Background and Objective Systemic sclerosis (SSc) is a chronic autoimmune disease characterised by excessive deposition of extracellular matrix (ECM) components, immune activation and neoangiogenesis. The pathogenesis of scleroderma is still poorly elucidated; however, an increasing burden of evidence suggests involvement of Toll-like Receptors (TLRs). In our working hypothesis, nucleic acid-containing immune complexes (ICs) isolated from scleroderma patients bearing different autoantibody antigenic specificities might activate TLRs, thus inducing several mediators involved in SSc pathogenesis. Therefore, the aim of this study was to characterise the role of TLR3 and TLR9 as potential mediators in the initiator phase of scleroderma.
Materials and Methods Cutaneous fibroblasts were isolated from skin biopsies obtained from healthy donors and cultured in adequate conditions up to the eight passage. ICs were isolated using polyethylene glycol precipitation from sera of healthy controls and scleroderma patients carrying different autoantibody specificities (antibodies against centromere proteins [ACA], DNA topoisomerase I [ATA], RNA polymerase [ARA] and Th/To [anti-Th/To]). Levels of TLR3, TLR9, IFN-a and IFN-b mRNA expression in different experimental conditions were analysed by RT-PCR. Fibroblasts were transiently silenced for TLR3 using a specific small interfering RNA (siRNA); silencing was confirmed by RT-PCR and Western Blotting. TLR3-silenced and unsilenced cells were incubated with ICs of different sources or TLR agonists (poly(I:C) and LPS). Adhesion molecule (ICAM-1) expression was evaluated by cell-ELISA; interleukin (IL)-6 and IL-8 secretion in the supernatants was measured by commercial ELISA assays.
Results In skin fibroblasts from healthy subjects, incubation with ICs isolated from SSc patients upregulated both TLR3 and TLR9 mRNA expression, with a higher increase in TLR9 levels. Consistently, a greater raise was observed for IFN-a than IFN-b mRNA expression levels.
Both mRNA and protein TLR3 levels were significantly reduced by specific silencing.
ICAM-1 expression was significantly increased in cells treated with both TLR agonists and ICs from SSc patients but not healthy controls. Similarly, IL-6 and IL-8 secretion was elevated in the same experimental conditions.
TLR3 specific silencing significantly affected the production of ICAM-1, IL-6 and IL-8.
Conclusions Our data suggest that activation of TLRs by ICs isolated from SSc patients leads to fibroblast activation, with upregulation of adhesion molecules and secretion of pro-inflammatory and pro-fibrotic interleukins.
Further work is needed to better investigate the role of TLRs as potential pathogenetic mediators in SSc.
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