Background/Purpose: Systemic Sclerosis (SSc) has complex aetiology with many potential driving forces, one of which is the microenvironment in lesional skin, in which resident mesenchymal stem cells (MSCs), are exposed to aberrantly expressed growth factors and cytokines, and excessively stiffened and abundant extracellular matrix. We hypothesise that MScs are persistently activated within this microenvironment contributing to chronic aberrant tissue repair and fibrosis. The effects of skin blister fluid (BF) and IL-31, the maximally induced cytokine in SSc BF, were studied in models of interaction between the disease microenvironment and MSCs. MSCs were studied in situ using amitotic nuclear division as a marker of their activation.

Methods: Staining of tissue spreads for metakaryotic nuclear divisions was used as a method to identify MSCs in SSc tissues. SSc BF sampled from forearm skin, as well as control BF (each diluted 1:125 in 0.2% DMEM) and IL-31, were studied as possible agonists in scratch wound assays and Western blots for SSc associated proteins. MSCs derived from both adipose and skin biopsy tissue were induced to differentiate into osteoblasts on soft and stiff extracellular matrix representations. Levels of pro-fibrotic factors were measured by qPCR in MSCs exposed to microenvironment representations.

Results: Metakaryotic nuclear divisions were observed in cells associated with fibrillar collagen in the deep dermal layer of SSc skin. SSc BF enhanced migration of MSCs compared with control BF, (~1 and ~0.7 mm² respectively p=0.231) and induced αSMA expression in MSCs ~ 3 times more than media only and ~ 1.5 times more than control BF. Collagen I and CTGF expression were also induced in MSCs by SSc BF. In addition, IL-31 enhanced migration of SSc and control dermal fibroblasts in the scratch wound assay (1.3mm² SEM=0.08 and 0.45mm² SEM= 0.07 in 0.2% DMEM and 50ng/ml IL-31 respectively at 20 hours, p=0.0001), inhibited by Wortmannin and U0126 (1.4mm² and 1.3mm² respectively, p=0.0001). The same was seen with MSCs, by 24 hours the area of the scratch in IL-31 treated wells was 0.9mm² compared to 1.5mm² for Wortmannin treatment (p=0.027). IL-31 did not fully reproduce the protein and migratory effects of blister fluid on MSCs. IL-31 had no effect on Collagen I expression (relative density of 0.7 vs 1.0 and 2.4 for 0.2% DMEM and SSc BF respectively). Matrix stiffness enhanced osteogenic differentiation of MSCs, this effect was enhanced further with IL-31 and TGFβ but abolished when treated with CCG1423 (an inhibitor of the matrix sensing protein MRTFA). Stiff matrices also elevated expression of a-SMA and Collagen I mRNA.

Conclusion: Proliferating metakaryotic cells were found to be present in the deep dermis of SSc patients and they may represent activated stem cells. SSc patients BF and SSc associated cytokine IL-31 enhanced pro-fibrotic differentiation of MSCs. Our findings suggest that MSCs may be important in SSc pathogenesis and that factors within the dermal interstitial fluid generate a microenvironment inducing them to create or induce excessive pro-fibrotic cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/modelling-the-interaction-between-disease-microenvironment-and-mesenchymal-cells-in-systemic-sclerosis/