Oxidized Low-Density Lipoproteins increases nivolumab-induced cardiotoxicity through TLR4/NF-KB and NLRP3 pathways

Abstract

Abstract Background Atherosclerosis is now recognized as a chronic inflammatory disease. Oxidized low-density lipoprotein (Ox-LDL) is oxidatively modified form of LDL with a key role in induction and progressio of atherosclerosis. Recent findings reported that cardiovascular events (myocarditis and atherosclerosis) were higher after initiation of immune check-point inhibitors (ICIs), potentially mediated by accelerated progression of atherosclerosis. Purpose We evaluated whether ox-LDL-induced apoptosis through toll-like receptor-4 (TLR4)/Nuclear factor κB (NF-κB) signaling pathway and NLRP3 inflammasome during exposure of human cardiomyocytes to nivolumab turning the light on the mechanisms of cell inflammation induced by OxLDL in cardiotoxicity of ICIs. Methods Human fetal cardiomyocytes (HFC cell line) in co-culture with hPBMC, were exposed to clinically relevant concentration of nivolumab (100 nM) alone or combined to OxLDL at 1, 10 and 50 μg/mL for 24h. After the incubation period, we performed the following tests: determination of cell viability, through analysis of mitochondrial dehydrogenase activity, study of lipid peroxidation (quantifying cellular Malondialdehyde and 4-hydroxynonenal), intracellular Ca2+ homeostasis and apoptosis. Moreover, pro-inflammatory studied were also performed (activation of NLRP3 inflammasome, expression of TLR4 and NF-kB). In order to evaluate the pathways involved in OxLDL damages, TLR4 and NLRP3 inhibitor (TAK-242 and dapansutrile, respectively) were added during cell viability and apoptosis studies. Results Nivolumab exerts cytotoxic and pro-apoptotic effects in co-coltures of cardiomyocytes and hPBMC. OxLDL increases significantly the nivolumab-induced cardiotoxicity in a manner that is sensitive to TLR4 and NLRP3. Incubation of cardiomyocytes with ox-LDL (10 and 50 μg/mL) for 24 hours increased TLR4 and NF-κB expressions. Ox-LDL had pro-apoptotic effects in a concentration-dependent manner with the involvement of lipid peroxidation but not of intracellular calcium. Conclusion Ox-LDL exacerbates cardiotoxicity during exposure to nivolumab through pro-inflammatory mechanisms. These results place the first step to preclinical studies aimed to reduce ox-LDL during treatment with ICIs through pharmacological inhibition or by changing diet and lifestyle. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Ricerca Corrente project