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Imiquimod-Induced Psoriasis in Mice Depends on the IL-17 Signaling of Keratinocytes

  • Sonja Moos
    Affiliations
    Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
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  • Alma N. Mohebiany
    Affiliations
    Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
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  • Ari Waisman
    Affiliations
    Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
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  • Florian C. Kurschus
    Correspondence
    Correspondence: Florian C. Kurschus, Department of Dermatology, Heidelberg University Hospital; Im Neuenheimer Feld 440, D-69120 Heidelberg; Germany.
    Affiliations
    Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany

    Department of Dermatology, Heidelberg University Hospital, Heidelberg, Germany
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Open ArchivePublished:January 23, 2019DOI:https://doi.org/10.1016/j.jid.2019.01.006
      The pathology of psoriasis strongly depends on IL-17A. Monoclonal antibodies blocking either the cytokine or its receptor are among the most efficient treatments for psoriatic patients. Keratinocytes can be activated upon exposure to IL-17A and tumor necrosis factor-α and secrete secondary cytokines and chemokines in the inflamed skin. In psoriasis and its imiquimod-induced mouse model, a strong skin infiltration of neutrophils and inflammatory monocytes can be observed. However, to date, it is not clear how exactly those cellular populations are attracted to the skin and how they contribute to the pathogenesis of the disease. To define the crucial cell type responding to IL-17 and initiating the downstream pathology in psoriasis-like dermatitis, we used mice specifically lacking the IL-17 receptor (IL-17RA) in different cell types. Deletion of IL-17RA in T cells or myeloid had no impact on disease development. Only deletion of this receptor in keratinocytes reflected the full-body deletion of IL-17RA, resulting in strongly reduced dermatitis development. Imiquimod treatment of those IL-17 signaling–deficient mice maintained high monocytic infiltration but failed to attract neutrophils into the skin. We conclude that keratinocytes are a critical cellular target for IL-17A–mediated neutrophil attraction and psoriasis development.

      Abbreviations:

      IL-17RA (receptor chain A), IMQ (imiquimod), KC (keratinocyte)

      Introduction

      Psoriasis is a chronic inflammatory skin disease characterized by skin tissue infiltration of T cells, monocytes, and neutrophils concomitant with strong hyperproliferation of keratinocytes (KCs) and parakeratosis. Several cytokines have been implicated in the pathogenesis of the disease, including tumor necrosis factor-α (TNFα), IL-17A, IL-23, and IL-22 (
      • Di Cesare A.
      • Di Meglio P.
      • Nestle F.O.
      The IL-23/Th17 axis in the immunopathogenesis of psoriasis.
      ), and neutralizing therapies against IL-17A, TNFα, or IL-12/IL-23-p40 are highly efficient in the treatment of patients (
      • Kurschus F.C.
      • Moos S.
      IL-17 for therapy.
      ).
      The IL-17 family consists of six members (IL-17A–F), of which four cytokines, namely, IL-17A, -F, -C, and -E, all signal through a heterodimeric receptor containing the IL-17 receptor chain A (IL-17RA) (
      • Toy D.
      • Kugler D.
      • Wolfson M.
      • Vanden Bos T.
      • Gurgel J.
      • Derry J.
      • et al.
      Cutting edge: interleukin 17 signals through a heteromeric receptor complex.
      ,
      • Yao Z.
      • Fanslow W.C.
      • Seldin M.F.
      • Rousseau A.M.
      • Painter S.L.
      • Comeau M.R.
      • et al.
      Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor.
      ). Of these, IL-17A and its most homologous family member, IL-17F, signal through to a receptor complex that includes not only IL-17RA but also IL-17RC. Although IL-17RA is expressed ubiquitously (
      • Yao Z.
      • Fanslow W.C.
      • Seldin M.F.
      • Rousseau A.M.
      • Painter S.L.
      • Comeau M.R.
      • et al.
      Herpesvirus saimiri encodes a new cytokine, IL-17, which binds to a novel cytokine receptor.
      ), expression of IL-17RC is rather restricted to epithelial cell types (
      • Haudenschild D.
      • Moseley T.
      • Rose L.
      • Reddi A.H.
      Soluble and transmembrane isoforms of novel interleukin-17 receptor-like protein by RNA splicing and expression in prostate cancer.
      ,
      • Kuestner R.E.
      • Taft D.W.
      • Haran A.
      • Brandt C.S.
      • Brender T.
      • Lum K.
      • et al.
      Identification of the IL-17 receptor related molecule IL-17RC as the receptor for IL-17F.
      ). Nevertheless, several groups reported signaling of different IL-17 members on macrophages, T cells, or neutrophils (
      • Ishigame H.
      • Kakuta S.
      • Nagai T.
      • Kadoki M.
      • Nambu A.
      • Komiyama Y.
      • et al.
      Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses.
      ,
      • Taylor P.R.
      • Roy S.
      • Leal Jr., S.M.
      • Sun Y.
      • Howell S.J.
      • Cobb B.A.
      • et al.
      Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2.
      ). Signaling by IL-17 in synergy with TNFα induces not only antibacterial defensins and cytokines but also chemokines such as CXCL1, CXCL2, and CXCL8, which mediate neutrophil tissue infiltration (
      • Ye P.
      • Rodriguez F.H.
      • Kanaly S.
      • Stocking K.L.
      • Schurr J.
      • Schwarzenberger P.
      • et al.
      Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.
      ).
      Psoriasis can be modeled in mice by daily topical application of Aldara cream containing 5% imiquimod (IMQ) (Meda, Solna, Sweden). The IMQ-induced dermatitis model phenocopies important features of psoriasis, including scaling, erythema, and acanthosis (
      • 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.
      ), as well as gene expression profiles that resemble those of psoriatic patients (
      • Swindell W.R.
      • Johnston A.
      • Carbajal S.
      • Han G.
      • Wohn C.
      • Lu J.
      • et al.
      Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.
      ). Similar to psoriasis, IL-17 signaling plays an important role in this model (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ,
      • 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.
      ). However, in contrast to psoriasis patients treated with IL-17 pathway–neutralizing drugs, who often show a complete remission (
      • Langley R.G.
      • Elewski B.E.
      • Lebwohl M.
      • Reich K.
      • Griffiths C.E.
      • Papp K.
      • et al.
      Secukinumab in plaque psoriasis—results of two phase 3 trials.
      ,
      • Papp K.A.
      • Leonardi C.
      • Menter A.
      • Ortonne J.P.
      • Krueger J.G.
      • Kricorian G.
      • et al.
      Brodalumab, an anti-interleukin-17-receptor antibody for psoriasis.
      ,
      • Waisman A.
      To be 17 again—anti-interleukin-17 treatment for psoriasis.
      ), mice deficient for IL-17RA are not fully resistant to disease development but show residual disease scores (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ). In line with this, it was recently shown that IL-17–independent pathways also play a role in this model (
      • Walter A.
      • Schafer M.
      • Cecconi V.
      • Matter C.
      • Urosevic-Maiwald M.
      • Belloni B.
      • et al.
      Aldara activates TLR7-independent immune defence.
      ). Nevertheless, a major infiltration of neutrophils and activated monocytes/macrophages and an elevation of T helper type 17 cells are also found in the IMQ model (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ,
      • 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.
      ). Additionally, many features of KC malfunctions, like hyperproliferation and disturbed epidermal differentiation, are similarly present in the IMQ model (
      • 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.
      ).
      We used this model to address exactly how, and by which cell types and pathways, IL-17 initiates disease. We showed that cell type-specific deletion of the IL-17RA in KCs results in reduced IMQ-induced diseases, similar to what is seen in mice that lack this receptor altogether. In contrast, once IL-17RA was removed in T cells, neutrophils, or macrophages, no change was seen in disease severity, indicating that these cells are not (direct) relevant targets for IL-17. Disease amelioration in mice lacking IL-17RA in keratinocytes was paralleled by decreased IL-1, IL-22, and CXCL2 expression levels and diminished neutrophil infiltration into the skin. Nevertheless, these mice still showed high monocyte infiltrations into their skin, indicating that their role is most likely secondary in pathology perpetuation.

      Results

      IL-17RA expression by T cells, neutrophils, and macrophages is dispensable for development of IMQ-induced dermatitis

      We recently showed that full-body deletion of IL-17RA leads to reduced signs of IMQ-induced psoriasis in mice (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ). To investigate which individual cell type is the target of IL-17 in this model for psoriasis, we crossed the IL-17RAfl/fl line (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ) to different tissue-specific Cre lines. Mice expressing CD4-Cre crossed to the IL-17RAfl/fl strain lack IL-17RA in all αβ T cells due to deletion in the CD4+CD8+ double-positive stage during thymic maturation (
      • Lee P.P.
      • Fitzpatrick D.R.
      • Beard C.
      • Jessup H.K.
      • Lehar S.
      • Makar K.W.
      • et al.
      A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.
      ). We treated these mice, termed IL-17RACD4-Cre, with IMQ and analyzed the development of dermatitis over a time frame of 6 days, using Cre-negative littermates (IL-17RAfl/fl) as controls. We found no change in disease susceptibility of the IL-17RACD4-Cre mice compared with the control animals (Figure 1a). As expected, mice with full-body deficiency of IL-17RA (IL-17RAdel/del) were mostly protected from disease development, and mice treated with a cream not containing IMQ (sham) stayed normal in all parameters (Figure 1a). To elucidate whether IL-17 signaling in myeloid cells such as neutrophils or macrophages plays a role in IMQ-induced dermatitis, we crossed IL-17RAfl/fl mice to LysM-Cre–expressing mice, which were shown to induce recombination in 100% of neutrophils and in about 80%–90% of macrophages (
      • Clausen B.E.
      • Burkhardt C.
      • Reith W.
      • Renkawitz R.
      • Forster I.
      Conditional gene targeting in macrophages and granulocytes using LysMcre mice.
      ). Treatment of the resulting IL-17RALysM-Cre mice with IMQ showed that dermatitis occurred independent of IL-17RA expression in neutrophils and macrophages (Figure 1b). We conclude that IL-17 signaling by T cells or neutrophils and macrophages does not play a critical role in the development of IMQ-induced dermatitis, in contrast to what we observed in mice with full-body deletion of IL-17RA.
      Figure thumbnail gr1
      Figure 1Development of IMQ-induced dermatitis does not depend on IL-17RA in T cells, neutrophils, or macrophages. Mice were treated for 5 days with Aldara cream (5% IMQ) or sham cream, and their back skin was scored daily for 6 days. (a) Single (erythema, scaling, and thickness) and cumulative scores of IL-17RAfl/fl, IL-17RAdel/del, and IL-17RACD4-Cre mice. The scores are representative of two independent experiments. Sham-treated groups, n = 1; IMQ-treated groups, n = 3–5. (b) Single (erythema, scaling, and thickness) and cumulative scores of IL-17RAfl/fl and IL-17RALysM-Cre mice. Sham-treated groups, n = 3; IMQ-treated groups, n = 5–6. P ≤ 0.05 ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. IL-17RA, IL-17 receptor chain A; IMQ, imiquimod.

      KCs are the critical responder cells to IL-17 signaling after IMQ treatment

      Because KCs were shown to respond to IL-17 signaling in psoriasis (
      • Perera G.K.
      • Di Meglio P.
      • Nestle F.O.
      Psoriasis.
      ) and in the IMQ model (
      • Ha H.L.
      • Wang H.
      • Pisitkun P.
      • Kim J.C.
      • Tassi I.
      • Tang W.
      • et al.
      IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms.
      ), we next tested the impact of deleting IL-17RA in KCs by crossing the IL-17RAfl/fl mice to K14-Cre expressing mice (
      • Hafner M.
      • Wenk J.
      • Nenci A.
      • Pasparakis M.
      • Scharffetter-Kochanek K.
      • Smyth N.
      • et al.
      Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein.
      ). After application of IMQ of IL-17RAK14-Cre mice, we found that all investigated parameters, including erythema formation due to vasodilation, scaling, and thickening caused by acanthosis, were strongly reduced compared with the control group (Figure 2a and b ). The remaining disease score of IL-17RAK14-Cre mice was very similar to that observed in IL-17RAdel/del mice, suggesting that IL-17 signaling in keratinocytes is crucial for disease development in this model (Figure 2a and b). Histological analysis of the back skin showed that both cohorts, the mice lacking IL-17RA in KCs or in the whole body, showed massively reduced histopathological features of psoriasis (Figure 2c and d). Immune cell infiltrates of the epidermis, degradation of the epidermal structure, hyperproliferation of KCs, and parakeratosis leading to acanthosis were also significantly decreased compared with the IMQ-treated control mice (Figure 2c and d). Similarly, we found that IMQ-treated ears of IL-17RAK14-Cre and IL-17RAdel/del mice showed significantly less thickening compared with IMQ-treated control mice (Figure 2e), concomitant with less acanthosis, immune cell infiltration, and hyperproliferation of KCs (Figure 2f and g). We conclude that IL-17RA signaling on KCs is crucial for development of IMQ-induced dermatitis because the conditional deletion of IL-17RA in KCs mimics the reduced disease severity of the body-wide IL-17RA–deficient mice.
      Figure thumbnail gr2
      Figure 2IL-17RA on keratinocytes is crucial for development of IMQ-induced dermatitis. (a) Mice were treated for 6 days with Aldara cream (5% IMQ) or sham cream, and their back skin was scored daily for 7 days. Single (erythema, scaling, and thickness) and cumulative scores of IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice are representative of four independent experiments. Sham-treated groups, n = 1–3; IMQ-treated groups, n = 3. (b) Phenotypical presentation of mouse back skin after sham treatment or IMQ-treatment of IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice. Pictures were taken on day 5. (c) Hematoxylin and eosin staining of back skin sections of sham- or IMQ-treated IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice. Samples were taken on day 5. Scale bar = 100 μm. (d) Microscopic quantification of epidermal hyperplasia in back skin of IMQ-treated IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice on day 5. Epidermal thickness was determined using CellˆP software (Olympus, Shinjuku, Japan). n = 6. (e) Ears were treated for 4 days with Aldara cream (5% IMQ) or sham cream, and the ear thickness was measured daily for 5 days. Sham groups, n = 1 or 2; IMQ-treated groups, n = 4–6. (f) Hematoxylin and eosin staining of ear sections of sham- or IMQ-treated IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice. Samples were taken on day 5. Scale bar = 100 μm. (g) Microscopic quantification of epidermal hyperplasia in the ears of IMQ-treated IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice on day 5. Epidermal thickness was determined using CellˆP software (Olympus). n = 4–6. P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. IL-17RA, IL-17 receptor chain A; IMQ, imiquimod; ns, not significant.

      Neutrophil, but not monocyte, infiltration to IMQ-treated tissue depends on IL-17RA expression in KCs

      Triggering of the IL-17 signaling pathway was shown to result in the attraction of inflammatory myeloid cells to the site of the cytokine production (
      • Ye P.
      • Rodriguez F.H.
      • Kanaly S.
      • Stocking K.L.
      • Schurr J.
      • Schwarzenberger P.
      • et al.
      Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.
      ). Therefore, we investigated cell infiltration in the ears of the IMQ-treated mice by flow cytometry. Analysis of ear infiltrates showed a strong infiltration of CD11b+ cells in IMQ-treated control mice compared with sham-treated control mice, whereas both IMQ-treated IL-17RAdel/del and IL-17RAK14-Cre mice displayed a reduced level of myeloid CD11b+ cell infiltration compared with IMQ-treated controls (Figure 3a and b ). Further analysis of the CD11b-positive myeloid cell population showed that neutrophil (Ly6Cint/Ly6G+ cells) numbers and relative percentages in the mononuclear populations were massively enhanced compared with the respective sham-treated controls (Figure 3c and d). We found significantly lower numbers of infiltrating neutrophils in the ears of the IMQ-treated IL-17RAdel/del and IMQ-treated IL-17RAK14-Cre mice compared with IMQ-treated control mice. In contrast to neutrophils, infiltration of Ly6C+Ly6G monocytes did not show any dependency of IL-17RA signaling, because the IL-17RAdel/del and IL-17RAK14-Cre mice showed comparable infiltrates as the control mice (Figure 3c and d). Therefore, the found reductions in CD11b-positive myeloid cells are due to the lack of infiltrating neutrophils in IL-17RAdel/del and IL-17RAK14-Cre mice but are not caused by the lack of monocyte infiltration. We further analyzed myeloid cell infiltration by immunofluorescence staining using myeloperoxidase as a marker for neutrophils and F4/80 as marker for inflammatory monocytes and macrophages to the IMQ-treated back skin. Similar to the flow cytometry data, we found that neutrophils were highly present in control mice but scarce in mice lacking IL-17RA in KCs or in the whole body, whereas no difference in F4/80+ cells could be observed between these mouse strains versus the control animals (Figure 3e). These findings show that attraction of neutrophils, but not of monocytes, to the IMQ-treated tissue is dependent on IL-17 signaling in keratinocytes, suggesting that secondary cytokines and chemokines, which are responsible for neutrophil attraction to the IMQ-treated tissue, are either directly or indirectly produced by IL-17–activated keratinocytes in this model.
      Figure thumbnail gr3
      Figure 3Infiltration of neutrophils, but not of monocytes, to IMQ-treated tissue depends on IL-17RA on keratinocytes. (a–d) Flow cytometric analysis of myeloid cell infiltrates into sham- or IMQ-treated ears of IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice on day 6. Sham-treated groups, n = 4; IMQ-treated groups, n = 3–5. (a) Dot plot showing CD11b+ gated cells. (b) Quantification of CD11b+ cells, shown as the percentage of living cells and as total cell numbers per two ears per mouse. Sham groups, n = 3; IMQ-treated groups, n = 3–5. (c) Dot plot showing Ly6C versus Ly6G gated out of CD11b+ cells. (d) Quantification of Ly6Cint/Ly6G+ neutrophils and Ly6C+/Ly6G monocytes, shown as the percentage of CD11b+ cells and as total cell numbers per two ears per mouse. Sham groups, n = 3; IMQ-treated groups, n = 3–5. (e) Immunofluorescence stainings for MPO (green), F4/80 (red), and DAPI (blue) of IMQ-treated back skin of IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice on day 6. Scale bar = 100 μm. The #symbols indicate neutrophils, and unspecific MPO staining of hair follicles is marked by asterisks. P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001. IL-17RA, IL-17 receptor chain A; IMQ, imiquimod; MPO, myeloperoxidase; ns, not significant.

      Absence of IL-17RA signaling in KCs results in reduced expression of neutrophil-attracting stimuli

      To test whether neutrophil attraction to IMQ-treated tissue depends on IL-17 signaling in keratinocytes, we performed expression analysis for a selected set of cytokines, chemokines, and chemokine receptors previously implicated in neutrophil infiltration and psoriatic inflammation. We found that the expression of the chemokine Cxcl2 and of the chemokine receptor Ccr1 was up-regulated only in control mice treated with IMQ but not in the IL-17RA signaling–deficient mice (Figure 4). Both CXCL2 and CCR1 were previously shown to be important in the attraction of neutrophils to sites of inflammation. Similarly, expression of Il1a and Il1b was up-regulated after IMQ treatment in controls but only very little in the skin of IL-17RAK14-Cre and IL-17RAdel/del mice. IL-22, a cytokine that was shown to be expressed specifically by T cells and innate lymphoid cells of group 3, was also highly expressed in the IMQ treated control group but not the IL-17RA mutant mice.
      Figure thumbnail gr4
      Figure 4Reduced expression of neutrophil-attracting stimuli in the absence of IL-17RA signaling on keratinocytes. Real-time PCR analysis of the indicated genes in the ears of IMQ-treated IL-17RAfl/fl, IL-17RAdel/del, and IL-17RAK14-Cre mice; n = 4–6.The sham-treated group is pooled of all three genotypes; n = 4. Data were analyzed using the 2–ΔΔCT method. Expression level is shown relative to the housekeeping gene Hprt. P ≤ 0.05, ∗∗∗P ≤ 0.001. IL-17RA, IL-17 receptor chain A; IMQ, imiquimod; ns, not significant.
      Our results show that IL-17 signaling by KCs is responsible for the influx of neutrophils to the skin and to the pathogenesis of psoriasis-like disease in mice, thus making KCs a preferable cell type for manipulation in a clinical setting in psoriasis patients.

      Discussion

      We investigated the target cell type for IL-17 in a mouse model for psoriasis and found that KC expression of IL-17RA is critical for the full induction of skin inflammation. Mechanistically, we showed that the attraction of neutrophils to the skin was prevented in the absence of IL-17 signaling in KCs. In contrast, we found that monocytic infiltration into the skin was independent of IL-17 signaling in KCs.
      What exactly triggers the inflammation in the skin of psoriasis patients is not clear. One possibility is that T cells recognize an autoantigen in the skin, and after their activation, they secrete key cytokines such as IL-17A, IL-22, and TNFα (
      • Prinz J.C.
      Human leukocyte antigen-class I alleles and the autoreactive T cell response in psoriasis pathogenesis.
      ). These cytokines trigger the local cells, such as the KCs, leading to the production of chemokines that initiate neutrophil infiltrations to the skin. As described, IMQ-induced dermatitis in mice only partially models the disease in humans, one major difference being its distinct etiology: by skin irritation via TLR7 activation (after IMQ treatment) versus a possible autoimmune etiology for plaque psoriasis. Nevertheless, the local presence of high IL-17A levels and similar downstream events in the tissue such as neutrophil attraction are common features of both psoriasis and the IMQ model.
      To understand the pathology of psoriasis and the effectiveness of its treatment by IL-17 pathway–neutralizing drugs, it is essential to understand how IL-17A exerts its harmful action. In psoriasis, not only KCs but also fibroblast or endothelial cells may be targets of IL-17. Even though it is thought that IL-17RC is not expressed on hematopoietic cells, a direct reaction of macrophages, neutrophils, and T cells to IL-17A has been described (
      • Ishigame H.
      • Kakuta S.
      • Nagai T.
      • Kadoki M.
      • Nambu A.
      • Komiyama Y.
      • et al.
      Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses.
      ,
      • Taylor P.R.
      • Roy S.
      • Leal Jr., S.M.
      • Sun Y.
      • Howell S.J.
      • Cobb B.A.
      • et al.
      Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2.
      ). We were able to exclude T cells and macrophages/neutrophils as IL-17 targets in psoriasis-like disease, and although we did not formally prove that KCs are the only target cell population important for the development of IMQ-mediated dermatitis, our data strongly suggest that KCs are the major important target cells for the IL-17 response in this model.
      A recent study showed that depletion of neutrophils in the IMQ model lowered disease development similarly as seen here by the use of IL-17RAdel/del or IL-17RAK14-Cre mice (
      • Sumida H.
      • Yanagida K.
      • Kita Y.
      • Abe J.
      • Matsushima K.
      • Nakamura M.
      • et al.
      Interplay between CXCR2 and BLT1 facilitates neutrophil infiltration and resultant keratinocyte activation in a murine model of imiquimod-induced psoriasis.
      ). Neutrophils, which are often found in plaque psoriasis in the upper epidermis layers in the form of Munro microabscesses (
      • Perera G.K.
      • Di Meglio P.
      • Nestle F.O.
      Psoriasis.
      ), may propagate disease by increasing neovascularization. It was shown that neutrophils secrete VEGF in response to G-CSF exposure (
      • Ohki Y.
      • Heissig B.
      • Sato Y.
      • Akiyama H.
      • Zhu Z.
      • Hicklin D.J.
      • et al.
      Granulocyte colony-stimulating factor promotes neovascularization by releasing vascular endothelial growth factor from neutrophils.
      ). This is in line with a strong decrease in erythema formation in IL-17RAdel/del or IL-17RAK14-Cre mice treated with IMQ. Neutrophils also secrete LL-37 (
      • Cowland J.B.
      • Johnsen A.H.
      • Borregaard N.
      hCAP-18, a cathelin/pro-bactenecin-like protein of human neutrophil specific granules.
      ), a defensin, which plays an important role in psoriasis (
      • Lande R.
      • Gregorio J.
      • Facchinetti V.
      • Chatterjee B.
      • Wang Y.H.
      • Homey B.
      • et al.
      Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
      ). Treatment of psoriasis patients with the IL-17A–neutralizing antibody secukinumab also leads to a fast down-regulation of CXCL1 and CXCL8 in the skin with a concomitant loss of neutrophils (
      • Reich K.
      • Papp K.A.
      • Matheson R.T.
      • Tu J.H.
      • Bissonnette R.
      • Bourcier M.
      • et al.
      Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.
      ).
      We found a high presence of infiltrated inflammatory monocytes despite absence of neutrophil infiltration and only residual disease in IL-17RAdel/del and IL-17RAK14-Cre mice treated with IMQ. However, which role these cells play in the model is not clear. Although these cells were shown to secrete CXCL1 and CXCL2, which attract neutrophils, their contribution in this process was not apparent. Their presence in the skin in the absence of major disease symptoms raises the possibility that it is necessary for the KCs to secrete factors such as IL-1 for their full activation. Indeed, it was reported that stearic acid present in the Aldara cream leads to an IL-1–mediated innate inflammatory process independent of the IL-17 pathway (
      • Walter A.
      • Schafer M.
      • Cecconi V.
      • Matter C.
      • Urosevic-Maiwald M.
      • Belloni B.
      • et al.
      Aldara activates TLR7-independent immune defence.
      ). It is likely that this pathway caused residual disease in IL-17RAdel/del and IL-17RAK14-Cre mice with a maintained monocytic infiltration, although Ila and Il1b mRNA levels were found up-regulated only in controls at day 5 of IMQ treatment.
      Acanthosis was shown to be dependent on IL-22, because it affects the differentiation of keratinocytes (
      • Boniface K.
      • Bernard F.X.
      • Garcia M.
      • Gurney A.L.
      • Lecron J.C.
      • Morel F.
      IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes.
      ,
      • Ma H.L.
      • Liang S.
      • Li J.
      • Napierata L.
      • Brown T.
      • Benoit S.
      • et al.
      IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation.
      ,
      • Wolk K.
      • Witte E.
      • Wallace E.
      • Docke W.D.
      • Kunz S.
      • Asadullah K.
      • et al.
      IL-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis.
      ). In line with that, we recently showed that the IL-22 inhibitor IL-22BP is down-regulated in skin of psoriasis patients compared with skin from healthy patients, enabling IL-22’s function in the disease (
      • Voglis S.
      • Moos S.
      • Kloos L.
      • Wanke F.
      • Zayoud M.
      • Pelczar P.
      • et al.
      Regulation of IL-22BP in psoriasis.
      ). Here, we found that the cytokine IL-22 was not up-regulated in mice lacking an IL-17 response in KCs after IMQ treatment. It is possible, therefore, that IL-17–activated KCs induce the infiltration of IL-22–producing cells or their generation in situ.
      In summary, our data show that KCs are the main target cells for IL-17 action in the skin in psoriasiform dermatitis, steering neutrophil infiltration and downstream pathology.

      Materials and Methods

      Mice

      Experiments were performed with the indicated mouse lines. To prevent signaling of IL-17RA in specified tissues, IL-17RAfl/fl mice with the exons 4–7 flanked by loxP sites (
      • El Malki K.
      • Karbach S.H.
      • Huppert J.
      • Zayoud M.
      • Reissig S.
      • Schuler R.
      • et al.
      An alternative pathway of imiquimod-induced psoriasis-like skin inflammation in the absence of interleukin-17 receptor a signaling.
      ) were crossed to the following Cre lines: CD4-Cre (
      • Lee P.P.
      • Fitzpatrick D.R.
      • Beard C.
      • Jessup H.K.
      • Lehar S.
      • Makar K.W.
      • et al.
      A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.
      ), LysM-Cre (
      • Clausen B.E.
      • Burkhardt C.
      • Reith W.
      • Renkawitz R.
      • Forster I.
      Conditional gene targeting in macrophages and granulocytes using LysMcre mice.
      ), or K14-Cre (
      • Hafner M.
      • Wenk J.
      • Nenci A.
      • Pasparakis M.
      • Scharffetter-Kochanek K.
      • Smyth N.
      • et al.
      Keratin 14 Cre transgenic mice authenticate keratin 14 as an oocyte-expressed protein.
      ). To obtain body-wide deletion of IL-17RA, the floxed mice were crossed to a Deleter-Cre–expressing line (
      • Schwenk F.
      • Baron U.
      • Rajewsky K.
      A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells.
      ). All mice were generated on C57BL/6 genetic background or backcrossed at least 10 times. The mice were bred and housed in the animal facility of the University Medical Center of the Johannes Gutenberg-University of Mainz under specified pathogen-free conditions. All mouse experiments were carried out in accordance with the relevant guidelines and regulations for animal welfare by the federal state of Rhineland-Palatinate, Germany. Experiments were done with approval from the Landesuntersuchungsamt Rheinland-Pfalz (individual animal experimentation application no. G13-1-096), and all efforts were made to minimize suffering of the mice.

      IMQ-induced psoriasis mouse model

      Mice were depilated on the back skin 2 days before the treatment and then daily treated with either Aldara (containing 5% IMQ, purchased from Meda [Solna, Sweden]) or sham cream on the back skin and ears for 5 or 6 consecutive days. For the back skin, disease severity was assessed every day with a scoring system considering scaling, erythema, and skin thickness, similar to the human Psoriasis Area and Severity Index, but not taking the area into account because it is defined by the experimenter. A cumulative score was generated from the three mentioned parameters. Erythema and scaling were scored from 0 to 4, with 0 indicating no severity and 4 indicating high severity. Thickness of the skin was scored based on the increase in the thickness compared with day 1 (1 for 20%–40%, 2 for 40T–60%, 3 for 60%–80% and 4 for > 80%). Additionally, ear thickness was measured every day.

      Flow cytometry

      Ears were mechanically disrupted and digested with 0.25 mg/ml Liberase (Roche, Basel, Switzerland) and 0.05 mg/ml DNase (Roche). Subsequently, cells were filtered through a 40-μm cell strainer (BD Falcon, San Jose, CA). The resulting single cell suspension was incubated with Fc-blocking reagent (BioXCell, West Lebanon, NH) before the surface staining with the following fluorescence-conjugated antibodies: αCD11b (clone: M1/70; eBiosciences, San Diego, CA), αLy6C (clone: AL-21; BD Biosciences, San Jose, CA) and αLy6G (clone: 1A8; BioLegend, San Diego, CA). Dead cells were excluded using Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific, Waltham, MA). Stained cells were acquired using a BD FACS Canto II and analysis was performed using FlowJo (Ashland, OR) software.

      Histology

      For hematoxylin and eosin staining, tissue samples were fixed in 4% formaldehyde and embedded in paraffin, and sections of 5–6 μm were cut before the staining was performed. Images were taken using the microscope IX81 (Olympus, Shinjuku, Japan). For immunohistochemistry, cryo sections of 7–8 μm were cut. The following antibodies were used: primary antibodies: α-myeloperoxidase (polyclonal; Abcam, Cambridge, UK) and αF4/80 (clone: BM8, eBiosciences, San Diego, CA), secondary antibodies: goat-αRat (polyclonal; Sigma-Aldrich, St. Louis, MO), and goat-αRabbit (polyclonal, Sigma-Aldrich). Nuclei were counterstained with Hoechst 33342 (Invitrogen, Waltham, MA). Images were taken using the fluorescent microscope DMi8 (Leica, Wetzlar, Germany).

      RNA isolation of skin tissue and quantitative real-time PCR

      RNA from ear tissue was extracted using TriFast (VWR International, Radnor, PA), and cDNA was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Genes of interest were quantified with GoTaq qPCR Master Mix (Promega, Madison, WI), with Hprt as the reference gene. All primers were purchased from Qiagen.

      Statistical analysis

      Graphs were created using GraphPad Prism (GraphPad Software, San Diego, CA). Statistical significance was calculated with one-way analysis of variance with Bonferroni post hoc test. Values of P ≤ 0.001, P ≤ 0.01, and P ≤ 0.05 were considered statistically significant. Data are represented as means with standard deviations.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      We thank Petra Adams, Elena Zurkowski, Michaela Blanfeld, and Bettina Kalt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) CRC/TR-156 to Ari Waisman and Florian C. Kurschus.

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      Linked Article

      • Psoriasis Pathogenesis: Keratinocytes Are Back in the Spotlight
        Journal of Investigative DermatologyVol. 139Issue 5
        • Preview
          Psoriasis is a T helper type 17–mediated immune disease. Initial triggers that lead to T helper type 17 production and inflammatory cell recruitment into skin are being delineated. Autoantigens that stimulate T helper type 17 cells are also being identified. A new and important piece of the puzzle indicates that keratinocytes not only amplify inflammation, but that they are essential for a full-blown IL-17–mediated psoriatic phenotype in mice.
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