AB0201 THE PATHOGENIC EFFECTS OF IMMUNE COMPLEXES CONTAINING SCLERODERMA-SPECIFIC AUTOANTIBODIES IN ENDOTHELIAL CELLS

Abstract

Background In systemic sclerosis (SSc), autoantibodies provide the most reliable tool to predict disease subset and the pattern of organ involvement. Objectives Consistently with the diagnostic and prognostic role of these autoantibodies, our group has previously demonstrated that immune complexes (ICs) containing scleroderma specific autoantibodies elicit a pro-inflammatory and pro-fibrotic signalling cascade in healthy skin fibroblasts, thus contributing to SSc aetiopathogenesis. In addition, we showed that SSc-ICs upregulate Toll-like Receptors (TLRs) and that pre-treatment with DNase and RNase prevent SSc-IC-mediated effects in fibroblasts, thus suggesting that SSc-ICs might engage TLRs via their nucleic acid components. The objective of the present study is to investigate the pathogenicity of SSc-ICs in endothelial cells (ECs). Methods Human umbilical vein ECs (HUVECs) have been isolated from umbilical vein and cultured in adequate conditions. ICs have been purified from sera of SSc patients bearing different autoantibody specificities (antibodies against centromeric proteins, DNA topoisomerase I, RNA polymerase and Th/To), patients with systemic lupus erythematosus (SLE), primary anti-phospholipid syndrome (PAPS) or healthy controls using polyethylen glycol precipitation. Cell cultures have been incubated with pathologic and control ICs and TLR3 [Poly(I:C)] and TLR4 (LPS) agonists. Several parameters have been assessed: mRNA levels of endothelin-1 (ET-1) and type I interferons (IFN-α and IFN-β) have been investigated by real-Time PCR; ICAM-1 expression has been evaluated by cell-ELISA and the secretion of IL-6, IL-8, tumour growth factor (TGF)-β1 and Pro-CollagenIα1 in culture supernatants has been measured by commercial ELISA kits. The involvement of intracellular signalling pathways culminating with the activation of p38MAPK has been assessed. Furthermore, skin fibroblasts from healthy controls were stimulated with supernatants from HUVEC incubated with scleroderma and control ICs. The mRNA levels of collagen (col)Iα1 and matrix metalloproteinases (MMP)-1 and the secretion of TGF-β1 were evaluated in fibroblasts. Statistical analysis was performed comparing ICs from SSc and disease controls to NHS-ICs. Results Stimulation of HUVEC with scleroderma and control ICs resulted in the modulation of study mediators, as detailed in Table 1. When skin fibroblasts from healthy controls were stimulated with supernatants from HUVEC incubated with scleroderma and control ICs, we observed a significant modulation of mediators (Table 2). Conclusion These data provide the first demonstration of the pathogenicity of ICs isolated from scleroderma patients with different autoantibody specificities in ECs. According to our preliminary data, the activation of ECs induced by SSc-ICs might ultimately lead to a pro-fibrotic phenotype in healthy skin fibroblasts. SSc-ICs might thus be at the crossroad of scleroderma pathogenesis, contributing to the recruitment of the many cells involved in disease onset. Reference [1] Raschi E, Chighizola CB, Cesana L, et al. Arthritis Res Ther2018;20:187 Disclosure of Interests Cecilia Chighizola Speakers bureau: Inova Diagnostics, Daniela Privitera: None declared, Maria Orietta Borghi: None declared, Pier Luigi Meroni Consultant for: Inova, Thermofisher, Teofarma, Elena raschi: None declared