Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by excessive extracellular matrix deposition in the skin, lungs, and other internal organs (1, 2). A growing body of evidence suggests that overproduction of extracellular matrix components by activated fibroblasts results from complex interactions between various cells, including leukocytes and fibroblasts, and via several soluble mediators, such as cytokines, chemokines, and growth factors (1, 2).
An emerging hypothesis for the pathogenesis of fibrotic disorders suggests that an imbalance between Th1 cytokines and Th2 cytokines leads to abnormal responses to tissue injury. Th2 cytokines such as interleukin-4 (IL-4), IL-6, and IL-13 stimulate the synthesis of collagen by human fibroblasts (3). In contrast, Th1 cytokines such as interferon-γ (IFNγ) and tumor necrosis factor α suppress collagen production by fibroblasts in vitro (3). Therefore, in general, a relative shift toward Th2 cytokine production rather than Th1 cytokine production can induce tissue fibrosis.
In patients with SSc, circulating T cells and T cells infiltrating the skin or lungs demonstrate a predominantly Th2 profile (4–6). Furthermore, a recent study suggested that the frequency of circulating Th17 cells is strikingly increased in patients with SSc (7). Th17 cells, which were discovered in 2007, are the third T helper cell subset that can produce IL-17A (8, 9). Th17 cells also secrete IL-17F, IL-21, and IL-22. The differentiation factors transforming growth factor β (TGFβ) plus IL-6 or IL-21, the growth and stabilization factor IL-23, and the transcription factors RORγt, RORα, and STAT-3 have been considered to be involved in the development of Th17 cells. This T helper cell subset may contribute not only to inflammation but also to fibrosis via production of IL-17A and other cytokines (10). In fact, elevated serum IL-17A levels and augmented IL-17A expression in peripheral blood lymphocytes and lesional skin have been reported in patients with SSc (7, 11, 12), although reduced plasma IL-17A levels were detected in a recent study (13).
Skin fibrosis induced by daily intradermal bleomycin injections is widely used as a representative animal model of SSc (14). Although a variety of factors have been reported to contribute to the fibrotic process, the exact mechanism remains unclear. In a previous study, administration of recombinant IFNγ, a representative Th1 cytokine, attenuated bleomycin-induced skin fibrosis (15). IFNγ-deficient mice have modest bleomycin-induced lung fibrosis (16). IL-4 signaling has been reported as critical for the development of increased skin thickness in the TSK-1 (TSK/+) mouse model, a genetic model of SSc (17, 18). In contrast, IL-4 is not required for the development of bleomycin-induced lung fibrosis (19). However, both bleomycin- and IL-1β–induced lung fibrosis are dependent on IL-17A (10). The specific roles of IFNγ, IL-4, and IL-17A in bleomycin-induced skin fibrosis have not been investigated using mice deficient for each of these cytokines.
In the current study, we demonstrated that IL-17A, but not IFNγ or IL-4, has pivotal roles in the development of skin fibrosis in a murine model of bleomycin-induced skin fibrosis. Furthermore, skin thickness was attenuated by IL-17A deficiency in TSK/+ mice, another model of skin fibrosis (20).
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By using cytokine-deficient mice, we determined that IL-17A, but not IFNγ or IL-4, is necessary for maximal bleomycin-induced skin fibrosis. Among mice deficient for IFNγ, IL-4, or IL-17A, only IL-17A–deficient mice demonstrated reduced skin fibrosis following bleomycin treatment. Daily intradermal bleomycin injections triggered IL-17A production in the lesional skin. Additionally, IL-17A was critical for the induction of TGFβ and CTGF in fibrotic skin during bleomycin treatment. The addition of recombinant IL-17A to a skin fibroblast cell line increased the production of collagen, TGFβ, and CTGF. Furthermore, the loss of IL-17A also significantly reduced skin thickness in another model of SSc, TSK/+ mice, suggesting the critical roles of IL-17A in skin fibrosis.
To our knowledge, this study is the first to identify specific roles for IL-17A in the bleomycin-induced skin fibrosis model, via the use of transgenic mice or blocking antibodies. Recently, IL-17A has been implicated in the development of tissue fibrosis and SSc. For example, IL-17A was shown to be critically involved in the development of bleomycin-, silica-, or IL-1β–induced lung fibrosis (10, 28, 29), and serum IL-17A levels were increased in mice treated with daily intradermal bleomycin injections (30). Elevated serum IL-17A levels and augmented IL-17A expression in peripheral blood lymphocytes and lesional skin have also been reported in patients with SSc (11, 12). Another study demonstrated that patients with SSc had markedly increased numbers of circulating Th17 cells (7, 31). In addition, a crucial role for IL-17A in sclerodermatous chronic graft-versus-host disease has been reported (32). Our findings support these observations and extend them, by providing a potential and possibly important function for IL-17A in promoting skin fibrosis.
To clarify the mechanism behind IL-17A regulation of fibrosis, we first determined whether bleomycin could induce IL-17A production. Splenic T cells from WT mice treated intradermally with bleomycin had augmented IFNγ, IL-6, IL-17A, and TGFβ1 mRNA expression; the increase in IL-17A expression was most striking. In addition, daily intradermal bleomycin injections markedly increased the IL-17A concentration in lesional skin, with a peak on day 7. These findings are similar to those of a recent study that showed elevated IL-17A expression in the bronchoalveolar lavage fluid and lung tissue of mice with bleomycin-induced lung fibrosis (10). In our study, daily bleomycin injections increased the potential source of Th17 cells in splenocytes. These findings suggest that the augmented IL-17A production induced by bleomycin treatment cooperated with other proinflammatory or profibrogenic cytokines to promote the development of skin fibrosis.
Although IL-17A has been shown to promote fibroblast proliferation, the direct effect of IL-17A on collagen synthesis in humans has not been reported (11). Therefore, IL-17A may be contributing to the development of skin fibrosis indirectly via induction of other molecules critical for fibrosis or inflammation. To examine this possibility, we examined mRNA expression of profibrogenic molecules in the bleomycin-induced fibrotic skin of IL-17A−/− mice, using real-time RT-PCR. The expression of TGFβ, CTGF, and ICAM-1 mRNA was dramatically reduced by the loss of IL-17A. Because TGFβ and CTGF are central players in the fibrotic process (1), induction of these cytokines by IL-17A is likely important for the development of skin fibrosis in vivo.
ICAM-1 is also considered to be important for skin fibrosis and/or skin inflammation in both bleomycin-induced skin fibrosis and in TSK/+ mice (30, 33). Our results are consistent with those from a prior study that revealed roles for IL-17A in ICAM-1 induction in human endothelial cells (11). Furthermore, human IL-17A has been shown to induce cytokines such as IL-6, IL-8, and granulocyte colony-stimulating factor from fibroblasts (34). Our data agree with these previous findings and indicate that IL-17A may be contributing indirectly to bleomycin-induced skin fibrosis via induction of adhesion molecules and various profibrogenic and proinflammatory cytokines.
Consistent with these in vivo data, we observed that recombinant IL-17A enhanced the expression of both TGFβ and CTGF in a cultured mouse skin fibroblast cell line. Furthermore, the addition of IL-17A increased collagen synthesis in these fibroblasts. In a recent study, mRNA levels of Colα2(I) and TGFβ were shown to be increased in cultured fibroblasts derived from mouse skin (30). Although our current results are similar to those of the previous study, we also detected increased CTGF expression in cultured fibroblasts following the addition of IL-17A. However, it is unclear whether the augmented collagen synthesis was directly caused by IL-17A. Another previous study showed that intratracheal administration of IL-17A induced lung fibrosis that was TGFβ dependent (10). Furthermore, an analysis of T helper cell subsets demonstrated that Th17 cells were the main producers of TGFβ1 both in vivo and in vitro (35). Therefore, our results for fibroblasts may also be reflecting the effects of increased TGFβ production. Further studies will be needed to determine whether IL-17A is directly affecting CTGF production by fibroblasts, and whether CTGF is contributing to IL-17A–induced collagen synthesis.
IL-17A and Th17 cells have been considered to be critical mediators of various autoimmune diseases and inflammatory bowel or skin diseases, in addition to their roles in host defense (8, 9). However, fibrotic changes are uncommon in these disorders. This may be due to different functions and/or expression of IL-17A or Th17 cells that is dependent on each disease, organ, phase, and environment (expression of other cytokines). In fact, recent studies showed significant plasticity of Th17 cells and suggested the possibility that the pathogenetic function of these cells may be mediated through a Th17-to-Th1 phenotype (IFNγ producer) transition in other autoimmune conditions such as autoimmune colitis and diabetes (36, 37). In contrast, Th17 cells require TGFβ for sustained expression of IL-17A (37). Therefore, in a TGFβ-rich environment such as fibrotic disorders (including SSc), Th17-to-Th1 conversions may be infrequent compared with the conversions that occur in other autoimmune or inflammatory disorders. Furthermore, TGFβ is highly expressed by Th17 cells and acts in an autocrine manner to maintain Th17 cells (35). Therefore, the function of IL-17A and Th17 may be different depending on the profile of cytokines and Th cell subsets that contribute to the development of each disease.
Recently, a list of criteria to use in selecting the most promising molecular targets for SSc trials was proposed (38). According to those criteria, the antifibrotic effects of specific molecules should be confirmed in at least 2 complementary animal models of SSc. Although in the current study we mainly analyzed the roles of IL-17A in a model of bleomycin-induced SSc, similar findings were also observed in TSK/+ mice. Bleomycin-induced skin fibrosis is characterized by dense inflammatory cell infiltration in lesional skin. Inflammatory cells have been considered to contribute to the development of skin fibrosis by stimulating profibrogenic cytokine production from fibroblasts. In contrast, TSK/+ mice are characterized by the absence of inflammation and by endogenous activation of fibroblasts. Thereby, the bleomycin-induced model and the TSK/+ mouse model mimic the early progressive stage and the late stage of SSc, respectively (27). Thus, our data indicate that IL-17A has fibrotic effects in 2 mouse models of SSc with different pathologic mechanisms.
We believe that further studies that include the administration of blocking antibody will be needed to clarify the roles of IL-17A in the development of SSc. Nonetheless, our findings suggest that inhibition of IL-17A represents a promising therapeutic target for antagonizing fibrotic skin disorders such as SSc. Because anti-human monoclonal antibodies against IL-17A or IL-17 receptor have been used for clinical trials in other autoimmune and inflammatory disorders (39–41), this strategy could be rapidly advanced into clinical trial testing in patients with SSc.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Hasegawa had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Okamoto, Hasegawa, Fujimoto.
Acquisition of data. Okamoto.
Analysis and interpretation of data. Okamoto, Hasegawa, Matsushita, Hamaguchi, Huu, Iwakura, Fujimoto, Takehara.