CXCL17 Attenuates Imiquimod-Induced Psoriasis-Like Skin Inﬂammation by Recruiting Myeloid-Derived Suppressor Cells and Regulatory T Cells

Published April 7, 2017, doi:10.4049/jimmunol.1601607 The Journal of Immunology

CXCL17 Attenuates Imiquimod-Induced Psoriasis-like Skin Inﬂammation by Recruiting Myeloid-Derived Suppressor Cells and Regulatory T Cells

Tomonori Oka, Makoto Sugaya, Naomi Takahashi, Takehiro Takahashi, Sayaka Shibata, Tomomitsu Miyagaki, Yoshihide Asano, and Shinichi Sato

CXCL17 is expressed in a variety of cancers and promotes tumor progression by recruiting myeloid-derived suppressor cells (MDSCs). MDSCs suppress tumor immunity by attracting regulatory T cells (Tregs) into tumor sites through CCL5. In this study, we examined the role of CXCL17 in skin disorders. CXCL17 mRNA levels in psoriasis skin, but not in lesional skin of atopic dermatitis or cutaneous T cell lymphoma, were significantly higher than those in normal skin. CXCL17 was mainly expressed in the epidermis, and IFN-g dose-dependently increased CXCL17 expression by human keratinocytes in vitro. As CXCL17 mRNA expression was increased by treatment with imiquimod (IMQ), we examined the effects of CXCL17 in IMQ-induced psoriasis-like skin inflammation. Injection of recombinant CXCL17 into the ear before and during IMQ application decreased ear thickness, inflammatory cytokine expression, and the number of infiltrating cells compared with PBS injection. Flow cytometric analysis and immunofluorescent staining revealed that the numbers of MDSCs, which are CD11b+Gr-1+, and that of Tregs, which are CD4+CD25+, were higher in the ear of the CXCL17-injected mice than in PBS-injected mice. MDSCs, but not Tregs, showed chemotaxis to CXCL17 in vitro. When mice were injected with anti-CCL5 Ab or anti-CCL4 Ab simultaneously with recombinant CXCL17, ear thickness and cytokine expression increased to a similar level of mice treated with PBS and control IgG, suggesting that these chemokines were important for anti-inflammatory effects. Taken together, CXCL17 attenuates IMQ-induced psoriasis- like skin inflammation by recruiting MDSCs and Tregs, which may be important for regulating excessive inflammation in psoriasis skin. The Journal of Immunology, 2017, 198: 000–000.

newly discovered 119-aa CXC chemokine, CXCL17, CXCL17 acts as a chemoattractant for monocytes, macrophages, was discovered by fine structure–based protein anal- and mature and immature dendritic cells (1, 6). Several studies A ysis and cDNA microarray analysis (1, 2). CXCL17 is have proposed that CXCL17 might accelerate tumor progression expressed in mucosal tissues such as trachea, stomach, and colon (1, 4, 6–8). CXCL17 was reported to be expressed in various types (1, 3). CXCL17 was reported to be highly expressed in epithelial of tumor cells such as breast, lung, hepatic, and colon cancer (1, 4, cells and vascular endothelial cells (1). CXCL17 is also expressed 6–8). In hepatic carcinoma, CXCL17 expression is an independent in malignancy: in hepatocellular carcinoma, CXCL17 was pro- indicator for poor prognosis (4). Moreover, CXCL17 was dem- duced mainly by tumor-infiltrating neutrophils and occasionally onstrated to recruit myeloid-derived suppressor cells (MDSCs) to by the tumor cells (4). The receptor of CXCL17 was recently tumor sites and promote tumorigenesis through angiogenesis in a detected as GPR35/CXCR8, which is expressed in mucosal tis- mouse model (7). Alternatively, CXCL17 is associated with an- sues, including the gastrointestinal tract and lung (5). The ImmGen titumor immune response during early stages of pancreatic car- database indicated that GPR35 is expressed by macrophages, cinogenesis through triggering the accumulation of dendritic cells dendritic cells, and granulocytes (5). at the tumor sites (9). In previous studies, other functions of CXCL17 have been reported: CXCL17 suppresses proinflammatory

Department of Dermatology, Graduate School of Medicine, University of Tokyo, cytokine production from murine macrophages and acts as an Tokyo 113-8655, Japan anti-inflammatory factor (10). CXCL17 was also reported to be ORCIDs: 0000-0001-7362-3207 (T.O.); 0000-0002-1618-329X (M.S.); 0000-0001- important as an antimicrobial mucosal chemokine in the patho- 5560-9778 (Y.A.); 0000-0001-5519-172X (S. Sato). genesis of interstitial lung disease (3). Received for publication September 14, 2016. Accepted for publication March 12, Psoriasis is a common chronic inflammatory cutaneous disease 2017. characterized by thickened and scaly skin patches caused by ab- This work was supported in part by a grant from the Ministry of Education, Culture, normal keratinocyte proliferation, vascular hyperplasia, and in- Sports, Science and Technology in Japan (16K10146). flammatory cell infiltration into the dermis and epidermis. This T.O. performed the research and collected and analyzed data; M.S. contributed to the design of the research, collected clinical samples and data, and wrote the paper; T.M., disease is long-lasting and can greatly influence a patient’s self- N.T., T.T., and S. Shibata collected clinical samples and data; and Y.A. and S. Sato esteem and sense of well-being. To understand the pathogenesis of financially supported and supervised the project. psoriasis and find a new therapeutic strategy, the mouse model is Address correspondence and reprint requests to Dr. Makoto Sugaya, Department of Dermatology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, very important. Topical application of imiquimod (IMQ), a ligand Bunkyo-ku, Tokyo 113-8655, Japan. E-mail address: [email protected] of TLR7 and TLR8, can induce and exacerbate skin inflammation Abbreviations used in this article: AD, atopic dermatitis; CTCL, cutaneous T cell resembling human psoriasis lesions (11, 12). In a mouse model of lymphoma; HPF, high-power field; IMQ, imiquimod; MDSC, myeloid-derived sup- IMQ-induced psoriasis-like skin inflammation, Th17 cells and pressor cell; NHEK, normal human epidermal keratinocyte; Treg, regulatory T cell. IL-17/IL-22–producing gd Tcellshavebeenshowntoplaya Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 pivotal role (13).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601607 2 CXCL17 ATTENUATES IMQ-INDUCED PSORIASIS-LIKE INFLAMMATION

Regulatory T cells (Tregs), which express CD4 and CD25 on Franklin Lakes, NJ). When the cells reached semiconfluence, the medium their cell surface, are engaged in suppressing immune responses was completely removed and 2 ml of serum-free medium was added to toward self and nonself. The transcription factor Foxp3 is a master each well. Simultaneously, recombinant human IL-4, TNF-a, or IFN-g (all products from R&D Systems, Minneapolis, MN) were added, and the cells regulator of Treg development and function (14). IL-10 contributes were incubated at 37˚C and 5% CO2. The concentration of IL-4 and TNF-a to suppression of autoimmune diseases, such as inflammatory was 10 ng/ml, and that of IFN-g was0.1,1,or10ng/ml.After24h, bowel diseases (15). IL-10 and Foxp3 can also serve as Treg ac- supernatant was collected, centrifuged to remove cell debris, and stored at 2 tivation markers (16, 17). A decrease in the number of Tregs or 80˚C until analyzed. Cells were processed by TRIzol reagent (Invitrogen, Carlsbad, CA) for isolation of total RNA, according to the manufacturer’s Treg dysfunction results in severe autoimmunity in both mice and instructions. humans (18). The number of Tregs in peripheral blood from patients with psoriasis is not decreased, but the dysfunction of Tregs Quantitative real-time RT-PCR assay is associated with psoriasis (19). MDSCs, a heterogeneous pop- mRNA was obtained from human and murine skin samples with an RNeasy ulation of progenitor and immature myeloid cells (20), contribute fibrous tissue mini kit (Qiagen, Valencia, CA) and from cells with TRIzol to immunosuppression in the tumor microenvironment (21, 22). reagent. cDNA was synthesized using ReverTra Ace qPCR RT master mix MDSCs potently inhibit T cell proliferation and cytokine pro- (Toyobo, Osaka, Japan). Expression levels of CXCL17, IL-17A, IL-22, duction in vitro. In mice, MDSCs are characterized by the coex- TNF-a, IL-12p35, IL-12/IL-23p40, IL-23p19, IL-10, Foxp3, CCL5, and arginase 1 mRNA were analyzed using the quantitative RT-PCR method pression of cell surface markers CD11b and Gr-1 (23). Arginase 1 with Thunderbird SYBR quantitative PCR mix (Toyobo) on an ABI Prism is reported to be associated with MDSC activation (24). 7000 sequence detector (Applied Biosystems, Foster City, CA). All sam- In this study, we have investigated the role of CXCL17 in ples were analyzed in parallel for GAPDH gene expression as an internal control. The relative change in the levels of genes of interest was deter- psoriasis and an IMQ-induced psoriasis mouse model. CXCL17 2 mined by the 2 DDCT method. Primers for murine GAPDH, human was highly expressed in the epidermis of psoriasis lesional skin. GAPDH, murine CXCL17, human CXCL17, murine IL-17A, murine IL-22, In vitro, IFN-g dose-dependently increased CXCL17 expression murine TNF-a, murine IL-12p35, murine IL-12/IL-23p40, murine IL-23p19, by human keratinocytes. IMQ-induced psoriasis-like skin in- murine IL-10, murine Foxp3, murine CCL5, and murine arginase 1 were as flammation was attenuated by CXCL17 treatment, accompanied follows: murine GAPDH, forward, 59-CGT GTT CCT ACC CCC AAT GT-39, 9 9 by increased infiltration of MDSCs and Tregs into the skin. We reverse, 5 -TGT CAT CAT ACT GGC AGG TTT CT-3 ; human GAPDH, forward, 59-ACC CAC TCC TCC ACC TTT GA-39,reverse,59-CAT ACC confirmed that MDSCs, but not Tregs, expressed Gpr35 mRNA AGGAAATGAGCTTGACAA-39; murine CXCL17, forward, 59-TGT and showed chemotaxis to CXCL17 in vitro. Furthermore, simul- TGC TTC CAG TGA TGC TC-39,reverse,59-GCT GTG CTT TTC TCT taneous injection of anti-CCL5 Ab or anti-CCL4 Ab attenuated the TTG G-39; human CXCL17, forward, 59-ACC GAG GCC AGG CTT CTA-39, anti-inflammatory effects of CXCL17 treatment, suggesting that reverse, 59-GGC TCT CAG GAA CCA ATC TTT-39; murine IL-17A, forward, 59-CAG CAG CGA TCA TCC CTC AAA G-39,reverse,59-CAG GAC CAG MDSCs recruited by CXCL17 induced Treg migration through GAT CTC TTG CTG-39; murine IL-22, forward, 59-AGC TTG AGG TGT CCL5 and CCL4. CXCL17 may be important for regulating ex- CCA ACT TC-39,reverse,59-GGT AGC ACT CAT CCT TAG CAC TG-39; cessive inflammation in psoriasis skin. murine TNF-a,forward,59-CCA CCA CGC TCT TCT GTC TAC-39,reverse, 59-AGG GTC TGG GCC ATA GAA CT-39; murine IL-12p35, forward, 59- ACT CTG CGC CAG AAA CCT C-39,reverse,59-CAC CCT GTT GAT GGT Materials and Methods CAC GAC-39; murine IL-12/IL-23p40, forward, 59-CTC ACATCT GCT GCT Mice CCA CAA G-39,reverse,59-AAT TTG GTG CTT CAC ACT TCA GG-39; murine IL-23p19, forward, 59-TGT GCC TAG GAG TAG CAG TCC TGA-39, C57BL/6 mice were purchased from SLC Japan (Tokyo, Japan). Mice were reverse, 59-TTG GCG GAT CCT TTG CAA GCA GAA-39 ; murine IL-10, 7–11 wk old for all experiments and were maintained in a specific forward, 59-TTT GAA TTC CCT GGG TGA GAA-39,reverse,59-ACA GGG pathogen-free facility. All animal experiments were approved by the An- GAG AAA TCG ATG ACA-39; murine Foxp3, forward, 59-CAC CCA GGA imal Research Committee of the University of Tokyo. AAG ACA GCA ACC-39,reverse,59-GCA AGA GCT CTT GTC CAT TGA- Tissue samples 39; murine CCL5, forward, 59-CAC TCC CTG CTG CTT TGC-39,reverse,59- CAC TTG GCG GTT CCT TCG-39; murine arginase 1, forward, 59-ATG mRNA was obtained from biopsy materials of lesional skin of psoriasis GAA GAG ACC TTC AGC TAC-39,reverse,59-GCT GTC TTC CCA AGA (n = 20), atopic dermatitis (AD; n = 10), cutaneous T cell lymphoma GTT GGG-39. (CTCL; n = 28), and normal skin adjacent to benign skin tumors (n = 11). Skin samples for immunohistochemistry were collected from psoriasis IMQ-induced psoriasis-like skin inflammation (n = 10), AD (n = 10), CTCL (n = 10), and normal (n = 10). The healthy controls had no history of allergy, psoriasis, AD, or CTCL. All samples C57BL/6 mice at 7–11 wk old received a daily topical dose of 31.25 mg were collected during daily clinical practice. The Medical Ethics Com- commercially available IMQ cream (5% Beselna cream; Mochida Phar- mittee of the University of Tokyo approved all described studies, and maceuticals, Tokyo, Japan), which contains 31.25 mg IMQ, on the right ear the study was conducted according to the Declaration of Helsinki princi- for 6 consecutive days (day 0 to day 5) as previously described (12). ples. Informed consent was obtained to use skin samples from patients and Vaseline containing 25% isostearic acid (Tokyo Chemical Industry, Tokyo, healthy controls. Japan) was used as a vehicle control. In some experiments, mice were anesthetized and 20 ml of PBS, either alone or containing 100 ng of Cell culture recombinant mouse CXCL17 (R&D Systems), was injected intradermally into the right ear using a 27-gauge needle on days 0, 2, and 4 before and HaCaT cells, a human keratinocyte cell line, were provided by Dr. T. Kuroki during IMQ application. We calibrated the change of ear thickness during (Institute of Molecular Oncology, Showa University, Tokyo, Japan) with and after IMQ application. When anti-CCL3 Ab (R&D Systems), anti- € permission from Dr. N. Fusenig (Institut fur Zell- und Tumorbiologie, CCL4 Ab (PeproTech, Rocky Hill, NJ), or anti-CCL5 Ab (PeproTech) Deutsches Krebsforschungszentrum, Heidelberg, Germany). HaCaT cells 2 was injected simultaneously with CXCL17, the mice were divided into were cultured in 75-cm cell culture flasks (Corning, NY) at 37˚C, 5% CO2 three groups. One group was injected with 20 ml of PBS containing 2 mgof in MEM (Sigma-Aldrich, St. Louis, MO) containing 10% FBS and anti- 2 control IgG alone, another group with 100 ng of CXCL17 and 2 mgof biotics. J774 cells were cultured in 75-cm cell culture flasks (Corning) at control IgG, and the third group with 100 ng of CXCL17 and 2 mg of anti- 37˚C, 5% CO2 in RPMI 1640 basic medium (Thermo Fisher Scientific, CCL3 Ab, anti-CCL4 Ab, or anti-CCL5 Ab. On day 7, mice were sacrificed Waltham, MA) containing 10% FBS and antibiotics. Normal human epi- and skin tissue was collected and processed for isolating mRNA, histo- dermal keratinocytes (NHEKs) were purchased from Kurabo Industries logical analysis, fluorescent immunostaining, or flow cytometric analysis. (Osaka, Japan). NHEKs were cultured in 75-cm2 cell culture flasks (Corning) at 37˚C, 5% CO2 in HuMedia-KB2 (Kurabo Industries) sup- Histological analysis of murine skin plemented with human keratinocyte growth supplement sets (Kurabo In- dustries). When confluence was achieved, the cells were trypsinized, Sections from murine ear skin were embedded in OCT compound, snap- washed, and resuspended in the medium at 5 3 105 cells/ml, and 2 ml was frozen in liquid nitrogen, and stored at 280˚C. Six-micrometer cryosections added to each well of the six-well plates (Becton Dickinson Labware, of snap-frozen skin were cut using a cryostat. Sections were fixed with The Journal of Immunology 3 cold acetone for 5 min and stained with H&E. The number of infil- Detection of Gpr35 mRNA trating lymphocytes and neutrophils was counted per high-power field + 2 (HPF; 3400) of a light microscope. Each section was examined inde- MDSCs, Tregs, CD4 CD25 cells, and J774 cells were processed by pendently by two investigators in a blinded manner. TRIzol reagent (Invitrogen) for isolation of total RNA, according to the manufacturer’s instructions. cDNA was synthesized using ReverTra Ace Immunohistochemical staining for human samples qPCR RT master mix (Toyobo). Murine Gpr35 primers were forward, 59- GGG GTA CTG GCT CTC CCT AC-39, reverse, 59-CCC AAG AGT CAA We performed immunohistochemical staining for CXCL17 in lesional CGT GCT TT-39. Amplifications were performed in a Life ECO thermal skin of psoriasis (n =10),AD(n = 10), and CTCL (n = 10). Normal skin cycler (Hangzhou Bioer Technology, Hangzhou, China) with the following adjacent to benign skin tumors (n = 10) served as control. Briefly, 5-mm- profile: 94˚C for 2 min, 94˚C for 30 s, 57˚C for 30 s, 72˚C for 2 min for 35 thick tissue sections from formaldehyde-fixed and paraffin-embedded cycles with a final extension at 72˚C for 2 min. The PCR product was samples were dewaxed and rehydrated. These sections were then analyzed under UV light after electrophoresis in 1.5% agarose (Nippon- stained with rabbit anti-human CXCL17 polyclonal Ab (R&D Systems), gene, Tokyo, Japan) stained with ethidium bromide. followed by ABC staining (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used for visualizing the staining, and counter- Statistical analysis staining with Mayer’s hematoxylin was performed, according to the manufacturer’s instructions. The positivity of staining was ranked as Statistical analysis was performed using Prism version 6 software negative, weak positive, and strong positive as shown in Fig. 1B and (GraphPad Software, San Diego, CA). The Mann–Whitney U test was used Table I. for determining the levels of significance of differences in sample means, and the Kruskal–Wallis test was used for multiple comparisons. A p value Fluorescent histochemical staining ,0.05 was considered statistically significant. Fluorescent immunostaining for CD11b, Gr-1, and Foxp3 was performed using IMQ-applied ear skin to identify infiltrating MDSCs and Tregs. For Results double staining, 6-mm-thick tissue sections were first fixed with cold ac- CXCL17 expression is increased in psoriasis lesional skin etone for 20 min. These sections were incubated for 1 h at room temperature with rat anti-mouse Ly-6G (Gr-1) Ab (eBioscience, San Diego, To investigate CXCL17 involvement in skin diseases, we first CA), followed by incubation with FITC-conjugated goat anti-rat IgG (BD examined mRNA expression of this chemokine in normal skin and Pharmingen, San Jose, CA) for 1 h. They were further stained with PE- AD, CTCL, or psoriasis lesional skin. CXCL17 expression levels conjugated anti-mouse CD11b Ab (BioLegend, San Diego, CA). FITC- conjugated anti-mouse Foxp3 Ab (eBioscience) was used to detect Tregs. were significantly elevated in psoriasis skin compared with normal The nuclear staining was performed using mounting medium with 4˚C skin (Fig. 1A). Alternatively, CXCL17 mRNA expression in AD DAPI (Vector Laboratories). or CTCL lesional skin was comparable to that of normal skin. Immunohistochemical staining for CXCL17 in skin lesions of ELISA AD, CTCL, or psoriasis and normal skin revealed that epidermal Immunoreactive CXCL17 in the supernatant of NHEKs was quantified by keratinocytes in psoriasis strongly expressed CXCL17 (Fig. 1B, human ELISA kits (MyBioSource, San Diego, CA). Optical densities were Table I). Thus, CXCL17 expression was increased in psoriasis measured at 450 nm, with the correction wavelength set at 570 nm, using a Bio-Rad model 550 microplate reader (Bio-Rad Laboratories, Hercules, lesional skin. CA). The concentrations were calculated from the standard curve generated IFN-g increases CXCL17 expression by keratinocytes by a curve-fitting program according to the manufacturer’s instructions. To study regulation of CXCL17 expression by keratinocytes, we Flow cytometric analysis investigated CXCL17 expression by real-time RT-PCR using To purify cells infiltrating into the skin, full-thickness skin without s.c. HaCaT cells and NHEKs after 24 h culture with or without stim- tissue was incubated for 1.25 h at 37˚C in RPMI 1640 containing 400 mg/ml ulation of IL-4, TNF-a, or IFN-g in vitro. Real-time RT-PCR Liberase TL (Roche, Basel, Switzerland), after which 0.1% DNase I analysis revealed that IFN-g induced HaCaT cells to express sig- (Sigma-Aldrich) was added (final concentration 0.05%) for an additional 15 min. Single-cell suspensions were prepared by sequential filtering nificantly increased levels of CXCL17 mRNA (Fig. 2A). Stimu- through 100-, 70-, and 40-mm nylon mesh, and centrifugation at 1000 3 g lation with IL-4 (10 ng/ml) or TNF-a (10 ng/ml) did not change for 20 min at room temperature, as previously described (25). The cells CXCL17 mRNA expression. When NHEKs were cultured with were washed twice in FACS buffer (1% BSA plus 0.1% sodium azide) IFN-g, CXCL17 mRNA levels were significantly higher than and stained for 30 min at 4˚C with allophycocyanin-conjugated mAb against CD11b and PE/Cy7-conjugated mAb against Gr-1, or with those of NHEKs cultured with medium alone (Fig. 2B). We also allophycocyanin-conjugated mAb against CD4 and PE/Cy7-conjugated confirmed that IFN-g enhanced CXCL17 mRNA expression by mAb against CD25 (all products from BioLegend). Finally, the cells NHEKs in a dose-dependent manner (Fig. 2C). Moreover, IFN-g were analyzed on a FACSVerse flow cytometer (BD Biosciences, San Jose, increased CXCL17 protein in the culture supernatant of NHEKs in CA). Data were analyzed with FACSuite (BD Biosciences). a dose-dependent manner (Fig. 2D). Thus, IFN-g stimulation in- Isolation of MDSCs and Tregs creased CXCL17 expression by keratinocytes. Isolation of MDSCs was performed by using an MDSC isolation kit CXCL17 treatment attenuates IMQ-induced psoriasis-like (Miltenyi Biotec, Bergisch Gladbach, Germany), and isolation of Tregs was skin inflammation done by using a CD4+CD25+ Treg isolation kit (Miltenyi Biotec) from spleen of C57BL/6 mice, according to the manufacturers’ instructions. At IMQ-induced skin inflammation has been widely used as a mouse the same time of Treg isolation, CD4+CD252 cells were also isolated. psoriasis model (13). CXCL17 mRNA expression in murine ear 2 d after IMQ application was significantly elevated compared Chemotaxis assay with that of vehicle-treated ear (Fig. 3A), which was consistent MDSCs and Tregs isolated from spleen of C57BL/6 mice were used for a with high CXCL17 expression in human psoriasis lesional skin chemotaxis assay. The chemotaxis assay was done by using a 48-well micro (Fig. 1). Recombinant CXCL17 injection into the ear did not chemotaxis chamber and filters with 5-mm pores (Neuro Probe, Gai- thersburg, MD). MDSCs or Tregs (5 3 103 cells per 48-well plate) were induce any clinical or pathological changes (data not shown). placed on top of the 5-mm pore size filters in octuplicate, whereas RPMI Therefore, the effect of CXCL17 in IMQ-induced psoriasis-like 1640 with or without recombinant mouse CXCL17 protein (R&D Sys- skin inflammation was examined by injecting recombinant tems) was placed into the lower chamber. The concentration of CXCL17 CXCL17 or PBS into the ear before and during IMQ applica- was 10, 100, or 200 ng/ml. Following 18 h at 37˚C, migrated cells that had fallen to the bottom of the plate were counted under a microscope. The tion. Recombinant CXCL17 treatment decreased ear thickness number of migrated cells was counted per 25 HPFs (3200). Two inde- significantly compared with PBS on days 4 and 7 after IMQ pendent experiments were performed with similar results. application (Fig. 3B). Microscopic evaluation revealed that the 4 CXCL17 ATTENUATES IMQ-INDUCED PSORIASIS-LIKE INFLAMMATION

and Foxp3 (Fig. 4B), suggesting an increase in Tregs in the skin. Arginase 1 and CCL5 mRNA levels were also significantly increased in the CXCL17-injected ear compared with control mice (Fig. 4C), indicating enhanced migration of MDSCs in the ear. Thus, CXCL17 treatment attenuated IMQ-induced psoriasis-like skin inflammation assessed by ear thickness, cytokine expression, and the number of infiltrating inflammatory cells. CXCL17 also increased mRNA expression levels of markers for Tregs and MDSCs. CXCL17 treatment increases the number of infiltrating MDSCs and Tregs As mRNA expression levels of Treg and MDSC markers were increased in the CXCL17-injected ear, we further confirmed their presence in the skin. Immunofluorescent staining revealed that the number of CD11b+Gr-1+ cells was significantly increased in the CXCL17-injected ear compared with that of the PBS-injected ear (Fig. 5). The number of Foxp3+ cells was also significantly elevated in the CXCL17-injected ear compared with that of the PBS- injected ear (Fig. 5). We next examined the number of CD11b+Gr-1+ cells and CD4+CD25+ cells infiltrating into the CXCL17-injected ear by flow cytometry. The number of CD11b+Gr-1+ cells and that of CD4+CD25+ cells in the CXCL17-injected ear were significantly elevated compared with those of the PBS-injected ear (Fig. 6). Thus, CXCL17 treatment increased the number of infiltrating MDSCs and Tregs in the skin. MDSCs, but not Tregs, express Gpr35 mRNA and migrate to recombinant mouse CXCL17 in a dose-dependent manner We first confirmed that J774 and MDSCs expressed Gpr35 mRNA, but Tregs or CD4+CD252 cells did not express Gpr35 mRNA (Fig. 7A). We next performed a chemotaxis assay to investigate whether MDSCs and Tregs migrate toward CXCL17. The number of MDSCs that showed chemotaxis to CXCL17 (10, 100, 200 ng/ml) was increased dose-dependently (Fig. 7B). Alternatively, Tregs did not respond to CXCL17 (Fig. 7B). FIGURE 1. CXCL17 expression is increased in psoriasis lesional skin. (A) CXCL17 mRNA expression levels in the lesional skin of psoriasis (n = 20), Simultaneous injection of anti-CCL5 Ab or anti-CCL4 Ab with AD (n = 10), or CTCL (n = 28) and normal skin (n = 11) were measured by recombinant CXCL17 into the ear attenuates the effects of real-time RT-PCR. The measured values from individual patients were CXCL17 treatment plotted by dots. *p , 0.05. (B) Immunohistochemistry of CXCL17 by CCL5 expressed by MDSCs is important to induce Treg migration ABC staining with diaminobenzidine in normal skin and lesional skin of psoriasis, AD, or CTCL. (26). To elucidate the role of CCL5 in the CXCL17-induced anti- inflammatory response, we injected anti-CCL5 Ab simultaneously with recombinant CXCL17 into the ear before and during IMQ CXCL17-injected ears on day 7 after IMQ treatment were thinner application. When CXCL17 and control IgG were injected, the ear compared with those of PBS-injected mice (Fig. 3C). The num- thickness was significantly decreased compared with that of the bers of lymphocytes and neutrophils infiltrating into the CXCL17- ear treated with PBS and control IgG (Fig. 8A), which was con- injected ear were significantly lower compared with those of PBS- sistent with the previous results (Fig. 3B). Remarkably, the ear injected mice (Fig. 3D). thickness after treatment with CXCL17 and anti-CCL5 Ab was The CXCL17-injected ear expressed significantly lower mRNA comparable to that of PBS and control IgG (Fig. 8A), suggesting that levels of IL-17A, IL-22, TNF-a, IL-12p35, IL-12/23p40, and anti-CCL5 blocking attenuated CXCL17-induced anti-inflammatory IL-23p19, all of which are inflammatory cytokines associated response. Simultaneous injection of anti-CCL5 Ab with recombinant with human psoriasis, compared with PBS-injected ear (Fig. 4A). CXCL17 into the ear significantly increased mRNA expression Alternatively, CXCL17 treatment increased mRNA levels of IL-10 levels of IL-17A, IL-22, TNF-a, IL-12p35, IL-12/23p40, and IL-23p19 compared with injection of control IgG with recombinant CXCL17, and to similar levels of mice treated with PBS and Table I. Immunohistochemical staining for CXCL17 control IgG (Fig. 8B). As CCL3 and CCL4 are also expressed by MDSCs (27), we also injected anti-CCL3 Ab and anti-CCL4 Ab Negative Weak Positive Strong Positive in a similar way. The ear thickness after treatment with CXCL17 Normal 10 0 0 and anti-CCL4 Ab was comparable to that of PBS and control IgG AD 6 4 0 (Fig. 8D). Alternatively, injection of anti-CCL3 Ab did not change CTCL 6 3 1 the anti-inflammatory effect of CXCL17 (Fig. 8C). Simultaneous Psoriasis 0 3 7 injection of anti-CCL5 Ab or anti-CCL4 Ab with recombinant The Journal of Immunology 5

FIGURE 2. IFN-g increases CXCL17 expression by keratinocytes. (A) CXCL17 mRNA expression by HaCaT cells cultured for 24 h with IL-4 (10 ng/ml), TNF-a (10 ng/ml), IFN-g (10 ng/ml), or medium alone. (B) CXCL17 mRNA expression by NHEKs cultured for 24 h with IL-4 (10 ng/ml), TNF-a (10 ng/ml), IFN-g (10 ng/ml), or medium alone. (C) CXCL17 mRNA expression by NHEKs cultured for 24 h with IFN-g (0.1, 1, 10 ng/ml) or medium alone. (D) CXCL17 protein levels measured by ELISA in the supernatant of NHEKs cultured for 24 h with IFN-g (0.1, 1, 10 ng/ml) or medium alone. All experiments were done three times and representative results are shown. All values represent the mean 6 SEM (n =6).*p , 0.05.

CXCL17 significantly decreased IL-10 and Foxp3 mRNA ex- IFN-g levels are augmented in sera and skin lesions in psoriasis pression levels compared with injection of control IgG with patients (28–30). These are consistent with enhanced CXCL17 ex- recombinant CXCL17, and to similar levels of mice treated with pression in the epidermis of psoriasis lesional skin (Fig. 1). Lesional PBS and control IgG (Fig. 8E). On the contrary, injection of anti- skin of AD or CTCL, both of which are Th2-dominant diseases CCL3 Ab did not change IL-10 and Foxp3 mRNA expression (31–36), did not express high levels of CXCL17. levels (Fig. 8E). Thus, injection of anti-CCL5 Ab or anti-CCL4 CXCL17 treatment attenuated IMQ-induced skin inflammation Ab attenuated the anti-inflammatory effects of CXCL17 on IMQ- assessed by ear thickness, histology, the number of infiltrating cells, induced psoriasis-like dermatitis. as well as cytokine expression (Figs. 3B–D, 4A). Increased mRNA levels of IL-10 and Foxp3 (Fig. 4B) and the increased number of Tregs in the skin (Figs. 5, 6) suggested that Tregs were important Discussion for CXCL17-induced anti-inflammatory effects. CXCL17, how- In this study, we showed high CXCL17 expression in the epidermis ever, could suppress immune responses by another pathway. For of psoriasis lesional skin. In vitro, IFN-g dose-dependently in- example, CXCL17 was reported to suppress the production of creased CXCL17 expression by human keratinocytes. IMQ-induced proinflammatory cytokines and factors from murine macrophages psoriasis-like skin inflammation was attenuated by CXCL17 and act as an anti-inflammatory factor (10). We also stimulated treatment, accompanied by increased infiltration of MDSCs and J774, a murine macrophage cell line, with CXCL17 and evaluated Tregs into the skin. MDSCs expressed Gpr35 mRNA and cytokine production, only to find that CXCL17 did not signifi- responded to CXCL17 in vitro. Alternatively, Tregs did not ex- cantly inhibit cytokine production (data not shown). Therefore, press Gpr35 mRNA, showing no chemotaxis to CXCL17. Fur- anti-inflammatory effects of CXCL17 may mainly be attributed to thermore, simultaneous injection of anti-CCL5 Ab or anti-CCL4 Tregs infiltrating in the skin. Ab attenuated the anti-inflammatory effects of CXCL17 treat- MDSCs were originally identified by the CD11b+Gr-1+ phe- ment. Collectively, the results of this study suggest that CXCL17 notype in tumor-bearing mice (23). In healthy individuals, MDSCs induced by IFN-g attenuates IMQ-induced psoriasis-like skin in- consist of myeloid progenitors that differentiate into dendritic flammation by recruiting MDSCs, which in turn induce Tregs cells, granulocytes, and macrophages. In the setting of malig- through CCL5 and CCL4. nancy, MDSCs cannot differentiate, migrating into cancer tissues, IFN-g stimulation increased CXCL17 mRNA expression by either where they suppress T cell–mediated tumor immunity (37, 38). HaCaT cells or NHEKs, but IL-4 or TNF-a did not (Fig. 2A, 2B). Increased mRNA levels of arginase 1 (Fig. 4C) and the increased 6 CXCL17 ATTENUATES IMQ-INDUCED PSORIASIS-LIKE INFLAMMATION

FIGURE 3. CXCL17 treatment attenuates IMQ-induced psoriasis-like skin inflammation. (A) CXCL17 mRNA expression in murine ear 2 d after IMQ or vehicle application. All values represent the mean 6 SEM (n = 6). *p , 0.05. (B) The change of epidermal thickness of the ear during and after treatment by IMQ with recombinant CXCL17 or PBS injection into the ear. Recombinant CXCL17 or PBS was injected on days 0, 2, and 4 before and during IMQ application. All values represent the mean 6 SEM (n = 6). *p , 0.05. (C) Representative H&E stain of skin on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. (D) The number of infiltrating lymphocytes and neutrophils in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. The number of infiltrating cells was counted per HPF. All experiments were done using six mice for each condition three times and representative results are shown. All values represent the mean 6 SEM (n = 6). *p , 0.05. number of MDSCs in the skin (Figs. 5, 6) suggested that MDSCs were consistent with previous reports showing that CXCL17 in- were involved in CXCL17-induced anti-inflammatory effects. duced chemotactic responses in the THP-1 human monocyte cell Furthermore, we clearly showed that MDSCs expressed Gpr35 line (5) and J774 mouse monocyte cell line (10), and that mouse mRNA, showing chemotaxis to CXCL17 (Fig. 7). The results spleen cells responding to CXCL17 were CD11b+Gr-1+F4/802 The Journal of Immunology 7

FIGURE 4. CXCL17 treatment decreases inﬂammatory cytokine expression and increases expression levels of markers for Tregs and MDSCs. (A) Expression levels of inﬂammatory cytokines associated with psoriasis, such as IL-17A, IL-22, and TNF-a, in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. (B) IL-10 and Foxp3 mRNA expression levels in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. (C) Arginase 1 and CCL5 mRNA expression levels in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. All experiments were done using six mice for each condition three times and representative results are shown. All values represent the mean 6 SEM (n =6).*p , 0.05. 8 CXCL17 ATTENUATES IMQ-INDUCED PSORIASIS-LIKE INFLAMMATION

FIGURE 5. CXCL17 treatment increases the number of infiltrating MDSCs and Tregs in the ear as assessed by immunofluorescent staining. (A) Representative histologic images showing CD11b+ cells, Gr-1+ cells, CD11b+Gr-1+ cells, and Foxp3+ cells pointed out by white arrows in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. (B) The number of infiltrating CD11b+ cells, Gr-1+ cells, CD11b+Gr-1+ cells, and Foxp3+ cells in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. The number of infiltrating cells was counted per HPF. All experiments were done using six mice for each condition three times and representative results are shown. Values represent the mean 6 SEM (n = 6). *p , 0.05. cells (7). CXCL17 was reported to recruit MDSCs into the cancer are key chemokines responsible for Treg migration as well as tissues (7). MDSCs, in turn, produce high levels of the CCR5 li- suppression of immune responses in IMQ-induced murine pso- gands such as CCL3, CCL4, and CCL5 and directly attract Tregs riasis model. in a CCR5-dependent manner (27). In the present study, injection We have examined the effects of MDSC depletion by i.p. injection of anti-CCL5 Ab neutralized the effects of CXCL17 attenuating of anti–Gr-1 Ab and those of Treg depletion by i.p. injection of anti- IMQ-induced skin inflammation (Fig. 8A, 8B). Additionally, CD25 Ab on the ability of CXCL17 attenuating IMQ-induced skin injection of anti-CCL4 Ab blocked the effects of CXCL17 at- inflammation. Because i.p. injection of anti–Gr-1 Ab depleted neu- tenuating IMQ-induced skin inflammation (Fig. 8D). Injection of trophils as well as MDSCs, injection of anti–Gr-1 Ab attenuated skin anti-CCL5 Ab or anti-CCL4 Ab decreased IL-10 and Foxp3 inflammation (data not shown). The effect of CXCL17 was blunted. expression levels (Fig. 8E). Alternatively, injection of Ab against When Tregs were depleted by i.p. injection of anti-CD25 Ab, ear CCL3, which is another CCR5 ligand, did not change the ef- inflammation was too severe to see the anti-inflammatory effect fects of CXCL17 attenuating IMQ-induced skin inflammation of CXCL17 (data not shown). Thus, depletion of MDSCs or Tregs (Fig. 8C). Moreover, injection of anti-CCL3 Ab did not decrease blunted the effect of CXCL17, although it was difficult to analyze IL-10 and Foxp3. Therefore, CCL5 and CCL4, but not CCL3, the true impact. The Journal of Immunology 9

FIGURE 6. CXCL17 treatment increases the number of infiltrating MDSCs and Tregs assessed by flow cytometric analysis. (A) Flow cytometric analysis of cells infiltrating in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. The percentage of CD11b+Gr-1+ cells and CD4+CD25+ cells in live cells infiltrating in the ear is shown. (B) The number of MDSCs (CD11b+Gr-1+ cells) and Tregs (CD4+CD25+ cells) in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 or PBS into the ear. All experiments were done using six mice for each condition three times and representative results are shown. All values represent the mean 6 SEM (n = 6). *p , 0.05 by Mann–Whitney U test.

Our study clearly showed the immune suppressive roles of Although psoriasis was initially considered to be mediated by CXCL17, MDSCs, and Tregs in the setting of skin inflammatory Th1 cells, they have been regarded as less important since the diseases, which have been reported in cancer tissues (1, 7, 10, discovery of Th17 cells (50). However, IFN-g, in synergy with 39–45). Only limited numbers of previous reports discussed sup- TNF-a, induces the production of various proinflammatory pressive function of MDSCs in inflammatory disorders such as cytokines and chemokines, such as IL-6, IL-8, and IL-1, by alopecia areata and arthritis (46–48). Very recently, MDSCs were epidermal keratinocytes (51–53). These cytokines further acti- reported to be expanded in psoriasis, infiltrating into lesional skin vate T cells, macrophages, and neutrophils, which would con- (49). They concluded that MDSC-mediated immunosuppression tribute to the development of psoriasis (54–56). In our study, contributed to regulating hyperactivated T cells in psoriatic skin, IFN-g stimulation increased CXCL17 expression by human which our results completely supported. keratinocytes (Fig. 2), suggesting that IFN-g could regulate excessive inflammation in psoriasis skin. An old clinical trial with recombinant IFN-g, however, failed to clear psoriasis skin lesions (57), indicating that proinflammatory effects of this cytokine might overcome its immune suppressive effects. In- jection of recombinant CXCL17 would be a promising strategy to regulate excessive inflammation and clear severe skin lesions in psoriasis patients. In addition to the negative feedback regulating excessive immune responses in psoriasis by recruiting MDSCs, CXCL17 could play another role. It was reported that CXCL17 had potent antimicrobial activities (3). The mechanism of antimicrobial action involved peptide-mediated bacterial membrane disruption. CXCL17 is strongly expressed in mucosal tissues (1, 3), which are the first protection against infection. In this study, epidermis of psoriasis skin highly expressed CXCL17. It is well known that psoriasis skin expresses various antimicrobial peptides such as b-defensins, cathelicidins, and psoriasin (58, 59). Resistance to cutaneous infection is observed in patients with psoriasis. Alter- natively, those with AD or CTCL often suffer from severe skin infection, which can occasionally lead to sepsis, multiple organ failure, and death. Relative low CXCL17 expression in lesional skin of these diseases may be one of the underlying mechanisms of frequent infection. CXCL17 treatment may be useful for AD FIGURE 7. MDSCs express Gpr35 mRNA and migrate to CXCL17 patients not only because it may decrease excessive skin inflam- dose-dependently. (A) Agarose gel electrophoresis of PCR fragments am- 2 mation but also because it may contribute to resistance to skin plified using Gpr35 primers from Tregs, CD25 T cells, MDSCs, and infection. J774. (B) The number of Tregs or MDSCs that migrated to CXCL17 (10, 100, 200 ng/ml) or medium alone. The number of migrated cells was In summary, CXCL17, which is induced by IFN-g, attenuates counted per 25 HPFs (3200). The relative number to the mean number of IMQ-induced psoriasis-like skin inflammation. MDSCs express cells migrating to medium alone is shown. All experiments were done two Gpr35 mRNA, showing chemotaxis to CXCL17 in vitro. MDSCs or three times and representative results are shown. All values represent the and Tregs, with the latter being recruited by CCL5 and CCL4, mean 6 SEM (n = 8). *p , 0.05 by Mann–Whitney U test. contribute to the negative feedback regulating excessive immune 10 CXCL17 ATTENUATES IMQ-INDUCED PSORIASIS-LIKE INFLAMMATION

FIGURE 8. Simultaneous injection of anti-CCL5 Ab or anti-CCL4 Ab with recombinant CXCL17 into the ear attenuates the effects of CXCL17 treatment. (A) The change of epidermal thickness of the ear during and after treatment by IMQ with injection of recombinant CXCL17 and anti-CCL5 Ab, recombinant CXCL17 and control IgG, or PBS and control IgG into the ear. Recombinant CXCL17 and anti-CCL5 Ab, recombinant CXCL17 and control IgG, or PBS and control IgG were injected on days 0, 2, and 4 before and during IMQ application. Values represent the mean 6 SEM (n = 6). *p , 0.05 compared with PBS and control IgG group. (B) Inflammatory cytokine mRNA expression levels in the ear on day 7 after IMQ treatment with injection of recombinant CXCL17 and anti-CCL5 Ab, recombinant CXCL17 and control IgG, or PBS and control IgG into the ear. *p , 0.05. (C) The change of epidermal thickness of the ear during and after treatment by IMQ with injection of recombinant CXCL17 and anti-CCL3 Ab, recombinant CXCL17 and control IgG, or PBS and control IgG into the ear. Recombinant CXCL17, PBS, and Abs were injected on days 0, 2, and 4 before and during IMQ application. Values represent the mean 6 SEM (n = 6). *p , 0.05 compared with PBS and control IgG group. (D) The change of epidermal thickness of the ear during and after treatment by IMQ with injection of recombinant CXCL17 and anti-CCL4 Ab, recombinant CXCL17 and control IgG, or PBS and control IgG into the ear. Values represent the mean 6 SEM (n = 6). *p , 0.05 compared with PBS and control IgG group. (E) IL-10 and Foxp3 mRNA expression levels in the ear on day 7 after IMQ treatment with injection of PBS and control IgG, recombinant CXCL17 and control IgG, recombinant CXCL17 and anti-CCL3 Ab, recombinant CXCL17 and anti-CCL4 Ab, or recombinant CXCL17 and anti-CCL5 Ab into the ear. 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