Ayumi Yoshizaki

Regulatory B cells control T-cell autoimmunity through IL-21-dependent cognate interactions

B cells regulate immune responses by producing antigen-specific antibodies. However, specific B-cell subsets can also negatively regulate T-cell immune responses, and have been termed regulatory B cells. Human and mouse regulatory B cells (B10 cells) with the ability to express the inhibitory cytokine interleukin-10 (IL-10) have been identified. Although rare, B10 cells are potent negative regulators of antigen-specific inflammation and T-cell-dependent autoimmune diseases in mice. How B10-cell IL-10 production and regulation of antigen-specific immune responses are controlled in vivo without inducing systemic immunosuppression is unknown. Using a mouse model for multiple sclerosis, here we show that B10-cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. Moreover, the ex vivo provision of CD40 and IL-21 receptor signals can drive B10-cell development and expansion by four-million-fold, and generate B10 effector cells producing IL-10 that markedly inhibit disease symptoms when transferred into mice with established autoimmune disease. The ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.

IL-10–Producing Regulatory B10 Cells Inhibit Intestinal Injury in a Mouse Model

B cells mediate multiple functions that influence immune and inflammatory responses. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to immediate intestinal injury. Dextran sulfate sodium-induced intestinal injury serves as an experimental animal model for human ulcerative colitis. The contribution of B cells to DSS-induced intestinal injury is unclear. In this study, we show that DSS-induced intestinal injury was more severe in CD19-deficient (CD19(-/-)) mice than in wild-type mice. These inflammatory responses were negatively regulated by a unique IL-10-producing CD1d(hi)CD5(+) regulatory B cell subset (B10 cells) that was absent in CD19(-/-) mice and represented only 1% to 2% of splenic B220(+) cells in wild-type mice. Remarkably, adoptive transfer of these B10 cells from wild-type mice reduced inflammation in CD19(-/-) mice in an IL-10-dependent manner. These results demonstrate that IL-10 production from regulatory B10 cells regulates DSS-induced intestinal injury. These findings may provide new insights and therapeutic approaches for treating ulcerative colitis.

Cell Adhesion Molecules Regulate Fibrotic Process via Th1/Th2/Th17 Cell Balance in a Bleomycin-Induced Scleroderma Model

Mice s.c. injected with bleomycin, an experimental model for human systemic sclerosis, develop skin and lung fibrosis, which is mediated by inflammatory cell infiltration. This process is highly regulated by multiple adhesion molecules and does not require Ag sensitization. To assess the role of adhesion molecules in this pathogenetic process, bleomycin-induced fibrosis was examined in mice lacking adhesion molecules. L-selectin and/or ICAM-1 deficiency inhibited skin and lung fibrosis with decreased Th2 and Th17 cytokines and increased Th1 cytokines. In contrast, P-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, or P-selectin glycoprotein ligand 1 (PSGL-1) deficiency augmented the fibrosis in parallel with increased Th2 and Th17 cytokines and decreased Th1 cytokines. Furthermore, loss of L-selectin and/or ICAM-1 reduced Th2 and Th17 cell numbers in bronchoalveolar lavage fluid, whereas loss of P-selectin, E-selectin, or PSGL-1 reduced Th1 cell numbers. Moreover, Th1 cells exhibited higher PSGL-1 expression and lower expression of LFA-1, a ligand for ICAM-1, whereas Th2 and Th17 cells showed higher LFA-1 and lower PSGL-1 expression. This study suggests that L-selectin and ICAM-1 regulate Th2 and Th17 cell accumulation into the skin and lung, leading to the development of fibrosis, and that P-selectin, E-selectin, and PSGL-1 regulate Th1 cell infiltration, resulting in the inhibition of fibrosis.

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

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Clinical Significance of Serum HMGB-1 and sRAGE Levels in Systemic Sclerosis: Association with Disease Severity

IntroductionThe high mobility group box 1 protein (HMGB-1)/advanced glycation end products (RAGE) system is recently shown to play an important part in immune/inflammatory disorders. However, the association of this system in systemic sclerosis (SSc) remains unknown.Materials and MethodsTo determine clinical association of serum levels of HMGB-1 and soluble RAGE (sRAGE) in patients with SSc, sera from 70 patients with SSc and 25 healthy controls were examined by enzyme-linked immunosorbent assay. Sera from tight-skin mice and bleomycin-induced scleroderma mice, animal models for SSc, were also examined. Skin HMGB-1 and RAGE expression was assessed by immunohistochemistry.Results and DiscussionSerum HMGB-1 and sRAGE levels in SSc were higher than those in controls. Similarly, HMGB-1 and sRAGE levels in animal SSc models were higher than those in control mice. SSc patients with elevated HMGB-1 and sRAGE levels had more frequent involvement of several organs and immunological abnormalities compared to those with normal levels. Furthermore, HMGB-1 and sRAGE levels correlated positively with modified Rodnan total skin thickness score and negatively with pulmonary function test.ConclusionsHMGB-1 and sRAGE expression in the sclerotic skin was more intense than normal skin. These results suggest that elevated serum HMGB-1 and sRAGE levels are associated with the disease severity and immunological abnormalities in SSc.

Immunization with DNA topoisomerase I and Freund's complete adjuvant induces skin and lung fibrosis and autoimmunity via interleukin-6 signaling.

OBJECTIVE The presence of anti-DNA topoisomerase I (anti-topo I) antibody correlates positively with disease severity in patients with systemic sclerosis (SSc). However, the role of induction of anti-topo I antibody production and its potential contribution to the pathogenesis of SSc remain unclear. The aim of this study was to examine the role of anti-topo I antibody in the pathogenesis of SSc. METHODS To assess the contribution of anti-topo I antibody to the pathogenetic process, dermal sclerosis, pulmonary fibrosis, and cytokine production were examined in mice treated with topo I and either Freunds complete adjuvant (CFA) or Freunds incomplete adjuvant (IFA). RESULTS Treatment with topo I and CFA, in contrast to treatment with topo I and IFA, induced skin and lung fibrosis with increased interleukin-6 (IL-6), transforming growth factor β1, and IL-17 production and decreased IL-10 production. Anti-topo I antibody levels were greater in mice treated with topo I and CFA than in mice treated with topo I and IFA. Furthermore, treatment with topo I and CFA increased Th2 and Th17 cell frequencies in bronchoalveolar lavage fluid, whereas treatment with topo I and IFA increased Th1 and Treg cell frequencies. Moreover, loss of IL-6 expression ameliorated skin and lung fibrosis, decreased Th2 and Th17 cell frequencies, and increased Th1 and Treg cell frequencies. CONCLUSION This study is the first to show that treatment with topo I and CFA induces SSc-like skin and lung fibrosis and autoimmune abnormalities. We also suggest that IL-6 plays important roles in the development of fibrosis and autoimmune abnormalities in this novel SSc model.

CD19, a Response Regulator of B Lymphocytes, Regulates Wound Healing through Hyaluronan-Induced TLR4 Signaling

Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-beta. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-beta through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing.

Increased Serum Soluble OX40 in Patients with Systemic Sclerosis

Objective To determine levels of serum soluble OX40 (also termed CD134, a member of the tumor necrosis factor receptor superfamily) and their clinical associations in patients with systemic sclerosis (SSc). Methods Serum soluble OX40 levels were examined by ELISA in 53 patients with SSc, 15 patients with systemic lupus erythematosus (SLE), and 32 healthy individuals. Results OX40 levels were significantly elevated in SSc patients (125.7 ± 5.7 pg/ml) compared to patients with SLE (80.7 ± 1.7 pg/ml; p < 0.005) and controls (88.2 ± 3.0 pg/ml; p < 0.0001). Elevated OX40 levels were found to be associated with disease duration of less than 2 years (p < 0.05). Conclusion Our results suggest that serum soluble OX40 levels correlate with the early-onset of SSc disease.

Elevated serum interleukin-27 levels in patients with systemic sclerosis: association with T cell, B cell and fibroblast activation

Objective To determine serum levels of interleukin 27 (IL-27) in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. Methods Serum levels of IL-27 in 91 patients with SSc and the production of IL-27 by isolated monocytes were examined by ELISA. The expression of IL-27 receptor in the skin fibroblasts, B cells and T cells was quantified by real-time PCR. The effect of IL-27 on immunoglobulin G (IgG) production of B cells, IL-17 production of CD4 T cells and proliferation and collagen synthesis of fibroblasts was also analysed. Results Serum IL-27 levels were raised in patients with SSc compared with healthy controls and correlated positively with the extent of skin and pulmonary fibrosis and immunological abnormalities. IL-27 levels also correlated positively with serum levels of hyaluronan, recently identified as an endogenous ligand for Toll-like receptors. The retrospective longitudinal analysis showed a tendency for serum IL-27 levels to be attenuated during the follow-up period. IL-27 production by cultured monocytes was increased by hyaluronan stimulation. IL-27 receptor expression was upregulated in the affected skin fibroblasts, B cells and CD4 T cells of patients with SSc. Moreover, IL-27 stimulation increased IgG production of B cells, IL-17 production of CD4 T cells and proliferation and collagen synthesis of fibroblasts in patients with SSc compared with those in healthy controls. Conclusion These results suggest that IL-27 and its signalling in B cells, T cells and fibroblasts contributes to disease development in patients with SSc.

Serum interleukin 9 levels are increased in patients with systemic sclerosis: association with lower frequency and severity of pulmonary fibrosis.

Objective. To determine serum interleukin 9 (IL-9) levels and their clinical associations in patients with systemic sclerosis (SSc). Methods. Serum IL-9 levels were examined by ELISA in 71 patients with SSc, 15 with systemic lupus erythematosus (SLE), 15 with dermatomyositis (DM), 39 with atopic dermatitis, and 28 healthy individuals. Results. Serum IL-9 levels were significantly elevated in SSc patients (84.6 ± 76.0 pg/ml) compared with healthy individuals (40.4 ± 41.7 pg/ml; p < 0.001), and patients with SLE (50.7 ± 52.0 pg/ml; p < 0.05) or DM (50.6 ± 55.8 pg/ml; p < 0.05) or atopic dermatitis (41.8 ± 38.8 pg/ml; p < 0.001). Among SSc patients, there were no differences in serum IL-9 levels between those with limited cutaneous SSc and those with diffuse cutaneous SSc. Patients with SSc and raised IL-9 levels less often had pulmonary fibrosis and decreased percentage vital capacity than those with normal IL-9 levels. IL-9 levels were positively correlated with percentage vital capacity in patients with SSc. Conclusion. Serum IL-9 level was increased in patients with SSc, and was associated with lower frequency and severity of pulmonary fibrosis in SSc. IL-9 could be a protective factor against the development of pulmonary fibrosis in this disease, and as such would be a possible therapeutic target.