BackgroundThe pathogenesis and etiology of systemic sclerosis (SSc) are complex and poorly understood. To date, several studies have demonstrated that the activation of the immune system undoubtedly plays a pivotal role in SSc pathogenesis. Activated immune effector T cells contribute to the release of various pro-inflammatory cytokines and drive the SSc-specific autoantibody responses. This, and a profibrotic environment, are all-important components of abnormal active immune responses that can lead to pathological disorders of SSc. CD11a is essential to inflammatory and immune responses, regulating adhesive and co-stimulatory interactions between CD4+ T cells and other cells. Although CD11a is overexpressed in SSc patients, the mechanisms leading to this overexpression and its consequences remain unclear. DNA methylation, a main epigenetic modification, plays an important role in the regulation of gene expression and is involved in the pathogenesis of autoimmune diseases. This work aims to investigate the effect of DNA demethylation on CD11a expression in SSc CD4+ T cells and to determine its functional significance. CD11a expression was measured using RT-PCR and flow cytometry. Bisulfite sequencing was used to determine the methylation status of the CD11a regulatory region. CD4+ T cells were co-cultured with antigen-presenting cells, B cells, or fibroblasts with and without anti-CD11a, and proliferation of CD4+ T cells, IgG production by B cells, and expression levels of COL1A2 mRNA by fibroblasts were evaluated.ResultsElevated CD11a expression levels were observed in CD4+ T cells from SSc patients; these levels were found to be positively correlated with disease activity. The methylation levels of the CD11a regulatory sequences were lower in SSc patients than in controls and inversely correlated with CD11a mRNA expression. Treatment of CD4+ T cells with 5-azacytidine (5-azaC) decreased CD11a promoter methylation and caused CD11a overexpression. SSc CD4+ T cells and 5-azaC-treated CD4+ T cells showed increased proliferation of CD4+ T cells, increased production of IgG by co-cultured B cells, and induced expression of COL1A2 mRNA by co-cultured fibroblasts. These stimulatory effects were abrogated by anti-CD11a.ConclusionsDemethylation of CD11a regulatory elements and subsequent CD11a overexpression in CD4+ T cells may mediate immunological abnormalities and fibrotic processes in SSc.
Ganoderma lucidum has featured in traditional Chinese medicine for >1,000 years. Ganoderma polysaccharides (GL-PS), a major active ingredient in Ganoderma, confer immune regulation, antitumor effects and significant antioxidant effects. The aim of the present study was to investigate the efficacy and mechanism of GL-PS-associated inhibition of ultraviolet B (UVB)-induced photoaging in human fibroblasts in vitro. Primary human skin fibroblasts were cultured, and a fibroblast photoaging model was built through exposure to UVB. Cell viability was measured by MTT assay. Aged cells were stained using a senescence-associated β-galactosidase staining (SA-β-gal) kit. ELISA kits were used to analyze matrix metalloproteinase (MMP) −1 and C-telopeptides of Type I collagen (CICP) protein levels in cellular supernatant. ROS levels were quantified by flow cytometry. Cells exposed to UVB had decreased cell viability, increased aged cells, decreased CICP protein expression, increased MMP-1 protein expression, and increased cellular ROS levels compared with non-exposed cells. However, cells exposed to UVB and treated with 10, 20 and 40 µg/ml GL-PS demonstrated increased cell viability, decreased aged cells, increased CICP protein expression, decreased MMP-1 protein expression, and decreased cellular ROS levels compared with UVB exposed/GL-PS untreated cells. These results demonstrate that GL-PS protects fibroblasts against photoaging by eliminating UVB-induced ROS. This finding indicates GL-PS treatment may serve as a novel strategy for antiphotoaging.
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