Activation of NLRP3 inflammasome/pyroptosis by protease inhibitor atazanavir at high concentrations is mediated through mitochondrial dysfunction

Abstract

AbstractProtease inhibitor (PI)-based antiretroviral therapy increases CD4 and CD8 cell counts through anti-apoptosis mechanism. However, there are emerging reports that PIs could have pro-apoptotic effects; thus, PIs may have biphasic effect on apoptosis. We hypothesized that PI-induced apoptosis may be mediated through PI-induced mitochondrial dysfunction resulting in increased production of reactive oxygen species (ROS).To test this hypothesis, we used human T lymphoblastoid cell line (CEM) cultured with increasing concentrations of atazanavir (ATV). We assessed mitochondrial function—i.e., cell growth, apoptosis, mitochondrial membrane potential (ΔΨ), ROS, and electron transport chain (ETC) proteins. Apoptosis pathway genes were interrogated using the Human Apoptosis RT² Profiler PCR Array kit (QIAGEN), followed by quantitative PCR for validation.CEM cells treated with 15, 22.5, and 30 µM of ATV resulted in significant reduction in cell growth and increased apoptosis. Further, high concentrations of ATV resulted in decreased mitochondrial ΔΨ, increased ROS production, and decreased protein expression of ETC I, II, III, and V. The following apoptosis pathway genes—caspase-1, BCL2A1, TP73 and TNFRSF1B—were differentially expressed. Caspase-1 is known to play a role in inducing NLRP3 inflammasome/pyroptosis pathway. We validated this with qPCR of the genes in the NLRP3 inflammasome pathway. Of note, NLRP3 inhibitor MCC950 and caspase-1 inhibitor YVAD reversed mitochondrial dysfunction and cell death. Our findings suggest that ATV-induced cell death is through the NLRP3 inflammasome/pyroptosis pathway. ATV-induced mitochondrial dysfunction plays an important role in the regulation of ATV-activated NLRP3 inflammasome pathway.