Th17 cells favor inflammatory responses while inhibiting type I collagen deposition by dermal fibroblasts: differential effects in healthy and systemic sclerosis fibroblasts

Systemic sclerosis (SSc) is an autoimmune disorder of unknown origin characterized by fibro-proliferative microangiopathy and progressive fibrosis of the skin and internal organs [1, 2]. Fibrosis results from an overproduction of extracellular matrix (ECM) components by fibroblasts, especially type I collagen, accompanied by impaired ECM degradation. In early SSc, dermal fibroblasts display an inappropriate phenotype essentially characterized by increased proliferative potential, increased synthetic capacity, resistance to inhibitory signals and decreased apoptosis [1, 2]. In addition to collagens and matrix metalloproteinases (MMP), fibroblasts release several pro-inflammatory chemokines, such as monocytes chemoattractant protein (MCP)-1 and interleukin (IL)-8, which may indirectly influence ECM remodeling [3]. Of interest, MCP-1 and IL-8 are increased in the skin and serum of SSc patients [4, 5] and appear to be critical in mediating bleomycin-induced lung and dermal fibrosis [6, 7].

The mechanisms leading to dysregulated activation of fibroblasts in SSc are only partially understood. T cells infiltrate SSc skin early and fibroblasts with high synthetic activity localize in close proximity to the inflammatory infiltrate (reviewed in [8]). T helper (Th) 2 polarized responses have been shown to be dominant in SSc skin and lung [9‐13]. Consistently, IL-4 and IL-13 were shown to have direct pro-fibrotic activities on fibroblasts both in vitro and in vivo[14]. In addition, we and others have reported that SSc individuals have increased Th17 cell counts in their peripheral blood and skin [15‐21].

Th17 cells are physiologically implicated in protection against extracellular bacteria and fungi [22] and are thought to have pathogenic roles in various autoimmune diseases [23‐25]. Th17 cells mainly produce IL-17A, in conjunction with IL-17 F, IL-21 and IL-22, and are enriched in the subset of T cells expressing the chemokine receptors CCR4 and CCR6 in the absence of CCR10 [26, 27]. They further express the lectin receptor CD161 [28]. IL-17A has been shown to participate in the development of skin and lung fibrosis induced by bleomycin in mice [29, 30]. In agreement with a potential profibrotic role, IL-17 was shown to enhance fibroblast proliferation in humans [15], as well as their production of pro-inflammatory cytokines (MCP-1, IL-6 and IL-8) and matrix metalloproteinases (MMP-1 and MMP-3) [31, 32], and ICAM-1 expression [15]. However, Kurasawa and colleagues could not show enhanced type I and type III procollagen mRNA expression in human fibroblasts cultured in the presence of IL-17 [15]. Moreover, Nakashima et al. recently provided evidence for an anti-fibrotic effect of IL-17A in human fibroblasts via upregulation of miR-129-5p and downregulation of connective tissue growth factor and α1(I) collagen [33]. In agreement with these findings, we observed that IL-17 decreased alpha-smooth muscle expression induced by transforming growth factor β (TGF-β) in human fibroblasts and that the number of IL-17A + cells in SSc skin correlated inversely with skin fibrosis [34]. Thus, the role of Th17 cells in SSc remains uncertain. The aim of the present study was to investigate whether Th17 cells could promote phenotypic changes in dermal fibroblasts and compare fibroblast responses in healthy and SSc individuals. Our data highlight the direct role of Th17 cells in collagen inhibition accompanied by the simultaneous enhanced production of mediators of inflammation. Furthermore, the data stress the intrinsic resistance of SSc fibroblasts to inhibitory signals delivered by Th17 cells.

In the present report, we show that Th17 cells elicit MCP-1, IL-8 and MMP-1 responses while simultaneously inhibiting type I collagen production in healthy and SSc dermal fibroblasts. Our data are consistent with a model in which Th17 cells participate in inflammatory events but not directly in enhanced collagen deposition. In this perspective, Th17 cells may be seen as cells with an important role in limiting the development of fibrosis. In line with our data, a recent work by Nakashima et al. indicated that IL-17A may have direct anti-fibrotic effects in human normal fibroblasts via upregulation of miR-129-5p and downregulation of connective tissue growth factor and type I collagen [15]. According to these authors, SSc fibroblasts may escape the negative control of IL-17A because of a reduced expression of the IL-17RA [15]. In our experimental settings, diffuse SSc fibroblasts expressed increased IL-17RA mRNA levels but, in partial agreement with Nakashima et al., we observed that collagen production by SSc fibroblasts was more resistant to inhibition by Th17 cells. Additional in vivo evidence consistent with this model was obtained when we studied the number of IL-17A + cells in the skin of SSc individuals and found that the total skin thickness score was higher when IL-17A + dermal cells were less numerous [34]. Of interest, Th17 cell numbers can be increased both in vitro and in vivo by iloprost, a PGI₂ analog used in the clinical management of SSc digital ulcers, which may have beneficial effects on the disease course [39]. These data and our model are distinctly different from data and conclusions generated in rodents, in which IL-17 was shown to favor in vivo collagen deposition in models of bleomycin-induced skin as well as lung fibrosis [29, 30, 40]. Furthermore, in the thigh skin of mice lacking IL-17 the spontaneous fibrotic skin was reduced [30], and finally IL-17 neutralization decreased lung inflammation and fibrosis induced by silica [41]. The discrepancy between studies in humans and mice stresses species-specific differences in the responses induced by IL-17, as thoroughly discussed recently [8].

Our data clearly show that IL-17A directly promotes the production of pro-inflammatory mediators and MMP-1 by dermal fibroblasts from healthy and SSc individuals. Within the limits of the cohort investigated in this study, no differences were observed between limited and diffuse SSc individuals in this respect. These effects were largely amplified when supernatants from Th17 cell clones, producing high levels of IL-17, were assessed. Neutralizing experiments confirmed a critical role for IL-17A, at least in the case of IL-8, and revealed additive/synergic effects of IL-17 and TNF. Along this line of evidence, IL-17 was shown to enhance TNF-induced synthesis of IL-1, IL-6 and IL-8 by normal skin fibroblasts and osteoarthritis fibroblast-like synoviocytes [42]. MCP-1 and IL-8 are increased in skin and serum of SSc patients [5, 43] and reported to be critical in mediating lung and dermal fibrosis in bleomycin-treated mice [6, 7]. However, whether these mediators have direct pro-fibrotic activities in humans is controversial. An increase in α1(I) collagen mRNA was reported by northern blot hybridization in human dermal fibroblasts activated by MCP-1 [5], while later reports could not confirm these findings [44]. Similarly, MCP-1 was reported to increase the expression of MMP-1 and MMP-2, critical matrix degrading enzymes, but also the levels of their inhibitor TIMP-1 [45]. The role of these mediators in tissue fibrosis observed in mice may be related more to chemoattractant and angiogenetic properties than to a direct pro-fibrotic activity on fibroblasts or to its role in favoring priming of Th2 cells [46, 47].

We found that IL-17A enhanced MMP-1 production in dermal fibroblasts, as previously reported in human cardiac fibroblasts and fibroblast-like synoviocytes [48‐51]. MMPs participate in tissue remodeling, directly acting on ECM but also modulating the activity of many important mediators regulating matrix deposition [52]. Despite its role as a degrading enzyme, MMP-1 levels have been paradoxically shown to be highly increased in human lung fibrosis [53], and variably reported to be increased, unchanged or decreased in SSc [54‐57]. Thus, the exact role of MMP-1 in the development of fibrosis remains to be established.

We showed that IL-17A induced the production of pro-inflammatory chemokines preferentially via NF-κB and p38 signaling pathways, while inducing MMP-1 via JNK. Consistent with our data, IL-17 was previously shown to promote IL-6/IL-8 production via NF-κB/Akt and NF-κB/MAPK pathways in rheumatoid arthritis synovial fibroblasts and colonic myofibroblasts, respectively [58, 59] and in partial agreement with our findings, IL-17 induced MMP-1 production via activation of c-Fos/c-Jun AP1 and NF-κB in addition to MAPK signaling in cardiac fibroblasts [49].

Th17 cell clones were obtained after enrichment of cells expressing the chemokine receptor CCR6 and CCR4 in the absence of CCR10 [26, 27] and the lectin receptor CD161 [28]. By applying this strategy, we obtained more than 70% of cells producing IL-17A. Compared to the expected numbers, the cloning procedure resulted in a slight enrichment of clones co-producing IL-17 and IFN-γ (Th1/Th17 cells), suggesting a relationship between the Th1 and Th17 differentiation programs. In line with these results, a functional plasticity connecting Th1 and Th17 cells was recently reported both in vitro and in vivo[28, 38, 60], although IL-17+/IFN-γ + cells were shown to have a transcription profile closer to Th17 than to Th1 cells [38].

Of note, SSc fibroblasts were more prone to produce pro-inflammatory mediators (MCP-1, IL-8) and less sensitive to collagen inhibition when cultured in the presence of Th17 cell clone supernatants than their healthy counterpart. This suggests that SSc fibroblasts may escape or limit the anti-fibrotic effects induced by Th17 cells, and further stresses the existence of intrinsic differences between normal and SSc fibroblasts. In this context, it is worth noting that the inhibition of type I collagen production induced by the Th17 clone supernatants was partially reversed by blockade of IL-17 or TNF mainly in HD but not SSc fibroblasts while IFN-γ neutralization had opposite effects. Again, the joint blockade of IL-17, TNF and IFN-γ resulted in maximal effects, specifically in SSc but not HD fibroblasts. In agreement with previous evidence [36, 61], the present data strongly suggest that, compared to normal fibroblasts, SSc fibroblasts are more resistant to inhibitory mediators present in the Th17 cell clone supernatants.

In conclusion, our data are consistent with a model in which Th17 cells may participate in enhancing inflammation while simultaneously limiting fibrosis. It is worth noting that the contribution of Th17 cells to inflammatory conditions remains in many instances a matter of debate. As an example, the role of IL-17 in the initiation, progression and stabilization of atherosclerosis is currently controversially interpreted with evidence in favor of its proatherogenic potential and evidence in favor of its atheroprotective role [62]. Our findings stress for the first time the concomitant dual role of Th17 cells in the context of matrix deposition and may provide the functional basis for novel approaches to harness fibrotic diseases.