Despite the ability of combination antiretroviral therapy to dramatically suppress viremia, the brain continues to be a reservoir of HIV low-level replication. Adding further complexity to this is the co-morbidity of drug abuse with HIV-associated neurocognitive disorders and neuroHIV. Among several abused drugs, the use of opiates is highly prevalent in HIV-infected individuals, both as an abused drug as well as for pain management. It has been shown that both HIV/HIV proteins and abused drugs such as morphine contribute to neuroinflammation, which, in turn, involves activation of the inflammasomes. Mounting evidence has also shown that noncoding RNAs function as epigenetic and post-transcriptional regulators controlling vital cellular functions, including activation, thus altering the dynamics of various biological processes. While both HIV proteins and opioids have been shown to change the gene expression profiles of CNS cells, there is a gap of knowledge on the detailed molecular mechanism(s) underlying the co-operative effects of HIV Transactivator of transcription (Tat) protein and morphine on astrocyte activation. The central hypothesis of this proposal is that HIV Tat and morphine, via distinct regulatory mechanism(s) augment astrocyte activation leading, in turn, to exacerbated neuroinflammation. Our preliminary data on whole-genome bisulfite sequencing in the frontal cortices of SIV- infected rhesus macaques show increased DNA hypomethylation of the NLRP6 (NOD-like receptor family, pyrin domain-containing protein 6) promoter with a concomitant upregulation in NLRP6 expression. Additionally, exposure of human and mouse primary astrocytes to HIV Tat and morphine also resulted in decreased expression of microRNA-152 that was accompanied by increased expression of NLRP6. Interestingly, we also found that exposure of these primary astrocytes to either HIV Tat or morphine resulted in increased cellular activation with increased expression of proinflammatory cytokines (IL1? and IL18) via pyroptosis. Based on the central hypothesis and the reliable preliminary data, this proposal led to the following specific aims:
Specific Aim 1 : Determine the molecular mechanism(s) involved in microRNA-152 mediated NLRP6 inflammasome activation in HIV Tat and morphine-exposed astrocytes in vitro;
Specific Aim 2 : Determine the epigenetic mechanism(s) involved in promoter DNA hypomethylation of the NLRP6 in HIV Tat and morphine-exposed astrocytes in vitro;
Specific Aim 3 : Validate the combinatorial effects of HIV Tat and morphine on NLRP6 inflammasome mediated astrocyte activation, in vivo. Understanding the mechanisms responsible for astrocyte activation induced by HIV and morphine will set the stage for the future development of novel therapeutics aimed at dampening HIV and opiate-mediated neuroinflammatory responses.
This proposed research is relevant to public health because drug abuse among HIV-infected individuals poses a significant health burden to the Society. One of the hallmark features of morphine abuse is augmented astrocyte activation in the setting of HIV infection. Herein, we proposed to investigate the molecular mechanisms underlying the effects of both HIV and morphine on astrocyte activation in the context of NLRP6 inflammasome signaling using various state-of-the-art approaches that could have ramifications for the future development of novel therapeutic approaches to mitigate central nervous system complications of NeuroHIV.
