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

Abstract

Protease 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 ({Delta}{Psi}), ROS, and electron transport chain (ETC) proteins. Apoptosis pathway genes were interrogated using the Human Apoptosis RT{superscript 2} Profiler PCR Array kit (QIAGEN), followed by quantitative PCR for validation. CEM cells treated with 15, 22.5, and 30 {micro}M of ATV resulted in significant reduction in cell growth and increased apoptosis. Further, high concentrations of ATV resulted in decreased mitochondrial {Delta}{Psi}, 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.