SAT0193 Macrophages Responding To Mechanical Stress in Scleroderma

Background

Inflammation, vasculopathy and tissue fibrosis are key features of scleroderma (SSc). While healthy forearm skin which has a Young9s 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 elucidated^3,4.

Objectives

To 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.

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 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%).

Results

We 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.

Conclusions

MRTF-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.

References

Sacksen et al., Arthritis Rheum 2013. Shiwen et al., PLoS One 2015. Stifano et al., Curr Rheumatol Rep 2015. Chia et al., Curr Opin Rheumatol 2015.

Disclosure of Interest

None declared