AB0157 SYNERGISTIC EFFECT OF AT1R-AUTOANTIBODIES AND EXTRACELLULAR VESICLES IN SSC PATHOGENESIS

Abstract

BackgroundSystemic sclerosis, a rare chronic inflammatory disease, are characterized by immune system activation, vasculopathy, and fibrosis of the body organs. Emerging evidence have so far indicated that autoantibodies (abs) directed against G protein-coupled receptors (GPCRs) particularly contribute to the SSc pathogenesis and inducethe release of inflammatory and profibrotic proteins by immune cells such as monocytes [1-3]. Increased levels of autoantibodies against the angiotensin II type 1 receptor (AT1R abs) have been found in SSc patients [4-5], which are associated with increased secretion of extracellular vesicles (EVs) [6]. Interestingly, upregulated CCL18 levels could be associated with AT1R-EVs in SSc patients [8]. EVs play an important role in the pathogenesis of diseases by packing and transfer of AT1R to different tissues and immune cells, exemplary shown by activated cardiomyocytes leading to higher responsiveness to angiotensin II of recipient cells and vessels [9]. Taken together, the importance of studying anti-GPCR-abs together with GPCR-EVs in the pathogenesis of SSc becomes evident [10].ObjectivesHere we decipher the immune response of peripheral blood monocytes mediated by anti-AT1R-abs and EVs, in the pathogenesis of SSc.MethodsMonoclonal AT1R ab (AT1R mab) has been generated by hybridoma technique, sequenced and recombinantly expressed in HEK cells. Human peripheral blood monocytes and monocytic cell lines were stimulated by the recombinant monoclonal anti-human AT1R ab and, in comparison, in the presence or absence of EVs precipitated from sera of SSc patients versus sera of HD. The response of the monocytes was measured via CCL18 secretion by ELISA.ResultsWe compared CCL18 release, a profibrotic cytokine, of monocytic cells upon stimulation for 24 h with the AT1R mab in presence or absence of sera EVs (SSc vs. HD). The recombinant monoclonal anti-human AT1R antibody induced secretion of CCL18 by monocytes. Our data indicate that EVs together with AT1R mab have an effect on monocyte activation and CCL18 secretion. Remarkably, combination of SSc-EVs, but not of HD-EVs, with the recombinant AT1R mab showed an additive effect on monocyte activation and CCL18 response (Figure 1).Figure 1.CCL18 levels released by monocytic cells after stimulation with EVs and AT1R mab or isotype.Stimulation with SSc EV + AT1R mab (n=4), HD EV + AT1R mab (n=3), AT1R mab control, SSc EV + isotype (n=4), HD EV + isotype (n=3) and isotype control. One-way ANOVA was used to test for statistical significance (*p<0.05, **p<0.01, ***p<0.001).ConclusionThe secretion of pro-fibrotic CCL18 by human monocytes in response to a monoclonal AT1R antibody as well as to SSc IgG indicates that anti-AT1R abs are involved in the SSc pathogenesis. Further, this effect could also be due to SSc-EVs potentially presenting anti-GPCR abs to their receptors on immune cells.