Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
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.
To cite this abstract in AMA style:Tam A, Xu S, Lopez H, Khan K, Ahmed Abdi B, Rosario H, Arumalla N, Gibson M, Denton C, Abraham D, Smith BD, Stratton RJ. 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 Transcription Factor-a [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). 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/. Accessed October 27, 2020.
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