Abstract: Chronic and sustained neuroinflammation is a hallmark feature underlying HIV-associated neurocognitive disorders (HAND). Factors contributing to this inflammation have been suggested to involve low-level HIV replication/HIV proteins and/or toxicity of cART istelf. It is well recognized that drug abuse, specifically opiate abuse, is a common comorbidity among individuals with HAND. Intriguingly, opiates have also been shown to exacerbate neuroinflammation either through direct effect on immune cells such as microglia and/or by decreasing the effectiveness of cART against HIV replication. It can thus be envisioned that within the CNS, combinations of HIV proteins, abused drugs and cART create a toxic milieu promoting exacerbated neuroinflammation. The detailed molecular pathways underlying this phenomenon however, remain elusive. Our preliminary studies using each of these factors demonstrate that: 1) HIV TAT can increase microglial activation via the NLRP3 inflammasome signaling; 2) Opiates such as morphine activate microglia via dysregulated autophagy pathway & 3) Combination of clinically used antiretrovrials (TFV, FTC, DTG) increase ER stress activity of microglia leading to their activation. Furthermore, we also demonstrated that combined exposure of TAT, morphine & cART in microglia resulted in exacerbated production of the pro- inflammatory mediator IL-6 (compared with cells treated with individual factor) and a concomitant disruption of the mitochondrial membrane in vitro. We thus hypothesize that combinations of HIV TAT, morphine & cART can activate microglia via the inflammasome, autophagy and ER stress pathways. Using in vitro and in vivo (HIV Tg rats) approaches we will test the hypothesis via two specific aims - SA1: Investigate the molecular mechanism(s) underlying TAT, morphine & ARVs (3 drug regimen) mediated activation of microglia in vitro; and SA2: To determine the mechanisms underlying Tat, morphine and cART-mediated neuroinflammation in vivo in a rodent model of HAND. Two experienced PIs (Drs. Guo & Buch) will co-lead this project to accomplish the proposed goals. This proposal is in response to NIDA RFA-DA-17-013 on “Mechanisms of Immune Activation and Inflammation: HIV Infection, ART, and Drugs of Abuse (R01)”.