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In this study, we investigated the potential of using curcumin (CUR) as an adjuvant to enhance the delivery of antiretroviral drug elvitegravir (EVG) across the BBB, and alleviate oxidative stress and inflammatory response, which are the major hallmark of HIV neuropathogenesis. In a mouse model, we compared the biodistribution of EVG alone and in combination with CUR using intraperitoneal (IP) and intranasal (IN) routes. IN administration showed a significantly higher accumulation of EVG in the brain, while both IP and IN routes led to increased EVG levels in the lungs and liver. The addition of CUR further enhanced EVG brain delivery, especially when administered via the IN route. The expression of neural marker proteins, synaptophysin, L1CAM, NeuN, and GFAP was not significantly altered by EVG or CUR alone or their combination, indicating preserved neural homeostasis. After establishing improved brain concentration and safety of CUR-adjuvanted EVG in mice in acute treatment, we studied the effect of this treatment in HIV-infected U1 macrophages. In U1 macrophages, we also observed that the addition of CUR enhanced the intracellular concentration of EVG. The total area under the curve (AUC tot ) for EVG was significantly higher in the presence of CUR. We also evaluated the effects of CUR on oxidative stress and antioxidant capacity in EVG-treated U1 macrophages. CUR reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) levels and elevated antioxidant enzyme expression. Furthermore, the combination of CUR and EVG exhibited a significant reduction in proinflammatory cytokines (TNFα, IL-1β, IL-18) and chemokines (RANTES, MCP-1) in U1 macrophages. Additionally, western blot analysis confirmed the decreased expression of IL-1β and TNF-α in EVG + CUR-treated cells. These findings suggest the potential of CUR to enhance EVG permeability to the brain and subsequent efficacy of EVG, including HIV neuropathogenesis., (© 2023. The Author(s).)

We have recently demonstrated that long-term exposure of cigarette smoke condensate (CSC) to HIV-uninfected (U937) and -infected (U1) macrophages induce packaging of pro-inflammatory molecules, particularly IL-1β, in extracellular vesicles (EVs). Therefore, we hypothesize that exposure of EVs derived from CSC-treated macrophages to CNS cells can increase their IL-1β levels contributing to neuroinflammation. To test this hypothesis, we treated the U937 and U1 differentiated macrophages once daily with CSC (10 µg/ml) for 7 days. Then, we isolated EVs from these macrophages and treated these EVs with human astrocytic (SVGA) and neuronal (SH-SY5Y) cells in the absence and presence of CSC. We then examined the protein expression of IL-1β and oxidative stress related proteins, cytochrome P450 2A6 (CYP2A6), superoxide dismutase-1 (SOD1), catalase (CAT). We observed that the U937 cells have lower expression of IL-1β compared to their respective EVs, confirming that most of the produced IL-1β are packaged into EVs. Further, EVs isolated from HIV-infected and uninfected cells, both in the absence and presence of CSC, were treated to SVGA and SH-SY5Y cells. These treatments showed a significant increase in the levels of IL-1β in both SVGA and SH-SY5Y cells. However, under the same conditions, the levels of CYP2A6, SOD1, and catalase were only markedly altered. These findings suggest that the macrophages communicate with astrocytes and neuronal cells via EVs-containing IL-1β in both HIV and non-HIV setting and could contribute to neuroinflammation., (© 2023. The Author(s).)

In this study, we investigated whether intravenously self-administered nicotine with menthol and audiovisual cue modulates nicotine-metabolizing CYP2A6, oxidative stress modulators, and cytokines/chemokines in plasma extracellular vesicles (EVs) in rats. We assigned rats to self-administered nicotine with: (a) audiovisual cue (AV), (b) menthol, and (c) menthol and AV cue. We found increased levels of CD9 in plasma EVs after self-administered nicotine with menthol and AV cue. Moreover, expression of CYP2A6 in plasma EVs was significantly increased after self-administered nicotine in response to menthol and AV cue. However, despite an upward trend on SOD1 and catalase, increase was not found to be statistically significant, while total antioxidant capacity was found to be significantly increased in plasma and plasma EVs obtained after self-administered nicotine with menthol and AV cue. Among cytokine and chemokine profiling, we found a significant increase in the levels of MCP-1 after self-administered nicotine with menthol and AV cue and complete packaging of IL-1β in EVs. Taken together, the study provides evidence that nicotine in response to menthol and AV cues can package altered levels of CYP2A6, and cytokines/chemokines in plasma EVs that may contribute to cell-cell communication, nicotine metabolism, and inflammation upon cigarette smoking., (© 2021. The Author(s).)

Cellular CYP2E1 is well-known to mediate alcohol- (ALC) and acetaminophen- (APAP) induced toxicity in hepatic and extra-hepatic cells. Although exosomes have been gaining importance in understanding mechanism of intra- and inter-cellular communication, the functional role of drug metabolizing cytochrome P450 (CYP) enzymes in human plasma exosomes are yet to be explored. In our previous study, we reported that human plasma-derived exosomes contain substantial level of functional CYP2E1. In the current project, we investigated the potential role of plasma exosomal CYP2E1 in mediating ALC- and APAP-induced toxicity. We treated hepatic and extra-hepatic (monocytic) cells with exosomes ± ALC/APAP. We observed that the plasma exosomes containing CYP2E1 cargo further exacerbate ALC- and APAP-induced toxicity in both hepatic and monocytic cells. Further, both exosomes- and ALC/APAP-induced toxicity was reduced/abolished by a selective inhibitor of CYP2E1 enzyme activity (diallyl ether). However, only ALC-, but not exosome-induced toxicity was reduced/abolished by CYP2E1 siRNA. These findings suggest that ALC/APAP-induced toxicity in the presence of exosomes are mediated, at least in part, by CYP2E1 enzyme. To validate these in vitro findings, we characterized plasma exosomal contents in a binge-drinking animal model and their effect on ALC/APAP-induced toxicity in monocytic cells. Our results showed that ALC exposure caused a significant induction of the plasma exosomal CYP2E1 level in a binge drinking murine model. These exosomes containing increased levels of CYP2E1 caused significant toxicity in monocytic cells compared to exosomes derived from control mice. Overall, our results showed an important role of exosomal CYP2E1 in exacerbating ALC- and APAP-induced toxicity. The study is significant in terms of understanding the role of exosomal CYP2E1 in cell-cell interactions, and their effects on drug-induced toxicity.

Smoking aggravates HIV-1 pathogenesis and leads to decreased responses to antiretroviral therapy. In this study, we aim to find a molecular mechanism that would explain smoking-induced HIV-1 replication. Benzo(a)pyrene (BaP), a major carcinogen in cigarette, requires metabolic activation through cytochrome P450s (CYPs) to exert its toxic effects. We hypothesized that CYP-mediated BaP metabolism generates reactive oxygen species (ROS), and the resultant oxidative stress aggravates HIV-1 replication. As expected, we observed ~3 to 4-fold increase in HIV-1 replication in U1 cells and human primary macrophages after chronic BaP exposure. We also observed ~30-fold increase in the expression of CYP1A1 at mRNA level, ~2.5-fold increase in its enzymatic activity as well as elevated ROS and cytotoxicity in U1 cells. The knock-down of the CYP1A1 gene using siRNA and treatment with selective CYP inhibitors and antioxidants significantly reduced HIV-1 replication. Further, we observed a nuclear translocation of NF-κB subunits (p50 and p65) after chronic BaP exposure, which was reduced by treatment with siRNA and antioxidants/CYP inhibitors. Suppression of NF-κB pathway using specific NF-κB inhibitors also significantly reduced HIV-1 replication. Altogether, our results suggest that BaP enhances HIV-1 replication in macrophages by a CYP-mediated oxidative stress pathway followed by the NF-κB pathway.