Studies on molecular aspects of inflammation : Involvement of purinergic P2X7R in collagen production in systemic sclerosis and chronic heart failure Neutrophil Extracellular Traps release in synovial fluid from microcrystal and non-microcrystal arthritis

Background: The purinergic P2X7 receptor (P2X7R) is a nucleotide-gated ionotropic channel chiefly involved in the inflammatory response triggered by release of ATP from damaged cells. It is largely expressed in inflammatory cells and plays a key role in promoting the release of pro-inflammatory cytokines such as IL-1β. Increasing evidence suggest that it is also expressed in fibroblasts and may play a pivotal role in the development of tissue fibrosis in different body districts. Therefore, we hypothesized a possible P2X7R involvement in the pathogenesis of two different diseases, Systemic sclerosis (SSc; scleroderma) and Chronic Heart Failure (CHF), that include the development of tissue fibrosis among their hallmarks. SSc is a connective tissue disease characterized by generalized fibrosis of the skin and internal organs, for which no effective treatments are currently available. CHF is a complex multistep disorder representing the final result of a number of cardiovascular diseases characterized by cardiac remodelling. Objective and methods: The aims of this study are to investigate the expression and the function of P2X7R in cultured human dermal fibroblasts from SSc patients in comparison with healthy dermal fibroblasts as control or in cardiac fibroblasts from CHF patients in comparison with human atrial fibroblast cell line as control, and also to analyze putative underlying mechanistic pathways involved in the fibrotic process. SSc fibroblasts were isolated from the skin biopsies of SSc patients and healthy volunteers; while CHF fibroblasts were isolated from human atrial fragments obtained from patients undergoing cardiac surgical intervention and the cell line Normal Human Cardiac Fibroblasts-Atrium (NHCF-A) was used as control. In these cells, P2X7R expression were evaluated by flow cytometry and RT q-PCR. P2X7R function and the ensuing effects were assessed by measuring: -cytosolic Ca2+ entry, by single cell fluorescence microscopy; -collagen production, by EIA assay; -α-SMA expression, by immunofluorescence; -fibroblasts migration, by using a wound healing scratch assay. Intracellular pathways potentially involved in fibrogenic process were investigated by measuring: -the effect of P2X7R and/or ERK1/2 inhibitors in different experiments; -the expression of ERK 1/2 by Western Blot analysis; -pro-inflammatory cytokines/growth factor release by ELISA. Results: P2X7R was overexpressed in SSc fibroblasts, compared to control fibroblast, and its stimulation correspond to a markedly higher Ca2+ permeability and collagen production. Moreover, increased α-SMA expression/organization and cell migration were observed in lipopolysaccharides (LPS)-primed SSc fibroblasts after P2X7R stimulation with respect to control fibroblasts. P2X7R-stimulation with LPS plus 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) induced IL-6 release in SSc fibroblasts, however IL-6 stimulation did not affected collagen production. Instead, collagen secretion was completely abrogated by P2X7R-inhibition with periodate-oxidized ATP (oATP) or by ERK 1/2-inhibition with FR-180204. Cardiac fibroblasts from CHF patients showed, with respect to control cardiac fibroblasts, a higher surface expression of P2X7R and an enhanced receptor function in terms of Ca2+ influx and collagen production. Unlike from SSc fibroblasts, P2X7R activation did not induce cytokine changes. Moreover, TNF-α induced a further increase of P2X7R expression. Finally, phosphorylated-ERK was more expressed in CHF fibroblasts compared to control cell and strictly correlated with the collagen production. Conclusions: In conclusions, our data provide evidence that both in dermal fibroblasts from SSc patients and in cardiac fibroblasts from CHF patients the expression and the function of the purinergic P2X7R are increased compared to healthy controls and are related to an increased collagen secretion. All together these results suggest a causative role of the P2X7R in the pathophysiology of the SSc and CHF and suggest P2X7R as a new attractive target for pharmacological modulation under these pathological conditions.