Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters.

AIMS: Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. RESULTS: In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE2) in plasma when compared with either HIV or alcohol alone. INNOVATION: This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. CONCLUSION: These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to immune toxicity, and further exacerbation with alcohol use. Antioxid. Redox Signal. 28, 324-337.

Preparation and characterization of anti-HIV nanodrug targeted to microfold cell of gut-associated lymphoid tissue.

The human immunodeficiency virus 1 (HIV-1) still remains one of the leading life-threatening diseases in the world. The introduction of highly active antiretroviral therapy has significantly reduced disease morbidity and mortality. However, most of the drugs have variable penetrance into viral reservoir sites, including gut-associated lymphoid tissue (GALT). Being the largest lymphoid organ, GALT plays a key role in early HIV infection and host-pathogen interaction. Many different treatment options have been proposed to eradicate the virus from GALT. However, it becomes difficult to deliver traditional drugs to the GALT because of its complex physiology. In this regard, we developed a polymer-based Pluronic nanocarrier containing anti-HIV drug called efavirenz (EFV) targeting Microfold cells (M-cells) in the GALT. M-cells are specialized epithelial cells that are predominantly present in the GALT. In this work, we have exploited this paracellular transport property of M-cells for targeted delivery of Pluronic nanocarrier tagged EFV, bioconjugated with anti-M-cell-specific antibodies to the GALT (nanodrug). Preliminary characterization showed that the nanodrug (EFV-F12-COOH) is of 140 nm size with 0.3 polydispersion index, and the zeta potential of the particles was -19.38±2.2 mV. Further, drug dissolution study has shown a significantly improved sustained release over free drugs. Binding potential of nanodrug with M-cell was also confirmed with fluorescence microscopy and in vitro uptake and release studies. The anti-HIV activity of the nanodrug was also significantly higher compared to that of free drug. This novel formulation was able to show sustained release of EFV and inhibit the HIV-1 infection in the GALT compared to the free drug. The present study has potential for our in vivo targeted nanodrug delivery system by combining traditional enteric-coated capsule technique via oral administration.

DJ1 expression downregulates in neuroblastoma cells (SK-N-MC) chronically exposed to HIV-1 and cocaine.

BACKGROUND: HIV-associated neurological disorder (HAND) has long been recognized as a consequence of human immunodeficiency virus (HIV) infection in the brain. The pathology of HAND gets more complicated with the recreational drug use such as cocaine. Recent studies have suggested multiple genetic influences involved in the pathology of addiction and HAND but only a fraction of the entire genetic risk has been investigated so far. In this regard, role of DJ1 protein (a gene linked to autosomal recessive early-onset Parkinson's disease) in regulating dopamine (DA) transmission and reactive oxygen species (ROS) production in neuronal cells will be worth investigating in HIV-1 and cocaine exposed microenvironment. Being a very abundant protein in the brain, DJ1 could serve as a potential marker for early detection of HIV-1 and/or cocaine related neurological disorder. METHODS: In vitro analysis was done to observe the effect of HIV-1 and/or cocaine on DJ1 protein expression in neuroblastoma cells (SK-N-MC). Gene and protein expression analysis of DJ1 was done on the HIV infected and/or cocaine treated SK-N-MC and compared to untreated cells using real time PCR, Western Blot and flow cytometry. Effect of DJ1 dysregulation on oxidative stress was analyzed by measuring ROS production in these cells. RESULTS: Gene expression and protein analysis indicated that there was a significant decrease in DJ1 expression in SK-N-MC chronically exposed to HIV-1 and/or cocaine which is inversely proportional to ROS production. CONCLUSION: This is the first study to establish that DJ1 expression level in the neuronal cells significantly decreased in presence of HIV-1 and/or cocaine indicating oxidative stress level of DA neurons.

Effect of mild-to-moderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals.

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals.

Immunoneuropathogenesis of HIV-1 clades B and C: role of redox expression and thiol modification.

Previous studies have shown that, during infection, HIV-1 clade B and clade C differentially contribute to the neuropathogenesis and development of HIV-associated neurocognitive disorders (HANDs). The low-molecular-weight tripeptide glutathione (GSH) alters the redox balance and leads to the generation of reactive oxygen species, which play a significant role in the neuropathogenesis of HANDs. We hypothesized that the HIV-1 clade B and clade C viruses and their respective Tat proteins exert differential effects on monocyte-derived immature dendritic cells (IDCs) and neuroblastoma cells (SK-N-MC) by redox activation, which leads to immunoneuropathogenesis. The GSH/GSSG ratio and mRNA expression levels and protein modification of glutathione synthetase (GSS), glutathione peroxidase 1 (GPx1), superoxide dismutase 1 (SOD1), and catalase (CAT) were analyzed in IDCs infected with HIV-1 clade B or clade C as well as in cells treated with the respective Tat proteins. The results indicated that HIV-1 clade B virus and its Tat protein significantly increased the production of reactive oxygen species and reduced the GSH/GSSG ratio and subsequent downregulation of gene expression and protein modification of GSS, GPx1, SOD1, and CAT compared to infection with the clade C virus or treatment with the clade C Tat protein. Thus, our studies demonstrate that HIV-1 clades B and C exert differential effects of redox expression and thiol modification. HIV-1 clade B potentially induces oxidative stress, leading to more immunoneuropathogenesis than infection with HIV-1 clade C.

Cocaine enhances HIV-1 infectivity in monocyte derived dendritic cells by suppressing microRNA-155.

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

Inhibition of nuclear factor erythroid 2-related factor 2 exacerbates HIV-1 gp120-induced oxidative and inflammatory response: role in HIV associated neurocognitive disorder.

The HIV epidemic continues to be the most severe public health problem and concern within USA and across the globe. In spite of the highly active antiretroviral therapy, HIV infected subjects experience major neurological complications that range from HIV associated dementia to moderate neurocognitive and motor impairments collectively termed as HIV associated neurocognitive disorders (HAND). Astrocytes play an important role in the neuropathogenesis of HAND. Further, in the recent years it has been shown that oxidative stress plays a major role in the neuropathogenesis of HAND. Nuclear factor erythroid 2-related factor 2 (Nrf2), a leucine zipper redox-sensitive transcription factor, is an important regulator of cell survival and adaptive mechanisms and has been shown to possess a protective role in a variety of neurological and inflammatory disorders. Earlier we have shown that Nrf2 is upregulated in response to HIV-1 gp120 and such upregulation of Nrf2 may be a protective mechanism against the HIV-induced oxidative stress. We hypothesize that Nrf2-mediated antioxidant pathways are important in regulating the HIV-induced oxidative stress and that the disruption of Nrf2 makes the cells more susceptible to HIV gp120-induced deleterious effects. Our results indicate that when astrocytes are exposed to gp120 there is an increase in the expression of NOX2, a subunit of NADPH oxidase, and also an upregulated expression of nuclear factor kappa B, tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9). However, the degree of expression was significantly higher in those cells where Nrf2 was silenced by siRNA. Taken together, these results suggest a possible protective role of Nrf2 in regulating the levels of pro-oxidative and pro-inflammatory molecules in HAND.

HIV-1 Tat upregulates expression of histone deacetylase-2 (HDAC2) in human neurons: implication for HIV-associated neurocognitive disorder (HAND).

Histone deacetylases (HDACs) play a pivotal role in epigenetic regulation of transcription and homeostasis of protein acetylation in histones and other proteins involved in chromatin remodeling. Histone hypoacetylation and transcriptional dysfunction have been shown to be associated with a variety of neurodegenerative diseases. More recently, neuron specific overexpression of HDAC2 has been shown to modulate synaptic plasticity and learning behavior in mice. However, the role of HDAC2 in development of HIV-associated neurocognitive disorders (HAND) is not reported. Herein we report that HIV-1 Tat protein upregulate HDAC2 expression in neuronal cells leading to transcriptional repression of genes involved in synaptic plasticity and neuronal function thereby contributing to the progression of HAND. Our results indicate upregulation of HDAC2 by Tat treatment in dose and time dependant manner by human neuroblastoma SK-N-MC cells and primary human neurons. Further, HDAC2 overexpression was associated with concomitant downregulation in CREB and CaMKIIa genes that are known to regulate neuronal activity. These observed effects were completely blocked by HDAC2 inhibition. These results for the first time suggest the possible role of HDAC2 in development of HAND. Therefore, use of HDAC2 specific inhibitor in combination with HAART may be of therapeutic value in treatment of neurocognitive disorders observed in HIV-1 infected individuals.

Identification of transcription start sites and preferential expression of select CB2 transcripts in mouse and human B lymphocytes.

Marijuana cannabinoids, the endocannabinoids, and cannabinoid cell receptors have been shown to play important roles in immune regulation particularly as potent modulators of anti-inflammatory cytokines. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB(2)), which is predominantly expressed in B lymphocytes. However, the promoter region and mechanisms of CB(2) gene regulation are unknown in this immune cell type. Utilizing a combination of bioinformatics, 5' rapid amplification of cDNA ends (5' RACE), real-time reverse transcription-polymerase chain reaction, DNA sequencing, and luciferase reporter assays, we show that human B cells express one CB(2) transcript while mouse B cells express three CB(2) transcripts, with specific transcript selection occurring during B cell activation by lipopolysaccharide. Alignment of our sequenced RACE products to either the mouse or human genome, along with the GenBank submitted mRNA sequences, revealed that the transcripts we isolated contained previously unidentified transcriptional start sites (TSS). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB(2) exon 1 transcripts showed an eightfold increase of promoter activity over baseline. These data show for the first time that human B cells use only one TSS for CB(2) while mouse B cells use multiple TSSs and that the mouse TSSs are in a genomic area with promoter activity, thus suggesting the location of the gene promoter region. Defining these TSSs also provides clues to the various gene regulatory factors involved in the expression of CB(2) during B cell activation.