Alcohol Influences HAND Via Astrocyte-TLR4 and cPLA2 Signaling

Background and Objective: Over the past few decades, ~25 million people died with human immunodeficiency virus (HIV)-1 disease. About 70% of HIV patients suffer from HIV-associated neurocognitive disorders (HAND). The prevalence of alcohol abuse among HIV-1+ve individuals is estimated to be 2-3 times that of the general population in the USA. HIV patient brains harbor up to 20% HIV-infected astrocytes, thus making them critical players in HAND. Previously, we reported that HIV-1 and/or alcohol (EtOH) activated astrocytes induced inflammation via cytosolic phospholipase A2 (cPLA2) activation. How EtOH regulates HIV-1-mediated inflammatory episodes initiated at the cell surface level is still unclear. Toll-like receptor (TLR) signaling in immune cells, astrocytes, microglia and neurons may play roles in pathogenesis of multiple diseases including HIV-1. Hypothesis: We propose that TLR4 may serve as critical regulator of alcohol-mediated inflammatory responses in HAND directly or by controlling cPLA2 signaling. Materials and Methods: To investigate the temporal order of events, primary human astrocytes were cultured and treated with HIV-1 (10ng/ml) and/or EtOH (50mM). TLR4, COX2 and CCL2 mRNA levels were measured by RT2PCR at 8h whereas protein levels were analyzed by ELISA, western blot and immunocytochemistry on 24h. We also explored the phosphorylation studies of cPLA2 and TLR4 downstream molecules such as IRAK4 and NF-kB by western blot and immuno-staining analysis on 30 min. TLR4-RNAi and cPLA2-specific inhibitor AACOCF3 were employed to carried out TLR4 and cPLA2-specific responses. Results: We showed EtOH, HIV-1, IL-1b and anti-retroviral (ARV) drugs significantly upregulated TLR4 in human astrocytes. Our results established that EtOH+/- HIV-1 activated TLR4 signaling leads to IRAK4 phosphorylation followed by NF-kB activation, ultimately leading to excessive production of inflammatory mediators such as COX2 & CCL2. EtOH and/or HIV-1 increased inflammatory molecules in MyD88-dependent manner. TLR4-RNAi studies reversed EtOH and/or HIV-1-regulated effects. Moreover, on silencing TLR4, the increase in EtOH+/-HIV-1-induced cPLA2 phosphorylation was not observed. Conclusions: Our study demonstrated that TLR4 regulates inflammatory responses in primary human astrocytes directly or by controlling cPLA2 cascade in HAND. Hence, TLR4 could be the critical regulator of alcohol-induced astrocyte inflammation with HIV-1.