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Functional Interplay between IL-9 and Peptide YY Contributes to Chronic Skin Inflammation

Open AccessPublished:July 15, 2022DOI:https://doi.org/10.1016/j.jid.2022.06.021
      Complex interactions between keratinocytes and various cell types, such as inflammatory cells and stromal cells, contribute to the pathogenesis of chronic inflammatory skin lesions. In proinflammatory cytokine‒mediated disease settings, IL-9 plays a pathological role in inflammatory dermatitis. However, IL-9‒related mechanisms remain incompletely understood. In this study, we established tamoxifen-induced keratinocyte-specific IL-9RA-deficient mice (K14CRE/ERTIl9raΔ/Δ mice) to examine the role of IL-9 in multicellular interactions under chronic skin inflammatory conditions. Studies using an imiquimod-induced psoriasis-like model showed that K14CRE/ERTIl9raΔ/Δ mice exhibited a significantly reduced severity of dermatitis and mast cell infiltration compared with control K14WTIl9rafl/fl mice. Transcriptome analyses of psoriasis-like lesions showed that the level of peptide Y-Y (Pyy), a member of the neuropeptide Y family, was markedly downregulated in K14CRE/ERTIl9raΔ/Δ epidermis. Pyy blockade suppressed epidermal thickening and mast cell numbers in imiquimod-treated wild-type mice. Together with in vitro studies indicating that Pyy induced IL-9 production and chemotactic activity in bone marrow‒derived mast cells, these findings suggest that Pyy-mediated interplay between keratinocytes and mast cells contributes to psoriasiform inflammation. Further investigation focusing on the IL-9‒Pyy axis may provide valuable information for the development of new treatment modalities for inflammatory dermatitis.

      Graphical abstract

      Abbreviations:

      BMMC (bone marrow‒derived mast cell), IL-9R (IL-9 receptor), IMQ (imiquimod), KC (keratinocyte), Npy (neuropeptide Y), Pyy (peptide Y-Y), rIL (recombinant IL), STAT (signal transducer and activator of transcription), Th (T helper), WT (wild-type)

      Introduction

      Inflammatory skin diseases are caused by dysregulated interactions between keratinocytes (KCs) and several different types of cells, such as inflammatory, immune, stromal, and vascular endothelial cells. These dysregulated intercellular interactions lead to abnormal KC differentiation with infiltration of various cells, as observed in inflammatory dermatitis (
      • Dainichi T.
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      The epithelial immune microenvironment (EIME) in atopic dermatitis and psoriasis.
      ;
      • Langan S.M.
      • Irvine A.D.
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      Atopic dermatitis.
      ). During the past decade, innovative biologic therapies targeting pathogenic cytokines have improved the clinical outcomes of inflammatory dermatoses, such as psoriasis and atopic dermatitis (
      • Liu T.
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      • Ying S.
      • Tang S.
      • Ding Y.
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      The IL-23/IL-17 pathway in inflammatory skin diseases: from bench to bedside.
      ;
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      ). Relevant studies in experimental murine models have further revealed the pathological significance of networks involving proinflammatory cytokines, including IL-17, IL-22, IL-23, and TNF-α, which mediate aberrant KC and inflammatory cell interactions in inflammatory cutaneous lesions (
      • Bieber T.
      Interleukin-13: targeting an underestimated cytokine in atopic dermatitis.
      ;
      • Chiricozzi A.
      • Romanelli P.
      • Volpe E.
      • Borsellino G.
      • Romanelli M.
      Scanning the immunopathogenesis of psoriasis.
      ;
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      • Le S.
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      • et al.
      New frontiers in psoriatic disease research, part I: genetics, environmental triggers, immunology, pathophysiology, and precision medicine.
      ). Given the limited effectiveness of biologic therapies for chronic inflammatory dermatoses, a further understanding of the mechanisms of inflammatory circuits contributing to cutaneous lesions is required.
      Recent reports revealed high expression of the proinflammatory cytokine IL-9 in the skin lesions of patients with psoriasis and atopic dermatitis (
      • Schlapbach C.
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      Human TH9 cells are skin-tropic and have autocrine and paracrine proinflammatory capacity.
      ;
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      IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
      ). In addition, the serum level of IL-9 is positively correlated with the severity of dermatitis in these inflammatory skin diseases (
      • Ma L.
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      • Guan X.H.
      • Shu C.M.
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      Possible pathogenic role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis.
      ;
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      • Singh S.
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      Interleukin-9 serves as a key link between systemic inflammation and angiogenesis in psoriasis.
      ). Experiments using a psoriasis-like murine model further supported the pathological involvement of IL-9 in the development of inflammatory skin lesions through the activation of a T helper (Th) 17 pathway (
      • Singh T.P.
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      • Wallbrecht K.
      • Gruber-Wackernagel A.
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      Involvement of IL-9 in Th17-associated inflammation and angiogenesis of psoriasis.
      ). IL-9 preferentially binds to heterodimeric receptor complexes consisting of an IL-9RA and common γ-chain (γc or IL-2RG), the latter of which is also involved in the intracellular signaling of IL-2, IL-4, IL-7, IL-15, and IL-21 (
      • Kovanen P.E.
      • Leonard W.J.
      Cytokines and immunodeficiency diseases: critical roles of the gamma(c)-dependent cytokines interleukins 2, 4, 7, 9, 15, and 21, and their signaling pathways.
      ). Through the IL-9 receptor complex, Jak1 and Jak3 associate with IL-9RA and γc, respectively, and activate multiple signaling pathways, such as Jak‒signal transducer and activator of transcription (STAT) (STAT1, STAT3, and STAT5), phosphoinositide 3-kinase, and MAPK, eventually inducing various biological processes (
      • Neurath M.F.
      • Finotto S.
      IL-9 signaling as key driver of chronic inflammation in mucosal immunity.
      ). As indicated by the expression profiles of the IL-9 receptor complex, several different types of cells produce biological effects through IL-9 in skin lesions. In addition, Th17 cells, Th9 cells, type 2 innate lymphoid cells, and mast cells are suggested to participate in pathological cutaneous foci as IL-9 producers (
      • Lozano-Ojalvo D.
      • Berin C.
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      ;
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      Resolution of inflammation by interleukin-9-producing type 2 innate lymphoid cells.
      ;
      • Schlapbach C.
      • Gehad A.
      • Yang C.
      • Watanabe R.
      • Guenova E.
      • Teague J.E.
      • et al.
      Human TH9 cells are skin-tropic and have autocrine and paracrine proinflammatory capacity.
      ). Given that IL-9 regulates the metabolic reprogramming of human KCs (
      • Marathe S.
      • Dhamija B.
      • Kumar S.
      • Jain N.
      • Ghosh S.
      • Dharikar J.P.
      • et al.
      Multiomics analysis and systems biology integration identifies the roles of IL-9 in keratinocyte metabolic reprogramming.
      ) and that IL-9 receptor (IL-9R) is highly expressed in KCs in atopic dermatitis (
      • Hong C.H.
      • Chang K.L.
      • Wang H.J.
      • Yu H.S.
      • Lee C.H.
      IL-9 induces IL-8 production via STIM1 activation and ERK phosphorylation in epidermal keratinocytes: a plausible mechanism of IL-9R in atopic dermatitis.
      ), IL-9 may play a certain role in unresolved skin inflammation. However, the interplay between KCs and other cells in inflammatory skin lesions is complex. Therefore, it remains to be determined how IL-9 acts on KCs to sustain chronic inflammatory conditions, such as psoriasis.
      In this study, we established and analyzed experimental models of psoriasis using KC-specific conditional-knockout mice deficient in all exons encoding Il9ra (K14CRE/ERTIl9rafl/fl mice, K14CRE/ERTIl9raΔ/Δ mice when treated with tamoxifen). We selected this model because of the advantages of using KC-specific conditional-knockout mice to explore the mechanisms of persistent cutaneous inflammation (
      • Wang Z.
      • Mascarenhas N.
      • Eckmann L.
      • Miyamoto Y.
      • Sun X.
      • Kawakami T.
      • et al.
      Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes.
      ). Our results of in vivo and in vitro experiments suggested that IL-9 and peptide Y-Y (Pyy), a member of the neuropeptide Y (Npy) family (
      • De Silva A.
      • Bloom S.R.
      Gut hormones and appetite control: a focus on PYY and GLP-1 as therapeutic targets in obesity.
      ;
      • Holzer P.
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      • Farzi A.
      Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut-brain axis.
      ), were produced by mast cells and KCs, respectively, and cooperatively sustained chronic inflammation. Numerous mast cells are often present in inflammatory skin diseases, indicating the pivotal roles of IL-9 and Pyy in the maintenance of inflammatory circuits in these lesions. Further studies focusing on the mechanisms by which the IL-9‒Pyy axis regulates the interplay between KCs and mast cells may lead to the development of a therapeutic approach to improve inflamed cutaneous conditions.

      Results

      IL-9R is upregulated in psoriatic lesions

      Before this study, we examined the expression profile of IL-9R in clinical specimens of psoriasis lesions. Immunohistochemistry results showed that IL-9RA was upregulated in psoriatic skin lesions (Supplementary Figure S1a). We then performed experiments using wild-type (WT) mice, in which psoriasis-like lesions were induced by daily application of imiquimod (IMQ) on the shaved back skin area (Figure 1a). As expected, IMQ-treated skin lesions exhibited redness, scaling, and thickening, mimicking psoriasis (Figure 1b and Supplementary Figure S1b). Histopathologically, epidermal thickness and inflammatory cell infiltration were observed (Figure 1b). The expression levels of Il9ra and Il2rg were markedly elevated in skin lesions, as assessed by immunohistochemistry and RT-qPCR analyses (Figure 1c and d). IL-9RA was expressed in several types of epidermal KCs in psoriasis-like lesions, whereas basal KCs in control mice primarily expressed IL-9RA, even under steady-state conditions (Figure 1c). Gene transcripts of cytokines and chemokines, including Il1b, Il6, Il17a, Tnfa, Ccl20, and Cxcl2, were upregulated in the skin lesions of IMQ-treated mice (Figure 1d). The levels of Stat3 and involucrin were also elevated in IMQ-treated skin lesions (data not shown). Further histochemical examinations indicated that IL-9 was present in toluidine blue‒ and c-Kit‒positive mast cells, suggesting that mast cells in skin lesions were potential producers of IL-9 in this model (Figure 1e and f). We failed to find significant differences in cutaneous manifestations between the same IMQ models of TCR β-chain‒deficient mice and WT mice, implying minimal involvement of T cells with TCRαβ in this model (Supplementary Figure S1c).
      Figure thumbnail gr1
      Figure 1IL-9R is highly expressed in the skin lesions of experimental psoriasis. (a) Experimental protocol of the IMQ-induced psoriasis model. IMQ cream or Vaseline as control was applied on the shaved back skin of wild-type mice daily for 6 days. n = 3 mice per group. (b) Representative photographs of back skin lesions on day 4 (upper). Representative microphotographs of H&E-stained FFPE tissue sections of skin lesions on day 6 (lower). Bar = 50 μm. (c) Confocal microscopy of frozen sections of skin lesions immunostained with an anti‒IL-9RA pAb (upper) or rabbit IgG as a control (lower). DAPI and IL-9RA were visualized in blue and green (Alexa 488), respectively. Dashed lines indicate the interface between the epidermis and dermis in skin tissues. Bar = 20 μm. (d) Transcript levels of Il9ra, cytokines, and chemokines in IMQ-induced skin lesions, as determined by RT-qPCR analysis. (e) Representative microphotographs of FFPE tissue sections of skin lesions immunostained with an anti‒IL-9 mAb or stained with TB. Arrowheads indicate IL-9+ cells and mast cells in IL-9‒immunostained and TB-stained sections, respectively. Bar = 50 μm. (f) Confocal microscopy of frozen skin sections of skin lesions immunostained with anti‒c-Kit and anti‒IL-9 mAbs. DAPI, c-Kit, and IL-9 were visualized in blue, green (Alexa 488), and red (Alexa 594), respectively. Arrows indicate c-Kit+IL-9+ mast cells. Bar = 20 μm. Data represent the mean ± SD of triplicate specimens in d. P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Similar results were obtained in two independent experiments. FFPE, formalin-fixed paraffin-embedded; IL-9R, IL-9 receptor; IMQ, imiquimod; TB, toluidine blue.

      IL-9 modulates psoriasis-like lesions

      To determine the pathological significance of IL-9 in psoriatic lesions, we further examined the effect of excess IL-9 in the IMQ-treated skin lesions of WT mice. We intradermally injected recombinant IL (rIL)-9, rIL-17a, or PBS as a vehicle control into the IMQ-treated areas of back skin (Figure 2a). The severity of dermatitis was significantly aggravated in the recombinant IL-9‒injected group compared with that in the rIL-17a‒injected group and the PBS-injected group (Figure 2b and Supplementary Figure S2a). The administration of recombinant IL-9 markedly promoted epidermal thickening and increased Ki-67‒positive cells compared with the administration of rIL-17a and PBS, suggesting a possible role of IL-9 in the control of KC proliferation (Figure 2b‒d). Interestingly, excess recombinant IL-9 increased the number of mast cells compared with that in PBS control lesions, whereas the number of mast cells in the recombinant IL-9‒injected group was similar to that in the rIL-17a‒injected group (Figure 2e and Supplementary Figure S2b). Conversely, we investigated the effect of administering an IL-9‒specific mAb into the IMQ-treated lesions of WT mice (Figure 2f). The results showed that epidermal thickening and the numbers of Ki-67‒positive cells and mast cells were considerably reduced by the administration of the IL-9‒specific mAb in the lesions (Figure 2g–j and Supplementary Figure S2c and d). Collectively, these results suggest a central role of IL-9 in the pathogenesis of psoriatic skin inflammation associated with KC and mast cell activities.
      Figure thumbnail gr2
      Figure 2Functional modulation of IL-9 influences experimental psoriasis. (a) Experimental protocol of the IMQ-induced psoriasis model with additional administration of reagents to skin lesions. IMQ cream was applied to the shaved back skin of wild-type mice daily for 6 days, and corresponding back skin areas were intradermally injected with 500 ng of rIL-9, rIL-17, or vehicle (PBS) on day 1, day 3, and day 5. n = 3 mice per group. (b) Representative photographs of back skin lesions on day 4 (upper). Representative microphotographs of FFPE tissue sections of skin lesions on day 6 stained with H&E (middle) or immunostained with an anti‒Ki-67 pAb (lower). Bar = 100 μm. (c) Thicknesses of the epidermis in H&E-stained sections. (d) Numbers of keratinocytes expressing Ki-67. (e) Numbers of mast cells in TB-stained sections (b). (f) Experimental protocol of the IMQ-induced psoriasis model, with additional administration of an anti‒IL-9 mAb to skin lesions. IMQ cream was applied to the shaved back skin of wild-type mice once daily for 6 days, and the back skin was intradermally injected with 20 μg of an anti‒IL-9 mAb or IgG2a isotype as a control on day 0, day 2, and day 4. n = 3 mice per group. (g) Representative photographs of back skin lesions on day 5 (upper). Representative microphotographs of FFPE tissue sections of skin lesions on day 6 stained with H&E (middle) or immunostained with an anti‒Ki-67 pAb (lower). Bar = 100 μm. (h) Thicknesses of the epidermis in H&E-stained sections. (i) Numbers of keratinocytes expressing Ki-67. (j) Numbers of mast cells in TB-stained sections (d). The number of positive cells was counted per five microscopic fields for each specimen under the magnification ×400 in d and i and ×200 in e and j. Data of a and f are shown in b–e and g–j, respectively. Data represent the mean ± SD. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Similar results were obtained in two independent experiments. FFPE, formalin-fixed paraffin-embedded; HPF, high power field; IMQ, imiquimod; n.s, not significant; rIL-9, recombinant IL-9; TB, toluidine blue.

      Epidermal IL-9R is required for the development of psoriasis-like lesions

      To further address the functional role of IL-9 in KCs, we generated K14CRE/ERTIl9rafl/fl mice with a conditional deletion of the entire Il9ra gene in KCs (Supplementary Figure S3a). Treatment of K14CRE/ERTIl9rafl/fl mice with tamoxifen induced the KC-specific deletion of Il9ra (K14CRE/ERTIl9raΔ/Δ mice), whereas control K14WTIl9rafl/fl mice retained Il9ra expression, even after treatment with tamoxifen (Supplementary Figure S3b). Experiments using the IMQ-treated psoriasis model showed that K14CRE/ERTIl9raΔ/Δ mice exhibited a significant decrease in psoriatic manifestations compared with K14WTIl9rafl/fl mice (Figure 3a–e). Consistent with Figure 2, K14CRE/ERTIl9raΔ/Δ mice displayed reduced numbers of Ki-67+ KCs and mast cells in skin lesions compared with K14WTIl9rafl/fl mice (Figure 3f and g). This was further supported by the evidence that IMQ-treated K14CRE/ERTIl9raΔ/Δ mice showed a decrease in ear thickness compared with K14WTIl9rafl/fl mice (Supplementary Figure S3c). These findings indicate that IL-9RA‒expressing KCs are a prerequisite for the development of psoriatic cutaneous lesions.
      Figure thumbnail gr3
      Figure 3Keratinocyte-specific deletion of IL-9R alleviates experimental psoriasis. (a) Experimental protocol of the IMQ-induced psoriasis model as shown in a except for pretreatment with TMX. Fourteen days before the experiment, K14WTIl9rafl/fl mice and K14CRE/ERTIl9rafl/fl mice were intraperitoneally injected with TMX (1 mg) for 5 consecutive days. Then, IMQ cream or Vaseline as control was applied to shaved back skin areas and right ears daily for 6 days. n = 3 to 4 mice per group. (b) Representative photographs of back skin lesions on day 5 (upper). Representative microphotographs of H&E-stained FFPE tissue sections of skin lesions on day 6 (lower). Bar = 50 μm. (c) Confocal microscopy of frozen skin sections immunostained with an anti‒IL-9RA pAb. DAPI and IL-9RA were visualized in blue and green (Alexa 488), respectively. Dashed lines indicate the interface of the epidermis and dermis. Bar = 20 μm. (d) Individual scores of erythema, scales, and thickness and cumulative scores to evaluate psoriatic back skin lesions. (e) Thicknesses of the epidermis in H&E-stained sections. (f) Representative microphotographs of FFPE tissue sections of skin lesions stained with an anti‒Ki-67 pAb (left). Numbers of keratinocytes expressing Ki-67 (right). Bar = 100 μm. (g) Representative microphotographs of FFPE tissue sections of skin lesions stained with TB (left). Numbers of mast cells in TB-stained sections (right). Arrowheads indicate mast cells. Bar = 50 μm. The number of positive cells was counted per five microscopic fields for each specimen under the magnification ×400 in f and ×200 in g. Data represent the mean ± SD. P < 0.05 and ∗∗P < 0.01. Similar results were obtained in three independent experiments. FFPE, formalin-fixed paraffin-embedded; IL-9R, IL-9 receptor; IMQ, imiquimod; K14, keratin 14; TB, toluidine blue; TMX, tamoxifen; WT, wild-type.

      IL-9 promotes Pyy in KCs in psoriasis-like lesions

      We next assessed the transcriptional profiles of IMQ-treated K14CRE/ERTIl9raΔ/Δ and K14WTIl9rafl/fl epidermal tissues by RNA-sequencing analysis (Figure 4a). The results showed that the transcripts encoding molecules associated with the recruitment of mast cells, such as Cxcl2 and Cxcl10 (
      • Brightling C.E.
      • Ammit A.J.
      • Kaur D.
      • Black J.L.
      • Wardlaw A.J.
      • Hughes J.M.
      • et al.
      The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle.
      ;
      • Schwarzer M.
      • Hermanova P.
      • Srutkova D.
      • Golias J.
      • Hudcovic T.
      • Zwicker C.
      Germ-free mice exhibit mast cells with impaired functionality and gut homing and do not develop food allergy.
      ), were downregulated in psoriasis-like lesions in K14CRE/ERTIl9raΔ/Δ epidermis (Figure 4a and b and Supplementary Table S3). Furthermore, K14CRE/ERTIl9raΔ/Δ KCs displayed markedly downregulated transcript and protein levels of Pyy (a member of the Npy family) compared with K14WTIl9rafl/fl KCs (Figure 4b–d). Study of primary culture indeed indicated that IL-9 could promote the secretion of Pyy from epidermal cells of WT mice (Figure 4e). Then, we examined the clinical specimens of psoriasis and clearly detected Pyy in psoriatic KCs but unlikely in CD1a+ Langerhans cells (Figure 4f). In the murine model of contact hypersensitivity induced by DNFB, the results for K14CRE/ERTIl9raΔ/Δ skin lesions were similar to those in the IMQ model regarding inflammatory responses, mast cell involvement, and Pyy production (Supplementary Figure S4a–e). Collectively, these results suggest a previously unidentified ability of KCs to produce Pyy in response to IL-9 during persistent pathological inflammation.
      Figure thumbnail gr4
      Figure 4IL-9 regulates Pyy in epidermal keratinocytes of psoriatic lesions. (a) RNA-seq analysis of TMX-treated K14WTIl9rafl/fl keratinocytes and K14CRE/ERTIl9raΔ/Δ keratinocytes derived from the ears of the IMQ model shown in a. Total RNA was purified from the epidermis of the ears (n = 6) of each mouse. Relative expression values are indicated in color. (b) Transcript levels of Pyy and Cxcl2 in the ear epidermis of the IMQ model, as assessed by RT-qPCR analysis. (c) Confocal microscopy of frozen tissue sections immunostained with an anti-Pyy mAb in K14WTIl9rafl/fl and K14CRE/ERTIl9raΔ/Δ skin lesions. DAPI and Pyy were visualized in blue and green (Alexa 488), respectively. Dashed lines indicate the interface of the epidermis and dermis. Bar = 20 μm. (d) Representative microphotographs of Pyy mRNA in situ hybridization in FFPE tissue sections of K14WTIl9rafl/fl and K14CRE/ERTIl9raΔ/Δ skin lesions. Bar = 10 μm. (e) The levels of Pyy secreted from ear epidermal cells of WT mice as assessed by ELISA. Cells were maintained in a medium with or without rIL-9 (100 ng/ml) for 3 days. (f) Confocal microscopy of frozen tissue sections of human psoriatic lesions immunostained with an anti-Pyy pAb (green), anti-pancytokeratin mAb conjugated with AF594 (red), anti-CD1a mAb conjugated with AF647 (white), and DAPI (blue). Dashed lines indicate the interface of the epidermis and dermis. Bar = 10 μm. The data shown in a were obtained from a single experiment, and b–e show representative data from three independent experiments (n = 3‒4 mice per group). The experiment shown in a–e followed the protocol of the IMQ model in . Data represent the mean ± SD of triplicate specimens in b and e. P < 0.05. FFPE, formalin-fixed paraffin-embedded; FPKM, fragments per kilobase of exon per million mapped reads; IMQ, imiquimod; i.p., intraperitoneal; K14, keratin 14; K77, keratin 77; RNA-seq, RNA sequencing; Pyy, peptide Y-Y; rIL-9, recombinant IL-9; TMX, tamoxifen; WT, wild-type.

      Functional blockade of Pyy relieves psoriasis-like lesions

      To understand the role of Pyy in the experimental psoriasis model, we investigated the effect of the local administration of a Pyy-specific antibody in the IMQ-treated back lesions of WT mice (Figure 5a). After the intradermal injection of a rabbit mAb specific to mouse Pyy into the cutaneous lesions, these mice displayed less severe dermatitis than control mice administered the same amount of IgG (Figure 5b and c). For example, epidermal thickening was improved by injection of the anti-Pyy mAb compared with that of controls (Figure 5b and d). In addition, the involvement of mast cells in the lesions injected with the anti-Pyy mAb was decreased compared with that in controls (Figure 5b and e). These results indicate that KC-derived Pyy plays a pivotal role in the development of psoriasiform cutaneous lesions. It has recently been reported that epidermal cell proliferation may be regulated by Pyy in a paracrine manner during skin remodeling (
      • Ichijo R.
      • Kabata M.
      • Kidoya H.
      • Muramatsu F.
      • Ishibashi R.
      • Abe K.
      • et al.
      Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs.
      ). In an in vivo wound healing model, tissue repair after skin injury was significantly delayed in K14CRE/ERTIl9raΔ/Δ mice, suggesting a possible role of the IL-9‒Pyy axis in skin repair (Figure 5f).
      Figure thumbnail gr5
      Figure 5Functional blockade of Pyy resolves experimental psoriasis. (a) Experimental protocol of the IMQ-induced psoriasis model with additional administration of reagents to skin lesions. IMQ cream was applied on the shaved back skin of wild-type mice daily for 6 days, and the corresponding back skin areas were intradermally injected with 50 μl PBS containing 20 ng of a rabbit anti-mouse Pyy mAb (clone D1K3Q) or rabbit IgG (clone DA1E) as an isotype control on day 0, day 2, and day 4. n = 3‒4 mice per group. (b) Representative photographs of back skin lesions on day 6 (upper). Representative microphotographs of FFPE tissue sections of skin lesions stained with H&E (middle) or TB (lower). Arrowheads indicate mast cells. Bar = 50 μm. (c) Cumulative scores to evaluate skin lesions. (d) Thicknesses of the epidermis in H&E-stained sections. (e) Numbers of mast cells in TB-stained sections. (f) In vivo wound healing assay of skin in TMX-induced K14WTIl9rafl/fl and K14CRE/ERTIl9raΔ/Δ mice. The time course of the wound healing index (%) is shown. The number of positive cells was counted per five microscopic fields for each specimen under the magnification ×200 in e. Data represent the mean ± SD. P < 0.05 and ∗∗P < 0.01. Similar results were obtained in two independent experiments. FFPE, formalin-fixed paraffin-embedded; IMQ, imiquimod; K14, keratin 14; Pyy, peptide Y-Y; TB, toluidine blue; TMX, tamoxifen.

      Epidermal Pyy regulates mast cells

      We further examined the capacity of Pyy to regulate mast cells in vitro using bone marrow‒derived mast cells (BMMCs) (Supplementary Figure S5a and b). Pyy, Npy, and pancreatic polypeptides of the Npy family share certain class A (rhodopsin-like) G protein-coupled cell-surface receptors, including NPY1R, NPY2R, NPY4R, and NPY5R (
      • Rinne M.
      • Tanoli Z.U.
      • Khan A.
      • Xhaard H.
      Cartography of rhodopsin-like G protein-coupled receptors across vertebrate genomes.
      ). Among these receptors, Npy2r and Npy4r were abundantly present in BMMCs but downregulated in an activated state induced by ionomycin and lipopolysaccharide) (Figure 6a and Supplementary Figure S5c). Epidermal cells of WT mice also expressed Npy2r and Npy4r, although IL-9 did not appear to be involved in their regulation (Supplementary Figure S5d). Pyy1-36 (the full-length form) augmented the production of IL-9 from BMMCs after stimulation with ionomycin and lipopolysaccharide (Figure 6b). Moreover, Pyy significantly induced the migration of BMMCs, as suggested by chemotaxis assays (Figure 6c). Because Pyy preferentially binds to NPY2R (
      • Østergaard S.
      • Kofoed J.
      • Paulsson J.F.
      • Madsen K.G.
      • Jorgensen R.
      • Wulff B.S.
      Design of Y2 receptor selective and proteolytically stable PYY(3–36) analogues.
      ), we applied an NPY2R-specific antagonist (BIIE0246) to control the IMQ-induced skin lesions of WT mice (Figure 6d). The results showed that local administration of BIIE0246 alleviated the manifestations of psoriatic lesions (Figure 6e–h). Given the expression profiles of IL-9 in IMQ-challenged lesions (Figure 1), Pyy-mediated inflammation may contribute to psoriasis-like lesions, in which mast cells play a role as a potential IL-9 producer.
      Figure thumbnail gr6
      Figure 6Activation of mast cells by Pyy in experimental psoriasis. (a) Expression of Npy2r and Npy4r in BMMCs after stimulation with Pyy1‒36, ION, and LPS, as assessed by RT-qPCR analysis. Expression of Npy1r and Npy5r in BMMCs under the same conditions shown in c. (b) IL-9 production from BMMCs after stimulation with ION and LPS with or without Pyy1‒36, as measured by CBA. (c) Chemotaxis assay of BMMCs. Representative microphotographs of hematoxylin-stained cells in porous membranes of chambers for Pyy1‒36, Pyy3‒36, and control (migrated cells indicated by arrowheads; left). Numbers of migrated cells (n = 4, right) are shown. Bar = 50 μm. (d) Experimental protocol of the IMQ-induced psoriasis model with the administration of a Pyy receptor antagonist (BIIE0246) to skin lesions. IMQ cream was applied to the shaved back skin of wild-type mice once daily for 6 days, and the back skin was intradermally injected with 50 μl PBS containing 0.1% DMSO with or without 250 ng BIIE0246 until day 5. (e) Photographs of back skin lesions (upper). Representative microphotographs of FFPE tissue sections of skin lesions stained with H&E (middle) or TB (lower). Arrowheads indicate mast cells. Bar = 50 μm. (f) Cumulative scores to evaluate psoriatic lesions. (g) Thicknesses of the epidermis in H&E-stained sections. (h) Numbers of mast cells in TB-stained sections. The number of positive cells was counted per five microscopic fields for each specimen under the magnification ×200 in c and h. Data represent the mean ± SD of triplicates in a and b and n = 3 mice per group in e, f, g, and h. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Similar results were obtained in 2‒4 independent experiments. BMMC, bone marrow‒derived mast cell; CBA, cytometric bead assay; FFPE, formalin-fixed paraffin-embedded; IMQ, imiquimod; ION, ionomycin; LPS, lipopolysaccharide; Pyy, peptide Y-Y; TB, toluidine blue.

      Discussion

      In this study, we describe the relevance of IL-9 and Pyy in the interactions between KCs and mast cells underlying inflammatory skin diseases. Extensive studies have revealed that chronic inflammatory dermatoses are characterized by unfavorable KC proliferation and differentiation. As a central player of persistent skin inflammation, IL-9 has also been implicated in the pathogenesis of psoriasis and other inflammatory skin diseases (
      • Baeck M.
      • Herman A.
      • de Montjoye L.
      • Hendrickx E.
      • Chéou P.
      • Cochez P.M.
      • et al.
      Increased expression of interleukin-9 in patients with allergic contact dermatitis caused by p- phenylenediamine.
      ;
      • Gutin L.
      • Tammaro A.
      • Fishelevich R.
      • Gaspari A.A.
      Elevation of IL-9 in extreme patch test reactions suggests it is an inflammatory mediator in allergic contact dermatitis.
      ;
      • Hong C.H.
      • Chang K.L.
      • Wang H.J.
      • Yu H.S.
      • Lee C.H.
      IL-9 induces IL-8 production via STIM1 activation and ERK phosphorylation in epidermal keratinocytes: a plausible mechanism of IL-9R in atopic dermatitis.
      ;
      • Kienzl P.
      • Polacek R.
      • Reithofer M.
      • Reitermaier R.
      • Hagenbach P.
      • Tajpara P.
      • et al.
      The cytokine environment influence on human skin-derived T cells.
      ;
      • Ma L.
      • Xue H.B.
      • Guan X.H.
      • Shu C.M.
      • Zhang J.H.
      • Yu J.
      Possible pathogenic role of T helper type 9 cells and interleukin (IL)-9 in atopic dermatitis.
      ;
      • Midde H.S.
      • Priyadarssini M.
      • Rajappa M.
      • Munisamy M.
      • Mohan Raj P.S.
      • Singh S.
      • et al.
      Interleukin-9 serves as a key link between systemic inflammation and angiogenesis in psoriasis.
      ;
      • Ruiz-Romeu E.
      • Ferran M.
      • de Jesús-Gil C.
      • García P.
      • Sagristà M.
      • Casanova J.M.
      • et al.
      Microbe-dependent induction of IL-9 by CLA+ T cells in psoriasis and relationship with IL-17A.
      ;
      • Sismanopoulos N.
      • Delivanis D.A.
      • Alysandratos K.D.
      • Angelidou A.
      • Vasiadi M.
      • Therianou A.
      • et al.
      IL-9 induces VEGF secretion from human mast cells and IL-9/IL-9 receptor genes are overexpressed in atopic dermatitis.
      ). Studies in K14CRE/ERTIl9raΔ/Δ mice with inflammatory skin diseases clarified the functional significance of IL-9RA in KCs during the development of corresponding skin lesions.
      The expression profile of IL-9RA in the normal skin of WT mice suggests that basal KCs expressing IL-9R efficiently sense IL-9 from surrounding IL-9‒producing cells, possibly to preserve cutaneous immunity in a steady state. In this context, mast cells, eosinophils, neutrophils, type 2 innate lymphoid cells, and effector CD4+ T cell subsets, such as Th17 and Th9 cells, activate robust IL-9 production for KCs. Given that murine psoriasis-like lesions preferentially expressed IL-9R, mast cells likely serve as a key link between local inflammation and disease progression in inflammatory skin lesions. Although the expression of IL-9RA appeared to be limited in normal basal KCs, it was widely present in every layer of the epidermis in psoriasis-like lesions. Therefore, the number of KCs sensing IL-9 in the epidermis was considerably increased in inflammatory skin lesions. The mechanism by which IL-9R is upregulated in KCs during persistent inflammation has not been fully elucidated, although inflammatory skin lesions were widely affected by surrounding inflammatory cells, including IL-9‒producing cells. In general, IL-9 disrupts the integrity of the epidermis, in which contacts of KCs are regulated mainly by claudin-1 and E-cadherin (
      • Doshi A.
      • Khamishon R.
      • Rawson R.
      • Duong L.
      • Dohil L.
      • Myers S.J.
      • et al.
      Interleukin 9 alters epithelial barrier and E-cadherin in eosinophilic esophagitis.
      ;
      • Hu J.
      • Gao N.
      • Zhang Y.
      • Chen X.
      • Li J.
      • Bian F.
      • et al.
      IL-33/ST2/IL-9/IL-9R signaling disrupts ocular surface barrier in allergic inflammation.
      ). IL-9‒mediated interference of epidermal barrier function may exacerbate inflammatory skin lesions.
      According to the results from murine models in this study, IL-9 facilitated the production of Pyy by KCs, which may subsequently induce a vicious inflammatory circuit with mast cells and mediate the pathological activities of neighboring KCs. Pyy has been investigated as a regulator of appetite through the gut‒brain axis, primarily on the basis of its expression profile in enteroendocrine cells (
      • Holzer P.
      • Reichmann F.
      • Farzi A.
      Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut-brain axis.
      ). Dipeptidyl peptidase IV cleaves two N-terminal amino acids of full-length Pyy (Pyy1‒36) to generate the 3–36 fragment (Pyy3‒36), which also activates Npy receptors, especially NPY2R (
      • Chandarana K.
      • Gelegen C.
      • Irvine E.E.
      • Choudhury A.I.
      • Amouyal C.
      • Andreelli F.
      • et al.
      Peripheral activation of the Y2-receptor promotes secretion of GLP-1 and improves glucose tolerance.
      ). In this study, Pyy1‒36 but not Pyy3‒36 increased IL-9 production from ionomycin- and lipopolysaccharide-stimulated BMMCs (data not shown). Moreover, both Pyy1‒36 and Pyy3‒36 enhanced BMMC migration, although further analyses are required to address the differential roles of Pyy1‒36 and Pyy3‒36 in the cutaneous inflammatory environment. Given that Pyy has the potential to preserve epidermal integrity (
      • Dumont Y.
      • Bastianetto S.
      • Duranton A.
      • Breton L.
      • Quirion R.
      Immunohistochemical distribution of neuropeptide Y, peptide YY, pancreatic polypeptide-like immunoreactivity and their receptors in the epidermal skin of healthy women.
      ;
      • Ichijo R.
      • Kabata M.
      • Kidoya H.
      • Muramatsu F.
      • Ishibashi R.
      • Abe K.
      • et al.
      Vasculature-driven stem cell population coordinates tissue scaling in dynamic organs.
      ), the protective effect of locally administered BIIE0246 on IMQ-induced cutaneous lesions may be attributable to the regulation of mast cells and KCs, in which NPY2R-mediated phosphoinositide 3-kinase and MAPK signaling are impaired by BIIE0246 (
      • Lay A.C.
      • Barrington A.F.
      • Hurcombe J.A.
      • Ramnath R.D.
      • Graham M.
      • Lewis P.A.
      • et al.
      A role for NPY-NPY2R signaling in albuminuric kidney disease.
      ). Previous studies have shown that Npy promotes mast cell secretion of histamine and other factors related to chronic inflammation (
      • Arzubiaga C.
      • Morrow J.
      • Roberts 2nd, L.J.
      • Biaggioni I.
      • Neuropeptide Y.
      a putative cotransmitter in noradrenergic neurons, induces mast cell degranulation but not prostaglandin D2 release.
      ;
      • Chandrasekharan B.
      • Nezami B.G.
      • Srinivasan S.
      Emerging neuropeptide targets in inflammation: NPY and VIP.
      ;
      • Lagraauw H.M.
      • Westra M.M.
      • Bot M.
      • Wezel A.
      • van Santbrink P.J.
      • Pasterkamp G.
      • et al.
      Vascular neuropeptide Y contributes to atherosclerotic plaque progression and perivascular mast cell activation.
      ). Rat peritoneal mast cells are also activated by Pyy to secrete histamine, implying a role of neuropeptides in mast cell‒mediated inflammation (
      • Grundemar L.
      • Håkanson R.
      Neuropeptide Y, peptide YY and C-terminal fragments release histamine from rat peritoneal mast cells.
      ). In addition to mast cells and KCs, Npy receptors are expressed in macrophages, endothelial cells, and adipocytes, suggesting the involvement of these cells in inflammatory circuits mediated by the IL-9‒Pyy axis (
      • Chandrasekharan B.
      • Nezami B.G.
      • Srinivasan S.
      Emerging neuropeptide targets in inflammation: NPY and VIP.
      ). Of note, a human mast cell line was shown to produce CCL5, TNF, GM-CSF, and IL-3 in response to neuropeptides, such as vasoactive intestinal polypeptide and substance P (
      • Kulka M.
      • Sheen C.H.
      • Tancowny B.P.
      • Grammer L.C.
      • Schleimer R.P.
      Neuropeptides activate human mast cell degranulation and chemokine production.
      ).
      In summary, we report a previously unidentified role of IL-9 in regulating epidermal Pyy, which potentially stimulates mast cell production of IL-9 and mobilization. Studies on the relationship between IL-9 and Pyy may contribute to a further understanding of the primary defense mechanism of the skin and provide insights into the pathogenesis of inflammatory skin diseases.

      Materials and Methods

      Clinical specimens

      Human skin samples were obtained with written informed consent. Formalin-fixed paraffin-embedded samples and fresh-frozen tissues of clinical specimens were used for immunohistochemistry following the guidelines of the Declaration of Helsinki and with the approval of the institutional review board of Sapporo Medical University Hospital (Sapporo, Japan) (number 292-137 and number 312-53).

      Mice

      Il9rafl/+ mice (accession number CDB0034E: http://www2.clst.riken.jp/arg/mutant%20mice%20list.html) with a C57BL/6 background were established to generate K14CRE/ERTIl9rafl/fl mice (Supplementary Figure S1). K14CRE/ERT mice and TCRβ-deficient mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were kept under specific pathogen-free conditions in the animal facility of Sapporo Medical University (Sapporo, Japan), and female mice aged 6–12 weeks were used unless otherwise stated. All experiments using mice were performed with approval by the institutional animal care and use committees of the RIKEN (Kobe, Japan) and Sapporo Medical University.

      Experimental psoriasis model

      IMQ cream (5%) was applied on the shaved back skin (50 mg per application) and ear (10 mg per application) of mice for 6 consecutive days. The severity of the back skin inflammatory lesion was scored as previously described (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Ear thickness was measured using a digital thickness gauge (Ozaki Mfg., Tokyo, Japan).

      Antibodies and reagents

      The antibodies and reagents used in this study are summarized in Supplementary Table S1.

      RNA-sequencing analysis

      To analyze the transcriptomes of KCs, mouse ears were soaked in 1 mg/ml dispase II (Wako, Tokyo, Japan) at 37 °C for 1 hour, and then epidermal cell layers were obtained under a stereo microscope. Total RNA was extracted from the epidermal cell layers using Trizol and subjected to RNA sequencing analysis with the Illumina NovaSeq6000 platform (RIKEN Genesis, Tokyo, Japan). Data were analyzed by bioinformatics software (RIKEN Genesis) and Heatmapper software (The University of Alberta, Alberta, Canada).

      Primary culture of KCs

      For primary culture, epithelial cell layers of ears were cultured in 0.5 ml of Epilife (Thermo Fisher Scientific, Waltham, MA) supplemented with penicillin and streptomycin in a 48-well plate. All cells were cultured at 37 °C in a humidified atmosphere with 5% carbon dioxide for up to 3 days. Pyys in the culture supernatants were measured by an ELISA kit following the manufacturer’s instruction (Wako, Tokyo, Japan).

      RT-qPCR analysis

      RT-qPCR analysis was performed with gene-specific primers as described previously (Supplementary Table S2) (
      • Kobayashi K.
      • Kamekura R.
      • Kato J.
      • Kamiya S.
      • Kamiya T.
      • Takano K.
      • et al.
      Cigarette smoke underlies the pathogenesis of palmoplantar pustulosis via an IL-17A-induced production of IL-36γ in tonsillar epithelial cells.
      ). Relative expression levels of target genes against the level of Gapdh were analyzed.

      Immunohistochemistry and confocal imaging

      Frozen tissue sections fixed in ice-cold acetone were immunostained and analyzed as previously described (
      • Kobayashi K.
      • Kamekura R.
      • Kato J.
      • Kamiya S.
      • Kamiya T.
      • Takano K.
      • et al.
      Cigarette smoke underlies the pathogenesis of palmoplantar pustulosis via an IL-17A-induced production of IL-36γ in tonsillar epithelial cells.
      ;
      • Yamashita K.
      • Kawata K.
      • Matsumiya H.
      • Kamekura R.
      • Jitsukawa S.
      • Nagaya T.
      • et al.
      Bob1 limits cellular frequency of T-follicular helper cells.
      ). Confocal images were acquired by laser scanning microscopy (LSM 780, Carl Zeiss, Jena, Germany).

      In situ hybridization

      In situ hybridization analysis of formalin-fixed paraffin-embedded tissue sections was performed with an RNAscope assay kit following the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA). Signals of mRNA transcripts were visualized by 3,3'-diaminobenzidine, and then sections were counterstained with hematoxylin. An in situ hybridization probe to detect mouse Pyy is described in Supplementary Table S2.

      Generation and activation of BMMCs

      To obtain BMMCs, nuclear cells from the bone marrow of C57BL/6 WT mice were cultured in RPMI 1640 medium supplemented with 20% fetal bovine serum containing 30 ng/ml rIL-3 and 100 ng/ml recombinant stem cell factor at 37 °C in a humidified atmosphere with 5% carbon dioxide for 4 weeks. Next, 4 × 105 cells were stimulated with 1 μg/ml ionomycin and 1 μg/ml lipopolysaccharide in the presence or absence of 20 μg/ml Pyy in 200 μl of RPMI 1640 medium containing 20% fetal bovine serum in a 96-well plate for 24 hours (
      • Stassen M.
      • Müller C.
      • Arnold M.
      • Hültner L.
      • Klein-Hessling S.
      • Neudörfl C.
      • et al.
      IL-9 and IL-13 production by activated mast cells is strongly enhanced in the presence of lipopolysaccharide: NF-kappa B is decisively involved in the expression of IL-9.
      ). Then, supernatants were measured by cytometric bead assay to detect IL-9 using a FACS CANTO II flow cytometer (BD Bioscience, San Jose, CA) following the manufacturer’s instructions.

      Chemotaxis assay

      The membrane-bound chamber (Chemotaxicell, 8.0 μm, Kurabo, Tokyo Industries, Japan) was inserted into a 24-well cell culture plate. BMMCs (1 × 105 cells) were added to the upper chamber, and RPMI 1640 medium with or without 100 ng/ml Pyy was placed into the lower chamber. After incubation at 37 °C in a humidified atmosphere with 5% carbon dioxide for 24 hours, the membranes were washed with PBS, fixed in formalin, and stained with hematoxylin. The numbers of stained cells were counted in five random fields (×200) under a microscope.

      In vivo wound healing assay

      Two circular full-thickness wounds (6 mm) were created on the shaved back skin, and the area of the hole was analyzed by ImageJ software (National Institutes of Health, Bethesda, MD). The wound healing index (%) was calculated as the ratio of the individual area per initial area.

      Statistics

      The Mann–Whitney U test and unpaired t-test were used to analyze the difference between two groups. For comparisons of multiple groups, one-way ANOVA with Tukey’s multiple comparison test was used. Statistical tests were performed using GraphPad Prism software (GraphPad, San Diego, CA). P < 0.05 was considered significant.

      Data availability statement

      RNA sequencing data generated in this manuscript are available from the Gene Expression Omnibus repository hosted by National Center for Biotechnology Information (accession number GSE190867).

      Conflict of Interest

      All authors state no conflict of interest.

      Acknowledgment

      This study was funded by Grants-in-Aid from the Japan Society for Promotion of Science to SK (19K17778) and SI (18H02632). This work was also supported by a research grant from Bristol-Myers Squibb to SI.

      Author Contributions

      Conceptualization: SI; Data Curation: SK, II, SI; Formal Analysis: SK, II; Funding Acquisition: SK, SI; Investigation: SK, II, SI; Methodology: MY, RK, KK, TS, HT, TK, YK, TA, KI, TH; Project Administration: SI; Resources: SK, II, SI; Supervision: HU; Validation: SK, II; Visualization: SK, II, SI; Writing - Original Draft Preparation: SI; Writing - Review and Editing: SK, II, SI

      Supplementary Materials

      • Supplementary Table S2

        PCR Primers and Probes for RT-qPCR and ISH, Including Primers for RT-qPCR Using SYBR Green, TaqMan probes, and Primers for the Genotyping of Conditional Il9ra-Deficient Mice

        Abbreviation: ISH, in situ hybridization.

        See also Supplementary Figure S3 to overview the sites of primers for genotyping of conditional Il9ra-deficient mice.

      • Supplementary Table S3

        Summary of Transcripts Expressed in IMQ-Induced Epidermis Obtained from Tamoxifen-treated K14WTIl9rafl/fl and K14CRE/ERTIl9raΔ/Δ Mice, as Analyzed by RNA-Seq

        Abbreviations: FPKM, fragments per kilobase of exon per million mapped reads; IMQ, imiquimod; K14, keratin 14; RNA-Seq, RNA sequencing.

      Figure thumbnail fx2
      Supplementary Figure S1IL-9R expression in psoriasis skin lesions. (a) Expression of IL-9R in clinical specimens of psoriasis, as analyzed by immunohistochemistry of FFPE tissue sections with an anti‒IL-9RA pAb or rabbit IgG as a control. Psoriatic skin lesion and normal skin are shown in the left and right panels, respectively. Corresponding microphotographs of H&E-stained sections are shown. Bar = 50 μm. (b) Individual scores of erythema, scales, and thickness and cumulative scores to evaluate skin lesions of the IMQ model in wild-type mice, as depicted in a. Vaseline was used as vehicle control. (c) Back skin lesions and scores of the IMQ model in TCR β-chain‒deficient mice (Tcrb‒/‒ mice) and wild-type mice following the same protocol as indicated in a. Back skin lesions on day 4 are shown in the upper panels, and graphs of individual scores are shown in the lower panels. Data represent the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Similar results were obtained in two independent experiments. FFPE, formalin-fixed paraffin-embedded; IL-9R, IL-9 receptor; IMQ, imiquimod; WT, wild-type.
      Figure thumbnail fx3
      Supplementary Figure S2IL-9 influences IMQ-induced skin lesions of wild-type mice. (a) Individual scores of skin lesions in wild-type mice locally injected with rIL-9, rIL-17a, or PBS (vehicle control), as shown in a. Asterisks indicate significant differences between values in the rIL-9 group and PBS control group. (b) Representative microphotographs of TB-stained sections of skin lesions, as shown in a. Arrowheads indicate mast cells. Bar = 50 μm. (c) Individual scores of skin lesions in wild-type mice locally injected with an anti‒IL-9 mAb or control IgG2a subtype, as shown in f. (d) Representative microphotographs of TB-stained sections of skin lesions, as shown in g. Arrowheads indicate mast cells. Bar = 50 μm. Data represent the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. IMQ, imiquimod; rIL, recombinant IL; TB, toluidine blue.
      Figure thumbnail fx4
      Supplementary Figure S3Strategy for establishing Il9rafl/fl mice and K14CRE/ERTIl9rafl/fl mice. (a) Editing strategy of the gene encoding IL-9RA (Il9ra gene) by CRISPR/Cas9. Il9rafl/fl mice were designed to have a specific deletion of the entire Il9ra gene using the Cre/loxP system by CRISPR/Cas9 genome editing technology. Briefly, two gRNAs targeting the 5' and 3' regions spanning nine exons of the Il9ra gene and two ssODNs, which contained loxP sequences by electroporation, were introduced into zygotes of C57BL/6 mice (Sankyo Laboratory Service, Tokyo, Japan). These embryos were transferred to foster mothers of ICR mice and maintained as F0 mice. F0 mice were subsequently mated with C57BL/6 mice to generate F1 mice to obtain Il9rafl/+ mice. By mating Il9rafl/+ mice, we generated and maintained Il9rafl/fl mice. Then, Il9rafl/fl mice were crossed with transgenic K14CRE/ERT mice (The Jackson Laboratory, Bar Harbor, ME) with a TMX-inducible Cre recombinase driven by the human K14 promoter to establish K14CRE/ERTIl9rafl/fl mice and K14WTIl9rafl/fl mice used as controls. To obtain K14CRE/ERTIl9raΔ/Δ mice, K14CRE/ERTIl9rafl/fl mice were treated with TMX (1 mg, i.p.) for 5 consecutive days from 14 days before the experiment. (b) Representative results of PCR-based genotyping for conditional Il9ra-deficient mice. PCR primers used for genotyping are summarized in . (c) Thicknesses of ear lesions following the same protocol as that shown in a. The Δear thickness was calculated as the changes in ear thickness (ear thickness on day 6 − ear thickness on day 0), as measured using a digital thickness gauge. Data represent the mean ± SD (n = 3). ∗P < 0.05. Ex, exon; FW, forward; gRNA, guide RNA; ICR, Institute of Cancer Research; i.p., intraperitoneal; K14, keratin 14; RV, reverse; ssODN, single-stranded oligodeoxynucleotide; TMX, tamoxifen, WT, wild-type.
      Figure thumbnail fx5
      Supplementary Figure S4DNFB-induced contact hypersensitivity model. (a) Protocol of the DNFB-induced contact hypersensitivity model in K14WTIl9rafl/fl mice and K14CRE/ERTIl9raΔ/Δ mice. To sensitize mice, 50 μl of 0.5% DNFB in acetone/olive oil (4:1) were applied to the shaved abdominal skin on day −3. On days 0, 2, and 4, 50 and 20 μl of 0.2% DNFB were added to the shaved back skin and right ear, respectively. As a control, the left ear was treated with 20 μl of acetone/olive oil (4:1). Fourteen days before performing this experiment, mice in both groups were treated with TMX (1 mg, i.p.) for 5 consecutive days (n = 3). (b) Representative photographs of back skin lesions on day 9 (upper) and day 11 (middle) and ears on day 11 (lower). (c) Ear thickness over time. The Δear thickness was calculated as the changes in ear thickness (ear thickness on the examined day − ear thickness on day 0). (d) Numbers of mast cells in back skin lesions in TB-stained sections. The data shown represent the mean numbers of mast cells per five microscopic fields (×200). (e) Transcript levels of Pyy in epidermal cells, as assessed by RT-qPCR analysis. Epidermal cells were obtained from ear skin lesions on day 11. Data represent the mean ± SD (n = 3 mice per group). ∗P < 0.05 and ∗∗P < 0.01. Similar results were obtained in two independent experiments. i.p., intraperitoneal; K14, keratin 14; Pyy, peptide Y-Y; TB, toluidine blue; TMX, tamoxifen; WT, wild-type.
      Figure thumbnail fx6
      Supplementary Figure S5Role of Pyy in inflammatory skin diseases. (a) Representative microphotographs of BMMCs used in this study. Cells were stained with May–Giemsa solution. Bar = 10 μm. (b) Representative expression profile of FcϵRI in BMMCs assessed by flow cytometry. Spleen cells were used as a negative control. (c) Expression of Npy1r and Npy5r in BMMCs after stimulation with ION, LPS, and Pyy1‒36, as measured by RT-qPCR. (d) Expression of Npy receptors in the psoriasis-like epidermis of ears. Data represent the mean ± SD (n = 3). ∗P < 0.05. Similar results were obtained in two independent experiments. BMMC; bone marrow‒derived mast cell; ION, ionomycin; LPS, lipopolysaccharide; Npy, neuropeptide Y; n.s, not significant; Pyy, peptide Y-Y; SSC-A, side scatter area.

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