasiform dermatitis is driven by IL-36-mediated DC-keratinocyte crosstalk.
X.-Y.
Luxenberg
Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides.
J. Exp. Med.
Nørby
P. L.
Steiniche
Thorn Clausen
Kragballe
Epidermal overexpression of interleukin-19 and -20 mRNA in psoriatic skin disappears after short-term treatment with cyclosporine a or calcipotriol.
Doecke
Interleukin (IL)-19, IL-20 and IL-24 are produced by and act on keratinocytes and are distinct from classical ILs.
Kumari
Ulvmar
Karagianni
Uthoff-Hachenberg
Kollias
Toftgard
Tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice.
P. A.
Zhong
Kasman
Modrusan
Danilenko
The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis.
Tohyama
Hanakawa
Hirakawa
IL-17 and IL-22 mediate IL-20 subfamily cytokine production in cultured keratinocytes via increased IL-22 receptor expression.
H. L.
J. J.
Circulating Th17, Th22, and Th1 cells are increased in psoriasis.
Kuchroo
V. K.
Th17 cell pathway in human immunity: lessons from genetics and therapeutic interventions.
Brauner
Nikamo
Gallais Serezal
Guerreiro-Cacais
A. O.
Jagodic
Eidsmo
Ståhle
IL-22 binding protein regulates murine skin inflammation.
DOI: 10.1111/exd.13225
Cloning of murine IL-22 receptor alpha 2 and comparison with its human counterpart.
The authors have no financial conflicts of interest.