Background and objectives Systemic sclerosis is an autoimmune disease that is characterised by inflammation and fibrosis. Perivascular infiltrates of T cells are found in the skin and the T cell bias is predominantly Th2 with an increase of IL-13 in the skin and blood. We have found that Interleukin-13 (IL-13) is elevated and mediates increase deposition of ECM in SSc but the underlying molecular mechanism of IL-13 mediated ECM increases is unknown. The aim here was to investigate the molecular mechanism of IL-13 mediated ECM induction in dermal fibroblasts and to determine the role of miR135b, a miR with a putative role in IL-13 effects.
Methods Dermal fibroblasts were taken from punch biopsies from healthy controls or SSc patients and cultured in vitro with or without the addition of recombinant IL-13. siRNA was carried out with 100nM of siRNA against STAT6 with scramble siRNA using DharmaFECT reagent. Collagen1A1, TIMP-1 and alphaSMa gene expression was analysed by qRT-PCR with 18S as the internal housekeeping gene for normalisation. miR135b mimics were transfected into dermal fibroblasts using DharmaFECT and a control miR was also transfected for comparison. Bleomycin was injected into mice to cause skin fibrosis or saline control for comparison and the mice were sacrificed and skin biopsies taken and immunohistochemistry was performed with specific IL-13 antibodies and florescent labelled secondary antibodies and imaged using confocal microscopy.
Results IL-13 dose dependently increased the levels of collagen1A1 in dermal fibroblasts. However, there was no increase in the other pro fibrotic molecules TIMP-1 or the myofibroblast marker alpha smooth muscle actin by IL-13 by qPCR. Furthermore there was no increase in the pro fibrotic molecule TGF-β1. siRNA mediated silencing of STAT6 in dermal fibroblasts attenuated the induction of collagen1A1 mediated by IL-13 indicating STAT6 is crucial in this effect. Prediction software suggested that STAT6 has a binding site for miR135b in its 3’UTR and transfection of miR135b reduced STAT-6 mediated collagen induction in these cells. Measurement of miR135 post transfection confirmed that the miR was transfected into the cells. Bleomycin induced fibrosis in the mouse model was used and using immunohistochemistry it was found that in the skin IL-13 was elevated compared to vehicle treated mice, indicating T cell infiltration in this model of systemic sclerosis.
Conclusions IL-13 utilises STAT6 to mediate induction of collagen in dermal fibroblasts. STAT6 is an important molecule in mediating fibrosis and is targeted by miR135b and elevated levels of miR135b diminishes STAT6-mediated effects in these cells. IL-13 from T cells is also elevated in bleomycin induced fibrosis models.