Role of the NLRP3 inflammasome in myocarditis induced by immune checkpoint inhibitors cancer immunotherapy.

The inflammasome NLRP3 is involved in the inflammatory processes of several cardiac diseases. Recently, GBP5 and 6 transcripts have been found to be specifically deregulated in immune-related myocarditis, but the mechanism of action remains unclear. Cardiomyocyte dysfunction during the cardiotoxic reaction probably results from the combined action of cytokines secreted by T lymphocytes reactivated by ICIs and inflammatory molecules released by endocardial and vascular endothelial cells. Our hypothesis is that cardiac dysfunction in immune-mediated myocarditis is linked to activation of the NLRP3 inflammasome in cardiac cells in this inflammatory context. Our aim is to unravel the regulatory mechanisms of the NLRP3 inflammasome in response to cytokines known to be secreted during ICI-related myocarditis, and to characterise the role of paracrine effects of endothelial cells in cardiomyocyte dysfunction. hiPSC-derived cardiomyocytes (hiPSC-CM) and endothelial cells (hiPSC-EC) were stimulated by different inflammatory cytokines (IFN-γ, RANTES, Granzyme B and IL-1β). We studied several groups: healthy individual (1 clone from 1 individual), melanoma patients who reported immuno-mediated myocarditis (13 clones from 5 patients), and melanoma patients who did not report cardiotoxicity after immunotherapy (7 clones from 3 patients). IFN-γ affects the viability of cardiac cells by activating the JAK/STAT inflammatory pathway, and by overexpressing NLRP3 inflammasome transcripts such as nlrp3, nlrc5 and caspase-1. This effect is associated to overexpression of the MHC and PD-L1 proteins, as described in humans. IFN-γ alone did not induce significant differences between groups on these targets. We have developed a flow cytometry multiplexing protocol to characterise apoptosis associated with caspase activity, in particular NLRP3 inflammasome-associated caspase-1, metabolic deregulation and oxidative stress. The results are currently being analysed after stimulation by inflammatory cytokines IFN-γ, RANTES, Granzyme B and IL-1β. hiPSC-CM and hiPSC-EC respond to IFN-γ by activating inflammatory pathways, in particular the NLRP3 inflammasome, leading to cell death. Our work unravels the mechanisms associated with cardiotoxicity, triggered by inflammatory cytokines released during the cardiotoxic response to ICIs, thus clarifying the role of the two cardiac cell types in the cardiac dysfunction observed in immuno-mediated myocarditis in patients. [ABSTRACT FROM AUTHOR]