In scleroderma (systemic sclerosis, SSc), persistent activation of myofibroblast leads to severe skin and organ fibrosis resistant to therapy. Increased mechanical stiffness in the involved fibrotic tissues is a hallmark clinical feature and a cause of disabling symptoms. Myocardin Related Transcription Factor-A (MRTF-A) is a transcriptional co-activator that is sequestered in the cytoplasm and translocates to the nucleus under mechanical stress or growth factor stimulation. Our objective was to determine if MRTF-A is activated in the disease microenvironment to produce more extracellular matrix in progressive SSc. Immunohistochemistry studies demonstrate that nuclear translocation of MRTF-A in scleroderma tissues occurs in keratinocytes, endothelial cells, infiltrating inflammatory cells, and dermal fibroblasts, consistent with enhanced signaling in multiple cell lineages exposed to the stiff extracellular matrix. Inhibition of MRTF-A nuclear translocation or knockdown of MRTF-A synthesis abolishes the SSc myofibroblast enhanced basal contractility and synthesis of type I collagen and inhibits the matricellular profibrotic protein, connective tissue growth factor (CCN2/CTGF). In MRTF-A null mice, basal skin and lung stiffness was abnormally reduced and associated with altered fibrillar collagen. MRTF-A has a role in SSc fibrosis acting as a central regulator linking mechanical cues to adverse remodeling of the extracellular matrix.
BackgroundInflammation, vasculopathy and tissue fibrosis are key features of scleroderma (SSc). While healthy forearm skin which has a Young's modulus of 4–12kPa, SSc fibrotic skin measures 50–80kPa with its increased mechanical stiffness1. We have shown that mechano-sensing properties of fibroblasts in SSc are mediated by myocardin-related transcription factor A (MRTF-A)3. Monocytes/macrophages are likely to be involved in SSc pathogenesis but the effect of mechanical stress on these cells remain to be elucidated3,4.ObjectivesTo investigate if a mechanically-stressed microenvironment like that in SSc tissue, promotes macrophages towards a pathogenic phenotype, and whether MRTF-A is involved in this process.MethodsControl and scleroderma skin sections were immunostained with anti-CD68 and anti-MRTF-A antibodies (n=3). Human PBMC-derived macrophages were cultured in RPMI/M-CSF on 4kPa and 50kPa collagen-fibronectin-coated plates to mimic “soft”/healthy and “stiff”/fibrotic skin, and activated with LPS (10ng/ml) or IL-10 (10ng/ml) for macrophages designated M(LPS) and M(IL-10) (n=4). MRTF-A expression was assessed by qPCR and conditioned media profiled by Luminex array for inflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) of wildtype and MRTF-A-null mice were maintained in RPMI/M-CSF on soft and stiff substrates. The data were analysed by two-way ANOVA and Tukey test (p<0.05, CI 95%).ResultsWe observed a greater number of CD68+ macrophages in diffuse SSc skin compared to control skin, mainly around perivascular regions. These macrophages also expressed MRTF-A. Human macrophages expressed MRTF-A mRNA and showed differential cytokine expression when cultured on soft and stiff substrates. M(LPS) on soft matrix expressed IFN-γ, which was undetectable with M(LPS) on stiff substrate (mean difference 0.2075±0.1576pg/ml, p<0.01). LPS- and IL-10 activation on soft substrate increased MCP-3 expression compared to controls (mean difference 68.51±49.19pg/ml, p<0.01, 92.88±49.22pg/ml, p<0.0001, respectively). M(LPS) on stiff compared to soft substrate showed lower MCP-3 expression (mean difference 57.01±49.22pg/ml, p<0.05). M(IL-10) on soft substrate showed higher MCP-1 expression compared to controls (mean difference 2448±2232pg/ml, p<0.05). M(IL-10) on stiff substrate decreased expression of MCP-1 (mean difference 2590±2233pg/ml, p<0.05) and increased fractalkine levels compared to soft substrate (mean difference 51.22±36.28pg/ml, p<0.01). Wildtype BMDMs on stiff compared to soft substrate displayed a more elongated morphology. MRTF-A-null BMDMs remained rounded on stiff substrate.ConclusionsMRTF-A is a mechanical stress-responsive factor which co-activates transcription of cytoskeletal and extracellular matrix-modifying genes. MRTF-A may couple mechanical stress to macrophage activation in SSc, where stiff matrix promotes macrophage secretion of cytokines and growth factors that exacerbate fibrosis.ReferencesSacksen et al., Arthritis Rheum 2013.Shiwen et al., PLoS One 2015.Stifano et al., Curr Rheumatol Re...
BackgroundMacrophages and fibroblasts are key effector cell types present in scleroderma tissue1. While the effect of scleroderma fibroblast conditioned medium on macrophages has been previously studied2, less is known about the effect of scleroderma macrophages on fibroblasts. Interleukin-6 (IL-6) is an important mediator of fibrosis and is overexpressed in scleroderma sera and cells such as skin fibroblasts, monocytes and endothelial cells3. Given the increase in IL-6 levels in scleroderma and presence of the IL-6 receptor on the cell surface of macrophages, we are now investigating the phenotype of macrophages exposed to IL-6. Here we present our work into the paracrine function of IL-6-treated macrophages in stimulating fibroblasts using a media transfer approach.ObjectivesTo investigate the effect of macrophage conditioned media on fibroblast activation.MethodsPBMC-derived macrophages from healthy control (n=3 females, mean age 50.8±21.9 years) and diffuse scleroderma (n=4 females, mean age 54.8±15.7 years, mean disease duration 73.2±90.3 months, 2 with antiScl70 and 2 with antiRNA polymerase antibodies) individuals were cultured in RPMI/10% FBS/M-CSF (4ng/ml)/P/S, quiesced in media with 1% BSA replacing the FBS, and left untreated (M0) or treated with IL-6 (50ng/ml, M (IL-6)) for 24 hours. The cultures were replaced with fresh media and collected after 24 hours. Conditioned media were applied to healthy control skin fibroblasts (24 hours) and fibroblast expression of fibrotic proteins was assessed by Western Blot, using β-tublin and TBP as loading controls. As control, fibroblasts from healthy volunteers were left untreated by culturing in non-conditioned media (fresh RPMI/ 1% BSA/M-CSF (4ng/ml)/P/S).ResultsFibroblast expression of collagen type I and connective tissue growth factor (CTGF) were not significantly different between untreated and macrophage conditioned medium treatment groups. Baseline levels of collagen type I were high in the fibroblasts cultured in non-conditioned media, and there was a trend towards increased CTGF expression in all conditioned media-treated groups compared to untreated fibroblasts in non-conditioned media. A 2.6-fold increase in α-smooth muscle actin (α-SMA) was observed in the healthy control M (IL-6)-conditioned medium-treated group compared to the group of fibroblasts cultured in non-conditioned media (one-way ANOVA with Sidak multiple comparison, p=0.048).ConclusionsAfter 24 hours treatment, control dermal fibroblasts treated with media of IL-6-polarised healthy control macrophages expressed higher levels of α-SMA compared to fibroblasts cultured in non-conditioned medium. A trend towards increased CTGF was also observed. These results suggest that paracrine factors in the IL-6-activated macrophage secretome may promote differentiation of fibroblasts into myofibroblasts, which is a key component of wound healing and scleroderma fibrosis.References Fuschiotti P. Current perspectives on the immunopathogenesis of systemic sclerosis. Immunotargets Ther. 2016;5:21–35.Denton ...
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