Shirish S. Barve - US grants

This is a proposal that evaluates the relationship between two CD4+ T cell destructive mechanisms, excessive alcohol consumption and HIV infection. Ethanol is believed to be a cofactor in HIV pathogenesis and both ethanol and HIV are known to cause the loss of T helper CD4+ lymphocytes leading to the impairment of multiple immune functions. Apoptotic cell death is a key mechanism involved in the depletion of CD4+ T lymphocytes during the HIV infection and is associated with an enhanced expression and subsequent triggering of the Fas receptor (FasR). Preliminary data from several experimental and clinical studies show that ethanol causes depletion of CD4+ T lymphocytes from peripheral blood, however, the mechanisms have not been characterized. The working hypothesis of this application is that alcohol consumption can be a cofactor in HIV pathogenesis by promoting enhanced oxidative stress and susceptibility of peripheral CD4 T lymphocytes to Fas-dependent apoptosis. Thus, the alcohol induced immunosuppressive effects should be attenuated by glutathione (GSH) enhancing prodrugs/precursors. The goals of this proposal are: 1) to elucidate the Fas-dependent mechanism involved in alcohol-induced immunosuppression and its role as a cofactor in HIV disease, and 2) to evaluate the efficacy of GSH enhancing prodrug/precursors N-acetylcysteine (NAC) and S-adenosyl methionine (SAMe) as possible therapeutic agents in counteracting alcohol and HIV infection mediated depletion of CD4+ T lymphocytes.

DESCRIPTION (provided by applicant): Alcohol abuse imposes a heavy burden on the U.S. health care system. Immune dysfunction associated with chronic alcohol abuse is linked to increased clinical morbidity, such as infectious diseases and neoplasia. A critical factor in the alcohol abuse associated immune impairment is the loss of CD4+ T lymphocytes. Based on our substantial preliminary data, we hypothesize that a decrease in S-adenosylmethionine (SAMe) caused by ethanol-induced inhibition of methionine adenosyl transferase (MAT2A) enhances the susceptibility of CD4+ T-cells to Fas- and activation (T cell receptor - TCR) - mediated apoptotic death. To test this hypothesis we will examine the effect of ethanol on PBMCs/CD4+ T-cells obtained from alcoholic patients and abstinent, healthy control subjects. The overall goals of this proposal are: (i) to elucidate the molecular mechanisms underlying ethanol mediated depletion of CD4+ T lymphocytes and ensuing immune dysfunction and (ii) to provide a basis for clinically relevant paradigms for the development of potential therapeutic intervention(s) using S-adenosylmethionine (SAMe) in alcoholic patients. Accordingly, the effects of ethanol on Fas- and TCR-induced apoptotic signaling components will be determined. We will perform systematic analyses of the effects of ethanol on MAT2A gene expression and activity and SAMe levels in CD4+ T-cells in order to determine the relevance of MAT2A and SAMe as molecular targets of ethanol-induced CD4+ T-cell toxicity. We will also determine the efficacy of exogenous SAMe supplementation in attenuating CD4+ T-cell death induced by ethanol. Overall, we expect our studies to elucidate critical molecular mechanisms underlying CD4+ T-cell depletion associated with chronic alcohol abuse. Moreover, the data from these studies would support our future goals by providing a basis for relevant clinical paradigms for the development of potential therapeutic intervention(s) for alcoholic patients.

DESCRIPTION (provided by applicant): It is clear that alcohol abuse can alter/suppress immune function, including CD4 T-lymphocyte depletion, which can accelerate HIV progression. Subjects who consume alcohol may be more likely to develop certain infectious diseases which can complicate HIV, examples being various types of pneumonia and tuberculosis. Moreover, subjects who consume alcohol are more likely to participate in high-risk behaviors that increase the likelihood of acquiring HIV. Patients who consume alcohol also may be less compliant with their HIV therapy, which can affect disease progression. The research focus of this grant is the potential impact of alcohol use/abuse as a co-factor for HIV therapy-induced hepatotoxicity. Since the introduction of highly active antiretroviral therapy (HAART) in 1996, the life expectancy of patients with HIV has increased significantly. However, HAART is associated with significant hepatotoxicity. The severity of liver toxicity ranges from the absence of symptoms to liver decompensation, and the reported incidence of severe liver toxicity after initiating HAART ranges from 2% to 18%. The mechanisms involved in HAART-derived hepatotoxicity are not well understood, which makes its management more difficult. Some likely mechanisms of hepatotoxicity include proinflammatory cytokine production mitochondrial toxicity, hypersensitivity reactions, steatosis and insulin resistance, immune reconstitution in HCV or HBV co-infected patients, and proteasome inhibition (with dysregulated cytokine metabolism and mitochondrial and proteasome dysfunction serving as the focus of this proposal). The long-term goals of this study are to: (1) define mechanisms of hepatotoxicity induced by a combination of drug therapy and alcohol abuse in HIV patients and (2) discover potential therapeutic interventions for HAART hepatotoxicity, especially for patients in whom alcohol use/abuse may be a contributing factor. Specific Aims of this proposal are to: 1. Evaluate the potential hepatotoxic interactions of alcohol and HAART medications using rapid in vitro hepatocyte screening technology;2. Determine the effects of anti- HIV drugs (selected agents based on preliminary toxicity studies from SO#1 and clinical hepatotoxicity risk), on the development and progression of hepatotoxicity in mice and the drug interactions with chronic alcohol intake;and 3. Evaluate in a human translational component: a) mitochondrial and proteasome function and ex vivo cytokine production in subsets of HIV patients, b) serial non-invasive measures of mitochondrial and proteasome function and cytokine production in patients who develop HAART hepatotoxicity, and c) liver histology findings including markers of proteasome and mitochondrial function, using a human translational approach from a cohort of HCV+HIV coinfected subjects enrolled in a prospective NIH study based at the University of Cincinnati. We postulate that the HAART and alcohol mediated metabolic complications of dysregulated proinflammatory cytokine production and mitochondrial and proteasome dysfunction converge on the liver in an overlapping fashion to induce hepatotoxicity. In this proposal, we test that hypothesis in a novel translational approach and we evaluate unique therapeutic interventions for this hepatotoxicity. PUBLIC HEALTH RELEVANCE: Alcohol abuse and HIV are important health problems. Liver disease is recognized as an increasingly important problem for the HIV population. Liver disease may be due to a variety of factors including co-infection with viral hepatitis, alcohol abuse, and antiretroviral hepatotoxicity. Liver disease is now a leading cause of death for patients with HIV. Hepatotoxicity due to HAART is also common, and up to 30% of patients on HAART experience World Health Organization grade 3 liver enzyme elevations. Furthermore, hepatotoxicity from antiretroviral drugs leads to adverse patient outcomes either from fulminant hepatic failure, or more commonly, AIDS following discontinuation of HAART. It is unknown why some, but not all HIV patients develop drug induced liver injury from HAART. Relevant to this proposal, alcohol/HAART interactions are emerging as critically important factors. Recent research has shown that alcohol abuse is associated with severe hepatotoxicity in patients on HAART. Importantly, alcoholic liver disease and HAART induced liver injury share many potential mechanisms of injury. These include cytokine dysregulation, mitochondrial dysfunction, and proteasomal dysfunction. Through these and other mechanisms, alcohol and antiretroviral medications converge on the liver in an overlapping fashion to produce hepatotoxicity. Here, we will study hepatotoxicity due to alcohol/HAART interactions to further define disease mechanisms and create therapeutic interventions.

CD4+ T cell depletion is a major component of the multiple detrimental effects of chronic alcohol consumption on the immune system. Chronic alcohol abuse leads to abnormal S-adenosylmethionine (SAM) metabolism and decreased hepatic SAM levels. Our earlier work in CD4+ T cells has demonstrated that ethanol significantly decreases lymphocytic methionine adenosyltransferase (MAT II) expression and activity, with resultant SAM deficiency, and also increases activation-induced Fas-mediated apoptotic death. Notably, exogenous SAM supplementation restored intracellular SAM and prevented apoptotic signaling and death in ethanol treated CD4+ T cells. These data not only showed a causal role for SAM deficiency in ethanol mediated apoptotic death but also provided the proof-of-concept for SAM therapy in preventing alcohol induced CD4+ T cell depletion and immune suppression. SAM functions as the critical methyl donor during epigenetic regulation leading to enzymatic modification of histones and DNA cytosine residues. Our recent work on ethanol treated primary human CD4+ T cells in vitro as well as CD4+ T cells obtained from alcoholic patients has provided convincing preliminary data showing that ethanol exposure can cause transcriptionally permissive modifications in histones associated with the FasL promoter. These histone modifications enhance transcriptional activation of the FasL promoter resulting in increased mRNA expression, apoptotic signaling and cell death. The overall objective of this proposal is to systematically examine genome wide as well as gene specific (promoter associated) histone and DNA modifications involved in alcohol-mediated apoptotic signaling and death in CD4+ T cells. We expect that the results of our study will provide critical molecular insights in the alcohol-induced immunotoxicity of CD4+ T cells. We predict that results from this research will also provide important insights into immune dysfunction/CD4+ T cell loss in alcohol consuming HIV-infected patients. The specific aims of this translational proposal investigating the epigenetic mechanisms involved in aberrant apoptotic signaling and cell death of CD4+ T lymphocytes in alcoholic patients are: Aim 1: Examine the effects of alcohol on cellular SAM levels, global histone and DNA modifications and apoptotic cell death in CD4+ T cells. Aim 2: Determine the gene specific (promoter associated) histone and DNA modifications occurring in response to alcohol-mediated SAM deficiency that impact the expression of apoptotic genes relevant to CD4+ T cell survival. RELEVANCE (See instructions): Alcohol abuse continues to be a significant burden on the US healthcare system. The studies proposed here will examine epigenetic modifications involved in alcohol-induced depletion of CD4+ T lymphocytes using both a genome-wide approach, as well as a hypothesis-based candidate gene approach. The data obtained from these studies will yield mechanistic insights underlying alcohol induced immune suppression and how alcohol may further depress immune function in HIV-infected patients.

The University of Louisville Hepatobiology and Toxicology COBRE is a multidisciplinary group of investigators focusing on the liver and liver injury, gut:liver interactions, and liver:environment/toxicant/drug interactions. The Bioanalytical Core is an integral part of the COBRE and will play a key role in the implementation and future development of COBRE-funded projects. The Core will provide the necessary expertise and state-of-the- art equipment for rigorous and comprehensive experimental evaluation of cell and tissue specimens to investigate the pathogenic mechanisms of liver disease. Besides providing requisite analytical services, a major goal of the Bioanalytical Core is to promote the education and training of the Center members, with particular attention to the COBRE junior investigators. The Bioanalytical Core will specifically communicate with COBRE junior investigators regarding their experimental requirements on a continual basis and appropriately participate in the planning, execution and data interpretation. The Bioanalytical Core will also interact with other COBRE Cores on a regular basis in order to avoid duplication of equipment or instrumentation, and to foster innovative protocol development and refinement, exchange of ideas, and integrative analysis of data. The Core will provide technical and analytical expertise with centralized state-of- the-art equipment including Cellomics ArrayScan(R) VTI HCS Reader for automated microscope based high content screening and the Seahorse XF96 extracellular flux analyzer for cellular bioenergetics and metabolism studies in real time. The Core facility is located in Clinical and Translational Research Building with 2,500 sq.ft of operating space which is contiguous with the 20,000 sq.ft laboratory space of the Hepatobiology and Toxicology COBRE. Dr. Barve, the Director of the Core, has extensive experience in various analytical and experimental approaches in the areas of liver injury and pathogenic mechanisms of liver disease. Before his move to the University of Louisville, he served for nearly a decade as the Director of the Molecular and Cytokine Core Laboratory of the NIH-funded GCRC at the University of Kentucky, and has considerable experience in running a core laboratory. Also, in this role, he supported the career development of several junior faculty. To function as a vital integrative component of the Center, providing services not only for its members but also for the Institution as a whole, the Bioanalytical Core has the following specific aims: 1) To foster the development and success of proposed COBRE projects and future pilots; 2) To play an active and significant role in the education and training of the investigators; 3) To assist and participate in the development of new experimental protocols and techniques; and 4) To become an integral and self-sustaining component of the COBRE.

There is increasing evidence that demonstrates a critical pathogenic role for the ?gut-liver axis? in the development of alcoholic liver disease (ALD). The major goal of this proposal is to conduct studies that determine the mechanisms underlying chronic alcohol induced pathogenic changes in the gut-liver axis leading to ALD. Chronic alcohol consumption-mediated enteric microbiota (dysbiosis) and impairment of the gut barrier function, leads to increased intestinal permeability and exposes the liver to gut-derived microbial products including endotoxin. The consequent hepatic steatosis and inflammation can in turn promote gut dysfunction. Our recent work has begun to identify specific alcohol-induced alterations in enteric microbiome, and shows that chronic alcohol consumption leads to a significant expansion of pathogenic gram-negative bacteria that may lead to the development of systemic endotoxemia. Significantly, the alcohol-induced intestinal and hepatic pathogenic changes were markedly attenuated by supplementation with the probiotic Lactobacillus rhamnosus Gorbach-Goldin (LGG). There is gathering evidence and increasing interest regarding the contributory role of epigenetic mechanisms in the development of ALD. In this regard, it has been observed that histone associated epigenetic modifications play a significant role in the development of hepatic pathology induced by chronic as well as binge alcohol exposure. Our recent work showed that binge alcohol treatment decreases hepatic class I, II, and IV HDACs with a sole increase in HDAC3, and a correspondent increase in the global hepatic histone H3 acetylation (H3Ac) status. Subsequently, we demonstrated that increased HDAC3 plays a critical role in the binge alcohol-induced suppression of the carnitine palmitoyltransferase 1? (Cpt1a) gene expression and development of hepatic steatosis. Besides altered hepatic HDAC expression, our recent preliminary data also show that chronic ethanol feeding leads to a significant increase in the intestinal HDAC1 expression. Accordingly, the current proposal examines the unifying hypothesis that chronic alcohol induced alterations in the intestinal as well as hepatic HDACs and histone modifications constitute critical epigenetic mechanisms driving the abnormal gut-liver axis and liver disease. Importantly, the proposal also pursues translational studies that examine the efficacy of nutrition based therapeutic interventions targeted at intestinal dysbiosis and HDAC inhibition in mitigating alcohol-induced gut-liver axis changes and liver disease. The specific aims of the proposal are: Aim 1 - Determine the effect of chronic alcohol on intestinal and hepatic HDAC expression and global histone modifications underlying gut-liver axis changes in ALD; Aim 2 - Determine the impact of alcohol-induced HDAC alterations on gene-specific histone modifications and consequent pathogenic expression affecting the gut-liver axis in ALD and; Aim 3 - Evaluate the efficacy of nutrition-based therapeutic interventions in ALD. We expect that the results of our study will provide critical molecular insights and facilitate the development of therapeutic interventions for ALD.  

? DESCRIPTION (provided by applicant): Long-term excessive use of alcohol is capable of damaging nearly every organ and system in the body. The adverse effects of chronic alcohol consumption on the brain include not only psychiatric disorders but also severe alcohol-related neuroinflammation and brain damage. Work done by us and others has shown that chronic alcohol exposure causes intestinal oxidative stress, epithelial barrier disruption, enhancement of gut leakiness, and consequent systemic endotoxemia and inflammation. Our recent work has begun to identify specific alcohol-induced alterations in enteric microbiota (dysbiosis), and shows that chronic alcohol consumption leads to a significant expansion of pathogenic gram-negative bacteria that may lead to the development of systemic endotoxemia. Moreover, our earlier work has shown that chronic alcohol exposure increases the expression and activity of phosphodiesterase-4 (PDE4), decreases cellular cAMP, and primes monocytes/macrophages, leading to exaggerated expression of endotoxin-inducible inflammatory cytokines, such as TNFa. Additionally, alcohol exposure leads to a deficiency in brain docosahexanoeic acid (DHA) which likely contributes to the development/augmentation of cerebral inflammation and neurodegenerative pathologies. We hypothesize that alcohol-mediated enteric dysbiosis, systemic endotoxemia and elevated inflammatory responses induce/exacerbate alcohol-associated neuropathologies, via TLR4- and PDE4-mediated signaling, DHA deficiency, microglial activation, neuroinflammation and injury. The proposed study is an intramural-extramural collaborative effort that combines the relevant expertise to investigate the complex interactive mechanisms involving intestinal dysbiosis, systemic endotoxemia and inflammatory responses (UofL), and cerebral DHA deficiency in alcohol-induced neuropathologies (NIAAA). Moreover, a translational aspect will examine the therapeutic potential of specific mechanism-based strategies in mitigating alcohol-induced injury. The Specific aims of the application are: Aim 1: Determine alcohol mediated changes in the gut bacterial composition and intestinal permeability and correlate these changes to the development of peripheral endotoxemia and brain inflammation. Aim 2: Examine the role of PDE4 in alcohol-induced brain inflammatory signaling associated with DHA deficiency and injury. Aim 3: Evaluate the therapeutic efficacy of three intervention strategies targeting (i) enteric dysbiosis, (ii) PDE4 activity, and (iii) DHA deficiency in attenuating alcohol mediated brain inflammation and injury.

HIV infected (HIV+) heavy drinkers are at risk for cardiovascular diseases (CVD) via myriad pathways. Heavy alcohol use causes microbial translocation (MT) and inflammation. Additionally, alcohol-related changes in the GI tract microbiome?termed dysbiosis?are central to this pro-inflammatory cascade and could serve as novel therapeutic targets for reducing CVD risk among HIV+ heavy drinkers. Alcohol-associated dysbiosis is characterized by a loss of gut bacterial biodiversity, a reduction in ?beneficial? bacteria, and/or an expansion of harmful or ?pro-inflammatory? bacteria (e.g., those which produce trimethylamine N-oxide (TMAO), a metabolite associated with increased CVD risk). While murine models show alcohol associated dysbiosis was attenuated by probiotics even in the presence of continued alcohol consumption, there remains insufficient knowledge to identify a therapeutic target in humans for two reasons: 1) most studies involve murine models; and 2) scant human studies have small sample sizes with few HIV+ participants, and lack longitudinal assessment of alcohol intake, inflammation, and CVD risk in relation to the GI microbiome. This application addresses these gaps. We hypothesize that alcohol-associated dysbiosis will: (1) be greater in HIV+ very heavy drinkers (AUDIT score?20) vs. heavy drinkers (AUDIT<20); (2) increase biomarker levels of intestinal permeability (e.g., intestinal fatty acid binding protein (IFABP), MT (e.g., endotoxin), inflammation (interleukin 6) and TMAO; and (3) increase serum biomarkers for and alter echocardiographic (Echo) measures of cardiac function (N-terminal pro Brain Naturetic Peptide, NT pro-BNP, and left ventricular ejection fraction, LVEF, respectively). To test these hypotheses, we will enroll 200 HIV+ participants from St. PETER HIV, a funded RCT using pharmacotherapy to reduce alcohol use, smoking, inflammation, and CVD risk. This application, Alcohol-associated CVD and Microbiome Evaluation Study (ACME HIV 1/2) leverages existing resources of St. PETER HIV: participant recruitment, measures of alcohol consumption, biospecimen and biomarker collection; expertise in alcohol, HIV, CVD, and microbial genomics. New data include: fecal samples to characterize the GI microbiome, serum biomarkers, and echo measures. In response to RFA-AA-17-014, we will partner with the Southern HIV & Alcohol Research Consortium (SHARC, ACME HIV 2/2) to corroborate our findings in Aims 1 and 2 and to conduct combined cross-cohort analyses. We will complete the following SPECIFIC AIMS among HIV+ heavy drinkers: AIM 1: To assess longitudinal qualitative and quantitative changes in the gut microbiome (dysbiosis) associated with very heavy alcohol consumption. AIM 2: To determine the effect of dysbiosis on intestinal permeability, MT, inflammation, and TMAO levels. AIM 3: To investigate the impact of dysbiosis on biomarkers for and echo measures of cardiac structure and function. Completion of these aims will lay groundwork for an intervention targeting alcohol-associated dysbiosis, which could profoundly reduce inflammation and favorably impact CVD risk among HIV+ heavy drinkers.

Heavy alcohol drinking and HIV-1 infection are independently associated with the development of abnormalities in the brain function and cognition, and increasing evidence indicates that the combinatorial effects of HIV infection and alcohol are likely to worsen these abnormalities. Significantly, both HIV- 1 infection and chronic alcohol abuse cause alterations in gut microbiome (dysbiosis) and increase intestinal permeability and microbial translocation (MT) which are major pathogenic factors driving local and systemic inflammation. The dysbiosis is characterized by loss of gut bacterial biodiversity, a reduction in beneficial bacteria, and/or an expansion of harmful or pro-inflammatory bacteria. However, there is limited understanding of (i) the interactive effects of heavy alcohol drinking and HIV-1 infection on gut dysbiosis and (ii) the longitudinal qualitative (microbial membership) and quantitative (relevant abundance) determinants of dysbiosis. The current proposal addresses these gaps with an overall goal to promote the development of rational therapies targeting gut dysbiosis. Accordingly, we pursue a unifying hypothesis that in HIV-infected individuals, heavy alcohol use compounds gut dysbiosis and consequent peripheral immune inflammation leading to exacerbation of neuro- inflammation and cognitive dysfunction. To test these hypotheses, we will enroll 100 HIV+ participants from ?30-to-90-day challenge? Florida SHARC study (AA020797). This is a funded prospective cohort study in which enrolled heavy drinkers are challenged to stop drinking for 30 to 90 days, assisted by contingency management (CM) payments and motivational interviewing. This application, Alcohol associated Comorbidities and Microbiome Evaluation in HIV (ACME HIV 2/2) leverages the existing resources of ?30-to-90-day challenge? Florida SHARC study: participant recruitment, measures of alcohol consumption, biospecimen and biomarker collection; expertise in alcohol, HIV and neuroimaging. New data include: longitudinal changes in gut dysbiosis, correlative changes in peripheral inflammation, and measures of cognition and neuroinflammation. In response to RFA-AA-17-014, we will partner with the St. PETER HIV study (ACME HIV 1/2) to corroborate our findings in Aims 1 and 2 and to conduct combined cross-cohort analyses. We will complete the following Specific Aims among HIV+ heavy drinkers: AIM 1: To assess longitudinal qualitative and quantitative changes in the gut microbiome (dysbiosis) associated with very heavy alcohol consumption. AIM 2: To determine the impact of HIV infection and alcohol abuse induced gut dysbiosis on intestinal permeability, microbial translocation (MT), and resultant peripheral endotoxemia, immune activation and inflammation. AIM 3: To investigate the impact of dysbiosis and peripheral inflammation on development of neuro-inflammation and cognitive function. Completion of these aims will lay groundwork for an intervention targeting alcohol-associated dysbiosis, which could profoundly reduce inflammation and improve cognitive functions in HIV+ heavy drinkers.

Although AIDS-defining illnesses have decreased, the prevalence of HIV-associated non-AIDS conditions such as HIV-Associated Neurocognitive Disorders (HAND) remains high and is estimated to be over 50%, particularly in aging individuals with long-standing HIV infection. However, the pathophysiology of HAND in aging HIV+ adults remains unresolved. Current evidence and our preliminary data suggest that interactions of altered gut microbiome (dysbiosis), gut-derived microbial translocation, and systemic inflammation contribute to neurodegenerative processes. It is becoming increasingly evident that in both HIV-1 infection and aging, alterations in gut microbiome (dysbiosis) and ensuing increase in intestinal permeability and microbial translocation (MT) are major pathogenic drivers of local and systemic inflammation. Importantly, aging- associated microbiota changes are shown to be connected to immunosenescence and inflammaging. Preclinical/clinical studies using bacterial 16S ribosomal RNA (rRNA) gene sequencing, indicate that microbial dysbiosis associated with HIV-1 infection or aging has several common pathogenic features. However, these studies were largely hypothesis-generating with limited sample sizes, and were not adequately powered to address microbiome endpoints after correction for multiple testing, and did not reveal the functional potential of the microbiota (pathogenic or beneficial), or yield bacterial resolution to species or strain level. The current proposal will address these limitations by using an adequately powered longitudinal study and will conduct 16S rRNA gene and Whole Genome Shotgun (WGS) metagenomic sequencing that will determine bacterial composition and diversity, provide identification at the species and strain level, and enable the functional characterization of the bacterial genes. Our overarching hypothesis is that the interactive effects of aging and HIV-1 infection at the level of gut dysbiosis and permeability, and ensuing local and systemic inflammation play a major pathogenic role in driving HIV infection and aging-associated neuroinflammation and cognitive dysfunction. To test these hypotheses, we will leverage and utilize HIV+ and healthy aging populations from ongoing NIH-sponsored longitudinal studies at the Universities of Louisville (UofL) and Florida (UF) with the following specific aims: Aim 1: To assess longitudinal qualitative and quantitative changes in the gut microbiome (dysbiosis) in older persons living with HIV-1 infection. Aim 2: To determine the impact of HIV-1 infection and age associated gut dysbiosis on (A) intestinal permeability and microbial translocation (MT), and resultant peripheral endotoxemia, and inflammation; and (B) multimodal MRI/MRS measures of neuroinflammation and cerebral metabolic disturbance. Aim 3: To investigate the impact of gut dysbiosis and peripheral and neuroinflammation, and cerebral metabolic disturbance on cognitive dysfunction and functional brain abnormalities (FMRI) relative to age and HIV status.

The goal of the active U01 is to examine the interactive effects of heavy alcohol use and HIV-1 infection at the level of gut dysbiosis and permeability, ensuing local and systemic inflammation and its pathogenic role in driving HIV infection and aging-associated neuroinflammation and cognitive dysfunction. This is being accomplished through longitudinal studies that systematically examine the role of alcohol use and HIV-1 infection altered microbiome on HIV pathogenesis and development of neurocognitive dysfunction using 16S rRNA gene sequencing and Whole Genome Shotgun (WGS) metagenomic sequencing analysis. The supplement is within the scope of the active award and extends the parent study by including an additional category of participants relevant to Alzheimer?s disease related dementia (ADRD). Specifically, the proposed work is focused on amnestic mild cognitive impairment (aMCI), an early stage of ADRD. Accordingly, the study will include HIV-infected adults with heavy alcohol use and aMCI. There is supporting evidence from pre-clinical rodent studies and few human studies for the role of gut microbiota and peripheral inflammation in the development or progression of Alzheimer?s disease (AD). Notably, there is a complete lack of comprehensive analysis and characterization of the gut microbial dysbiosis in heavy alcohol using HIV+ individuals with ADRD. By contrasting neurocognitive, neuroimaging, and biospecimen indices between heavy alcohol using HIV+ individuals with aMCI and heavy alcohol using HIV+ individuals without aMCI, we will determine whether microbiome abnormalities contribute to the more severe cognitive disturbances and possible development of ADRD. This will enable us to examine the effects of HIV and heavy alcohol use in individuals with and without aMCI/ADRD. Based on the one-year scope and budgetary limits, this supplemental study will differ from the parent project with respect to the inclusion of aMCI and only a single baseline assessment. These supplemental studies will leverage participant recruitment, validated clinical and demographic data, biospecimen/biomarker collection, and clinical expertise utilize from our ongoing NIH sponsored longitudinal studies at the Universities of Louisville (UofL) and Florida (UF) with the following specific aim: Determine HIV-1 infection- and heavy alcohol use-associated qualitative and quantitative changes in the gut microbiome (dysbiosis) and attendant intestinal permeability, microbial translocation (MT) and peripheral inflammation in adults with Alzheimer?s disease related dementias (ADRD).

The Integrated Metagenomics and Metabolomics Core (IMMC) will provide a platform that interconnects Projects 1 and 2. Specifically, IMMC will provide high-quality, metagenomics and targeted metabolomics data, to be integrated with the probiotics randomized clinical trial and clinical cohort outcomes for Projects 1 and 2, respectively. The IMMC has already acquired relevant metagenomics and metabolomics data (see Preliminary data sections E1.2 and F.1.A.2) that supports the experimental rationale and study design for Projects 1 and 2. To ensure optimal scientific output from the study samples, the IMMC has implemented standardized procedures for collection of samples from Projects 1 and 2 study sites, and established logistics to coordinate shipment of samples for analysis, manage inventory, and enable tracking of chain of custody. For Project 1, IMMC - sub-core 1 will conduct state of the art metagenomics analysis of fecal samples collected from study participants receiving probiotics supplementation, to profile the changes in the gut microbial communities. The culture- independent molecular methods will consist of 16S rRNA gene and whole genome shotgun (WGS) metagenomic sequencing that provide the most comprehensive microbiota profiling. Rigorous sequence data analysis will utilize a set of advanced computational algorithms, according to taxon- based or function-based data matrices. IMMC- sub-core 2 will conduct targeted metabolomics analysis of plasma/serum (Projects 1 and 2) to monitor host and gut-microbiota metabolomes. The IMMC will collaborate with Projects to develop meaningful study designs, implement effective sample collection and storage strategies, generate high-quality metagenomics and targeted metabolomics data, computationally summarize these data and support interpretation of the results. It will implement high-quality techniques, along with quality control standards, that produce meaningful results in a timely fashion. The IMMC will also standardize data generation methods across studies to enable integration across Projects and uncover robust discoveries. To accomplish these goals the IMMC has established the following aims: 1. Employ metagenomic approaches to generate and analytically summarize project microbiome data. 2. Perform targeted metabolomics analyses to profile plasma/serum metabolites. 3. Manage specimen and data biorepository and facilitate transfer between research Projects.

The overarching theme for this program project grant (PPG) is that alcohol associated gut dysbiosis and gut dysbiotic metabolites are cardiovascular disease (CVD) risk factors among people living with HIV infection (PLWH) who are heavy drinkers. The goals of this research are (1) to determine if a tailored probiotic (i.e., contains bacteria supporting butyrate synthesis) can mitigate alcohol associated gut dysbiosis and lower levels of microbial translocation, inflammation, and improve harmful dysbiotic metabolite profiles (e.g. trimethylamine N oxide, TMAO) and (2) to determine if these metabolites are associated with incident CVD and death among PLWH. We hypothesize that, among PLWH, a probiotic vs. placebo can mitigate alcohol associated gut dysbiosis and lower levels of microbial translocation, inflammation, and improve metabolite profiles (Project 1 RCT, n=250); and that harmful alterations of these metabolites will be associated with higher risk of incident CVD and death (Project 2 Cohort, n=2,900). Project 1 will be conducted at Pavlov State Medical University in St. Petersburg, Russia, the same site as our gut microbiome and metabolite studies (ACME HIV and TMAO HIV). Project 2 will leverage the Veterans Aging Cohort Study, an observational cohort of veterans living with and without HIV. The Projects will be supported by our Administrative Core at Vanderbilt University Medical Center and the Integrated Metagenomics and Metabolomics Core at the University of Louisville's Alcohol Research Center (ULARC). The latter is the core for ACME HIV and will generate the metagenomics and metabolomics for this PPG. The former will coordinate all study projects/cores and integrate the Vanderbilt SCHolars in HIV and Heart, Lung, Blood, and Sleep ReSearch NIH K12 training program into the PPG. Our preliminary data: (1) HIV infection is a CVD risk factor; (2) inflammation is associated with increased risk of CVD among PLWH; (3) among PLWH, heavy drinking is associated with increased CVD risk and correlated with measures of gut dysbiosis, characterized by loss of butyrate producing bacteria; (4) gut dysbiosis among PLWH who are heavy drinkers is correlated with higher levels of inflammation, TMAO, and adverse bile acid metabolite profiles; and (5) ULARC data in murine models show heavy drinking causes dysbiosis, that dysbiosis leads to increased biomarker levels of inflammation, and that probiotic administration targeting alcohol-associated gut dysbiosis attenuates the rise in these inflammatory biomarkers even in the presence of alcohol consumption. Cross project validation: Biospecimens from Project 1 will be used to validate significant findings in Project 2. Metabolites significantly associated with alcohol and CVD in Project 2, will be explored in Project 1 to see if probiotics favorably impact the levels of those metabolites. The Microbiome, METabolites, and Alcohol in HIV to reduce CVD (META HIV CVD) PPG will inform probiotics' role as standard adjunctive therapy complementing alcohol interventions among PLWH who are heavy drinkers and advance the understanding of how alcohol associated gut dysbiosis and its metabolites contribute to CVD and death.

This project (Project 2) will define specific pathogenic features of alcohol-induced gut dysbiosis and investigate its causative role (and underlying mechanisms) in systemic/hepatic immune dysregulation in alcoholic liver disease (ALD). Based on our compelling preliminary data from pre-clinical and clinical studies, we hypothesize that loss of butyrate producing bacteria is a significant pathogenic feature of gut dysbiosis in patients with ALD, which plays a major role in developing a pro-inflammatory T cell-IL-17 milieu leading to pathogenic changes in the gut-liver axis in ALD. This project will utilize a highly innovative animal model of human fecal microbiota transplant (FMT) from alcoholic hepatitis patients into conventional mice that recapitulates key features of human ALD, to identify mechanisms and develop targeted therapeutic interventions. Specifically, the project will test the efficacy of evidence-based nutritional therapeutic strategies that target gut dysbiosis, namely, (i) Tributyrin (Tb), a dietary butyrate pro-drug and known HDAC inhibitor and (ii) Faecalibacterium prausnitzi - a major human butyrate producing probiotic. The proposal leverages existing resources of the NIH funded AlcHepNet clinical consortium (U01AA026980) which include a large available cohort of ALD patients and control subjects, participant recruitment, validated demographic and clinical data, biospecimen and biomarker collection and clinical expertise in liver diseases. This proposed study will provide new insights into the mechanisms underlying ALD and identify new targets for ALD treatment, thereby contributing to the ULARC program and theme of nutrition and alcohol-induced organ injury. The specific aims of the proposal are: AIM 1) Determine the qualitative and quantitative loss of butyrate producing microbial communities as a defining pathogenic feature of alcohol-induced gut dysbiosis leading to immune dysregulation represented by the activation of the proinflammatory T cell-IL-17 axis in patients with ALD, AIM 2) Determine the causative role of alcohol-induced loss of butyrate producing bacteria on the activation of the proinflammatory T cell/IL-17 axis and neutrophil recruitment in hepatic inflammation/injury, and AIM 3) Evaluate the efficacy of nutritional interventions targeting the alcohol-induced loss of enteric butyrate producing bacteria and butyrate production in attenuating the pathogenic changes in the gut-liver axis in ALD.