The role of interleukin-17 in the pathogenesis of systemic sclerosis: Pro-fibrotic or anti-fibrotic?

Abstract

Systemic sclerosis is characterized by widespread fibrosis of the skin and internal organs, vascular impairment, and dysregulation of innate and adaptive immune system. Growing evidence indicates that T-cell proliferation and cytokine secretion play a major role in the initiation of systemic sclerosis, but the role of T helper 17 cells and of interleukin-17 cytokines in the development and progression of the disease remains controversial. In particular, an equally distributed body of literature supports both pro-fibrotic and anti-fibrotic effects of interleukin-17, suggesting a complex and nuanced role of this cytokine in systemic sclerosis pathogenesis that may vary depending on disease stage, target cells in affected organs, and inflammatory milieu. Although interleukin-17 already represents an established therapeutic target for several immune-mediated inflammatory diseases, more robust experimental evidence is required to clarify whether it may become an attractive therapeutic target for systemic sclerosis as well.

Introduction

Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology characterized by widespread fibrosis of the skin and internal organs, vascular impairment, and dysregulation of innate and adaptive immune system.^1,2 SSc has an unpredictable course, and available treatments only delay disease progression. In analogy to other autoimmune disorders, environmental, genetic, and immunological factors are all involved in the pathogenesis of SSc, but their relative contribution to the spectrum of scleroderma manifestations is not fully clarified. In view of its interplay with the immune system, the human microbiota also represents a potential player in the onset and progression of SSc. ³

Growing evidence indicates that T-cell proliferation and cytokine secretion play a major role in the initiation of SSc,^4–6 suggesting that T lymphocytes may orchestrate disease pathogenesis and the characteristic tissue fibrosis. ⁵ In particular, T helper 17 (Th17) cells represent a subset of T lymphocytes of major interest in SSc because of established roles in different fibro-inflammatory disorders. Th17 cells are increased in the peripheral blood of patients with SSc, accumulate at disease sites, and secrete the pro-inflammatory cytokine interleukin (IL)-17.^7,8 Yet, the exact contribution of Th17 cells and IL-17 to disease pathogenesis—whether pro-fibrotic or anti-fibrotic—remains a matter of debate.^9–11

Th17 cells may, in fact, display functionally distinctive features, and their inflammatory potential can be largely influenced by a number of environmental and immunological triggers. Looking at the gastrointestinal tract and at the commensal flora, for instance, non-inflammatory Th17 cells and IL-17A production are elicited by segmented filamentous bacteria via the engagement of the ILC3/IL-22/SAA1/2 axis, while Th17 cells with pro-inflammatory activity are induced by Citrobacter species. ¹² Hence, because of these nuanced inflammatory properties, it is possible that the role of Th17 cells and IL-17 in the pathogenesis of SSc and tissue fibrosis is not uniformly stimulatory or inhibitory, and likely depends on a complex and tightly regulated network of signals yet to be identified.

In the present review, we aim to discuss the most recent advances in our understanding of the contribution of Th17 cells in the pathogenesis of SSc, with particular focus on the debated pro-fibrotic and anti-fibrotic role of IL-17. A brief overview of IL-17 biology and of the potential therapeutic implications of blocking the IL-17 pathway will be also provided.

Immunobiology of IL-17

The IL-17 family comprises six cytokines: IL-17A (also named as IL-17, the first discovered and most studied member), IL17B, IL-17C, IL-17D (known as IL-27), IL-17E (also named as IL-25), and IL-17F.^13–15 IL-17 cytokines bind to their receptors (IL-17Rs) on target cells and exert pleiotropic functions^13,14,16,17 being involved in the sustainment of inflammation, autoimmunity, and tissue repair.^16,17 The IL-17Rs consist of five single-pass transmembrane receptors with conserved domains: IL-17RA, IL-17RB, IL-17RC, IL-17RD, and IL-17RE. ¹⁸ Functionally, the IL-17R is a heterodimeric complex composed of the IL-17RA subunit in combination with other subunits. IL-17A as well as IL-17F signal through the IL-17RA/IL-17RC heterodimer, but the IL-17A interaction with its receptors appears to be stronger. ¹⁸ IL-17RA is also used by IL-17B, IL-17C, and IL-17E. Of note, while the IL-17RA subunit is ubiquitously expressed, IL-17RC is mainly present on non-hematopoietic epithelial and mesenchymal cells. IL-17 exerts its in vivo functions through canonical and non-canonical signaling pathways. Canonical signaling induces both transcriptional and post-transcriptional mechanisms involved in autoimmunity and metabolic reprogramming of lymphoid tissues. Non-canonical signaling acts in synergy with other receptor systems—including transforming growth factor β (TGFβ), interferon γ (IFNγ), and tumor necrosis factor α (TNFα)—and is mainly responsible for tissue repair and regeneration. ¹⁸

IL-17 is primarily produced by Th17 cells, but several other cell types are able to produce it such as gamma delta (γδ) T cells, invariant natural killer T cells, group 3 innate lymphoid cells, natural killer cells, double-negative T cells, B lymphocytes, and mast cells.^7,8,19,20 Th17 cells arise from naive CD4⁺ T cells, and their maturation is influenced by TGFβ, IL-6, and IL-1β.^13,21 Conversely, cytokines secreted by Th1 and Th2 cells, such as IFN-c and IL-4, respectively, exert an inhibitory action on Th17 cells. ²¹ IL-23 is also crucial for Th17 differentiation, and the IL-23/IL-17 axis has been long regarded as a redundant pathway for perpetuating chronic inflammation. ²¹ Indeed, IL-23 has been shown to cause inflammation through IL-17-dependent and IL-17-independent pathways, increasing the complexity around Th17 pathophysiology and IL-17 signaling. ²² Th17 cells synthesize the IL-17A and IL-17F isoforms of IL-17 as well as additional pro-inflammatory cytokines such as IL-21 and IL-22. ¹⁴ Dual role of this cytokine armamentarium is to perpetuate inflammation via IL-17 and IL-22, and to inhibit regulatory T cells (Tregs) through IL-21, thus sustaining inflammatory responses. ¹⁴ In view of these pro-inflammatory properties, Th17 cells participate in a number of physiological processes including (1) neutrophil recruitment in inflammatory niches; (2) response to fungi, mycobacteria, and viruses; and (3) remodeling of the extracellular matrix (ECM).^13,15 Th17 cells have been also implicated in autoimmune disorders such as psoriasis and inflammatory bowel diseases, but conflicting data exist regarding their engagement in SSc. ²³ Similarly, regardless of its cellular source, the relevance of IL-17 in the pathogenesis of scleroderma remains to be fully addressed and the question whether it would be beneficial or detrimental to neutralize it in this specific pathological setting needs to be unequivocally answered. Animal models of scleroderma, in fact, essentially support a pro-fibrotic and a pro-inflammatory activity of IL-17A, but in vitro studies point toward an anti-fibrotic action.^24,25 Yet, although experiments with human fibroblasts in plastic plates have intrinsic limitations due to nonphysiological conditions of cell culture and, hence, result interpretation, in vivo murine models also require careful consideration because of intrinsic differences between humans and rodents with regard to immunological responses and fibrogenesis.

Evidence in favor of a pro-fibrotic role of IL-17 in SSc

Involvement of IL-17 and of its downstream pathways in the pathogenesis of SSc has been first hypothesized following the accumulation of experimental evidence showing the pro-fibrotic effects of this cytokine on a series of organ-specific chronic disorders.^26,27 In experimental models of liver cirrhosis, idiopathic pulmonary fibrosis, and heart failure, in fact, IL-17 participates in fibrogenesis via different mechanisms including direct activation of resident stromal cells and amplification of the inflammatory response.

In liver fibrosis, IL-17 and IL-17-producing cells contribute to disease pathology both by perpetuating inflammation in synergy with IL-1β, IL-6, and IL-23, and by inducing the expression of TGFβ and TGFβ receptor by hepatic stellate cells.^28–31 IL-17 also promotes the release of periostin by hepatocytes leading to fibroblast activation and collagen production. ³² Accordingly, inactivation of IL-17 signaling in hepatocytes reduces liver fibrosis in mouse models of hepatitis-induced liver injury. ³³ In the bronchial mucosa of patients with moderate-to-severe asthma, IL-17 is highly expressed and drives epithelial-to-mesenchymal transition when used to stimulate human small airway epithelial cells in in vitro cultures.^34,35 In patients with idiopathic pulmonary fibrosis as well as in bleomycin-induced murine models of lung fibrosis, Th17 cells increase the production of TGFβ and IL-17A, and co-culture of these cells with human lung fibroblasts results in increased deposition of collagen and other ECM proteins. ³⁶ Indeed, blocking IL-17 ameliorates lung fibrosis in murine models of post-bone marrow transplant pulmonary disease. ³⁷ Finally, IL-17 produced by γδ T cells and Th17 cells plays a role in different models of cardiac fibrosis, likely representing a central pro-fibrotic mediator of multiple mechanical, toxic, and inflammatory insults in these organs. IL-17, in fact, activates cardiac myofibroblasts in ischemia- or isoproterenol-induced mouse models of heart failure as well as in spontaneous or angiotensin II–induced models of hypertension.^38–44 Blockade of IL-17, of its receptor, or of IL-17 signaling in these experimental scenarios all reduces cardiac fibrosis along with improvement of cardiac contractile function.^38–44

Initial observation in support of a pathogenic role of IL-17 in SSc came from the pathological evidence of IL-17 producing cells in the inflammatory infiltrate of scleroderma lesions, including Th17 cells, topoisomerase-specific CD4⁺ Th cells, and γδ T cells.^27,45–47 Circulating Th17 cells and serum IL-17 concentrations were also found to be significantly increased in active scleroderma patients compared to healthy controls, and to correlate with disease activity as defined by the Valentini score.^48–50 Yet, when addressing direct pro-fibrotic effects of IL-17, only two in vitro studies out of a dozen were able to show a stimulatory activity on fibroblast growth and collagen production.^48,51 Most in vitro studies, in fact, seem to support a general pro-inflammatory role of IL-17 in SSc rather than an intrinsic pro-fibrotic one. In particular, when stimulated with IL-17A, dermal fibroblasts as well as endothelial cells increase the expression of adhesion molecules and the production of a variety of pro-inflammatory mediators such as chemokine ligand (CCL)2, CCL8, IL-1, IL-6, and matrix metalloproteinase 1 (MMP1).^{25,27,48,49,52,53} Indeed, these cytokines and chemokines are highly expressed in the skin of scleroderma patients, enhance the recruitment of inflammatory cells, and ultimately amplify pro-fibrotic responses in fibroblasts.^54–56

Various mouse models of SSc also indicate that IL-17 consistently favors the development of tissue fibrosis primarily through the amplification of a fibro-inflammatory response. Lung fibrosis induced by Saccharopolyspora rectivirgula and bleomycin, for instance, was attenuated in IL-17^−/− mice with less collagen deposition and expression of ECM-associated genes.^36,57 In the bleomycin-induced murine model of cutaneous scleroderma, administration of bleomycin increased the recruitment of Th17 cells to the skin as well as the local secretion of IL-17. ⁵⁸ Of note, IL-21 and B cell activating factor (BAFF)—two key cytokines in the proliferation and activation of T and B cells—also seemed to boost IL-17-mediated fibrosis downstream to IL-17 in this model.^59,60 In addition, IL-17 both promoted collagen deposition and prevented collagen degradation in a TGFβ-independent manner. ⁶¹ These findings were confirmed in the “tight skin mouse 1” (TSK-1) mouse model, wherein blockade of IL-17 reduced the severity of skin fibrosis. ⁶² In other models of IL-1 receptor antagonist deficient and “graft versus host disease” murine models of scleroderma, severe dermal and pulmonary fibrosis induced by bleomycin or splenocyte injection was reversed by knocking down IL-17. ⁵⁸ Similarly, IL-1β-treated IL-17A-deficient mice showed reduced pulmonary inflammation and fibrosis, and delivery of IL-17A resulted in increased TGFβ-dependent collagen deposition, suggesting that IL-1β and IL-17 exerted a synergistic effect on the expression of pro-fibrotic and inflammatory mediators. ³⁶

Taken together, this in vitro and in vivo evidence indicates that IL-17 clearly contributes to SSc pathogenesis and, specifically, to the characteristic fibrotic outcomes but likely sustains tissue fibrosis indirectly by amplifying inflammation and by enhancing TGFβ-driven collagen secretion. Indeed, pro-inflammatory properties of IL-17 are well established in literature and fibroblasts are known to respond to IL-17 by increasing the expression of inflammatory mediators, adhesion molecules, and MMPs.^{25,27,48,49,52,53} In theory, IL-17 may also contribute to SSc pathogenesis beside its effects on stromal cells by boosting the production of autoantibodies.^63,64 By enhancing the retention of B cells in germinal centers, in fact, IL-17 can favor the emergence of autoreactive clones and the generation of high-affinity autoantibodies with pro-fibrotic activity.^63,64

In conclusion, a body of experimental data sustain a role of IL-17 in the initiation and progression of scleroderma. However, two major points need to be stressed when considering translation of these results from murine models of SSc to humans. On one hand, in fact, because most of these models are focused on skin involvement, it is possible that different IL-17-mediated mechanisms—such as those described in the context of heart failure and liver and pulmonary fibrosis—may contribute to tissue fibrosis in the multiple organs potentially affected by SSc depending on the inflammatory context and on the distribution of IL-17 receptors on resident cells. On the other hand, because most evidence obtained in murine models points toward an indirect role of IL-17 in modulating tissue fibrosis, partial to no amelioration of clinical outcomes should be taken into account when targeting only this cytokine within a complex system of redundant and interconnected pathways.

Evidence in favor of an anti-fibrotic role of IL-17 in SSc

The role of IL-17 in SSc is complex, incompletely understood, and controversial. Although there is consistent evidence regarding the pro-fibrotic role of this cytokine, there are also data suggesting an opposite, anti-fibrotic effect. The role of IL-17 in regulating ECM expression is yet unexplained.

In a 2012 study, Nakashima et al. ⁶⁵ compared IL-17A, IL-17 receptor (IL-17RA), and IL-17F expression in patients with SSc and healthy controls. In addition to serum measurements, skin biopsies obtained from patients with SSc, dermatopolymyositis, and healthy individuals were analyzed. The results showed an increased level of serum IL-17A in patients with SSc compared to healthy subjects, while IL-17F levels were similar among different conditions. Furthermore, there was no correlation between IL-17A and IL-17F levels in individual patients. Correlations between IL-17A levels and certain clinical features have also been identified. In patients with an elevated concentration of IL-17A, the prevalence of pitting scars was significantly higher compared to patients with normal levels of IL-17A. The authors also identified increased expression of IL-17A mRNA in the affected skin of patients with SSc (but not in the normal skin of these patients) compared to controls, both in the superficial and deep dermis. However, this was not observed for IL-17F mRNA. Expression of IL-17RA in the skin of patients with SSc both in vitro (in fibroblast cultures) and in vivo (on skin biopsies) was lower than that observed in healthy individuals. In particular, in vitro expression of IL-17RA in SSc fibroblasts was downregulated by TGFβ1 signaling, and IL-17A levels increased as a result of a negative-feedback mechanism. This led to an increased accumulation of collagen and to fibrosis. Another observation of this study was that the expression of connective tissue growth factor (CTGF) mRNA—a factor involved in the induction of fibrosis and the pathogenesis of SSc—decreased under the influence of IL-17A but not of IL-17F. ⁶⁵

Although several studies have shown a higher Th17 cell count and increased IL-17 expression in the affected skin of patients with SSc compared to healthy subjects, it is noteworthy that no differences were observed in IL-17 expression between patients with diffuse or limited forms of the disease or between those with early forms or those with late forms.^52,65 There are also several studies that have shown that the modified Rodnan skin thickness score (mRSS) is lower in patients with elevated serum levels of IL-17A.^52,66

Other arguments in favor of the anti-fibrotic role of IL-17A are provided by data suggesting that the synthesis of α1 (I) collagen by fibroblasts is reduced under the influence of this cytokine. ⁶⁵ In particular, microRNA analysis showed that by increasing miR-129-5p—an inhibitor of α1 (I) collagen SSc fibroblasts—IL-17A decreases collagen expression. ⁶⁷ These effects were not observed using IL-17F. ⁶⁵ IL-17A also reduces the differentiation of fibroblasts into myofibroblasts under the action of TGFβ and directly stimulates the production of MMP1 by dermal fibroblasts.^52,68

In addition to type I collagen and other components of the ECM, activated fibroblasts in the skin of patients with SSc, especially those in the deep dermis, express α-smooth muscle actin (α-SMA). ⁶⁹ Data have shown that IL-17A does not induce the expression of this protein, but inhibits the stimulatory effect of TGFβ, both in fibroblasts of patients with SSc and in healthy individuals, ⁵² suggesting a direct negative influence on dermal fibrosis. ⁵² Whether IL-17 increases the expression of type I and type III procollagen mRNA in human fibroblast cultures has not been unequivocally demonstrated. ²⁷ By stimulating the synthesis of MMP1 by dermal fibroblasts, but not type I collagen, IL-17A also increases turnover of the ECM and alters collagen metabolism in SSc. ⁷⁰

Skin homeostasis is tightly regulated by the cooperation of keratinocytes and fibroblasts. Disruption of this homeostasis can lead to fibrosis. ⁷¹ Recent data from a study that used an original organotypic skin model proposed that IL-17A may influence the interaction between keratinocytes and fibroblasts in SSc. ⁷² Under the influence of IL-17 and keratinocyte-conditioned media, in fact, fibroblasts from patients with SSc increased MMP1 production and ECM degradation. This effect was reversed by the addition of TGFβ, supporting opposite functions of IL-17 and TGFβ in this specific model. ⁷² The authors concluded that despite its strong pro-inflammatory effect, IL-17 also has anti-fibrotic effects, which may be of particular importance in the pathogenesis of SSc.

All together, the above evidence suggests that IL-17A can play a dual role in inflammation (Figure 1). Although there is a body of literature supporting its pro-fibrotic effects, IL-17A appears at the same time able to limit, if not fully prevent, fibrosis in SSc patients.^70,73,74

Figure 1.

Dual effect of IL-17 in the pathogenesis of SSc: pro-fibrotic and anti-fibrotic. IL-17A is secreted by different immune cell types but prominently by Th17 cells. IL-17A may instruct plasma cell production of autoantibodies and, indirectly, activate quiescent fibroblasts.

IL-17A: interleukin-17A; CCL: chemokine (C-C motif) ligand; MMP: matrix metalloproteinase; IL-6: interleukin-6; IL-1: interleukin-1; CTGF: connective tissue growth factor; TGF: transforming growth factor; IL-17RA: interleukin-17 receptor; SMA: smooth muscle actin; ECM: extracellular matrix, ILC3: innate L-cell3; NK: natural killer; EMT: epithelial-mesenchymal transition.

Treatment perspectives

IL-17 represents an interesting therapeutic target for autoimmune diseases, and its direct neutralization is currently approved for treating psoriasis, psoriatic arthritis, inflammatory bowel diseases, and ankylosing spondylitis.^75–78 Indeed, IL-17 is also indirectly targeted by other immunosuppressive drugs already available in immunological settings, thus widening their anti-inflammatory activity. Anti-TNFα therapy, for instance, decreases IL-17 in rheumatoid arthritis and ankylosing spondylitis^79–81 by inducing Treg cells and by modulating the IL-6 signaling.^82,83 Rituximab does not affect TNFα or Treg cell responses, but strongly reduces IL-17 and IL-22 levels in patient sera. ⁸⁴ Allogeneic adipose-derived stem cells (ADSCs) in patients with active systemic lupus erythematosus reduced the number of Th17 cells and their ability to release IL-17. ⁸⁵ On the other hand, conflicting data have been reported on the ability of methotrexate to modify IL-17 levels. ⁸⁶

As reported above, IL-17 represents a therapeutic target of great interest also for SSc, and modulation of Th17 cells and IL17 pathways correlate with the clinical response to currently used therapeutic strategies. Truchetet et al., ⁸⁷ for instance, showed that prostacyclin analogs significantly increase IL-17A and IL-22 production in vitro and the frequency of Th17 cells in vivo. Yet, clinical information regarding the effects of targeting IL-17 in patients with SSc is still limited. The promising results obtained with tocilizumab in SSc and the comparative experience of anti-IL-17 and IL-6 monoclonal antibodies in other immune-mediated disorders may, indeed, anticipate a potential argument against the utility of blocking IL-17 in SSc.^88–90 Targeting IL-17, in fact, did not prove effective in diseases responsive to anti-IL-6 biologic agents such as rheumatoid arthritis, while showing positive results in disorders that failed to respond to anti-IL-6 therapy such as psoriatic arthritis.^91–93 At present, there are two ongoing phase I (Clinicaltrials.gov identifier: NCT04368403) and phase III (Clinicaltrials.gov identifier: NCT03957681) clinical trials assessing IL-17 inhibition in SSc with brodalumab. Brodalumab is a human anti-IL-17 receptor monoclonal antibody binding to human IL-17RA and blocking the biological activity of IL-17A and IL-17F.^94,95 The phase I trial is an open-label Japanese study of 210-mg subcutaneous brodalumab every 2 weeks in patients with moderate to severe progressive skin thickening and an mRSS of 10–30 but without severe lung disease. The primary and secondary aims of the study are to evaluate serum concentrations of brodalumab at different intervals until completion and to assess changes in the mRSS compared to baseline, respectively. The phase III trial is a placebo-controlled, double-blind comparative Japanese study of brodalumab with an open-label extension period in SSc patients with moderate to severe skin thickening. The primary aim of the study is to assess changes in the mRSS at week 24 and 52. Results of both trials are expected to be released in 2024. The two other anti-IL-17 drugs available on the market (ixekizumab and secukinumab) have not been tested in SSc yet. Looking ahead, because of the central role of IL-23 in the development of Th17 cells and in view of the non-redundant and independent functions of these two cytokines, dual inhibition of both IL-23 and IL-17 could offer even greater efficacy for treating SSc relative to targeting IL-17 alone.

Finally, due to potential influence of microbiota on the immune system and on IL-17 production, the efficacy of probiotics on gastrointestinal symptoms and immune responses in SSc patients has been recently evaluated. Maringhela TF et al. reported that the use of probiotics reduced Th17 cells in patients with SSc after 8 weeks of treatment. This finding indicates a possible effect of probiotics on the systemic immune system and might represent an innovative therapeutic approach for patients with SSc. ⁹⁶

Conclusive remarks

Dissecting the pleiotropic role of IL-17 is of crucial importance to drive definitive conclusions about the therapeutic potential of blocking its effects in scleroderma patients. In particular, additional in vivo data are eagerly awaited to better understand whether interfering with IL-17 might be beneficial to arrest collagen deposition and tissue fibrosis or, rather, detrimental in SSc. In this regard, understanding what regulates the pro-fibrotic as well as the anti-fibrotic outcomes of IL-17 is pivotal to properly tune the in vivo biology of this cytokine. As this dual activity possibly depends also on the distribution of IL-17 receptors on target organs and on different intracellular pathways on target cells, useful information may be derived in the future by clinical studies assessing the efficacy of IL-17 inhibitors on organ-specific fibrotic disorders such as liver and lung fibrosis.