Cardiotoxicity is mediated by the NLRP3 inflammasome in hiPSC-derived endothelial cells from patients with immune checkpoint inhibitor-related myocarditis

Abstract

In association with immune checkpoint inhibitors (ICI) anticancer therapy, myocarditis (ICI-M), have emerged with a mortality rate reaching 50%. The IFN-γ pathway and inflammasome regulatory proteins seem to be specifically involved in ICI-M, but the mechanism of action remains unclear. In the cardiovascular system, the endocardial and vascular endothelium is involved in the regulation of the contractile function of cardiomyocytes. Our hypothesis is that the cardiotoxicity induced by the infiltration of immune cells into the myocardium is partly due to an exacerbation of inflammasome activation in endothelial cells, which subsequently affects the cardiomyocytes function. Our aim is to characterize the role of endothelial cells in ICI-related myocarditis in a unique model of induced pluripotent stem cells (hiPSC)-derived cardiac cells. The hiPSC were generated from melanoma patients who received anti-PD-1/anti-CTLA-4 immunotherapy and followed at the Cardio-Oncology Centre (North Hospital, Marseille). Twelve hiPSC clones were generated from patients who developed myocarditis (ICI-M, 6 clones from 3 patients), and patients who did not develop cardiotoxicity (non-ICI-M, 6 clones from 3 patients). hiPSC-derived endothelial cells (hiPSC-EC) were differentiated from hiPSC clones and stimulated by 10 ng/mL of IFN-γ during 48 h. IFN-γ induced a decrease in cell viability, as well as an increase in the expression of transcripts of genes associated with the JAK/STAT and NLRP3 inflammasome pathways in hiPSC-EC. However, the decrease in cell viability was significantly greater in hiPSC-EC from the ICI-M group compared to the non-ICI-M group. IFN-γ activation of inflammatory intracellular signalling is greater in the ICI-M group, in particular overexpression of the jak1, jak2, stat3 and gbp6 transcripts. At the protein level, in this ICI-M group, the IFN-γ-induced increase of MHC-I and PD-L1 is greater than in the non-ICI-M group. IFN-γ stimulation revealed specific regulations in endothelial cells derived from hiPSC clones from patients with ICI-related myocarditis. We are now analysing the apoptotic and inflammatory activity of hiPSC-EC by real-time microscopy monitoring of caspases (Incucyte S3) and by assaying pro-inflammatory molecules in the supernatants (ELISA). This study will allow to establish the role played by endothelial cells in cardiac toxicity induced by ICI immunotherapy.