Background/Purpose: Skin involvement is one of the most prominent clinical features in scleroderma. There is a marked contrast in mechanical stiffness between healthy forearm skin which has a Young’s modulus of 4-12kPa, and scleroderma fibrotic skin at 50-80kPa. We have shown that mechano-sensing properties of fibroblasts in scleroderma are mediated by myocardin-related transcription factor A (MRTF-A). This study is to investigate if a mechanically-stressed microenvironment promotes macrophages towards a pathogenic phenotype, and whether MRTF-A mediates this process.

Methods: Control 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 unpolarised, or activated with LPS(10ng/ml) or IL-10(10ng/ml) for macrophages designated M(0), M(LPS) and M(IL-10) (n=4). MRTF-A expression was assessed by qPCR and Western blot. Conditioned media was profiled by Luminex array for inflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) of wildtype (WT) and MRTF-A-null mice were maintained in RPMI/M-CSF on soft and stiff substrates.

Results: We observed increased accumulation of CD68⁺ macrophages and MRTF-A⁺CD68⁺macrophages in perivascular areas of scleroderma skin compared to control skin. No significant differences were detected in MRTF-A expression by qPCR between control and scleroderma macrophages, although stiff substrate increased expression of MRTF-A protein. M(LPS) expressed TNF-α and IL-12 mRNA (10-fold lower in expression or undetectable in M(0), respectively), on soft substrate. Macrophages on stiff substrate showed a trend towards decreased LPS-induced TNF-α and IL-12 mRNA. M(LPS) on soft substrate expressed IFN-γ, which was undetectable with M(LPS) on stiff substrate (mean difference (∆) 0.2075±0.1576pg/ml, p<0.01). M(IL-10) on stiff substrate showed increased MCP-1 expression compared to soft (∆ 2590±2233pg/ml, p<0.05). M(LPS) on stiff compared to soft substrate showed increased MCP-3 expression (∆ 57.01±49.22pg/ml, p<0.05). M(IL-10) on stiff substrate showed a trend towards increased MCP-3 compared to soft substrate. M(IL-10) on stiff substrate showed increased fractalkine compared to soft substrate (∆ 51.22±36.28pg/ml, p<0.01). WT BMDMs displayed a more elongated morphology on stiff compared to soft substrate while MRTF-A-null BMDMs remained rounded on stiff substrate.

Conclusion: MRTF-A is a mechanical stress-responsive transcription factor which regulates cytoskeletal and ECM-related genes. MRTF-A may mediate mechanical stress and macrophage activation in scleroderma. A stiff microenvironment promoted macrophages to change from a pro-inflammatory phenotype towards a pro-healing phenotype marked by the loss or reduced expression of inflammatory markers IFN-γ, TNF-a and IL-12 alongside increased expression of monocyte chemoattractants MCP-1, MCP-3 and the pro-healing macrophage marker fractalkine.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modelling-healthy-and-scleroderma-fibrotic-skin-in-vitro-mechanical-stress-alters-macrophage-cytokine-expression-and-triggers-signalling-via-the-mechano-sensing-transcription-factor-myocardin-related/