Bleomycin-induced Systemic Sclerosis Research Articles

Attenuation of murine sclerodermatous models by the selective S1P1 receptor modulator cenerimod

Sphingosine-1-phosphate (S1P), a lipid mediator, regulates lymphocyte migration between lymphoid tissue and blood. Furthermore, S1P participates in several physiological phenomena including angiogenesis, inflammation, immune regulation, and neurotransmitter release. Moreover, S1P/S1P receptor signaling involves in systemic sclerosis (SSc) pathogenesis. This study aimed to investigate whether the selective S1P1 receptor modulator cenerimod attenuates murine sclerodermatous models. Cenerimod was orally administered to murine sclerodermatous chronic graft versus host disease (Scl-cGVHD) mice, either from day 0 to 42 or day 22 to 42 after bone marrow transplantation. Bleomycin-induced SSc model mice were administered cenerimod from day 0 to 28. Early cenerimod administration inhibited, and delayed cenerimod administration attenuated skin and lung fibrosis in Scl-cGVHD mice. Cenerimod suppressed the infiltration of CD4+ T cells, CD8+ T cells, and CD11b+ cells into the inflamed skin of Scl-cGVHD mice as opposed to control mice. In contrast, cenerimod increased the frequency of regulatory T cells in the spleen and skin of Scl-cGVHD mice. Additionally, cenerimod attenuated the mRNA expression of extracellular matrix and fibrogenic cytokines in the skin. Furthermore, cenerimod attenuated bleomycin-induced fibrosis in the skin and lung. Hence, the selective S1P1 receptor modulator cenerimod is a promising candidate for treating patients with SSc and Scl-cGVHD.

Salvianolic acid B attenuates experimental skin fibrosis of systemic sclerosis

Systemic sclerosis (SSc) is a connective tissue disease characterized mainly by fibrosis of skin and internal organs. Our previous study has shown that salvianolic acid B (SAB), a bioactive component extracted from Salvia miltiorrhiza (SM), was one of the essential ingredients in the traditional Chinese medicine Yiqihuoxue formula, which has been used to treat SSc-related dermal and pulmonary fibrosis. The aim of the present study was to evaluate the effect of SAB on skin fibrosis and explore its underlying anti-fibrotic mechanism. We found that SAB was capable of alleviating skin fibrosis in a bleomycin-induced SSc mouse model, alleviating skin thickness and reducing collagen deposition. in vitro studies indicated that SAB reduced SSc skin fibroblast proliferation and downregulated extracellular matrix gene transcription and collagen protein expression. TGF-β/SMAD and MAPK/ERK pathway activation were also shown to be suppressed in SAB treated fibroblasts. Moreover, RNA-seq revealed that the anti-fibrotic effect of SAB might be related to antioxidant activity, the cell cycle, and the p53 signaling pathway. Taken together, our results suggest that SAB has the ability to alleviate SSc-related skin fibrosis both in vivo and in vitro.

RGC32 Promotes Bleomycin-Induced Systemic Sclerosis in a Murine Disease Model by Modulating Classically Activated Macrophage Function.

Systemic sclerosis (SSc) is a multisystem autoimmune disorder that is characterized by inflammation and fibrosis in the skin and internal organs. Previous studies indicate that inflammatory cells and cytokines play essential roles in the pathogenesis of SSc; however, the mechanisms that underlie the inflammation-driven development of SSc are not fully understood. In this study, we show that response gene to complement 32 (RGC32) is abundantly expressed in mouse macrophages in the early stage of bleomycin-induced SSc. Importantly, RGC32 is required to induce the inflammatory response during the onset of SSc, because RGC32 deficiency in mice significantly ameliorates skin and lung sclerosis and inhibits the expression of inflammatory mediators inducible NO synthase (iNOS) and IL-1β in macrophages. RGC32 appears to be a novel regulator for the differentiation of classically activated macrophages (M1 macrophages). IFN-γ and LPS stimulation induces RGC32 expression in primary peritoneal macrophages and bone marrow-derived macrophages. RGC32 deficiency impairs the polarization of M1 macrophages and attenuates iNOS and IL-1β production. Mechanistically, RGC32 interacts with NF-κB proteins and promotes iNOS and IL-1β expression by binding to their promoters. Collectively, our data reveal that RGC32 promotes the onset of SSc by regulating the inflammatory response of M1 macrophages, and it may serve as a promising therapeutic target for treating SSc.

Wenyang Huazhuo Tongluo formula inhibits fibrosis via suppressing Wnt/\u03b2-catenin signaling pathway in a Bleomycin-induced systemic sclerosis mouse model

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. So far, no Western medicine treatment can completely inhibit or reverse the progress of SSc, while at the same time, our previous series of studies have shown that the treatment of SSc by the Wenyang Huazhuo Tongluo formula (WYHZTL), a Chinese herbal decoction, shows a delightful prospect. The aim of this study is to further investigate the mechanism of anti-fibrosis of WYHZTL formula in SSc mouse model.MethodsThe Bleomycin-induced SSc mouse model was treated with saline (BLM), high-dosage of WYHZTL formula (WYHZTL-H), medium-dosage of WYHZTL formula (WYHZTL-M), low-dosage of WYHZTL formula (WYHZTL-L) and XAV-939, a small molecule inhibitor of Wnt/β-catenin signaling pathway, by the intragastric administration and intraperitoneal injection, respectively. The mRNA and protein levels of Wnt/β-catenin signaling pathway associated genes, fibrosis markers and histopathology were detected by reverse transcription-quantitative polymerase chain reaction, Western blotting and hematoxylin/eosin-staining. The levels of Wnt1, CTGF and DKK1 protein in serum were detected by enzyme-linked immunosorbent assay.ResultsCompared with BLM group, the WYHZTL formula and XAV-939 could significantly inhibit the thickness of the skin tissue of the SSc mouse model. The mRNA expression levels of GSK3β and DKK1 in the WYHZTL formula and XAV-939-treated group were significantly higher than those in the BLM group, while Wnt1, β-catenin, TCF4, cyclin D1, survivin, VEGF, CTGF, FN1, collagen I/III were decreased. Compared with BLM group, the protein expression levels of GSK3β and DKK1 in the WYHZTL formula and XAV-939-treated group were upregulated, while Wnt1, β-catenin, cyclin D1, survivin, CTGF, FN1, collagen I/III were downregulated. WYHZTL formula and XAV-939 could inhibit expression of Wnt1 and CTGF, but promoted DKK1 in serum. Furthermore, WYHZTL-H seemed more effective than WYHZTL-M and/or XAV-939 on regulating Wnt1, β-catenin, TCF4, GSK3β, DKK1, cyclin D1, survivin, VEGF, CTGF, FN1 and collagen I/III.ConclusionThis present study demonstrates that WYHZTL formula has anti-fibrosis effect in Bleomycin-induced SSc mouse model in a dosage-dependent manner, and the molecular mechanism may be related to the inhibition of Wnt/β-catenin signaling pathway.

Pathogenic roles of B lymphocytes in systemic sclerosis

Systemic sclerosis (SSc) is a collagen disease characterized by autoimmunity and excessive extracellular matrix deposition in the skin and visceral organs. Although the pathogenic relationship between systemic autoimmunity and the clinical manifestations of SSc remains unknown, SSc patients show a variety of abnormal immune activation including the production of disease-specific autoantibodies and cytokine production. Many recent studies have demonstrated that immune cells, including T cells, B cells, and macrophages, have a variety of immunological abnormalities in SSc. So far, several groups and our group reported that B cells play a critical role in systemic autoimmunity and disease expression through various functions, such as cytokine production, lymphoid organogenesis, and induction of other immune cell activation in addition to autoantibody production. Recent studies show that B cells from SSc patients demonstrate an up-regulated CD19 expression, a crucial regulator of B cell activation, which induces chronic hyper-reactivity of memory B cells and SSc-specific autoantibody production and also causes fibrosis of several organs. Furthermore, in SSc-model mice, such as tight-skin mice, bleomycin-induced SSc model mice, and DNA topoisomerase I and complete Freund’s adjuvant-induced SSc model mice, have abnormal B cell activation which associates with skin and lung fibrosis. Indeed, B cell depletion therapy using anti-CD20 Ab, Rituximab, is considered to one potential beneficial treatment for patients with SSc. However, there is no direct evidence which can explain how B cells, especially autoantigen-reactive B cells, progress or regulate disease manifestations of SSc. Collectively, B cell abnormalities in SSc is most likely participating in fibrosis and tissue damage of SSc. If the relationship between SSc-specific tissue damage and B cell abnormalities is revealed, these findings lead to novel effective therapy for SSc.

Bone Marrow-Derived Mesenchymal Stem Cells Expressing Thioredoxin 1 Attenuate Bleomycin-Induced Skin Fibrosis and Oxidative Stress in Scleroderma

Systemic sclerosis (SSc) is an autoimmune disorder that affects multiple organs. It is characterized by a thickening of the dermis and connective tissue caused by collagen accumulation, and vascular injuries that induce hypoxia. The present study investigated the therapeutic potential of bone marrow-derived mesenchymal stem cells (BMSCs) expressing thioredoxin 1 (Trx-1) in treating SSc-mediated skin disease after transplantation into a bleomycin-induced murine model. Mice with bleomycin-induced SSc were subcutaneously injected with BMSCs or Trx-1-overexpressing BMSCs and exposed to hypoxic conditions for 48 hours. Two weeks later, skin tissue samples were collected to assess fibrosis, oxidative stress, and angiogenesis by western blotting, ELISA, and histologic and immunofluorescence approaches. Invivo experiments showed that Trx-1-overexpressing BMSCs inhibited hypoxia-induced apoptosis and inhibited fibrosis under hypoxic conditions, possibly by downregulating transforming growth factor-β. Trx-1-overexpressing BMSCs also promoted the formation of tubular-like structures by endothelial progenitor cells, indicating that Trx-1 can promote angiogenesis in bleomycin-induced SSc. These results demonstrate that the transplantation of Trx-1-overexpressing BMSCs restored normal skin tissue in a mouse model of bleomycin-induced SSc, highlighting the therapeutic potential of engineered BMSCs for treating SSc.

An Autotaxin/Lysophosphatidic Acid/Interleukin-6 Amplification Loop Drives Scleroderma Fibrosis.

We previously implicated the lipid mediator lysophosphatidic acid (LPA) as having a role in dermal fibrosis in systemic sclerosis (SSc). The aim of this study was to identify the role of the LPA-producing enzyme autotaxin (ATX), and to connect the ATX/LPA and interleukin-6 (IL-6) pathways in SSc. We evaluated the effect of a novel ATX inhibitor, PAT-048, on fibrosis and IL-6 expression in the mouse model of bleomycin-induced dermal fibrosis. We used dermal fibroblasts from SSc patients and control subjects to evaluate LPA-induced expression of IL-6, and IL-6-induced expression of ATX. We next evaluated whether LPA-induced ATX expression is dependent on IL-6, and whether baseline IL-6 expression in fibroblasts from SSc patients is dependent on ATX. Finally, we compared ATX and IL-6 expression in the skin of patients with SSc and healthy control subjects. PAT-048 markedly attenuated bleomycin-induced dermal fibrosis when treatment was initiated before or after the development of fibrosis. LPA stimulated expression of IL-6 in human dermal fibroblasts, and IL-6 stimulated fibroblast expression of ATX, connecting the ATX/LPA and IL-6 pathways in an amplification loop. IL-6 knockdown abrogated LPA-induced ATX expression in fibroblasts, and ATX inhibition attenuated IL-6 expression in fibroblasts and the skin of bleomycin-challenged mice. Expression of both ATX and IL-6 was increased in SSc skin, and LPA-induced IL-6 levels and IL-6-induced ATX levels were increased in fibroblasts from SSc patients compared with controls. ATX is required for the development and maintenance of dermal fibrosis in a mouse model of bleomycin-induced SSc and enables 2 major mediators of SSc fibrogenesis, LPA and IL-6, to amplify the production of each other. Our results suggest that concurrent inhibition of these 2 pathways may be an effective therapeutic strategy for dermal fibrosis in SSc.

B lymphocytes in systemic sclerosis: Abnormalities and therapeutic targets.

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs with an autoimmune background. Although the pathogenic relationship between systemic autoimmunity and the clinical manifestations remains unknown, SSc patients have immunological abnormalities including the production of disease-specific autoantibodies. Recent studies have demonstrated that B cells play a crucial role in systemic autoimmunity and disease expression via various functions in addition to autoantibody production. Recent studies show that B cells from SSc patients demonstrate an upregulated CD19 signaling pathway, which is a crucial regulator of B-cell activation, that induces SSc-specific autoantibody production in SSc. In addition, B cells from SSc patients exhibit an overexpression of CD19. Consistently, in CD19 transgenic mice, CD19 overexpression induces SSc-specific autoantibody production. SSc patients have also intrinsic B-cell abnormalities characterized by chronic hyperreactivity of memory B cells, possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a genetic model of SSc, show augmented CD19 signaling and chronic hyperreactivity. Furthermore, in bleomycin-induced SSc model mice, endogenous ligands for Toll-like receptors, induced by bleomycin treatment, stimulate B cells to produce various fibrogenic cytokines and autoantibodies. Remarkably, CD19 loss results in inhibition of B-cell hyperreactivity and elimination of autoantibody production, which is associated with improvement of fibrosis. Taken together, altered B-cell function may result in tissue fibrosis, as well as autoimmunity, in SSc. Although further studies and greater understanding are needed, B cells are potential therapeutic target in SSc.

Nucleosome in patients with systemic sclerosis: possible association with immunological abnormalities via abnormal activation of T and B cells.

To determine the serum levels of nucleosome in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. Serum nucleosome levels in 91 patients with SSc were examined by ELISA. The expression of Toll-like receptor (TLR) 9 in T and B cells was quantified by flow cytometric intracellular protein analysis. The effects of nucleosomes on lymphocytes were also analysed. Moreover, we assessed the effects of nucleosomes on fibrosis, using wild type and CD19-deficient bleomycin-treated mice, an experimental model for human SSc. Serum nucleosome levels were elevated in SSc compared with healthy controls and correlated positively with the extent of skin and pulmonary fibrosis and immunological abnormalities. The retrospective longitudinal analysis showed the serum nucleosome levels to be attenuated during the follow-up period. TLR9, which can be stimulated by nucleosome expression was upregulated in the affected T and B cells of patients with SSc. Moreover, nucleosome stimulation strongly increased interleukin (IL)-4 and IL-17 expression of T cells, B-cell IgG production and proliferation of lymphocytes in SSc compared with those in healthy controls. In bleomycin-induced SSc model mice, serum nucleosome levels were elevated compared with control mice. Furthermore, nucleosomes increased IgG production and proliferation of mouse B cells. Although TLR9 expression was similar between wild type and CD19-deficient splenic B cells, CD19 deficiency reduced these nucleosome effects. These results suggest that nucleosomes and its signalling in B and T cells contribute to disease development in SSc via TLR9.

Abnormal B lymphocyte activation and function in systemic sclerosis.

Systemic sclerosis (SSc) is characterized by tissue fibrosis and autoimmunity. Although the pathogenic relationship between autoimmunity and clinical manifestations of SSc remains unknown, SSc patients display abnormal immune responses including the production of disease-specific autoantibodies. Previous studies have demonstrated that B cells play a critical role in systemic autoimmunity and disease expression through various functions such as induction of the activation of other immune cells in addition to autoantibody production. CD19 is a crucial regulator of B cell activation. Recent studies demonstrated that B cells from SSc patients showed an up-regulated CD19 signaling pathway that induced SSc-specific autoantibody production in SSc mouse models. CD19 transgenic mice lost tolerance for autoantigen and generated autoantibodies spontaneously. B cells from SSc patients exhibited an overexpression of CD19 that induced SSc-specific autoantibody production in transgenic mice. Moreover, SSc patients displayed intrinsic B cell abnormalities characterized by chronic hyper-reactivity of memory B cells, which was possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a genetic model of SSc, showed augmented CD19 signaling. In bleomycin-induced SSc mouse models, endogenous ligands for toll-like receptor 4 induced by bleomycin stimulated B cells to produce various fibrogenic cytokines and autoantibodies. Remarkably, the loss of CD19 resulted in the inhibition of B cell hyper-reactivity and autoantibody production, which are associated with improvements in fibrosis and a parallel decrease in fibrogenic cytokine production by B cells. Taken together, the findings suggest that altered B cell function may result in tissue fibrosis as well as autoimmunity in SSc.

Amelioration of tissue fibrosis by toll-like receptor 4 knockout in murine models of systemic sclerosis.

Bleomycin-induced fibrosis and the tight skin (TSK/+) mouse are well-established experimental murine models of human systemic sclerosis (SSc). Growing evidence has demonstrated the pivotal role of Toll-like receptors (TLRs) in several autoimmune inflammatory diseases, including SSc. This study was undertaken to determine the role of TLR-4 in the fibrotic processes in these murine models. We generated a murine model of bleomycin-induced SSc using TLR-4(-/-) mice and TLR-4(-/-) ;TSK/+ mice. The mechanisms by which TLR-4 contributes to pathologic tissue fibrosis were investigated in these 2 models by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and flow cytometry. Dermal and lung fibrosis was attenuated in bleomycin-treated TLR-4(-/-) mice compared with their wild-type counterparts. Inflammatory cell infiltration, expression of various inflammatory cytokines, and pathologic angiogenesis induced by bleomycin treatment were suppressed with TLR-4 deletion. Furthermore, the increased expression of interleukin-6 (IL-6) in fibroblasts, endothelial cells, and immune cells in response to bleomycin in vivo and to lipopolysaccharide in vitro was notably abrogated in the absence of TLR-4. Moreover, TLR-4 deletion was associated with alleviated B cell activation and skew toward a Th2/Th17 response against bleomycin treatment. Importantly, in TSK/+ mice, another SSc murine model, TLR-4 abrogation attenuated hypodermal fibrosis. These results indicate the pivotal contribution of TLR-4 to the pathologic tissue fibrosis of SSc murine models. Our results indicate the critical role of TLR-4 signaling in the development of tissue fibrosis, suggesting that biomolecular TLR-4 targeting might be a potential therapeutic approach to SSc.

2-Methoxyestradiol inhibits bleomycin-induced systemic sclerosis through suppression of fibroblast activation

The most dominant feature of systemic sclerosis (SSc) is fibrosis, which is caused by overproduction of collagen by fibroblasts. 2-Methoxyestradiol (2-ME) has exhibited disease-modifying activity in animal models of rheumatoid arthritis and autoimmune encephalomyelitis and inhibitory effect in cell proliferation and collagen synthesis. Therefore, we hypothesized that 2-ME may exhibit antifibrotic effect in SSc. To investigate the antifibrotic effect of 2-ME in SSc. We established a bleomycin-induced SSc mice model by injection with bleomycin daily for 21 days. 2-ME (100mg/kg/d) was simultaneously administered for 14 days. On the end of Week1 (W1), W2, W3 and W4, skins and lungs were collected for histological examination and analysis of hydroxyproline content and mRNA level of α1(I) procollagen (COL1A1) and COL1A2. In skin fibroblasts derived from SSc patients and healthy subjects treated with 2-ME (1, 5, or 25 μM), we examined cell proliferation, expression of α-smooth muscle actin (SMA) and mRNA level of COL1A1, COL1A2, COL3A1, matrix metalloproteinase(MMP)-1 and tissue inhibitors of MMP (TIMP)-1. We found reduced dermal thickness and lung fibrosis and decreased hydroxyproline content and mRNA level of COL1A1 and COL1A2 in skin and lung in SSc mice treated with 2-ME. In cell study, we observed a dose- and time-dependent inhibitory effect on proliferation of SSc fibroblasts by 2-ME. We also detected reduced α-SMA expression, decreased mRNA level of COL1A1, COL1A2, COL3A1 and TIMP-1, and increased mRNA level of MMP-1 in SSc fibroblasts treated with 2-ME. 2-ME could suppress SSc tissue fibrosis, which may be attributable to its inhibitory effect on the excessive proliferation, differentiation and production of collagen in fibroblasts. 2-ME is rising as a prospective agent for control of fibrosis in SSc.

Alpha2‐antiplasmin regulates the development of dermal fibrosis in mice by prostaglandin F2α synthesis through adipose triglyceride lipase/calcium‐independent phospholipase A2

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor β (TGFβ) production. This study was undertaken to clarify how α2AP induces TGFβ production and the development of fibrosis. To clarify the detailed mechanism by which α2AP induces TGFβ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A(2) (iPLA(2)) and examined whether ATGL/ iPLA(2) is associated with α2AP-induced TGFβ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFβ and prostaglandin F(2α) (PGF(2α)). We found that α2AP binding to ATGL promoted PGF(2α) synthesis through iPLA(2) in fibroblasts, and the PGF(2α) synthesis that was promoted by α2AP induced TGFβ production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFβ and PGF(2α) in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF(2α) synthesis, while the administration of PGF(2α) to α2AP-deficient mice facilitated α2AP deficiency-attenuated fibrosis. These findings suggest that α2AP regulates the development of fibrosis by PGF(2α) synthesis through ATGL/iPLA(2). The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.

Potential roles of interleukin‐17A in the development of skin fibrosis in mice

Although transforming growth factor β (TGFβ) and connective tissue growth factor (CTGF) have been considered to play central roles in the pathogenesis of systemic sclerosis (SSc), other cytokines may also be crucial for the development of SSc. The aim of this study was to examine the roles of T helper cytokines in the development of skin fibrosis. To compare the roles of Th1, Th2, and Th17 cytokines, we examined bleomycin-induced SSc in mice deficient for interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-17A. The mechanism by which IL-17A contributes to bleomycin-induced fibrosis was investigated in vivo and in vitro. The outcome of mice lacking IL-17A was also investigated in TSK-1 mice. The loss of IL-17A significantly attenuated bleomycin-induced skin fibrosis, whereas a deficiency of IFNγ or IL-4 did not. Leukocyte infiltration and the expression of TGFβ and CTGF messenger RNA in bleomycin-injected skin were significantly reduced in IL-17A-deficient mice compared with wild-type (WT) mice. Daily bleomycin injections induced the expression of IL-17A in the skin and potent IL-17A producers in splenic CD4+ T cells from WT mice. Furthermore, a skin fibroblast cell line expressed increased TGFβ, CTGF, and collagen after the addition of recombinant IL-17A. IL-17A deficiency also attenuated skin thickness in TSK-1 mice. This study demonstrates that IL-17A contributes to skin fibrosis in 2 mouse models of SSc. These findings suggest that inhibition of IL-17A represents a therapeutic target for antagonizing fibrotic skin disorders such as SSc.

The specific free radical scavenger edaravone suppresses fibrosis in the bleomycin-induced and tight skin mouse models of systemic sclerosis

Patients with systemic sclerosis (SSc) exhibit enhanced production of free radicals due to ischemia and reperfusion injury following Raynaud's phenomenon, an initial clinical manifestation. Oxidative stress induces cytokine production, inflammatory cell recruitment, and tissue injury in several inflammatory diseases. The aim of this study was to examine the effect of edaravone, a free radical scavenger, on the development of fibrosis and autoimmunity in two different mouse models of SSc. The bleomycin-induced SSc model in mice and the tight skin mouse model were used to evaluate the effect of edaravone on fibrosis and immunologic abnormalities. To assess the reaction of fibroblasts to stimulation with free radicals, fibroblasts from these mice were cultured with NONOate, a nitric oxide-releasing agent, and hydrogen peroxide. Treatment with edaravone reduced fibrosis in mice with bleomycin-induced SSc and in TSK/+ mice. The production of free radicals was also attenuated by edaravone in both models. In addition, production of fibrogenic cytokines such as interleukin-6 and transforming growth factor β1, production of anti-topoisomerase I antibody, and the degree of hypergammaglobulinemia were reduced by edaravone. Furthermore, bleomycin induced the production of H2O2 and nitric oxide from inflammatory cells, and collagen production was increased in fibroblasts cultured with H2O2 and NONOate. This study is the first to show that edaravone has a significant inhibitory effect on fibrosis both in the bleomycin-induced SSc model and in TSK/+ mice. These results indicate that edaravone should be further evaluated for potential use as an antifibrotic agent in SSc.

Treatment with rapamycin prevents fibrosis in tight‐skin and bleomycin‐induced mouse models of systemic sclerosis

Rapamycin, a novel macrolide immunosuppressive drug, is increasingly used as an agent for posttransplant immunosuppression and treatment of autoimmune disease. The molecular mechanism related to rapamycin-mediated immunosuppression is that rapamycin binds to FK-506 binding protein 12, and the formed complex inhibits the function of the mammalian target of rapamycin (mTOR), which in turn reduces protein phosphorylation, cell cycle progression, and cytokine production. The aim of this study was to examine the effect of rapamycin against the development of fibrosis and autoimmunity in 2 different types of systemic sclerosis (SSc) model mice. Tight skin (TSK/+) mice and bleomycin- induced SSc model mice were used to evaluate the effect of rapamycin on fibrosis and immunologic abnormalities. Furthermore, the antifibrotic effect of rapamycin was assessed using TSK/+ mouse fibroblasts. Treatment with rapamycin reduced skin fibrosis of TSK/+ mice and skin and lung fibrosis of bleomycin-induced SSc model mice. The production of fibrogenic cytokines, such as interleukin-4 (IL-4), IL-6, IL-17, and transforming growth factor beta1, was attenuated by rapamycin. Hypergammaglobulinemia and anti-topoisomerase I antibody production were also reduced by rapamycin treatment in TSK/+ mice. In addition, mTOR expression levels were increased in TSK/+ mouse fibroblasts compared with those in wild-type mouse fibroblasts. Rapamycin treatment inhibited proliferation and collagen production of TSK/+ mouse fibroblasts in a dose-dependent manner. This study is the first to show that rapamycin has a significant inhibitory effect on fibrosis in both TSK/+ and bleomycin-induced SSc model mice. These results suggest that rapamycin might be an attractive candidate for clinical trials in SSc patients.

CD19 Regulates Skin and Lung Fibrosis via Toll-Like Receptor Signaling in a Model of Bleomycin-Induced Scleroderma

Mice subcutaneously injected with bleomycin, in an experimental model of human systemic sclerosis, develop cutaneous and lung fibrosis with autoantibody production. CD19 is a general "rheostat" that defines signaling thresholds critical for humoral immune responses, autoimmunity, and cytokine production. To determine the role of CD19 in the bleomycin-induced systemic sclerosis model, we investigated the development of fibrosis and autoimmunity in CD19-deficient mice. Bleomycin-treated wild-type mice exhibited dermal and lung fibrosis, hyper-gamma-globulinemia, autoantibody production, and enhanced serum and skin expression of various cytokines, including fibrogenic interleukin-4, interleukin-6, and transforming growth factor-beta1, all of which were inhibited by CD19 deficiency. Bleomycin treatment enhanced hyaluronan production in the skin, lung, and sera. Addition of hyaluronan, an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, stimulated B cells to produce various cytokines, primarily through TLR4; CD19 deficiency suppressed this stimulation. These results suggest that bleomycin induces fibrosis by enhancing hyaluronan production, which activates B cells to produce fibrogenic cytokines mainly via TLR4 and induce autoantibody production, and that CD19 deficiency suppresses fibrosis and autoantibody production by inhibiting TLR4 signals.