NLRP3 Inflammasome Products IL‐1beta and IL‐18 Have A Direct Effect on Cardiomyocytes

Abstract

ObjectiveWe previously demonstrated a significant role for NLRP3 inflammasome activation in cardiomyocytes in cardiac inflammation, remodeling and contractile dysfunction in response to stressors such as Angiotensin II infusion and pressure overload. There is limited data regarding responses of cardiomyocytes to interleukin‐1 beta (IL‐1beta) and interleukin 18 (IL‐18), which are generated as a result of NLRP3 activation. The goal of this study was to determine the effects of IL‐1beta and IL‐18 on cardiomyocytes.MethodsIsolated Neonatal Rat Ventricular Myocytes (NRVMs) were treated for various times with a range of doses of recombinant IL‐1beta or IL‐18. Western Blotting was used to measure the activation state of protein kinases and downstream signaling pathways. Quantitative Polymerase Chain Reaction (q‐PCR) was used to assess the expression of pro‐inflammatory cytokines and chemokines in cardiomyocytes.ResultsWestern blotting showed a significant increase in phosphorylation of multiple protein kinases and molecules that regulate apoptosis and other inflammatory functions after IL‐1beta and IL‐18 treatment, with maximal responses seen at 1ng/ml IL‐1beta and 10ng/ml IL‐18. Phosphorylated mitogen‐activated protein kinases (MAPKs) including JNK and P38 as well as p‐STAT3 were increased several fold at 10 and 30 mins of treatment with either cytokine. q‐PCR analysis of NRVM mRNA revealed increases in gene expression of multiple pro‐inflammatory cytokines and chemokines in cells treated with either IL‐1beta and IL‐18. CXCL‐10, TGF‐beta, IL‐18, and NLRP3 were all upregulated by both IL‐1beta or IL‐18. Interestingly, upregulation of CCL2, IL‐6, CXCL1, and TNF‐alpha resulted from treatment with IL‐1beta but not with IL‐18. We are currently analyzing the effects of IL‐1beta and IL‐18 on cell viability to further determine the direct effect of these products of the NLRP3 inflammasome in regulating cardiomyocyte apoptosis basally or in response to stressors.ConclusionsWe have previously shown that the cardiomyocyte is a source of proinflammatory cytokines and chemokines, including IL‐1beta and IL‐18. Our results suggest the possibility that IL‐1beta and IL‐18 activate a pro‐inflammatory positive feedback loop in cardiomyocytes which enhances and sustains cardiac inflammation, contributing to cardiac remodeling under stress conditions.