Combined in vivo and in cellulo approach to study the role of endothelial cells and the NLRP3 inflammasome in cardiac dysfunction associated with immune-mediated myocarditis

Introduction: Immunotherapy improves cancer outcomes but can cause myocarditis, with high mortality risk. Immune checkpoint inhibitor-induced myocarditis (ICI-M) involves T cell and macrophage infiltration in the myocardium, leading to tissue necrosis. The NLRP3 inflammasome pathway, triggered by IFN-γ, is overexpressed in ICI-M patients.

Objective: To study ICI-M pathophysiology using a preclinical model to investigate electrophysiological disorders linked to myocardial immune infiltration, and to explore molecular mechanisms in an IPSC cellular model from ICI-M patients.

Method: The preclinical ICI-M model was performed by injecting murine melanocytic cells into BALB/c mice, treated with anti-PD1/anti-CTLA-4 therapy. IPSC-derived cardiomyocytes (CM) and endothelial cells (EC) from ICI-M patients and healthy donors were exposed to IFN-γ.

Results: A decrease in tumour volume (p<0.01) was observed in mice with ECG disturbances, showing reduced QRS (p<0.01) and T wave (p<0.01) amplitude, as well as shorter repolarisation time (p<0.05). Impaired cardiac function correlated with increased CD3 transcript (p<0.01) and PD-L1 overexpression in the myocardium. In hiPSC-derived CM and EC, IFN-γ (10 ng/ml, 72 h) upregulated immune-response genes (PD-L1, MHC-II, p<0.001) and NLRP3 inflammasome genes (GBP5, GBP6, NLRC5, p<0.0001). More dysregulated genes were found in EC (FDR 0%, 2,210 vs. 789 in CM) after treatment. These genes are linked to immune response, apoptosis, and inflammasome activation. HiPSC-ECs showed a decreased expression of cell communication genes and an increased expression of JAK-STAT/MAP Kinases, NF-κB, and cytokine production genes. The inflammasome activated by IFN-γ differed between cell types, with NLRP3 in CM and AIM2 in EC. HiPSC-ECs from ICI-M patients showed specific immune-response regulations, including PD-L2, TLR2 overexpression, and pro-inflammatory cytokines (CCL2, CCL5, IL-1β), linked to NLRP3 inflammasome activation.

Conclusion: Mice treated with ICI show ECG disturbances linked to myocardial inflammation. Our model highlights the key role of endothelial cells in the IFN-γ response and NLRP3 inflammasome regulation in ICI-M patient cells.