Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition with an autoimmune background (1). The presence of autoantibodies is a central feature of SSc, since antinuclear antibodies (ANAs), such as anti–DNA topoisomerase I (anti–topo I) antibody, are detected in >90% of patients (2). Furthermore, abnormal activation of several immune cells has been identified in SSc (3). A recent study has shown that skin and lung fibrosis is ameliorated by treatment with cyclophosphamide, an immunosuppressive agent, indicating that immune activation leads to fibrosis through the stimulation of collagen production by fibroblasts (4). Indeed, SSc patients exhibit infiltration of inflammatory cells, especially CD4+ T cells, and elevated serum levels of various cytokines, especially fibrogenic Th2 and Th17 cytokines and transforming growth factor β1 (TGFβ1), a major fibrogenic growth factor, which correlate positively with disease severity (5, 6).
Autoimmune responses with high levels of circulating autoantibodies are commonly detected in patients with rheumatic diseases (7). Furthermore, specific autoantibodies are associated with clinical subsets of a particular autoimmune disease. Anti–topo I antibody is detected more frequently in SSc patients with diffuse cutaneous thickening than in those with limited cutaneous thickening (8). The presence of anti–topo I antibody correlates positively with dermal sclerosis, pulmonary fibrosis, and overproduction of inflammatory cytokines (9–11). These data indicate that serum levels of anti–topo I antibody are associated with disease severity and/or activity in patients with SSc. In addition, anti–topo I antibodies have been detected in mouse models of SSc (12, 13). These studies strongly suggest a close relationship of autoimmune responses to the pathogenesis of SSc. However, the role of induction of anti–topo I antibody production and its potential contribution to the pathogenesis of SSc remain unclear.
A recent study indicated that mice immunized with recombinant human topo I protein emulsified in Freund's complete adjuvant (CFA) and boosted with topo I emulsified in Freund's incomplete adjuvant (IFA) showed anti–topo I antibody production (14). However, these mice did not show dermal and pulmonary fibrosis (14). Previously, in an experimental autoimmune encephalomyelitis (EAE) model, treatment with myelin oligodendrocyte glycoprotein (MOG) and IFA, in contrast to treatment with MOG and CFA, did not induce interleukin-6 (IL-6) production (15). In the absence of IL-6, Th17 cell responses are impaired whereas Treg cell responses are dominant, suggesting that IL-6 is a critical factor that shifts the immune response from Treg cell responses toward pathogenic Th17 cell responses (16, 17). Indeed, in contrast to treatment with MOG and CFA, treatment with MOG and IFA did not trigger antigen-specific production of IL-17 (15). Moreover, treatment with MOG and IFA did not induce EAE symptoms (15, 18). These data suggest that IL-6 induced by CFA plays important roles in the pathogenesis of autoimmune diseases. However, the relative contributions of the SSc-specific antigen, topo I, and IL-6 induced by CFA to the development of SSc remain unknown.
In this study, we investigated the associations of topo I immunization and of IL-6 induced by CFA with the development of SSc, using wild-type (WT) and IL-6–deficient (IL-6−/−) mice. According to our results, treatment with both topo I and CFA increased IL-6 and IL-17 production and Th17 cell frequencies compared to treatment with both topo I and IFA. In contrast, IL-10 production and Treg cell frequencies were greater in WT mice treated with topo I and IFA than in WT mice treated with topo I and CFA. Furthermore, treatment with topo I and CFA induced dermal sclerosis, pulmonary fibrosis, and anti–topo I antibody production, although treatment with topo I and IFA induced only anti–topo I antibody production. In addition, IL-6 deficiency reduced dermal sclerosis, pulmonary fibrosis, and IL-17 and anti–topo I antibody production as well as the increased Th17 cell frequencies induced by treatment with topo I and CFA, while increasing IL-10 production and Treg cell frequencies. These results suggest that subcutaneous treatment with topo I and CFA induces dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities, which are mainly regulated by IL-6.
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The precise mechanisms involved in the pathogenesis of SSc remain unknown, although autoimmunity is considered to be involved (3). The presence of anti–topo I antibody has been clinically associated with a more severe form of SSc that exhibits diffuse cutaneous and lung involvement (3, 9, 10, 28). In addition, a recent study showed that 20% of anti–topo I antibody–positive patients lost anti–topo I antibodies during the disease course and had a favorable outcome, suggesting the clinical importance of anti–topo I antibody levels in patients with SSc (29). The topo I protein is a ubiquitous and indispensable enzyme involved in DNA replication and protein transcription (30). Human topo I has >93% sequence identity to the 766 amino acid residues of mouse topo I (GenBank accession no. L20632). It has been demonstrated that immunization using a mutated self antigen is capable of inducing an autoreactive response that is more potent than that using the bona fide autoantigen in mice (31). Therefore, we immunized mice with recombinant human topo I as described previously (14, 32). The present study is the first to demonstrate that subcutaneous injection of topo I with CFA induces skin and lung fibrosis, hypergammaglobulinemia, and anti–topo I antibody production in WT mice (Figures 1 and 3), generating many characteristics of human SSc. Furthermore, treatment with topo I and CFA increased the production of various fibrogenic cytokines (Figure 2). Collectively, these results indicate that mice treated with topo I and CFA show SSc-like fibrosis and might be a novel animal model of human SSc.
In this study, treatment with topo I and IFA, in contrast to treatment with topo I and CFA, did not induce skin and lung fibrosis (Figure 1). Previous studies have demonstrated that CFA is essential for development of autoimmune responses, such as autoimmune encephalomyelitis, in each of the induction protocols (18). In contrast, IFA injection can prevent induction by CFA (33). This may be explained by the fact that CFA treatment induces IL-6 production, whereas IFA injection cannot affect IL-6 production (15, 34, 35). Indeed, in our present study, WT mice treated with topo I and CFA exhibited significantly higher levels of IL-6 relative to mice treated with topo I and IFA (Figure 2). Moreover, a recent study has shown that both IL-6 induced by CFA and exogenous IL-6 injection augment autoantibody production in immunized mice (36). In the present study, the levels of IgG anti–topo I antibody, IgG1, and IgG3 in WT mice treated with topo I and CFA were greater than those in WT mice treated with topo I and IFA (Figure 3). These results suggest that IL-6 induced by CFA contributes to development of fibrosis and augments autoantibody production. Furthermore, a recent case report has shown that the anti–IL-6 receptor antibody tocilizumab decreased skin sclerosis in SSc patients (37). In fact, IL-6 deficiency inhibited the development of fibrosis with decreased autoantibody production in mice treated with topo I and CFA (Figures 4 and 5). Thus, immunization with topo I can induce dermal and pulmonary fibrosis and hypergammaglobulinemia, which requires IL-6 induced by CFA.
Previous studies have demonstrated a fibrogenic effect of Th2 cytokines, such as IL-4 and IL-6 (13, 24, 38). Th17 cytokines, such as IL-17, also have a fibrogenic effect on dermal, pulmonary, and cardiac fibroblasts (39, 40). Indeed, SSc patients exhibit elevated serum levels of these cytokines, which promote collagen synthesis (5, 6, 27, 39, 41, 42). Some studies have also shown that IFNγ, a Th1 cytokine, has an antifibrotic effect (26, 27, 43, 44). In addition, IL-10 produced by Treg cells has antifibrotic and antiinflammatory effects on fibrotic diseases (45). Previously, we and others confirmed that IL-4 and/or IL-17 stimulation increased proliferation and collagen production of dermal fibroblasts, while these processes were inhibited by IFNγ and IL-10 (27, 45). The results of the present study indicate differential expression levels of these cytokines in mice treated with both topo I and adjuvant (Figure 2). Treatment with topo I and CFA induced significantly higher production of IL-6, IL-17, and TGFβ1 in parallel with increased dermal and pulmonary fibrosis. Treatment with topo I and IFA enhanced IL-10 production, which was accompanied by inhibited fibrosis. Thus, the differential expression levels of these cytokines that were induced by topo I and adjuvant treatment might contribute to the development of skin and lung fibrosis.
It is possible that treatment with topo I and adjuvant alters the frequencies of fibrogenic Th2 and Th17 cells and antifibrogenic Th1 and Treg cells. This may result in differential production of cytokines, which may then directly or indirectly influence the development of dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities. A recent study indicated that CD4+ cells play an important role in fibrosis, although these cells are a minority population (27). Moreover, previous studies have demonstrated that IL-6 together with TGFβ1 is capable of inducing Th17 cells (15). Furthermore, in the absence of IL-6, Th17 cell responses are impaired, whereas Treg cell responses are dominant, suggesting that IL-6 is a critical factor that shifts the immune response from a Treg cell response toward a pathogenic Th17 cell response (16). Consistent with these findings, the results of the present study showed that treatment with topo I and CFA increased Th17 cell frequencies and decreased Treg cell frequencies in parallel with increased IL-6 and TGFβ1 overproduction (Figures 2 and 6). In contrast, treatment with topo I and IFA increased Treg cell frequencies and decreased Th17 cell frequencies. Furthermore, lack of IL-6 expression inhibited accumulation of Th17 cells and increased numbers of Treg cells in mice treated with topo I and CFA (Figure 6).
Recently, other studies suggested that IL-6 also promotes Th2 cell differentiation and simultaneously inhibits Th1 cell polarization (46, 47). In the absence of any polarizing cytokine, IL-6 directs the differentiation of the CD4+ cells to a Th2 phenotype but not to a Th1 phenotype, since cells differentiated in the presence of IL-6 produce high amounts of IL-4 but not IFNγ (46, 47). IL-4 promotes Th2 differentiation but inhibits Th1 differentiation, although IFNγ stimulates Th1 differentiation but suppresses Th2 differentiation (48–50). Indeed, in the present study, treatment with topo I and CFA increased Th2 cell frequencies and decreased Th1 cell frequencies in WT mice (Figure 6). Moreover, IL-6 deficiency inhibited augmentation of Th2 cell frequencies and increased Th1 cell frequencies. Collectively, treatment with topo I and CFA may lead to increased Th2 and Th17 cell frequencies in parallel with deteriorated skin and lung fibrosis, whereas topo I and IFA treatment leads to increased Th1 and Treg cell frequencies that are accompanied by inhibited fibrosis of skin and lung.
To date, few studies have addressed the role of induction of anti–topo I antibody production and its potential association with the pathogenesis of SSc. This is the first systematic study to reveal that treatment with topo I and CFA induces SSc-like dermal sclerosis, pulmonary fibrosis, and autoimmune abnormalities in mice. However, the exact role of anti–topo I antibody in fibrogenesis and overproduction of cytokines remains unclear. Future studies, in which anti–topo I antibodies are isolated from mice treated with topo I and adjuvant and transferred into recipient mice, will be needed to clarify the exact role of anti–topo I antibody in this pathogenesis. We also suggest that IL-6 plays important roles in the development of fibrosis and autoimmune abnormalities in this novel model of SSc induced by treatment with topo I and CFA. These results provide additional clues to understanding the complexity of the pathogenesis of 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. Sato 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. Yoshizaki, Sato.
Acquisition of data. Yoshizaki, Yanaba, Ogawa.
Analysis and interpretation of data. Yoshizaki, Ogawa, Asano, Kadono, Sato.