Jimmunol.1600236.full.pdf

Mast Cells Limit the Exacerbation of Chronic Allergic Contact Dermatitis in Response to Repeated Allergen Exposure This information is current as of January 22, 2019.

Vladimir-Andrey Gimenez-Rivera, Frank Siebenhaar, Carolin Zimmermann, Hanna Siiskonen, Martin Metz and Marcus Maurer J Immunol published online 2 November 2016 http://www.jimmunol.org/content/early/2016/11/01/jimmun ol.1600236

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Supplementary Material

Published November 2, 2016, doi:10.4049/jimmunol.1600236 The Journal of Immunology

Mast Cells Limit the Exacerbation of Chronic Allergic Contact Dermatitis in Response to Repeated Allergen Exposure Vladimir-Andrey Gimenez-Rivera,1 Frank Siebenhaar,1 Carolin Zimmermann, Hanna Siiskonen, Martin Metz, and Marcus Maurer

A

llergic contact dermatitis (ACD) is a chronic inflammatory skin disease caused by the sensitization and repeated exposure to contact allergens. ACD is characterized by itchy eczematous lesions that typically appear between 24 and 72 h after acute allergen contact. With subsequent exposures to the contact allergen, patients show more pronounced episodic and persistent eczema (i.e., skin redness, papules, vesicles, scaling, skin thickening, and fissures). ACD often starts at a young age, with a prevalence of 15% in 12–16-y olds, represents ∼20% of occupational skin disorders, and frequently persists for life (1). Avoidance of relevant alDepartment of Dermatology and Allergy, Charite´ - Universita¨tsmedizin Berlin, D-10405 Berlin, Germany 1

V.-A.G.-R. and F.S. contributed equally to this work.

ORCID: 0000-0002-4070-9976 (M. Metz). Received for publication February 9, 2016. Accepted for publication October 5, 2016. This work was supported by grants from The Paulo Foundation and the Finnish Society of Dermatology (to H.S.) and by Deutsche Forschungsgemeinschaft Grant ME 2668/3-2 (to M. Metz) and SPP1394. V.-A.G.-R. was the recipient of a fellowship from the European Academy of Allergy and Clinical Immunology. This work was supported by the Corporation in Science and Technology Action BM1007 (Mast Cells and Basophils – Targets for Innovative Therapies) of the European Community. Address correspondence and reprint requests to Prof. Marcus Maurer, Department of Dermatology and Allergy, Allergie-Centrum-Charite´, Charite-Universita¨tsmedizin Berlin, Charite´platz 1, D-10117 Berlin, Germany. E-mail address: marcus.maurer@ charite.de The online version of this article contains supplemental material. Abbreviations used in this article: ACD, allergic contact dermatitis; BMCMC, bone marrow–derived cultured MC; CCHS, chronic CHS; CHS, contact hypersensitivity; CPA, carboxypeptidase A; Cre+, MCPT5-Cre+iDTR+; dLN, draining lymph node; DNFB, dinitrofluorobenzene; DT, diphtheria toxin; MC, mast cell; OXA, oxazolone; Sash, KitW-sh/ W-sh; TRM, tissue-resident memory T; WT, wild-type. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600236

lergens results in remission but is difficult to achieve for many allergens (e.g., nickel); better approaches for avoiding ACD recurrence and episodic exacerbation are needed. Our understanding of the mechanisms that underlie the sensitization to allergens, the response to allergen exposure, and the resolution of inflammation in ACD patients is largely shaped by the results of mouse studies of contact hypersensitivity (CHS). In these CHS studies, mice are sensitized and then challenged with an experimental allergen (e.g., oxazolone [OXA] or dinitrofluorobenzene [DNFB]) by topical application to the skin. For example, CHS studies revealed the role of TLR signaling in the sensitization phase and the importance of several T cell subsets, as well as dermal dendritic cells, for the inflammatory response to allergen challenge (2). Importantly, the vast majority of CHS studies are acute CHS studies (i.e., limited to investigations of the sensitization phase and the acute inflammatory response to a single allergen challenge). A few CHS studies used continued repeated allergen exposure to investigate the mechanisms of subchronic CHS (3–6), and all of them were limited to a few weeks of allergen exposure. There are no models or studies of chronic CHS (CCHS; i.e., repeated chronic allergen challenge over several months); as a consequence, the mechanisms of CCHS and ACD remain ill-defined. Mast cells (MCs) are tissue-resident effector cells of innate immune responses to pathogens and IgE-mediated allergic inflammatory reactions. Two independent lines of evidence suggest that MCs may play important roles in the episodic exacerbation of chronic ACD and CCHS. First, MCs are predominantly located in organs that border our environment (e.g., airways, intestine, and skin). Large numbers of MCs are present in skin areas that are frequently exposed to contact allergens and that are predilection sites of ACD, such as the hands and the face (7). Also, chronically

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Allergic contact dermatitis is a chronic T cell–driven inflammatory skin disease that is caused by repeated exposure to contact allergens. Based on murine studies of acute contact hypersensitivity, mast cells (MCs) are believed to play a role in its pathogenesis. The role of MCs in chronic allergic contact dermatitis has not been investigated, in part because of the lack of murine models for chronic contact hypersensitivity. We developed and used a chronic contact hypersensitivity model in wild-type and MC-deficient mice and assessed skin inflammatory responses to identify and characterize the role of MCs in chronic allergic contact dermatitis. Ear swelling chronic contact hypersensitivity responses increased markedly, up to 4-fold, in MC-deficient KitW-sh/W-sh (Sash) and MCPT5-Cre+iDTR+ mice compared with wild-type mice. Local engraftment with MCs protected Sash mice from exacerbated ear swelling after repeated oxazolone challenge. Chronic contact hypersensitivity skin of Sash mice exhibited elevated levels of IFN-g, IL-17a, and IL-23, as well as increased accumulation of Ag-specific IFN-g–producing CD8+ tissue-resident memory T (TRM) cells. The CD8+ T cell mitogen IL-15, which was increased in oxazolone-challenged skin of Sash mice during the accumulation of cutaneous TRM cells, was efficiently degraded by MCs in vitro. MCs protect from the exacerbated allergic skin inflammation induced by repeated allergen challenge, at least in part, via effects on CD8+ TRM cells. MCs may notably influence the course of chronic allergic contact dermatitis. A better understanding of their role and the underlying mechanisms may lead to better approaches for the treatment of this common, disabling, and costly condition. The Journal of Immunology, 2016, 197: 000–000.

2 affected skin sites of ACD patients show markedly increased numbers of MCs (8). Second, MCs were reported to promote acute CHS responses to a single allergen challenge in some studies (9– 12), although not all (13, 14). MCs also were demonstrated to limit and reduce acute CHS responses to a single allergen challenge in one study (15) but not another (12). The aim of our study was to better characterize the mechanisms that drive and control CCHS to better understand chronic responses in ACD. To this end, we developed and used a murine model of CCHS, which mimics the recurrent sporadic allergen encounters that occur in ACD patients and lead to pronounced episodic exacerbation and persistence of their condition. This CCHS model, which involves repeated OXA challenges in sensitized mice every 30 d over several months, was applied to genetically MC-deficient mice and normal control mice to identify and characterize the relevance and role of MCs in CCHS.

Materials and Methods All mice used in the experiments were 6–8-wk-old females on a C57BL/6 background. Wild-type (WT) C57BL/6-Kit+/+ and C57BL/6-KitW-Sh/W-Sh (Sash) mice were bred and housed at our facilities, and MCPT5-Cre 3 iDTR (Cre+ or Cre2) mice were kindly provided by Dr. Axel Roers (University of Dresden, Dresden, Germany). Ly5.1 mice were purchased from the Jackson Laboratory. All mice were kept under specific pathogen– free conditions, and all experiments were conducted according to institutional regulations.

CCHS Animals were sensitized on day 25 with 20 ml of 6% w/v OXA (SigmaAldrich, Steinheim, Germany) in acetone by topical application to shaved abdominal skin. Inflammation was elicited at selected skin sites by topical application of 20 ml of 0.6% w/v OXA in acetone at 30-d intervals. We designated the inflammatory response after the first OXA challenge as acute CHS. Inflammatory responses induced by the third or subsequent challenges with OXA were designated as chronic CHS. The OXAchallenged sites and the number of challenges for each group are specified in the figure legends. For cross-sensitization experiments using DNFB (Sigma-Aldrich), animals were sensitized on day 85 with 0.5% v/v DNFB in acetone by topical application on shaved abdominal skin. On day 90, inflammation was elicited with 20 ml of 0.1% v/v DNFB in acetone and 0.6% w/v OXA in acetone on the right and left ears, respectively. Ear thickness was assessed with a micrometer caliper (Mitutoyo) before and for several days after each challenge.

In vivo anti-CD8 Ab treatment On days 5 and 6 after the induction of acute CHS, 250 mg of anti-CD8 Ab clone 2.43 (2, 16) in 500 ml of PBS or PBS alone was injected i.p.

Depletion of MCs with diphtheria toxin MCPT5-Cre 3 iDTR mice were injected weekly for 4 wk with diphtheria toxin (DT, i.p., 25 ng/g bodyweight in saline; Sigma-Aldrich) before allergen sensitization. Two days before the induction of CCHS, mice were injected intradermally in the ear pinnae with 40 ml of DT (5 ng/g bodyweight in saline). To maintain the MC deficiency, i.p. injections of DT (25 ng/g bodyweight in saline) were administered weekly from day 0 (OXA sensitization day) until day 120.

Adoptive transfer of MCs or CD8+ tissue-resident memory T cells For engraftment of MCs in the ears of Sash mice (Sash-Ear+BMCMCs), 4-wkold bone marrow–derived cultured MCs (BMCMCs, 2 3 106 cells per mouse) were cultured as previously described (17) and injected into the ear pinnae of mice (40 ml per ear). Only BMCMCs that exhibited .95% purity, as determined by the coexpression of CD117/FcεRI, were used for reconstitution experiments. Mice were used for experiments 4–6 wk after the engraftment of BMCMCs. For the adoptive transfer of CD8+ tissue-resident memory T (TRM) cells, donor Ly5.1 mice (WT animals expressing the CD45.1 haplotype) and recipient Sash and WT mice were sensitized (day 25) and challenged (day 0) with OXA. On day 5, the draining lymph nodes (dLNs) and total blood

of donor animals were collected, passed through a 70-mm cell strainer, and washed with PBS to produce a cell suspension. Blood was depleted of erythrocytes using an erythrocyte lysis buffer (eBioscience, Frankfurt, Germany) and a Lymphoprep centrifugation gradient (STEMCELL Technologies, Cologne, Germany). The cell suspensions from both organs were pooled, stained with anti-CD8b–PE Ab (1:400; BioLegend, London, U.K.) and purified (.90% purity) by FACS. The positive fraction was injected into the tail vein of recipient mice (2 3 107 cells per mouse). Three days after the adoptive transfer of CD45.1+CD8+ T cells, the ears of WT and Sash recipient mice were harvested, and the composition and numbers of CD45.1+CD8+ T cells expressing TRM cell markers were analyzed by FACS.

Cytokine array A total of 200 ml of protein lysate was extracted from ear skin, purified as described by Lan et al. (18), and analyzed using a Multi-Analyte ELISArray Kit (QIAGEN, Hilden, Germany).

Whole mount of ear skin and confocal microscopy Ears were perfused with 80 ml of TBS, harvested, fixed in 4% PFA for 20 min, split in ventral and dorsal halves, and digested with proteinase K (1:1000 in TBS; DAKO, Hamburg, Germany) for 1 h. For quantification of skin MCs, the ear halves were incubated with a mixture of DAPI (1:100) and Avidin–Texas Red (1:500; both from Roche, Mannheim, Germany) for 15 min at 37˚C. For identification of CD8+IFN-g+ double-positive cells, the ear halves were incubated with a mixture containing anti-CD8b–PE Ab (1:100) and anti-IFN-g–Biotin Ab (1:50; both from BioLegend) for 3 h at 37˚C. The tissue was blocked with Biotin and Avidin block for 15 min at 37˚C (DAKO) and then incubated with FITC-streptavidin (1:200; eBioscience) in TBS and 0.5% BSA. After separating the epidermal layer from the dermis, the dermal layer was washed in TBS and 0.5% BSA, mounted, and prepared for confocal imaging. Images were taken with Olympus FV1000MPE and Zeiss LSM 510 microscopes in single-photon settings. For visualization, 50 mm of the z-axis was scanned at 5-mm intervals and merged in the z-axis to construct the z-stacks. T cells and MCs in every stack were analyzed in terms of their numbers, localization, and distribution.

Cell preparations and flow cytometry The ear halves were split by separating the ventral half from the dorsal half, cut into small pieces, and incubated in a vial containing 0.2 mg/ml of Liberase TL (Roche) diluted in 1% streptomycin/penicillin in RPMI 1640. The reaction was stopped by adding RPMI 1640 with 10% FCS v/v. Cell sorting was performed with a FACS Aria II (BD Bioscience, Heidelberg, Germany). The phenotypic characterization of MCs and T cells was performed with a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). The following Abs (all from BioLegend) were used for the identification of T cell populations: CD45 (clone 30-F11), CD103 (clone 2E7), CD8b (clone YTS156.7.7), CD44 (clone IM7), TCRb (clone H57-597), CD3 (clone 145-2C11), and CD4 (clone GK1.5).

In vitro experiments on the degradation of IL-15 Peritoneal lavage from WT mice (three to five animals per experiment, three experiments) was collected, and the total cell number was adjusted to 1.5 3 107 cells per milliliter in PBS with Ca2+ and Mg2+. The cells were stimulated with 2 mM ionomycin for 30 min at 37˚C, after which the supernatant was isolated, diluted if appropriate, and treated with 100 mM chymostatin, 100 mM leupeptin, 400 mg/ml carboxypeptidase A (CPA) inhibitor from potato tuber (all from Sigma-Aldrich), or vehicle in addition to 8 ng of recombinant mouse IL-15 in PBS as a substrate for 2 h at 37˚C. Samples were kept in 280˚C until analysis of IL-15 levels with a mouse IL-15 ELISA (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.

Statistical analysis For all of the experiments, the data are shown as means of pooled values of independent experiments with error bars representing 6 SEM, unless otherwise indicated. The Mann–Whitney U test was used to analyze experiments in Figs. 1A, 2A, 2B, 5A, and 5B. The nonparametric Wilcoxon test was used to analyze experiments in Figs. 1C and 4A. The Student t test was used to analyze experiments in Figs. 3A, 6A, 6B, and 7A. For data shown in Fig. 5A, linear regression between left/right ear and strain was performed, whereas for data in Fig. 7B and 7C, ANOVA with the Tukey post test was performed. All statistical tests were performed with GraphPad Prism version 5.0b (GraphPad, La Jolla, CA).

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The Journal of Immunology

Results MCs control CCHS responses

Th1 cytokine levels and numbers of CD8+ T cells are increased in CCHS in the absence of MCs To better understand how MCs control CCHS responses, we analyzed the skin cytokine profile and the cellular infiltrate of MC-deficient CCHS ears. We found higher levels of IFN-g, IL-17a, and IL-23, but not IL-4, IL-6, or IL-10, in Sash mice compared with WT mice 24 h after induction of CCHS responses (Fig. 3A). In CCHS ears, the CD8+ T cells appeared to be a prominent source of IFN-g, indicating that these cells were proinflammatory (Fig. 3B). We also found higher numbers of CD8+ T cells, but not CD4+ T cells, in the CCHS ears of Sash mice and MC-depleted Cre+ mice (Fig. 4A). The majority of these CD8+ T cells were TRM cells, as determined by their coexpression of CD44high and CD103 (19) (Fig. 4B). The control of CCHS by skin MCs is local, not systemic, involves CD8+ TRM cells, and is Ag specific Do MCs control CCHS by controlling cutaneous CD8+ TRM cell numbers? To address this question, we sensitized mice to OXA, challenged them multiple times, and induced and compared CHS responses at skin sites that had not previously been challenged with OXA (naive skin sites) with skin sites that had been repeatedly challenged. MC-deficient mice showed CHS responses comparable to those of their WT controls in their naive skin sites but exacerbated CHS responses in their repeatedly challenged skin

sites (Fig. 5A), indicating that local, and not systemic, effects are critical for MC-mediated CCHS control. Next, we subjected skin sites in Sash mice to acute CHS and then depleted them of CD8+ cells (Sash2CD8), which reduced the numbers of cutaneous CD8+ TRM cells at these sites to levels similar to those observed in WT mice (Fig. 5B). Subsequent CCHS responses at these sites were normal and similar to those in WT mice (Fig. 5C), indicating that CD8+ T cells and their local increase are important for uncontrolled CCHS responses in the absence of MCs. Finally, we tested whether MCs control CCHS responses via effects on Ag-specific cells. To this end, we sensitized and repeatedly challenged Sash mice with OXA, sensitized them to DNFB, which does not cross-react with OXA, and then challenged them, at sites of previous OXA challenges, with DNFB or OXA (Fig. 5D). The DNFB-challenged sites showed controlled CHS responses comparable to those in WT and Sash2CD8 mice (Fig. 5D). In contrast, the OXA-challenged sites showed uncontrolled inflammation, suggesting that MCs control CCHS by effects on Ag-specific CD8+ T cells (i.e., OXA-specific CD8+ TRM cells). Skin MCs control CD8+ TRM cell accumulation at sites of allergen challenge How do skin MCs control the increase in CD8+ TRM cell numbers at CHS sites? The naive skin of Sash and WT mice contained no CD8+ TRM cells (Fig. 6A). During acute CHS, these cells gradually increased in both mice, more so in Sash mice than in WT mice (Fig. 6A). In parallel, CD8+ TRM cell numbers also increased in the dLNs of both mice, again more so in Sash mice than in WT mice (Fig. 6A). This suggested that MCs may control the increase in CD8+ TRM cells at CHS sites by controlling their recruitment to the skin and/or by controlling their generation in lymph nodes. To clarify this, we adoptively transferred CD45.1+CD8+ TRM cells to CD45.12 recipient Sash mice and WT controls and subjected these mice to CHS. We found significantly more donor-derived CD45.1+CD8+ TRM cells in the CHS ears of Sash mice compared with WT mice (Fig. 6B), indicating that MCs, through local mechanisms, restrict the recruitment of CD8+ TRM cells to allergenchallenged skin. Because Sash CHS ears also had higher numbers of endogenously generated TRM cells (i.e., CD45.12CD8+ TRM cells) (Fig. 6B), it suggests that MCs may also control the development of CD8+ TRM cells in CHS in dLNs. Next, we analyzed the ear skin levels of TGF-b, IL-7, and IL-15, which are known to be important for cutaneous CD8+ TRM cells (20, 21). Our results showed higher levels of IL-15 (Fig. 7A), but not of TGF-b and IL-7 (data not shown), in the challenged skin of Sash mice compared with WT mice.

FIGURE 1. CCHS reactions are more pronounced and persistent in the absence of MCs. Animals were sensitized to OXA (day 25, abdominal skin) and challenged with OXA (day 0, left ears; day 30, right ears; and day 60, left ears) or treated with vehicle (acetone) as control (day 0, right ears; day 30, left ears; and day 60, right ears). (A) MC-deficient Sash mice show uncontrolled ear swelling after chronic CHS to OXA (right panel), but not after acute CHS (left panel). Data are shown as means of pooled values of n = 4 experiments, and error bars indicate SEM of 20 animals per group. Dagger symbols (†) indicate the number of mice that showed signs of scaling or necrosis on the ears at the specified points in time. (B) Representative pictures showing the macroscopic state of the skin on day 68. A total of 12 of 35 Sash mice developed scaling and necrosis (†) at CCHS sites. (C) The area under the curve (AUC) was calculated for acute CHS (days 0–8) and chronic CHS (days 60–68). **p , 0.01, ***p , 0.001.

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CCHS responses to the third monthly challenge with OXA were stronger than acute CHS responses to a single OXA challenge in the ears of WT mice (Fig. 1A). Unexpectedly, CCHS responses were even more pronounced and of longer duration in genetically MCdeficient C57BL/6-KitW-sh/W-sh (Sash) mice. Compared with WT mice, Sash mice showed up to 4-fold increased ear swelling, as well as skin scaling and necrosis (Fig. 1A, 1B). In contrast, acute CHS responses were similar in Sash and WT mice (Fig. 1A, 1C). MC-depleted MCPT5-Cre+iDTR+ (Cre+) mice also developed more pronounced and persistent CCHS responses compared with controls (i.e., MCPT5-Cre2iDTR+ mice) (Fig. 2A), despite having not developed skin scaling or necrosis as it was observed in Sash mice. CCHS in Sash mice that had been locally repaired of their MC deficiency (Sash-Ear+BMCMCs) was less severe and of shorter duration than that in MC-deficient control ears, did not show scaling or necrosis, and was similar to CCHS in WT mice (Fig. 2B). Taken together, these findings suggested that local skin MCs control CCHS responses.

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To investigate further the possible mechanism how MCs could control the accumulation of TRM cells by modulating IL-15 levels during CHS in the skin, we incubated supernatants from ionomycinactivated peritoneal lavage cells including MCs, which closely resemble skin MCs, with IL-15 ex vivo and found a significant concentration-dependent decrease in IL-15 levels (Fig. 7B). The reduction in IL-15 levels was inhibited by the addition of MC protease inhibitors, such as chymostatin (inhibitor of chymase) and CPA inhibitor, to the supernatant before the substrate (Fig. 7C), suggesting that chymase and CPA are capable of degrading IL-15.

Discussion Our results show that MCs control allergic skin inflammation in a murine CCHS model of human ACD. To investigate the role of MCs in CCHS, we first had to develop a new and suitable murine model. Unlike acute CHS models (single

allergen challenge) and subchronic CHS models (multiple challenges for a few weeks), as used in previous studies, our CCHS model entails once-monthly allergen challenges over several months. This mimics the course of ACD in many patients, with chronic recurrent allergic skin inflammation triggered by episodic skin contact with relevant allergens. Our CCHS model shows many features of ACD, including increasingly more pronounced inflammatory responses upon repeated allergen challenges, Th1 skewing, and increased expression of IFN-g at sites of inflammation (22), as well as long-lasting accumulation of immune cells (e.g., MCs and T cells) at involved skin sites (23). Our results complement those of a previous study by Hershko et al. (4), who found that MCs contribute to subchronic CHS. In that study, sensitized mice were challenged with OXA three times per week for up to 10 times. MCs were found to control CHS by migrating to the dLNs and to the spleen and by controlling CD4+

FIGURE 3. In CCHS, Th1 cytokine levels are increased in the absence of MCs. The ears of mice were harvested 24 h after the induction of CCHS and processed for cytokine analysis (A) or for whole-mount immunohistochemistry (B). (A) Cutaneous levels of cytokines in Sash and WT mice during CCHS to OXA (day 61). Data are shown as the mean of pooled values of n = 3 experiments, and error bars indicate SEM of nine animals per group. (B) Sash skin CD8+ T cells are a prominent source of IFN-g in CCHS. Immunofluorescence staining (original magnification 340). Pictures from ear whole mount. The CD81IFNg panel is a merge of the CD8 and IFNg panels. DAPI (blue; nuclei), anti-CD8b Ab (red), anti–IFN-g Ab (green), or isotype Abs (Control). ***p , 0.001, *p , 0.05.

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FIGURE 2. Skin MCs control CCHS responses. (A) MC-depleted Cre+ mice show increased ear swelling after CCHS to OXA compared with MCPT5-Cre2iDTR+ control mice. Data are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of 12 animals per group. (B) Sash mice reconstituted locally with BMCMCs in the ears (Sash-Ear+BMCMCs) show normal inflammatory responses after CCHS to OXA. Data are shown as means of pooled values of n = 4 experiments, and error bars indicate SEM of 15 animals per group. **p , 0.01. Dagger symbols (†) indicate the number of mice that showed signs of scaling or necrosis on the ears at the specified points in time.

The Journal of Immunology

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FIGURE 4. In CCHS, TRM cells are increased in the absence of MCs. Ears of mice were harvested 30 d after the induction of CCHS (on day 90), when they were no longer macroscopically inflamed, and analyzed by FACS. (A) MC-deficient Sash mice contain more CD8+ cells, but not CD4+ cells, at sites of previous CCHS responses. The bar graphs show fold increases in CD8+ cells and CD4+ cells in relation to naive skin of the respective genotypes, in which cell numbers were low. Data are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of nine animals per group. (B) Most CD8+ T cells in the skin of MC-deficient Sash and Cre+ mice 4 wk after induction of CCHS are TRM cells (red dots). In the FACS gating strategy, the CD45+CD42TCRb+CD8b+ CD103+CD44high cells represented the CD8+ TRM cells. ## p , 0.01, #p , 0.05 versus naive skin; **p , 0.01 versus WT controls.

more pronounced than in WT mice, but only Sash mice, and not Cre+ mice, showed skin scaling and necrosis at CCHS sites. The most likely explanation for this is that the MC-depleted Cre+ mice contain more skin MCs than Sash mice. Under noninflammatory conditions, the depletion efficiency of skin MCs in Cre+ mice is .90% (12) (Supplemental Fig. 1A). However, in CCHS we found only a 75% reduction in skin MCs in Cre+ mice compared with a .90% reduction in Sash mice (Supplemental Fig. 1B). These results suggest that the depleting effect of DT is counteracted by the accumulation of local skin MCs, probably triggered by the induction of local inflammation. In line with this, WT mice

FIGURE 5. The control of CCHS by skin MCs is local, not systemic, involves CD8+ T cells, and is Ag specific. (A) The control of CCHS by skin MCs is local. All mice were sensitized to OXA and challenged multiple times. CHS was induced at skin sites that had been repeatedly challenged with OXA or at sites that had never been challenged with OXA. Data are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of 12 animals per group. ##p , 0.01 versus naive skin; **p , 0.01 versus WT controls. (B) Numbers of skin CD8+ T cells after treatment with anti-CD8 Ab. Eight days after the first challenge with OXA, the ear skin was harvested, and the numbers of CD8+ T cells were assessed by flow cytometry. One representative experiment of n = 3 independent repetitions. **p , 0.01 versus Sash. (C) The control of CCHS by skin MCs involves CD8+ T cells. Mice were sensitized (day 25, abdominal skin) and challenged (day 0, both ears; day 30, abdominal skin; and day 60, both ears) with OXA. Additionally, on days 5 and 6 after the first OXA challenge, some Sash mice were injected with anti-CD8 Ab (Sash2CD8). Control mice were injected with saline (Sash and WT). **p , 0.01 versus controls. (D) The control of CCHS by skin MCs is Ag specific. The mice in (B) were sensitized to a different allergen (DNFB) on the abdominal skin (on day 85) and subsequently challenged (on day 90) with DNFB on the right ears (left panel) and with OXA on the left ears (right panel). Sash, Sash2CD8, and WT mice show comparable ear swelling responses after CHS to DNFB (day 90–98) but exacerbated CCHS responses to OXA. ##p ,0.01, Sash versus Sash2CD8 mice. Data in (B and C) are shown as means of pooled values of n = 3 experiments, and error bars indicate SEM of 10 animals per group. Dagger symbols (†) indicate the number of mice that showed signs of scaling or necrosis on the challenged ears at the specified points in time. n.s., not significant.

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T cells via IL-2. In our chronic CHS study, cutaneous MCs at sites of allergen challenge control CHS by effects on TRM cells. In both studies, MCs protect from allergic inflammation induced by repeated allergen challenge. The differences in the mechanisms involved may reflect differences in the models used or point to the involvement of multiple mechanisms in MC-mediated suppression of allergic skin inflammation, which may change in relevance as CHS progresses from acute to subchronic to chronic. To our knowledge, this is the first CHS study to use two independent MC-deficient mouse models showing very similar results. In both MC-deficient mice, CCHS inflammation was significantly

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showed MC activation (degranulation) and MC accumulation at CCHS skin sites (Supplemental Fig. 2). The key findings that led us to explore the importance of CD8+ TRM cells for MC-mediated suppression of CCHS were that Th1 cytokine levels and numbers of CD8+ T cells at CCHS skin sites were increased in the absence of MCs. The critical role of these Ag-specific TRM cells is underlined by the outcome of our extensive characterization of CCHS skin infiltrates and our depletion experiments that, taken together, indicate that MCs control CCHS by controlling CD8+ TRM cells at sites of allergen challenge. MC-deficient

mice showed higher numbers of adoptively transferred and endogenously generated CD8+ TRM cells. This suggests that MCs may limit the recruitment of CD8+ TRM cells to the skin sites of CCHS and/or limit their generation in the dLNs. Gaide et al. (24) recently demonstrated the relevance of Ag-specific expansion of TRM cells in ACD, as well as in DNFB-induced CHS, in mice. Because TGF-b, IL-7, and IL-15 are known to be secreted locally in the skin and required for the maintenance of cutaneous CD8+ TRM cells (20, 21), we analyzed these cytokines in our CCHS model and found higher IL-15 levels in the ear skin of MC-deficient Sash mice compared

FIGURE 7. MC-deficient Sash mice have increased cutaneous levels of IL-15 at the time of CD8+ TRM cell accumulation in their skin, and MC enzymes are capable of degrading IL-15 in vitro. (A) Cutaneous levels of IL-15 7 d after acute CHS in Sash and WT mice. *p , 0.05 versus WT mice. (B) Supernatant from ionomycin-activated peritoneal lavage cells degrades IL-15 in a concentration-dependent manner. ***p , 0.001, vehicle versus supernatant. (C) Reduction in IL-15 levels is inhibited by chymostatin and CPA inhibitor but not by leupeptin. ***p , 0.001, **p , 0.01, vehicle versus supernatant or between supernatant with and without inhibitors. n.s., not significant.

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FIGURE 6. MCs control the recruitment of CD8+ TRM cells to sites of CCHS responses. (A) Sash mice show increased levels of CD8+ TRM cells in the ears (left panel) and dLNs (right panel) as early as 6 d after acute CHS. (B) Sash mice contain more donor and recipient CD8+ TRM cells than do WT mice after acute CHS. Statistical significance was calculated with the Student t test. For all experiments, data are shown as the means of pooled values of n = 3 experiments, and error bars indicate SEM of nine animals per group. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., not significant.

The Journal of Immunology

Acknowledgments We thank Dr. Axel Roers for providing MCPT5-Cre 3 iDTR mice. We also thank the staff of the microscope facility at the Max-Delbr€uck Center (Berlin, Germany) and the Danish Molecular Biomedical Imaging Center (Odense, Denmark) for technical support and Sina Heydrich and Mariella Bothke for excellent technical assistance.

Disclosures The authors have no financial conflicts of interest.

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with controls. Furthermore, we found that chymase and CPAs are able to degrade IL-15 in vitro, as also shown earlier for several other cytokines (25). MCs are the sole source of chymase and CPA (26), but the effects of other CPA-secreting cells cannot be ruled out. However, it seems that MCs may impair the survival or proliferation of skin CD8+ TRM cells in CCHS by contributing to the reduced local levels of IL-15, which is believed to be the mechanism that decreases the availability of IL-15 in situ (27, 28). Interestingly, our present findings and the ability of IL-15 to suppress chymase activity that was reported earlier (29) point to a novel feedback loop that regulates the levels of IL-15 and chymase. In addition to the possible enzymatic degradation of IL-15, MCs have a high expression of IL15Rs (30, 31), are highly responsive to IL-15 in vitro (32) and, under in vitro conditions, consume large amounts of IL-15 (33). However, the effects of MCs on other cells producing IL-15 (e.g., mononuclear cells) (34) cannot be ruled out. In conclusion, we show that the increased accumulation of CD8+ TRM cells in the skin of MC-deficient mice results in exacerbated inflammation upon repeated allergen challenge. This supports an immunoregulatory function for cutaneous MCs and encourages the further study of this role of MCs in TRM cell–mediated diseases, such as ACD or psoriasis (24, 35). Approaches that increase local skin MC numbers or enhance their functions could limit or reduce the exacerbation of symptoms in ACD patients by reducing pathogenic TRM cells in the skin.

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