IL-22 Is Required for Imiquimod-Induced Psoriasiform Skin Inflammation in Mice Astrid B. Van Belle, Magali de Heusch, Muriel M. Lemaire, Emilie Hendrickx, Guy Warnier, Kyri This information is current as Dunussi-Joannopoulos, Lynette A. Fouser, Jean-Christophe of September 29, 2021. Renauld and Laure Dumoutier J Immunol published online 30 November 2011 http://www.jimmunol.org/content/early/2011/11/30/jimmun

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 30, 2011, doi:10.4049/jimmunol.1102224 The Journal of Immunology

IL-22 Is Required for Imiquimod-Induced Psoriasiform Skin Inflammation in Mice

Astrid B. Van Belle,*,† Magali de Heusch,*,† Muriel M. Lemaire,*,† Emilie Hendrickx,*,† Guy Warnier,*,† Kyri Dunussi-Joannopoulos,‡ Lynette A. Fouser,‡ Jean-Christophe Renauld,*,†,1 and Laure Dumoutier*,†,1

Psoriasis is a common chronic autoimmune skin disease of unknown cause that involves dysregulated interplay between immune cells and keratinocytes. IL-22 is a cytokine produced by the TH1, TH17, and TH22 subsets that are functionally implicated in the psoriatic pathology. We assessed the role of IL-22 in a mouse model where psoriasiform skin inflammation is triggered by topical application of the TLR7/8 agonist imiquimod. At the macroscopic level, scaly skin lesions induced by daily applications of imiqui- mod in wild-type mice were almost totally absent in IL-22–deficient mice or in mice treated with a blocking anti–IL-22 Ab. At the microscopic level, IL-22–deficient mice showed a dramatic decrease in the development of pustules and a partial decrease in Downloaded from acanthosis. At the molecular level, the absence or inhibition of IL-22 strongly decreased the expression of chemotactic factors such as CCL3 and CXCL3 and of biomarkers such as S100A8, S100A7, and keratin 14, which reflect the antimicrobial and hyper- proliferative responses of keratinocytes. IL-22 also played a major role in neutrophil infiltration after imiquimod treatment. IL-23 was required for IL-22 production, and gd TCR lymphocytes represented the major source of IL-22 in lymph nodes from imiquimod-treated mice. However, T cells were not absolutely required for IL-22 production because imiquimod-induced IL- 22 expression in the skin is still preserved in Rag22/2 mice. Taken together, our data show that IL-22 is required for psoriasis-like http://www.jimmunol.org/ lesions in the mouse imiquimod model and is produced by both T cells and innate immune cells. The Journal of Immunology, 2012, 188: 000–000.

L-22 is a cytokine related to IL-10 for its structure and to IL- eration of keratinocytes and induced hyperplasia of reconstituted 17 for its cellular pattern of production (1). This cytokine can epidermis (4). I hardly be classified as a pro- or anti-inflammatory cytokine. These biological activities are expected to be beneficial during For instance, analysis of IL-22–deficient mice indicated that it wound healing and infectious processes but might contribute plays a pathogenic role in an arthritis model (2) while being to skin lesions of patients with psoriasis disease, one of the most by guest on September 29, 2021 protective in several colitis models (3). The main physiological common autoimmune-mediated chronic inflammatory skin dis- role of IL-22 appears to be related to wound healing and innate orders that affects ∼2% of the general population (10). Increased antimicrobial responses, because its receptor is mainly expressed levels of Th17 cells and their associated cytokines including IL- by nonhematopoietic cells at body barriers including epithelial 23, IL-17, and IL-22 have been found in human psoriatic skin (6, cells of the lung and gastrointestinal tract and keratinocytes (1). 11, 12). Elevated IL-22 concentrations were measured in plasma In vitro studies showed that IL-22 activates STAT3 (4–6) and from patients and IL-22 production correlated positively with upregulates the expression of proinflammatory and antimicrobial severity of the disease and negatively with response to therapy molecules such as S100A7, S100A8, S100A9, and b-defensins in suggesting a significant role of this cytokine in psoriasis (5, 7, 13). human keratinocytes (4–7), alone or in synergy with IL-17 or TNF T lymphocytes appeared to represent a major source of IL-22 in (8, 9). IL-22 also regulates the cellular differentiation and prolif- skin lesions (7) and treatment with cyclosporine A downregulated both IL-17 and IL-22 expression in those patients (14). *Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; †de Duve Insti- Psoriasis is not observed in animals other than humans, but tute, Universite´ catholique de Louvain, B-1200 Brussels, Belgium; and ‡Inflammation a large number of mouse models have been set up to mimic at least and Autoimmunity Research Unit, Pfizer BioTherapeutics Research and Development, some aspects of the human disease (15, 16). Psoriasis lesions Cambridge, MA 02140 1 are characterized by several histological features including 1) a J.-C.R. and L.D. share senior coauthorship. thickened epidermis caused by keratinocyte proliferation that is Received for publication August 4, 2011. Accepted for publication November 2, called acanthosis, 2) retention of nuclei in the stratum corneum 2011. (parakeratosis) that arises from an aberrant differentiation of This work was supported in part by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy keratinocytes, and 3) inflammatory cell infiltrates in the epidermis Programming, and by the Actions de Recherche Concerte´es of the Communaute´ and dermis (17, 18). IL-22–transgenic mice die soon after birth Franc¸aise de Belgique. L.D. is a Research Associate and A.B.V.B. is a Research with acanthosis and hyperkeratosis that are reminiscent of such Fellow with the Fonds National de la Recherche Scientifique, Belgium. psoriatic lesions (19). Acanthosis can also be induced in wild-type Address correspondence and reprint request to Dr. Jean-Christophe Renauld and Laure Dumoutier, Ludwig Institute for Cancer Research, Avenue Hippocrate 74, 1200 mice by intradermal injection of rIL-23, and this effect is partially Brussels, Belgium. E-mail addresses: [email protected] and laure.dumoutier@bru. abolished in IL-22–deficient mice (20). The role of IL-22 is also licr.org supported by a graft versus host disease (GVHD) mouse model Abbreviations used in this article: GVHD, graft versus host disease; Ngp, neutro- that is based on the transfer of CD4+CD45RBhi T lymphocytes, philic granule protein; qPCR, quantitative PCR. depleted of regulatory T cells, into immunodeficient mice. The Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 recipient mice develop acanthosis, elevated levels of antimicrobial

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102224 2 ROLE OF IL-22 IN IMIQUIMOD-INDUCED PSORIASIS peptides and inflammatory infiltrates that can be abolished by Diff-Quick solutions (Medion Diagnostics Ag, Du¨dingen, Switzerland). anti–IL-22 Abs (21). Interestingly, this same GVHD mouse model Percentages of neutrophils were counted on five different areas (with ∼200 can also result in colitis that is exacerbated by IL-22 inhibition, in cells) for each cytospin. Cells from lymph nodes were extracted by mechanical disruption and contrast to the effect in the skin, demonstrating the dual activity resuspended in Iscove Dulbecco medium. For intracellular staining, cells of this cytokine in inflammation (3). were incubated at 37˚C in the presence of GolgiStop solution (BD Bio- More recently, topical treatment with imiquimod, a ligand for sciences) and IL-23 (10 ng/ml) and IL-2 (400 U/ml). After 4 h, cells were TLR7 and TLR8 was reported as a novel mouse model for stained with Abs specific for cell surface Ags at 4˚C. Cells were then fixed, permeabilized, and processed for intracellular staining using the Cytofix/ psoriasis-like skin inflammation, inducing acanthosis, parakeratosis, Cytoperm Plus Kit (BD Biosciences). The mAbs specific to murine Ags and a mixed inflammatory infiltrate (22). Imiquimod is used in used in this study were MH22B2 (anti–IL-22) and MM17F3 (anti–IL- humans for topical treatment of genital and perianal warts caused 17A), which were produced in our laboratory (32), whereas PC61 (anti- by human papillomavirus (23), actinic keratosis, and superficial CD25), 17A2 (anti-CD3), GK1.5 (anti-CD4), H57-597 (anti-TCRb), and basal cell carcinoma (24, 25). Clinical application of imiquimod GL3 (anti-TCRgd) were purchased from BioLegend. can also induce psoriasis or exacerbate the disease in patients T lymphocyte adoptive transfer with a well-controlled psoriasis (26–30). Spleen cell suspensions from BALB/c mice were prepared by mechanical Recently, a pivotal role of the IL-23/IL-17 axis was demonstrated disruption and RBC lysis. The AutoMACS system from Miltenyi Biotec in the mouse skin inflammation induced by imiquimod (22). In this (Bergisch Gladbach, Germany) was used to isolate T lymphocytes, based on study, we show that IL-22–deficient mice are almost totally pro- CD5 expression following the manufacturer’s instructions. Briefly, cells tected from psoriasis lesions induced by imiquimod treatment. were incubated for 20 min at 4˚C with anti-CD5 Ab-coupled microbeads, washed, and separated by one or two passages on the AutoMACS instru- T lymphocytes played a key role in this inflammatory process and Downloaded from ment. Purification was checked by FACS analysis with an anti-CD5 Ab and gd T cells, in particular, were found to represent a major source of determined to be at least 97% of purity. A total of 7 3 106 CD5+ T cells both IL-22 and IL-17 in draining lymph nodes. However, we show were i.p. injected into Rag22/2 mice 3 d before the imiquimod treatment. that non-T cells from the innate immune system also largely RT-PCR contribute to IL-22 but not IL-17 production. Total RNA was isolated from two pieces of skin using TriPure isolation reagent (Roche, Mannheim, Germany). Reverse transcription was per- http://www.jimmunol.org/ Materials and Methods formed on 1 mg total RNA with oligo(dT) primer (Roche) and Moloney Mice and treatments murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA). All mice used in this study were bred in the animal facility of Brussels Quantitative PCR (qPCR) amplifications were performed from cDNA branch of the Ludwig Institute for Cancer Research under specific pathogen- corresponding to 25 ng total RNA using primer sets and TaqMan probes 2/2 b free conditions. Rag2 BALB/c mice were originally purchased from corresponding to murine -actin, IL-17A, IL-22, S100A7, and S100A8 with Taconic (Ejby, Denmark). IL-23 p192/2 mice were provided by B. Ryffel qPCR Mastermix TaqMan (Eurogentec, Seraing, Belgium). The sequences b 9 (University of Orle´ans, Orle´ans, France). Wild-type 129sv mice were of primers and probes were as follows: -actin, 5 -CTCTGGCTCCTAG- 9 9 9 9 originally purchased from Harlan (Horst, The Netherlands). IL-22–defi- CACCATGAAG-3 ,5-GCTGGAAGGTGGACAGTGAG-3 , and probe, 5 - 9 9 cient mice were generated in our laboratory as described previously (31). ATCGGTGGCTCCATCCTGGC-3 ; IL-17A, 5 -GCTCCAGAAGGCCC- 9 9 9 9 The IL-22–deficient mice were originally generated in the 129 background TCAG-3 ,5-CTTTCCCTCCGCATTGACA-3 , and probe, 5 -ACCTC- 9 9 by guest on September 29, 2021 and were subsequently backcrossed with BALB/c mice for 15 generations AACCGTTCCACGTCACCCTG-3 ; IL-22, 5 -CGCTGCCCGTCAACA- 2/2 9 9 9 9 or with C57Bl6 for 13 generations. Rag2 IL-22–deficient mice were CCCGG-3 ,5-CTGATCTTTAGCACTGACTCCTCG-3 , and probe, 5 - 2/2 9 9 generated by crossing Rag2 with IL-22–deficient BALB/c mice. The TGAGGTGTCCAACTTCCAGCAGCCG-3 ; S100A7, 5 -GAGGAGTTG- 9 9 9 experiments were performed in compliance with institutional guidelines AAAGCTCTGCTCTTG-3 ,5-GTGATGTAGTATGGCTGCCTGCGG-3 , 9 9 9 and were approved by the local ethical committee. and probe, 5 -GTGTGCCTCGCTTCATGGAC-3 ; and S100A8, 5 -CAC- 9 9 Mice at 8 wk of age were shaved on the back skin and received daily, for CATGCCCCCTCTACAAGAATGA-3 ,5-CTCTGCTACTCCTTGTGGCT- 9 9 9 7 consecutive days, topical applications of imiquimod (3.125 mg) from GT-3 , and probe, 5 -CCTTGCGATGGTGATAAAAGTGGGTGTGGC-3 . a commercially available cream (5%) (Aldara; 3M Pharmaceuticals). For CCL3, CXCL3, CCL20, neutrophilic granule protein (Ngp), and loricrin, Anti–IL-22 blocking mAbs used in this study were AM22.1, which was qPCR was done using MasterMix for SYBR Green (Eurogentec) and 9 9 produced in our laboratory, and IL22-103 as described previously (21). Both the following primers: CCL3, 5 -GTACCATGACACTCTGCAACC-3 9 9 9 Abs neutralize human and mouse IL-22 activities in vitro. 0.5 mg Abs were and 5 -GTCAGGAAAATGACACCTGGCTG-3 ; CXCL3, 5 -CCATCC- 9 9 i.p. injected 1 d before the imiquimod treatment and after 3 d of treatment. AGAGCTTGACGGTGAC-3 and 5 -GGCTCAGCTGGACTTGCCGC- 9 9 9 9 Isotype control Abs were a mouse IgG2ak and a human IgG1l, respectively. TC-3 ; CCL20, 5 -GTACTGCTGGCTCACCTCTG-3 and 5 -CTTCAT- CGGCCATCTGTCTTGTG-39; Ngp, 59-GACTGCGACTTCCTGGAGG- Single-cell suspension and FACS staining ATG-39 and 59-GTATCCTCTCGACTGCAATCCCTG-39; and loricrin, 59-CACATCAGCATCACCTCCTTCC-39 and 59-CCTCCTCCACCAGA- Back skin fragments from imiquimod-treated or untreated mice were GGTCTTTC-39. Samples were first heated for 10 min at 95˚C, before dissected and incubated at 4˚C overnight in dispase II at 1 U/ml (Roche). amplification as follows: 40 cycles of two-step PCR program at 95˚C After separation with forceps, dermis was incubated at 37˚C in type III for 10 s and 60˚C for 1 min. For SYBR Green qPCR, melting point collagenase at 200 U/ml (Worthington Biochemical). After 2 h, activity of analysis was carried out by heating the amplicon from 60 to 95˚C. A collagenase was stopped by addition of 2 mM EDTA. Isolated cells were standard curve with various concentrations of a cloned cDNA fragment filtered on 40-mm nylon filters to obtain a single-cell suspension. Epider- was performed for each gene for accurate quantification. mis cells were isolated by using TrypLE Select, according to the manu- facturer recommendations (CELLnTEC). Briefly, epidermis was incubated Histopathological analysis at room temperature for 20 min in TrypLE Select before the single-cell Paraffin tissue blocks of skin biopsies were prepared using routine methods suspension was filtered. For FACS staining, 1 million cells were pre- and sectioned to obtain consecutive levels. The sections were stained with incubated with 10 mg/ml purified rat anti-mouse CD16-CD32 mAb (Fc H&E. Microscope analysis of the staining was performed by two eval- Block; BD Pharmingen) before incubation with specific Abs for CD45 (30- uators by reading three sections from each mouse. Proliferation of kera- F11 conjugated with PerCP) and CD11B (M1/70 conjugated with Pe) and tinocytes was measured by BrdU incorporation. A total of 1 mg BrdU for Ly6G (RB6-8C5 conjugated with AlexaFluor 700) (all from Bio- (Roche) was injected 12 h before sacrifice. Back skin biopsies were fixed Legend). The Abs were added for 1 h at 4˚C at 2 mg/ml. Cells were gated, in formaldehyde and embedded in paraffin. Sections were deparaffinized, based on forward and side scatter and on CD45+ cells with a FACSFortessa boiled in citrate buffer (5 mM [pH 5.5]), and incubated with mouse anti- (BD Biosciences). Postacquisition analysis was performed using FlowJo BrdU, followed by HRP-labeled goat–anti-mouse IgG. software (Tree Star). + For cytospin preparation, CD45 cells were isolated from dermal single- Statistics cell suspension using magnetic beads (MACS; Milteny Biotec). A total of 30,000 CD45+ cells were spread on microscope slides by cytospin Results are presented as the mean 6 SEM for n = 4 mice/group. Statistical centrifugation (Cytospin 3; Shandon, Pittsburgh, PA) before staining with significance between groups was assessed by using one-tailed unpaired The Journal of Immunology 3

Student t test or one-way ANOVA with Tukey’s or Bonferroni’s multi- showninFig.1D, BrdU was incorporated into DNA within ker- comparison test, using the InStat software. atinocytes that are located throughout the different layers of the epidermis, reflecting dysregulated proliferation. In contrast, Results positive BrdU cells are restricted to the basal layer of the epi- IL-22–deficient mice are protected from the development of dermis in IL-22–deficient mice. At the molecular level, the imi- psoriasis-like lesions induced by imiquimod quimod treatment induces expression of keratinocyte-specific Consistent with a previous report (22), topical application of genes and of inflammatory markers, including cytokines, gran- imiquimod on the shaved back skin of wild-type mice for ulocyte markers, and antimicrobial peptides (Fig. 2). In the skin 7 d induced psoriasis-like lesions including redness, scales from IL-22–deficient mice, we observed a significant reduction in resulting from desquamation, and crust formation. Because it was the imiquimod induction of several inflammatory chemokines previously shown that mouse IL-22 expression is transiently in- such as CCL3 (MIP-1a) and CXCL3 (MIP-2b), of a granulocyte duced by imiquimod (22), we took advantage of IL-22–deficient marker (Ngp), of antimicrobial peptides (S100A7 and S100A8), mice to assess the functional role of this cytokine in this model of and of keratin 14, which is associated with hyperproliferative skin inflammation. As shown macroscopically in Fig. 1A, IL–22- keratinocytes. Expression of loricrin, the major protein compo- deficient mice were almost totally protected from imiquimod- nent from the cornified cell envelope of terminally differentiated induced skin inflammation, showing less desquamation and no keratinocytes, remained higher in imiquimod-treated IL-22–de- crusts in comparison with the wild-type animals. Microscopic ficient mice compared with wild-type, thereby demonstrating, at evaluation of skin sections from wild-type mice treated for the molecular level, that the absence of IL-22 prevents the de-

7 d showed characteristic changes of psoriasis lesions, such as differentiation of the epidermis (Fig. 2). Interestingly, IL-17 and Downloaded from acanthosis (thickening of the epidermis), parakeratosis (presence CCL20, which are, respectively, the hallmark cytokine produced of nuclei in the stratum corneum), desquamation, and dermal in- by, and chemokine for the trafficking of Th17 cells are similarly filtrate. By contrast, imiquimod application had qualitatively and induced by imiquimod in IL-22–deficient and wild-type mice. quantitatively fewer effects on IL-22–deficient mice, including This observation indicates that IL-22 is not required for the de- almost no pustules, a small, yet statistically significant, decrease in velopment of a Th17 immune response by imiquimod. Rather, IL-

acanthosis, as well as less desquamation and parakeratosis (Fig. 22 acts to induce the expression of proteins that point to a func- http://www.jimmunol.org/ 1B,1C). Hyperproliferation of keratinocytes was also monitored tion of IL-22 for granulocyte recruitment in this model of skin by BrdU incorporation. In imiquimod-treated wild-type mice, as inflammation. by guest on September 29, 2021

FIGURE 1. IL-22–deficient mice are protected against psoriasis-like lesions induced by imiquimod treatment. Imiquimod was applied daily to 129sv wild-type and IL-22–deficient mice. After seven days, pictures of mice were taken and mice were sacrificed to analyze skin lesions. A, Macroscopic phenotypical representation of psoriasiform lesions in 129sv wild-type or IL-22–deficient mice treated with imiquimod for 7 d (four representative mice are presented) or untreated (one representative mouse). B, H&E staining of back skin sections from wild-type and IL-22–deficient mice, treated or not with imiquimod (original magnification 320). One representative picture is shown for each treatment regimen. Acanthosis (a), parakeratosis (p), pustules (pu), and desquamation (d) were observed in wild-type mice treated with imiquimod. C, Acanthosis was evaluated by counting the number of epidermal cell layers throughout the skin section of four mice per group. The number of pustules divided by the length of the section is also represented. Bar graphs represent mean for four mice 6 SD. D, BrdU was injected into mice 12 h before sacrifice. BrdU incorporation in the back skin was detected by im- munohistochemistry. BrdU-positive cells were distributed across the epidermis in the wild-type mice, whereas, in IL-22–deficient mice, the staining was restricted to the basal layer of the epidermis (original magnification 320). Data are representative of three experiments with four mice per group. *p , 0.05 wild-type treated mice versus IL-22–deficient mice. 4 ROLE OF IL-22 IN IMIQUIMOD-INDUCED PSORIASIS Downloaded from http://www.jimmunol.org/

FIGURE 2. The IL-22 deficiency affects expression of biomarkers reflecting the inflammatory response or keratinocyte differentiation and proliferation. Imiquimod was applied daily to 129sv wild-type or IL-22– FIGURE 3. Neutrophil infiltration induced by imiquimod application is deficient mice. After 7 d, quantitative RT-PCR analysis was performed for IL-22–dependent. Imiquimod was applied daily to C57Bl6 wild-type mice by guest on September 29, 2021 each indicated gene. RNA was isolated from two different pieces of back or IL-22–deficient mice. After 5 d, a single-cell suspension is prepared skin. Data correspond to the mean 6 SEM from four mice (calculated from from the dermis. A, Flow cytometry analysis of dermal single-cell sus- + the two pieces of skin for each mouse). Data are representative of at least pensions. Cells were gated on CD45 cells, and expression of CD11B and three independent experiments. *p , 0.05 and ** p , 0.01 (one-way Ly6G was analyzed. Percentages of neutrophils are indicated for repre- ANOVA and Bonferroni multiple comparisons). sentative pools of cells from two mice in an experiment where each group contained four mice. Similar results were obtained in five different experiments performed either in C57BL/6 or in 129sv mice. B, Cytospins The absence of IL-22 activity prevented the dermal infiltration of CD45+ cells isolated from the dermis (original magnification 320). of neutrophils induced by imiquimod CD45+ cells were purified from the dermis of wild-type or IL-22–deficient mice treated for 5 d with imiquimod. Cells were stained with eosin–thia- To further analyze granulocyte infiltration after imiquimod treat- zin. Arrows indicate neutrophils. C, Percentages of neutrophils in wild- ment, we isolated cells from the dermis and analyzed them by flow type or IL-22–deficient mice that had been treated with imiquimod. cytometry, using the pan-leukocyte marker CD45 in combination Neutrophils were counted in five different areas for four different mice per with the CD11B and Ly6G granulocyte markers. After 5 d of group. Mean values and SEM were calculated for each group. These data imiquimod treatment, we observed an increase in the CD11B+ are representative of three different experiments. Ly6G+ cell population in wild-type mice but not in IL-22–defi- cient mice (Fig. 3A). To confirm this observation, cytospins were prepared with MACS-purified CD45+ cells from imiquimod- obtained with a second IL-22 mAb (data not shown). As observed treated mice. Eosin–thiazin staining showed increased neu- for IL-22–deficient mice, IL-22 Ab-treated mice had less des- trophils in wild-type mice compared with IL-22–deficient mice as quamation and almost no crusts. Histopathological analysis, as illustrated in Fig. 3B. Percentages of neutrophils counted on these presented in Fig. 4B, showed that mice receiving a control Ab had slides (Fig. 3C) correlated with those measured by flow cytometry, acanthosis, parakeratosis, desquamation, and inflammatory infil- confirming that IL-22 plays a key role on neutrophil infiltration trates, whereas mice that were treated with an IL-22–neutralizing in this model. Ab were partially protected, showing a decrease in acanthosis with almost no desquamation and parakeratosis. The protective effect Ab neutralization of IL-22 decreased psoriasis-like lesions of IL-22 neutralization was also observed at the molecular level. induced by imiquimod treatment Quantitative RT-PCR analysis of RNA from the skin demonstrated To confirm the effect of IL-22 in this model, we treated wild-type that IL-22 blockade led to a significant decrease in several in- mice with an IL-22–neutralizing or isotype control Ab, starting flammatory markers such as CCL3 and CXCL3 and the neutro- 1 d before the first application of imiquimod. As shown in Fig. 4A, philic marker Ngp (Fig. 4C). In addition, transcript levels for mice that received an IL-22–blocking Ab presented with reduced S100A7 antimicrobial peptide was also reduced with IL-22 Ab- skin lesions as compared with control mice. Similar results were treated mice. As observed for IL-22–deficient mice, administra- The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 5. Induction of IL-22 expression in imiquimod-induced lesions is IL-23 dependent. A, Imiquimod was applied daily to 129sv wild-type mice. After different periods of time, RNA was extracted from two dif- ferent pieces of skin, and quantitative RT-PCR was performed for IL-22, IL-23, CCL20, and b-actin. Data correspond to the mean 6 SEM from four mice per group. Data are representative of two independent experi- ments. B and C, Wild-type and IL-23 p192/2 mice are treated for the indicated period of time with imiquimod and RNA was extracted. Quan- by guest on September 29, 2021 titative RT-PCR was performed for IL-22, CCL20, and b-actin. Data FIGURE 4. IL-22 Ab neutralization prevented the development of correspond to the mean 6 SEM from four mice. Data are representative of imiquimod-induced skin lesion. Imiquimod was applied daily to 129sv at least three independent experiments. wild-type mice that were treated biweekly with 500 mg of a neutralizing IL-22 Ab (AM22.1) or isotype control Ab. After 7 d, pictures of mice were taken and mice were sacrificed to analyze skin lesions. A, Macroscopic between days 3 and 5. Because IL-23 is a major inducer of IL-22 phenotypical representation of psoriasiform lesions in two 129sv mice that expression, these data suggested that IL-23 was responsible for IL- received control Abs and in three mice that were treated with anti–IL-22 22 production in this model of skin inflammation. Indeed, the role Abs. B, H&E staining of back skin sections from imiquimod-treated mice of IL-23 was supported by the observation that p192/2 mice did (original magnification 320). One representative picture is shown. C, not express IL-22 upon imiquimod treatment (Fig. 5B). In con- Quantitative RT-PCR analysis was performed for each indicated gene. trast, the absence of IL-23 did not affect CCL20 expression, RNA was isolated from two different pieces of back skin. Data correspond consistent with the early production of this chemokine in this to the mean 6 SEM from four mice. Data are representative of at least two process (Fig. 5C). independent experiments. *p , 0.05 and ***p , 0.001 (one-way ANOVA and Bonferroni multiple comparisons). IL-22 is produced both by gd T lymphocytes and by non T cells upon imiquimod application to the skin tion of IL-22 Ab to imiquimod-treated mice did not suppress the To determine the cellular source of IL-22 in this model, we isolated expression of IL-17. In addition, the expression of the loricrin cells from the dermis, the hypodermis, and the axillary and inguinal differentiation marker was higher than with the administration of draining lymph nodes of wild-type 129sv mice treated with imi- the control Ab, indicating that blockade of IL-22 activity pre- quimod, and cells were subsequently stimulated in vitro with IL-23 vented dedifferentiation of the keratinocytes in the presence of and IL-2. Surface and intracellular stains were then evaluated by imiquimod (Fig. 4C). FACS, using cells from IL-22–deficient mice to confirm the specificity of the IL-22 Ab for detecting intracellular cytokine. IL-22 production upon imiquimod treatment is IL-23 dependent Significant numbers of IL-22–producing cells were observed in To better characterize the regulation of the Th17-associated lymph nodes from imiquimod-treated wild-type mice in contrast cytokines IL-22, IL-23, and CCL20 in this model, we investi- to those from untreated mice (Fig. 6A). All the IL-22+ cells were gated the kinetics of expression for the corresponding RNA in mice CD3+ and CD25+ (Fig. 6B), corresponding to activated T cells. that were treated everyday with imiquimod. As shown in Fig. 5A, Approximately two-thirds of the IL-22+ cells were gdTCR+ cells, CCL20 is induced early with a peak at day 1 or 2. IL-23p19 in- whereas one-third were abTCR+ cells (Fig. 6C). All of the IL-22+ duction was increased at day 1 and peaked between days 2 and 3. gdTCR+ cells also produced IL-17 and are negative for CD4 and Finally, IL-22 expression became detectable at day 2 and peaked CD8 (Fig. 6C,6D). By contrast, the IL-22+gdTCR2 population 6 ROLE OF IL-22 IN IMIQUIMOD-INDUCED PSORIASIS Downloaded from

FIGURE 7. IL-22 is produced by T lymphocytes and by non-T cells http://www.jimmunol.org/ after imiquimod treatment. Imiquimod was applied daily to Rag22/2 or Rag22/2 IL-22–deficient mice that had or had not previously received an i.p. injection of 7 3 106 CD5+ T lymphocytes from BALB/c mice. After 7 d, quantitative RT-PCR analysis was performed for IL-22 (A) and IL-17 (B). RNA was isolated from two different pieces of back skin, and mean expression was measured for each mouse. Data correspond to the mean 6 SEM from four mice. Data are representative of at least two independent experiments. by guest on September 29, 2021 type CD5+ cells were transferred into Rag22/2 IL-22–deficient mice, cells from the donor representing the only potential source FIGURE 6. IL-22 is produced by mostly gd TCR+ cells from draining of IL-22, IL-22 expression levels were fully restored to levels 2/2 lymph nodes after imiquimod treatment. Imiquimod was applied daily to detected in the skin of Rag2 mice with imiquimod treatment. 2 2 129sv wild-type mice. After 5 d, single cell suspensions from draining By contrast, IL-17 induction was completely absent in Rag2 / lymph nodes were stimulated with IL-2 (400 U/ml) and IL-23 (10 ng/ml) mice unless they received CD5+ T cells, indicating that IL-17 in the presence of monensin. After 4 h, cells were stained with Abs specific production is strictly T cell dependent. for IL-22 and IL-17A as well as the CD3, CD4, CD25, TCR b, and gd cell Taken together, these observations indicate that IL-22 is re- + surface markers. A, Dot plots show an IL-22 population of cells that is dundantly produced by cells of the adaptive and innate immune enriched in the single suspension of lymph node cells from imiquimod- systems and plays an important role in the inflammatory response treated mice compared with untreated mice. B, Histograms show cell surface marker for CD3 and CD25 on the IL-22+ cell population gated in as well as in the dysregulation of keratinocyte proliferation and A. C, Density plots show cell surface markers for TCR ab and gd and differentiation induced by imiquimod. intracellular staining for IL-17A on IL-22+ cells derived from mice treated with imiquimod. D and E, CD4 and CD8 expressions on IL-22+ cells that Discussion were either positive (D) or negative (E) for the gd TCR. Data correspond to The IL-23/Th17 pathway is considered to play a major role in a representative pool of cells from two mice in an experiment where each psoriasis because polymorphisms of genes encoding components of group contained four mice. Similar results were obtained in at least three IL-23 and its receptor have been associated with an increased risk independent experiments. of psoriasis (33–36), and because of the clinical effectiveness of Abs targeting IL-23/IL-12 p40 as well as IL-17 (37, 38). It has contains CD4+ and CD8+ cells (Fig. 6E), and only one-third of previously been shown that inhibition of IL-23 or IL-17 partially them coexpressed IL-17 (Fig. 6C), whereas all IL-17–expressing protected the skin from imiquimod-induced inflammation (22, 39). cells coexpressed IL-22 (data not shown). In this article, we show that blocking IL-22 can also protect mice To further address the role of T lymphocytes, we treated Rag2- from the skin inflammation induced by imiquimod. In addition, deficient mice with imiquimod. As shown in Fig. 7, comparable IL- we demonstrate that IL-22 production is strictly dependent on IL- 22 expression was detected in the skin of Rag22/2 and wild-type 23 but redundantly stems both from non-T cells and from mice, indicating that imiquimod induced IL-22 expression from T lymphocytes, particularly gd T cells as well as CD4+ and CD8+ innate cells and that T cells were not essential for this IL-22 ab Tcells in the draining lymph nodes. production. The adoptive transfer of wild-type CD5+ T cells into A major difference between IL-17 and IL-22 production in this IL-22–competent Rag22/2 recipient did not significantly increase model is that IL-17 is exclusively produced by T lymphocytes, IL-22 expression induced by imiquimod. However, when wild- whereas wild-type and Rag22/2 mice, which have neither ab nor The Journal of Immunology 7 gd T cells, showed a similar skin IL-22 induction upon imiquimod Among the different pathophysiological features of this model, application, highlighting the role of non-T cells. We were unable the absence of IL-22 had a particularly stringent effect on neu- to define the cell types responsible for this innate IL-22 production trophil recruitment into the skin, most likely via the regulation of in the skin. Potential innate IL-22 sources can be hypothesized, CXCL3 and CCL3 because induction of these chemokines was based on the numerous studies addressing this issue in the intes- partially prevented in the absence of IL-22. Neutrophil recruitment tinal mucosae. Dendritic cells from the dermis or Langerhans cells is a typical activity attributed to IL-17, and this observation further from the epidermis might be involved, but there is little evidence highlights the cooperation between both cytokines. However, this so far that this cell type represents a significant source of IL-22 might also result from the regulation of specific chemotactic (40). Innate lymphoid cells, which include but are not limited to factors. For instance, CXCL1 and CXCL5, which are often de- lymphoid inducer cells, represent the major source of IL-22 in the pendent on IL-17 for production, were poorly affected in IL-22– intestine (41–44) or in the spleen upon administration of TLR4 or deficient mice (data not shown), whereas CXCL3 and CCL3 are TLR5 agonists (45, 46), but such cells were not described in the not generally considered as typical mediators of IL-17 activity. skin so far. In the K5.Stat3C transgenic model, intradermal ad- In vitro studies focusing on the regulation of these chemokines ministration of IL-23 induces IL-22 expression by CD56+ cells would be required to determine whether the latter represent spe- from draining lymph nodes, pointing to NK cells as a potential cific IL-22–responsive genes or depend on IL-22 and IL-17 co- source of IL-22 (39), although such NK markers are also shared operation for their expression. Nevertheless, the pathogenic role of by a subset of the above mentioned innate lymphoid cells (41, 42, neutrophils in psoriasis is supported by clinical observations de- 44). In our model, we did not detect any IL-22 production by NK scribing disease remission during drug-induced agranulocytosis cells from draining lymph nodes, suggesting that another innate (54). Thus, our data showing that blocking IL-22 can both affect Downloaded from cell type, possibly residing in the skin might be involved. markers of keratinocyte dysregulation and of neutrophil infiltra- Among T cells, our data indicate that conventional Th17 cells tion further support the potential clinical effectiveness of IL-22 probably represent only a minority of the T lymphocytes involved inhibitors in psoriatic patients. in IL-22 production. The majority of IL-22+ T cells found in draining lymph nodes expressed a gd TCR and coexpressed IL-17. Disclosures

By contrast, most of gd-negative IL-22–producing cells did not K.D.J. and L.A.F. are currently employees of Pfizer. The other authors http://www.jimmunol.org/ + + coexpress IL-17 and included both CD4 and CD8 cells. The role have no financial conflicts of interest. of T lymphocytes in human psoriasis is well established (22, 47), but the Ags that are recognized remain ill-defined. The association of this disease with streptococcal throat infections supports the References 1. Renauld, J. C. 2003. Class II cytokine receptors and their ligands: key antiviral hypothesis that pathogenic T cells are specific determinants and inflammatory modulators. Nat. Rev. Immunol. 3: 667–676. common to streptococcal M protein and keratin (48), but this 2. Geboes, L., L. Dumoutier, H. Kelchtermans, E. Schurgers, T. Mitera, observation might only be relevant for a subset of patients. The J. C. Renauld, and P. Matthys. 2009. 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