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Is CCR6 Required for the Development of Psoriasiform Dermatitis in Mice?

Open ArchivePublished:September 04, 2018DOI:https://doi.org/10.1016/j.jid.2018.07.036

      Abbreviations:

      GDL (γδ low), IMQ (imiquimod), KO (knock out), PsD (psoriasiform dermatitis), WT (wild-type)
      To the Editor
      Intradermal injection of IL-23 and topical application of imiquimod (IMQ) are two widely adopted murine models of psoriasis. Both models result in psoriasiform dermatitis (PsD) in mice that resembles human psoriasis (
      • 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.
      ,
      • Zheng Y.
      • Danilenko D.M.
      • Valdez P.
      • Kasman I.
      • Eastham-Anderson J.
      • Wu J.
      • et al.
      Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis.
      ). CCR6 is important for epidermal trafficking of IL-17/22–producing cells (
      • Mabuchi T.
      • Singh T.P.
      • Takekoshi T.
      • Jia G.F.
      • Wu X.
      • Kao M.C.
      • et al.
      CCR6 is required for epidermal trafficking of gammadelta-T cells in an IL-23-induced model of psoriasiform dermatitis.
      ) and is required for the development of PsD in the IL-23 injection model since CCR6-deficient knockout (CCR6KO) mice fail to show significant dermal inflammation (
      • Hedrick M.N.
      • Lonsdorf A.S.
      • Shirakawa A.K.
      • Richard Lee C.C.
      • Liao F.
      • Singh S.P.
      • et al.
      CCR6 is required for IL-23-induced psoriasis-like inflammation in mice.
      ). Using the IMQ model, however,
      • Cochez P.M.
      • Michiels C.
      • Hendrickx E.
      • Dauguet N.
      • Warnier G.
      • Renauld J.C.
      • et al.
      Ccr6 Is dispensable for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22-producing cells.
      recently reported that CCR6 KO mice were still able to develop psoriatic lesions after application of a specific version of IMQ called Aldara (3M Pharmaceuticals, Maplewood, MN). Although epidermal IL-22 production was decreased, the authors found relatively little morphological change in the skin in CCR6KO versus wild-type (WT) control.
      Other recent reports, however, suggest that CCR6 is critical for development of IMQ-mediated PsD. First,
      • Robert R.
      • Ang C.
      • Sun G.
      • Juglair L.
      • Lim E.X.
      • Mason L.J.
      • et al.
      Essential role for CCR6 in certain inflammatory diseases demonstrated using specific antagonist and knockin mice.
      show that monoclonal antibodies to human CCR6 in a knock-in genetic model effectively block Aldara-mediated PsD. Second, using a small molecule antagonist of CCR6,
      • Campbell J.J.
      • Ebsworth K.
      • Ertl L.S.
      • McMahon J.P.
      • Newland D.
      • Wang Y.
      • et al.
      IL-17-secreting gammadelta T cells are completely dependent upon CCR6 for homing to inflamed skin.
      found that this inhibitor is effective in both IL-23– and IMQ-mediated PsD models in blocking skin inflammation. In the latter report, a generic version of IMQ (Fougera, Melville, NY) was used.
      The possibility exists that the reason for the apparent differences in the importance of CCR6 lies in other ingredients in different commercial preparations of IMQ cream. For example, the Aldara vehicle alone can induce caspase-1–dependent pyroptosis of keratinocyte and concomitant pro-inflammatory cytokines secretion (
      • Walter A.
      • Schafer M.
      • Cecconi V.
      • Matter C.
      • Urosevic-Maiwald M.
      • Belloni B.
      • et al.
      Aldara activates TLR7-independent immune defence.
      ). To test this possibility and explore the role of CCR6 in the IMQ model, we applied a non-Aldara preparation of IMQ cream to CCR6KO mice and assessed the degree of PsD by histologic and immunologic criteria.
      Mice were treated with 5% IMQ cream (Taro Pharmaceuticals, Hawthorne, NY) from day 0 to day 6 on each ear and then euthanized on day 7 (Figure 1a). Ear swelling, a marker for dermal inflammation and corresponding edema (
      • Hedrick M.N.
      • Lonsdorf A.S.
      • Shirakawa A.K.
      • Richard Lee C.C.
      • Liao F.
      • Singh S.P.
      • et al.
      CCR6 is required for IL-23-induced psoriasis-like inflammation in mice.
      ), was consistently reduced in CCR6KO versus WT mice throughout the entire course of the experiment (Figure 1a). Ear skin epidermal hyperplasia in the CCR6KO mice was reduced by 80% versus WT mice (Figure 1b–1c). Quantitative reverse transcriptase PCR showed that mRNA expression of IL-17A, IL-17F, and IL-22 was also markedly suppressed in CCR6KO animals (Figure 1d). We have previously shown that γδ low (GDL) T cells account for the majority of production of IL-17A and IL-22 in the IL-23 PsD model (
      • Mabuchi T.
      • Takekoshi T.
      • Hwang S.T.
      Epidermal CCR6+ gammadelta T cells are major producers of IL-22 and IL-17 in a murine model of psoriasiform dermatitis.
      ). Flow cytometry revealed an approximately 75% reduction in accumulation of epidermal GDL T cells following topical IMQ in CCR6KO (vs. WT) mice (Figure 2a, 2b). Moreover, production of IL-17A and IL-22 by GDL T cells was strikingly inhibited (Figure 2c, 2d). In summary, contrary to data provided by
      • Cochez P.M.
      • Michiels C.
      • Hendrickx E.
      • Dauguet N.
      • Warnier G.
      • Renauld J.C.
      • et al.
      Ccr6 Is dispensable for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22-producing cells.
      , every tested histologic and immunologic feature of PsD was significantly reduced in CCR6KO versus WT mice when IMQ (Taro Pharmaceuticals) was used topically instead of Aldara.
      Figure thumbnail gr1
      Figure 1CCR6 KO mice show reduction in IMQ-induced psoriasiform dermatitis. (a) IMQ was applied to mouse ears from day 0 to day 6. Mice were sacrificed on day 7. The ear thickness change between CCR6 KO mice and WT mice was significantly different. (b) H&E staining of ear skin of CCR6 KO and WT mice. Scale bar = 100 μm (left panel), 50 μm (right panel). (c) Epidermal thickness of CCR6 KO and WT mice, H&E and quantification. Scale bar = 10 μm. (d) On day 3, reverse transcriptase PCR analysis Il17a, Il17f, and Il22 mRNA in CCR6 KO mice compared with WT mice. H&E, hematoxylin and eosin; IMQ, imiquimod; KO, knockout; WT, wild-type.
      Figure thumbnail gr2
      Figure 2Recruitment and cytokine expressions of GDL T cells in WT and CCR6 KO mice after IMQ exposure. (a) IMQ was applied daily to mouse ears as indicated. Cells suspensions were generated as described (
      • Mabuchi T.
      • Takekoshi T.
      • Hwang S.T.
      Epidermal CCR6+ gammadelta T cells are major producers of IL-22 and IL-17 in a murine model of psoriasiform dermatitis.
      ), and GDL T cells in the epidermis were assessed by flow cytometry after staining with antibody against γδ TCR versus SSC and quantified in (b). (c, d) Percentages of IL-17A–positive GDL T cells and IL-22–positive GDL T cells from whole skin were calculated by using flow cytometry (n = 3, P < 0.05). GDL, γδ low; IMQ, imiquimod; KO, knockout; SSC, side scatter; TCR, T-cell receptor; WT, wild-type.
      Our experiments clearly demonstrate upregulation of key T-helper type 17 cytokines and robust epidermal hyperplasia in skin after exposure to this formulation of IMQ. In contrast to the results obtained using Aldara (
      • Cochez P.M.
      • Michiels C.
      • Hendrickx E.
      • Dauguet N.
      • Warnier G.
      • Renauld J.C.
      • et al.
      Ccr6 Is dispensable for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22-producing cells.
      ), our experiments demonstrated that CCR6 is required for maximal development of PsD. Of note, not only recruitment, but also function, of GDL T cells was impacted by the absence of CCR6. Although it is not yet clear how CCR6 impacts the expression of cytokines such as IL-17A by GDL T cells, this result using IMQ validates a similar conclusion we made using the IL-23 injection model (
      • Mabuchi T.
      • Singh T.P.
      • Takekoshi T.
      • Jia G.F.
      • Wu X.
      • Kao M.C.
      • et al.
      CCR6 is required for epidermal trafficking of gammadelta-T cells in an IL-23-induced model of psoriasiform dermatitis.
      ).
      Others have also observed that IMQ preparations from different sources can lead to differential experimental effects in murine psoriatic models (
      • Alvarez P.
      • Jensen L.E.
      Imiquimod treatment causes systemic disease in mice resembling generalized pustular psoriasis in an IL-1 and IL-36 dependent manner.
      ,
      • Luo D.Q.
      • Wu H.H.
      • Zhao Y.K.
      • Liu J.H.
      • Wang F.
      Original Research: Different imiquimod creams resulting in differential effects for imiquimod-induced psoriatic mouse models.
      ). Interestingly, toll-like receptor 7 KO mice also showed dermatitis upon Aldara cream application, despite abolished production of IL-23, suggesting other ingredients in IMQ cream induce dermatitis in a toll-like receptor 7–independent manner (
      • Ueyama A.
      • Yamamoto M.
      • Tsujii K.
      • Furue Y.
      • Imura C.
      • Shichijo M.
      • et al.
      Mechanism of pathogenesis of imiquimod-induced skin inflammation in the mouse: a role for interferon-alpha in dendritic cell activation by imiquimod.
      ). Isostearic acid, the second most abundant component in Aldara vehicle, can induce inflammasome activation and may partially account for the differential results (
      • Walter A.
      • Schafer M.
      • Cecconi V.
      • Matter C.
      • Urosevic-Maiwald M.
      • Belloni B.
      • et al.
      Aldara activates TLR7-independent immune defence.
      ). To test the hypothesis that origins of IMQ cream account for different effects, we performed a direct comparison study between the Aldara and IMQ (Taro Pharmaceuticals) formulations. The results showed IMQ (Taro Pharmaceuticals) induced significantly greater ear swelling in WT mice than CCR6KO mice, while there was no difference between WT mice and CCR6KO mice in terms of Aldara-induced ear swelling (Supplementary Figure S1a, S1b online). Quantitative histologic analysis showed that the epidermis of IMQ (Taro Pharmaceuticals)–treated WT mice was significantly thicker than that of IMQ (Taro Pharmaceuticals)–treated CCR6KO mice on day 5, while there was no difference in epidermal thickness between Aldara-treated WT and CCR6KO mice (Supplementary Figure S1c, S2 online). Epidermal collections of neutrophils (Munro’s microabscesses), a well-recognized feature of human psoriasis, were also notably reduced in a number of CCR6KO mice treated with IMQ (Taro Pharmaceuticals) compared to Aldara-treated WT and CCR6KO mice, suggesting a qualitative reduction in the extent of psoriasiform inflammation in these animals (Supplementary Figure S2).
      It should be noted that the specific bacterial and sanitary conditions in different facilities may account for differential responses to IMQ, especially because this agent acts on pathogen recognition receptors (eg, toll-like receptors). While indeed bacteria and the microbiome may influence the development of inflammatory skin disease, as illustrated with atopic dermatitis in dogs (
      • Bradley C.W.
      • Morris D.O.
      • Rankin S.C.
      • Cain C.L.
      • Misic A.M.
      • Houser T.
      • et al.
      Longitudinal evaluation of the skin microbiome and association with microenvironment and treatment in canine atopic dermatitis.
      ), our direct side by side comparison of Aldara versus generic IMQ was performed on mice housed under identical conditions.
      In conclusion, CCR6 remains crucial for the development of PsD in both the IL-23 and IMQ murine models, although to different degrees, depending on the specific methodology of testing, as suggested by our current results and results of others (
      • Campbell J.J.
      • Ebsworth K.
      • Ertl L.S.
      • McMahon J.P.
      • Newland D.
      • Wang Y.
      • et al.
      IL-17-secreting gammadelta T cells are completely dependent upon CCR6 for homing to inflamed skin.
      ,
      • Cochez P.M.
      • Michiels C.
      • Hendrickx E.
      • Dauguet N.
      • Warnier G.
      • Renauld J.C.
      • et al.
      Ccr6 Is dispensable for the development of skin lesions induced by imiquimod despite its effect on epidermal homing of IL-22-producing cells.
      ,
      • Robert R.
      • Ang C.
      • Sun G.
      • Juglair L.
      • Lim E.X.
      • Mason L.J.
      • et al.
      Essential role for CCR6 in certain inflammatory diseases demonstrated using specific antagonist and knockin mice.
      ). Notably, in both the IL-23 and IMQ models, the function of GDL T cells in terms of producing IL-17A and IL-22 in vivo is dependent on CCR6 expression. While topical application of IMQ is a convenient and reproducible model for PsD, the preparation and source of IMQ must be considered when interpreting data obtained from these models. Additional validation with additional systems, such as the IL-23 injection model, may be prudent in order to make the most generalizable interpretations.

      ORCID

      Conflict of Interest

      STH and YI have intellectual property rights related to a molecule that blocks the CCR6 receptor. The other authors state no conflict of interest.

      Acknowledgments

      This work was partially supported by NIAMS R01 R063091-01A1 to STH].

      Supplementary Material

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