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A Role for TGFβ Signaling in the Pathogenesis of Psoriasis

      Deregulation of transforming growth factor-β (TGFβ) signaling has been reported in human psoriasis. Our recent study using a keratin 5 promoter (K5.TGFβ1wt) showed that transgenic mice expressing wild-type TGFβ1 in the epidermis developed severe skin inflammation. Additional experimental data further support a direct role for TGFβ1 overexpression in skin inflammation. First, we temporally induced TGFβ1 expression in keratinocytes in our gene-switch TGFβ1wt transgenic mice and found inflammation severity correlated with TGFβ1wt transgene expression. Second, deletion of T cells in K5.TGFβ1wt mice significantly delayed skin inflammation and associated epidermal hyperplasia/hyperkeratosis. Third, therapeutic approaches effective for human psoriasis, that is, Etanercept and Rosiglitazone, are effective in alleviating the symptoms observed in K5.TGFβ1wt mice. Future studies will analyze specific mechanisms and identify key factors in TGFβ1-induced skin inflammation. Our mouse models will provide a useful tool for understanding the molecular mechanisms of inflammatory skin disorders in which TGFβ1 is overexpressed.

      Abbreviations

      IFN-γ
      interferon gamma
      IL
      interleukin
      LFA-1
      lymphocyte function-associated antigen 1
      MIP
      macrophage inflammatory protein
      TGFβ
      transforming growth factor-β
      TGFβRI
      type I TGFβ receptor
      TGFβRII
      type II TGFβ receptor
      Th
      T helper
      TNF-α
      tumor necrosis factor alpha

      Introduction

      Psoriasis is a common inflammatory skin disease that has a severe negative impact on a patient's quality of life and can be an economic burden. Histologically, psoriasis is characterized by epidermal hyperplasia and parakeratosis, dilated and prominent blood vessels in the upper dermis, and leukocyte infiltration in the dermis and epidermis (
      • Griffiths C.E.
      • Barker J.N.
      Pathogenesis and clinical features of psoriasis.
      ;
      • Gudjonsson J.E.
      • Johnston A.
      • Dyson M.
      • Valdimarsson H.
      • Elder J.T.
      Mouse models of psoriasis.
      ). Psoriatic lesions express high levels of IL-2, IFN-γ, and TNF-α, but not IL-4 and IL-10. Deletion of active T lymphocytes with toxin-coupled IL-2 (DAB389IL-2) or inhibition of migration and activation of T cells with an anti-LFA-1 antibody significantly reduces the severity of psoriasis (
      • Gottlieb S.L.
      • Gilleaudeau P.
      • Johnson R.
      • Estes L.
      • Woodworth T.G.
      • Gottlieb A.B.
      • et al.
      Response of psoriasis to a lymphocyte-selective toxin (DAB389IL-2) suggests a primary immune, but not keratinocyte, pathogenic basis.
      ;
      • Thaci D.
      Long-term data in the treatment of psoriasis.
      ). IL-4 or IL-10 therapy also significantly relieves the symptoms of human psoriasis (
      • Ghoreschi K.
      • Thomas P.
      • Breit S.
      • Dugas M.
      • Mailhammer R.
      • van Eden W.
      • et al.
      Interleukin-4 therapy of psoriasis induces Th2 responses and improves human autoimmune disease.
      ;
      • Asadullah K.
      • Sabat R.
      • Friedrich M.
      • Volk H.D.
      • Sterry W.
      Interleukin-10: an important immunoregulatory cytokine with major impact on psoriasis.
      ). Therefore, psoriasis was widely considered as a Th1 type disease (
      • Sabat R.
      • Sterry W.
      • Philipp S.
      • Wolk K.
      Three decades of psoriasis research: where has it led us?.
      ) until the discovery of IL-17-producing T cells (Th17); the new findings suggest that Th17 cells have a crucial function in the development of psoriasis (
      • Zaba L.C.
      • Cardinale I.
      • Gilleaudeau P.
      • Sullivan-Whalen M.
      • Suarez-Farinas M.
      • Fuentes-Duculan J.
      • et al.
      Amelioration of epidermal hyperplasia by TNF inhibition is associated with reduced Th17 responses.
      ;
      • Lowes M.A.
      • Kikuchi T.
      • Fuentes-Duculan J.
      • Cardinale I.
      • Zaba L.C.
      • Haider A.S.
      • et al.
      Psoriasis vulgaris lesions contain discrete populations of Th1 and Th17 T cells.
      ;
      • Di Cesare A.
      • Di Meglio P.
      • Nestle F.O.
      The IL-23/Th17 axis in the immunopathogenesis of psoriasis.
      ). Further, a recent study revealed cooperative effects between Th1 and Th17 cells in the pathogenesis of psoriasis (
      • Asadullah K.
      • Sabat R.
      • Friedrich M.
      • Volk H.D.
      • Sterry W.
      Interleukin-10: an important immunoregulatory cytokine with major impact on psoriasis.
      ;
      • Kryczek I.
      • Bruce A.T.
      • Gudjonsson J.E.
      • Johnston A.
      • Aphale A.
      • Vatan L.
      • et al.
      Induction of IL-17+ T cell trafficking and development by IFN-gamma: mechanism and pathological relevance in psoriasis.
      ).

      Alteration Of Tgfβ1 In Human Psoriasis

      Transforming growth factor β (TGFβ) is a multipotent cytokine. Among the three isoforms of TGFβ, TGFβ1 is the predominant isoform in the skin. TGFβ binds to type I and type II receptors (TGFβRI and TGFβRII). TGFβRI phosphorylates downstream mediators Smad2 and Smad3. Phosphorylated Smad2 and Smad3 complex with Smad4 to regulate TGFβ-responsive genes (
      • Owens P.
      • Han G.
      • Li A.G.
      • Wang X.J.
      The role of Smads in skin development.
      ). Expressions of TGFβ receptors and Smads are detected in epidermal keratinocytes (
      • Lange D.
      • Persson U.
      • Wollina U.
      • Ten Dijke P.
      • Castelli E.
      • Heldin C.H.
      • et al.
      Expression of TGF-beta related Smad proteins in human epithelial skin tumors.
      ;
      • Quan T.
      • He T.
      • Kang S.
      • Voorhees J.J.
      • Fisher G.J.
      Ultraviolet irradiation alters transforming growth factor beta/smad pathway in human skin in vivo.
      ;
      • Han G.
      • Lu S.L.
      • Li A.G.
      • He W.
      • Corless C.L.
      • Kulesz-Martin M.
      • et al.
      Distinct mechanisms of TGF-beta1-mediated epithelial-to-mesenchymal transition and metastasis during skin carcinogenesis.
      ).
      A significant reduction of TGFβ receptors in psoriatic epidermis has been reported (
      • Leivo T.
      • Leivo I.
      • Kariniemi A.L.
      • Keski-Oja J.
      • Virtanen I.
      Down-regulation of transforming growth factor-beta receptors I and II is seen in lesional but not non-lesional psoriatic epidermis.
      ;
      • Doi H.
      • Shibata M.A.
      • Kiyokane K.
      • Otsuki Y.
      Downregulation of TGFbeta isoforms and their receptors contributes to keratinocyte hyperproliferation in psoriasis vulgaris.
      ). As TGFβ1 is a potent growth inhibitor for keratinocytes, it has been suspected that reduced TGFβ signaling potentiates keratinocyte hyperproliferation in psoriasis epidermis. However, reduced TGFβ receptors could also be a result of an increased TGFβ1 ligand. Increased TGFβ1 in the epidermis and serum has been found in psoriatic patients (
      • Flisiak I.
      • Chodynicka B.
      • Porebski P.
      • Flisiak R.
      Association between psoriasis severity and transforming growth factor beta(1) and beta (2) in plasma and scales from psoriatic lesions.
      ), and the TGFβ1 serum level correlates with disease severity (
      • Nockowski P.
      • Szepietowski J.C.
      • Ziarkiewicz M.
      • Baran E.
      Serum concentrations of transforming growth factor beta 1 in patients with psoriasis vulgaris.
      ;
      • Flisiak I.
      • Zaniewski P.
      • Chodynicka B.
      Plasma TGF-beta1, TIMP-1, MMP-1 and IL-18 as a combined biomarker of psoriasis activity.
      ). Successful treatment resulted in reduced serum levels of TGFβ1 in patients with psoriasis (
      • Flisiak I.
      • Porebski P.
      • Flisiak R.
      • Chodynicka B.
      Plasma transforming growth factor beta1 as a biomarker of psoriasis activity and treatment efficacy.
      ). The mechanism responsible for increased serum levels of TGFβ1 in patients with psoriasis remains unclear; increased TGFβ1 could result from activated stromal cells (
      • Flisiak I.
      • Zaniewski P.
      • Chodynicka B.
      Plasma TGF-beta1, TIMP-1, MMP-1 and IL-18 as a combined biomarker of psoriasis activity.
      ). On the basis of clinical data, it is difficult to determine whether increased TGFβ1 has a causal function in psoriasis or whether it is simply a consequence of psoriasis pathogenesis.

      Tgfβ1 Overexpression In Keratinocytes Induces Skin Inflammation

      In the past, TGFβ1 was considered an anti-inflammatory cytokine with strong immune suppressive effects, as TGFβ1 knockout mice died of autoimmune diseases (
      • Shull M.M.
      • Ormsby I.
      • Kier A.B.
      • Pawlowski S.
      • Diebold R.J.
      • Yin M.
      • et al.
      Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.
      ;
      • Kulkarni A.B.
      • Huh C.G.
      • Becker D.
      • Geiser A.
      • Lyght M.
      • Flanders K.C.
      • et al.
      Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death.
      ). We developed TGFβ1 transgenic mice, targeting wild-type human TGFβ1 at the epidermis using a keratin 5 (K5) promoter (K5.TGFβ1wt). In this transgenic mouse model, TGFβ1 was overexpressed in the epidermis at levels similar to that of peak expression during cutaneous wound healing (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). K5.TGFβ1wt transgenic mice surprisingly developed severe inflammatory skin phenotypes, including focal lesions induced by ear tagging, erythematous plaques with a scaly appearance at around 1 month of age, and generalized scaly erythema when skin inflammation progressed. Histologically, K5.TGFβ1wt transgenic skin developed significant epidermal hyperplasia and hyperkeratosis with massive inflammatory cell infiltration, neovascularization, and epidermal basement membrane degradation (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). Epidermal hyperproliferation is mainly a secondary effect of inflammation and angiogenesis, because cultured K5.TGFβ1wt keratinocytes without a stromal component undergo growth arrest (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). Among infiltrated leukocytes, CD4+ and CD8+ T cells are found in large numbers in the dermis and at the dermal–epidermal junction in K5.TGFβ1wt skin. Molecular analysis showed that Th1 type cytokines predominated in K5.TGFβ1wt skin (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). The original characterization of this model identified many similarities to psoriasis. However, a recent study shows that despite an increase in IL17 in K5.TGFβ1wt lesional skin, these mice do not respond to IL23 antibody treatment and have high levels of IL4 and IgE (
      • Fitch E.L.
      • Rizzo H.L.
      • Kurtz S.E.
      • Wegmann K.W.
      • Gao W.
      • Benson J.M.
      • et al.
      Inflammatory skin disease in K5.hTGF-beta1 transgenic mice is not dependent on the IL-23/Th17 inflammatory pathway.
      ). Consistent with the known role of TGFβ1 in Treg cell functions, these mice also have increased Treg-related cytokines (
      • Fitch E.L.
      • Rizzo H.L.
      • Kurtz S.E.
      • Wegmann K.W.
      • Gao W.
      • Benson J.M.
      • et al.
      Inflammatory skin disease in K5.hTGF-beta1 transgenic mice is not dependent on the IL-23/Th17 inflammatory pathway.
      ). Therefore, the role of TGFβ1-induced skin inflammation seems to be more complicated than previously appreciated.
      Consistent with our findings, a pro-inflammatory effect of TGFβ1 has also been reported. In the skin, TGFβ1 is required for Langerhans cell development and maturation (
      • Borkowski T.A.
      • Letterio J.J.
      • Farr A.G.
      • Udey M.C.
      A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: the skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells.
      ,
      • Borkowski T.A.
      • Letterio J.J.
      • Mackall C.L.
      • Saitoh A.
      • Farr A.G.
      • Wang X.J.
      • et al.
      Langerhans cells in the TGF beta 1 null mouse.
      ), which can trigger skin inflammation. To eliminate the possibility that skin inflammation in these mice is a secondary effect of infection, we re-derived these mouse embryos in a specific pathogen-free facility and found the offspring developed onset phenotypes at the same age as did their parental mice (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). This suggests a direct effect of TGFβ1 overexpression in skin inflammation. To further assess this, we induced TGFβ1 in the epidermis of our gene-switch transgenic mice, in which transgene expression can be regulated by a topical application of RU486 (
      • Lu S.L.
      • Reh D.
      • Li A.G.
      • Woods J.
      • Corless C.L.
      • Kulesz-Martin M.
      • et al.
      Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation.
      ;
      • Li A.G.
      • Lu S.L.
      • Han G.
      • Kulesz-Martin M.
      • Wang X.J.
      Current view of the role of transforming growth factor beta 1 in skin carcinogenesis.
      ). When TGFβ1 was induced for 10 days, scaly erythema and plaques developed in transgenic but not in control mouse skin. Histopathology showed that bigenic skin recapitulated the pathological alterations observed in K5.TGFβ1wt skin (Figure 1). Continuous RU486 application maintained skin inflammation, and phenotype severity correlated with TGFβ1 expression levels in bigenic mice (data not shown). Leukocyte infiltration found in bigenic skin was similar to that in K5.TGFβ1wt skin. Particularly, CD4+ T cells resided in the dermis, whereas CD8+ T cells predominantly infiltrated the epidermis. Increased BM8+ macrophages and angiogenesis were also prominent in bigenic skin as compared with control skin (data not shown). Interestingly, epidermal thickness and leukocyte infiltration in bigenic skin declined markedly 1 week after withdrawal of RU486 (Figure 1), suggesting that skin inflammation is dependent on TGFβ1 overexpression.
      Figure thumbnail gr1
      Figure 1Effects of TGFβ1wt transgene induction in gene-switch-TGFβ1wt skin. The H&E staining of dorsal skin from TGFβ1wt gene-switch mice treated with EtOH (i) or RU486 (ii) for 10 days. Epidermal hyperplasia and corneal microabscess (arrow) were noticed on TGFβ1wt induction (ii) and recovered to normal skin after withdrawal of RU486 (iii). The bar in panel (i) represents 100μm for all sections. The dotted line in each section highlights the epidermal–dermal junction.

      Deletion Of T Cells Delays But Does Not Prevent Tgfβ1-induced Inflammation

      Studies reveal that TGFβ1 is required for naïve mouse CD4+ T cells to differentiate into Th17 cells (
      • Bettelli E.
      • Carrier Y.
      • Gao W.
      • Korn T.
      • Strom T.B.
      • Oukka M.
      • et al.
      Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells.
      ;
      • Mangan P.R.
      • Harrington L.E.
      • O’Quinn D.B.
      • Helms W.S.
      • Bullard D.C.
      • Elson C.O.
      • et al.
      Transforming growth factor-beta induces development of the T(H)17 lineage.
      ;
      • Veldhoen M.
      • Hocking R.J.
      • Atkins C.J.
      • Locksley R.M.
      • Stockinger B.
      TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells.
      ;
      • Yang L.
      • Anderson D.E.
      • Baecher-Allan C.
      • Hastings W.D.
      • Bettelli E.
      • Oukka M.
      • et al.
      IL-21 and TGF-beta are required for differentiation of human T(H)17 cells.
      ). As K5.TGFβ1wt skin showed increased IL17 but did not respond to anti-IL23 treatment (
      • Fitch E.L.
      • Rizzo H.L.
      • Kurtz S.E.
      • Wegmann K.W.
      • Gao W.
      • Benson J.M.
      • et al.
      Inflammatory skin disease in K5.hTGF-beta1 transgenic mice is not dependent on the IL-23/Th17 inflammatory pathway.
      ), it is possible that (1) the Th17–IL23 pathway is not involved in skin inflammation in this model; (2) IL23 may primarily activate Th17 cells, but this function is dispensable with TGFβ1-induced Th17 activation; or (3) Th17 cells initiate but cannot sustain skin inflammation in this model. More thorough studies in the future are needed to delineate these possibilities. To determine whether depletion of all T cells would have a stronger effect in alleviating skin inflammation in K5.TGFβ1wt mice, we crossed K5.TGFβ1wt mice with Rag1−/- mice, which lack mature lymphocytes (
      • Mombaerts P.
      • Iacomini J.
      • Johnson R.S.
      • Herrup K.
      • Tonegawa S.
      • Papaioannou V.E.
      RAG-1-deficient mice have no mature B and T lymphocytes.
      ). Immunohistochemical staining showed that CD4+T cells were largely depleted in K5.TGFβ1wt.Rag1−/- skin, but total leukocytes (CD45+, data not shown), which are mainly macrophages (F4/80) and granulocytes (Ly6G+) in K5.TGFβ1wt.Rag1−/- skin, were similar to K5.TGFβ1wt.Rag1+/- skin (Figure 2a). This finding suggests that overall leukocyte infiltration was not a secondary event of T-cell activation, but rather a direct chemoattractant effect of TGFβ1 in this model. Histologically, a significant reduction in epidermal hyperplasia and inflammation has been observed as early as 3 weeks in K5.TGFβ1wt.Rag1−/- compared with K5.TGFβ1wt.Rag1+/- littermates, and this effect is sustained over 4 months (Figure 2b). In accordance with phenotype changes, the expression levels of pro-inflammatory cytokines, including TNF-α and IL-1β in skin as determined by quantitative RT-PCR, were significantly decreased in K5.TGFβ1wt.Rag1−/- skin when compared with K5.TGFβ1wt.Rag1+/- skin (data not shown). The anti-inflammatory effect of T-cell depletion was gradually lost after 6 months of age in K5.TGFβ1wt/Rag1−/- mice (Figure 2b). These data suggest that T cells are important drivers of TGFβ1-mediated skin inflammation, especially during the early stages of inflammation. However, accumulated pro-inflammatory cytokines or chemokines from other inflammatory cells may be crucial in maintaining TGFβ1-mediated skin inflammation.
      Figure thumbnail gr2
      Figure 2T-cell depletion delays TGFβ1-induced inflammation. (a) Immunohistochemical staining for T (CD4+) cells, macrophages (F4/80+), and granulocytes (Ly6G+) in K5.TGFβ1wt.Rag1+/- and K5.TGFβ1wt.Rag1−/- skin. CD4+ T-cells were largely depleted in K5.TGFβ1wt.Rag1−/- skin. No significant difference in macrophage (F4/80+) and granulocyte (Ly6G+) staining was observed between K5.TGFβ1wt.Rag1+/- and K5.TGFβ1wt.Rag1−/- skin. (b) Histological analysis of skins from K5.TGFβ1wt.Rag1+/- and K5.TGFβ1wt.Rag1−/- mice at 2, 4, and 6 months of age. Skin from K5.TGFβ1wt.Rag1+/- mice shows profound inflammatory cell infiltration, epidermal hyperplasia, and basement membrane degradation at 2 and 4months of age, but the phenotype was almost reversed in K5.TGFβ1wt.Rag1−/- mice. Skin from both genotypes showed a similar histological alteration at 6months of age. The bar in panel (a) and (b) represents 40μm.

      Therapeutic Approaches For Psoriasis Reduced Inflammation In K5.tgfβ1wt Skin

      As the phenotypes of K5.TGFβ1wt mice are more similar to psoriasis than to any other inflammatory skin diseases, we wondered whether current therapies for psoriasis would alleviate K5.TGFβ1wt phenotypes. We first tested the efficacy of Enbrel (Etanercept), a soluble fusion protein composed of TNF-α receptors and the Fc portion of human IgG1, which competitively binds to TNF-α and prevents TNF-α from binding to endogenous receptors (
      • Zeichner J.A.
      • Lebwohl M.
      Potential complications associated with the use of biologic agents for psoriasis.
      ). We treated K5.TGFβ1wt mice with Enbrel at 6 weeks of age, when skin inflammation began to develop. Enbrel was injected intraperitoneally to K5.TGFβ1wt mice at a dosage of 0.4mg per mouse every other day for up to 6 weeks; controls were treated with normal saline. Beginning 3 weeks after Enbrel treatment, K5.TGFβ1wt mice showed a reduction of epidermal hyperplasia and fewer numbers of infiltrated T cells compared with control mice (Figure 3b). Improvements in the gross appearance of Enbrel-treated mice were obvious after 6 weeks of treatment (Figure 3a and b). Without treatment, the phenotype worsened. In contrast, 6 weeks after Enbrel treatment, K5.TGFβ1wt mice exhibited few scaly plaques or mild skin inflammation. Histology shows that the treated skin exhibited a marked reduction in epidermal hyperplasia (Figure 3b).
      Figure thumbnail gr3
      Figure 3Etanercept therapy relieves the inflammatory symptoms of K5.TGFβ1wt mice. (a) Typical gross appearances of K5.TGFβ1wt mice before and after Enbrel treatment for 6 weeks. Normal saline-treated mice were used as controls. Minor skin inflammation appeared on the ear of K5.TGFβ1wt mice at the age of 6 weeks (i & iii) and progressed to spread over most of the body area with scaly plaques or skin inflammation at the age of 12 weeks (ii). Enbrel therapy prevented the acceleration of skin phenotypes (iv). Arrows point to inflammation sites. (b) H&E staining of K5.TGFβ1wt skin sections at 6 weeks with and without Enbrel treatment (left) revealed a significant reduction of epidermal thickness. K5.TGFβ1wt skin exhibited an alleviative infiltration of CD4 and CD8 T cells and a mild reduction in epidermal thickness as early as 3 weeks after starting Enbrel therapy (middle and right panels). The bar in panel (b) represents 40μm.
      Second, we tested the efficacy of Rosiglitazone (Avandia) on K5.TGFβ1wt mice. Avandia, a peroxisome proliferator-activated receptor-γ agonist used to treat type II diabetes, is also used to treat psoriasis (
      • Pershadsingh H.A.
      Peroxisome proliferator-activated receptor-gamma: therapeutic target for diseases beyond diabetes: quo vadis?.
      ). We administered Avandia 0.04mgml−1 through drinking water in K5.TGFβ1wt mice or RU486-treated gene-switch TGFβ1 mice when skin inflammation was well developed, starting from when they were 2–3-months old up to 1 year of age. Transgenic littermates that received no Avandia in drinking water were used as controls. Terminal differentiation markers of the epidermis, loricrin and fillagrin, which were lost in TGFβ1wt skin, were restored after only 3 weeks of Avandia treatment (Figure 4a). Total (CD45+) leukocytes and CD4+ lymphocytes were reduced, whereas BM8+ macrophages were slightly reduced in TGFβ1wt skin 3 weeks after Avandia treatment (Figure 4b). With an 8-week treatment, lesions on treated transgenic mice were much less severe than in non-treated mice, and epidermal hyperplasia was appreciatively reduced (Figure 5a). Interestingly, at this stage, in addition to a considerable reduction of T cells and leukocytes in the lesion (data not shown), the number of macrophages stained by BM8 was significantly decreased in the skin of K5.TGFβ1wt mice (Figure 5b). These data further suggest that activated macrophages contribute significantly to the maintenance of TGFβ1-mediated skin inflammation. It should be noted that mRNA expression levels of many pro-inflammatory molecules, for example, TNF-α, IL-1α, IL-1β, IL-2, IL-6, and MIP-2 (IL-8 homolog), were markedly reduced after an 8-week treatment with Avandia (Figure 5c). As many of these inflammatory cytokines can be produced in multiple cell types, including inflammatory cells, keratinocytes, and fibroblasts, it is likely that Avandia targets multiple cell populations, which seems to be more effective than depleting only T cells. Indeed, the anti-inflammatory effect of Avandia in K5.TGFβ1wt mice persisted during treatment for up to 1 year of observation.
      Figure thumbnail gr4
      Figure 4Restoration of abnormal epidermal differentiation and reduction of lymphocytes and leukocytes in K5.TGFβ1wt mouse skin after short-term Rosiglitazone treatment. (a) Staining of the epidermal differentiation marker in K5.TGFβ1wt skin with and without Avandia treatment. Note that the loricrin and fillagrin lost in K5.TGFβ1wt skin reappeared (arrows) after 3-week Avandia treatment. (b) Short-term Avandia treatment significantly reduced the infiltration of total leukocytes (CD45) and T lymphocytes (CD4), but not of macrophages (BM8) in skin from K5.TGFβ1wt mice. The bar in panel (a) and (b) represents 40μm.
      Figure thumbnail gr5
      Figure 5Attenuation of the inflammatory phenotype in K5.TGFβ1wt mice with Rosiglitazone treatment. (a) K5.TGFβ1wt mice with 8 weeks of Avandia treatment showed a significant reduction in skin inflammation compared with non-treated transgenic mice. H&E staining shows a reduced epidermal thickness with Avandia treatment. (b) The number of macrophages stained by BM8 was significantly decreased in the skin of K5.TGFβ1wt mice treated with Avandia for 8 weeks. (c) mRNA expression levels of inflammatory cytokines and chemokines were all significantly reduced (n=5, P<0.01) in the skin of K5.TGFβ1wt mice treated with Avandia for 8 weeks, in comparison with non-treated K5.TGFβ1wt skins. The bar in panel (a) and (b) represents 40μm.

      Summary

      Although many of the pathological and molecular alterations in K5.TGFβ1wt mice are more similar to psoriasis than to any other inflammatory skin diseases, there are differences. First, the lack of a major growth factor targeted at the K5.TGFβ1wt epidermis accounts for less epidermal hyperproliferation than in human psoriasis or mouse psoriasis models expressing a potent growth factor, for example, in Stat3 or amphiregulin transgenic mice (
      • Cook P.W.
      • Brown J.R.
      • Cornell K.A.
      • Pittelkow M.R.
      Suprabasal expression of human amphiregulin in the epidermis of transgenic mice induces a severe, early-onset, psoriasis-like skin pathology: expression of amphiregulin in the basal epidermis is also associated with synovitis.
      ;
      • Sano S.
      • Chan K.S.
      • Carbajal S.
      • Clifford J.
      • Peavey M.
      • Kiguchi K.
      • et al.
      Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model.
      ). Second, the role of activated T cells in skin inflammation is limited to the initiation stage in K5.TGFβ1wt mice. Third, anti-IL23 treatment, which is an effective psoriasis therapy, does not alleviate skin inflammation in K5.TGFβ1wt mice. Finally, pathological alterations similar to atopic dermatitis are also reported in these mice (
      • Fitch E.L.
      • Rizzo H.L.
      • Kurtz S.E.
      • Wegmann K.W.
      • Gao W.
      • Benson J.M.
      • et al.
      Inflammatory skin disease in K5.hTGF-beta1 transgenic mice is not dependent on the IL-23/Th17 inflammatory pathway.
      ). As molecular and pathological alterations similar to psoriasis occur much earlier (3 weeks) (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ) than in atopic dermatitis (4–6 months) (
      • Fitch E.L.
      • Rizzo H.L.
      • Kurtz S.E.
      • Wegmann K.W.
      • Gao W.
      • Benson J.M.
      • et al.
      Inflammatory skin disease in K5.hTGF-beta1 transgenic mice is not dependent on the IL-23/Th17 inflammatory pathway.
      ), environmental factors contributing to the latter pathological alterations remain to be examined. Similar to all other psoriasis-like mouse models, it is a challenge to make a humanized psoriasis mouse model because of significant differences in skin anatomy and immune system between the two species and the complex nature of psoriasis. On the basis of current findings, it seems that TGFβ1 overexpression alone is insufficient to mimic all characteristics of psoriasis. Conversely, TGFβ1 overexpression may be a contributing factor in several inflammatory skin disorders. An examination of changes in TGFβ signaling among different inflammatory skin diseases will help delineate the conditions in which TGFβ signaling has a pathological role. Additional studies from multiple laboratories using our K5.TGFβ1wt mice will provide a more comprehensive understanding of the role of TGFβ1 overexpression in inflammatory skin diseases.

      Conflict of Interest

      The authors state no conflict of interest.

      ACKNOWLEDGMENTS

      The original study carried out in this laboratory was supported by NIH grants to XJW. The study on the effect of Avandia was partially supported by GSK. CW was supported by the National Institutes of Health under Ruth L Kirschstein National Research Service Award T32-CA106195.

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