Samantha M. Yeligar

People with HIV (PWH) are at increased risk of a broad range of infectious and non-infectious pulmonary diseases. Since the last ATS Workshop on HIV-associated pulmonary disease, large national and international cohorts have demonstrated ongoing gaps in our understanding of lung disease in this vulnerable population that range from underappreciation of disease incidence and prevalence to lack of targeted treatments. Translational work on the underlying mechanisms of disease has demonstrated novel, HIV-specific mechanisms that may lend themselves to tailored therapies. Twenty-four experts in HIV-associated lung disease from across the translational spectrum were invited to participate in this ATS-sponsored Workshop utilizing a modified Delphi method that included a series of pre- and post-workshop electronic surveys and a two-day virtual workshop. Lung disease among PWH is driven by complex and interrelated mechanisms including persistent immune dysregulation, oxidative stress, accelerated aging, and microbiome alterations. Despite these advances in our understanding, important gaps persist between mechanistic insights and clinical care that include limited HIV-specific trials, underrepresentation of PWH in studies of lung disease, and the absence of tailored care strategies. These gaps are further accentuated by substantial variation in the availability of data from the most affected populations, particularly in sub-Saharan Africa and among children and adolescents. Overcoming these challenges will require coordinated, globally representative research efforts that leverage large cohorts, integrate multi-omics and longitudinal phenotyping, use mechanistic insights to inform clinical trials, and consider implementation science approaches with the ultimate goal of developing tailored prevention and treatment strategies for HIV-associated pulmonary diseases.

Alcohol misuse is twice as prevalent among people living with HIV (PWH), and this increases the risk of pulmonary complications even in those receiving antiretroviral therapy. Our prior work showed that the alcohol metabolite, acetaldehyde, activates nuclear factor kappa B p65 (p65), leading to HIV replication and interleukin (IL)-1β activation in alveolar macrophages (AMs). Since the aforementioned processes are energy-demanding, which conversely impair mitochondrial functions, we hypothesized that acetaldehyde-induced p65 drives AMs to a mitochondrial hyperactive state to promote HIV replication and IL-1β release and induces oxidative stress and mitochondrial dysfunction. Since we found pioglitazone (PIO) to be a negative regulator of p65, we postulate that PIO suppresses HIV replication and IL-1β activation in AMs by restricting p65-induced mitochondrial hyperactivation. Murine AMs were exposed to acetaldehyde via the acetaldehyde generating system (AGS) and infected in vitro with EcoHIV, a chimeric ecotropic HIV construct. AGS + EcoHIV activated p65, resulting in enhanced ATP-linked mitochondrial respiration, proton leak, non-mitochondrial respiration and the generation of reactive oxygen species (ROS) in AMs. Inhibition of mitochondrial ATP synthesis with low-dose oligomycin attenuated AGS-induced HIV replication and AGS + EcoHIV-induced IL-1β release from AMs. PIO treatment, which attenuated AGS-induced p65 activation, suppressed proton leak, non-mitochondrial oxygen consumption, ROS, and IL-1β and p24 release. While p65-induced mitochondrial hyperactivation represents AMs’ adaptive response to the energy demands imposed by HIV replication and proinflammatory activation when exposed to acetaldehyde, PIO treatment may offer a novel therapeutic strategy to restore adequate mitochondrial bioenergetics in the AMs of PWH who misuse alcohol.

Approximately 50% of people living with Human Immunodeficiency Virus (HIV) in the United States misuse alcohol, and they are at increased risk of chronic lung inflammation despite antiretroviral therapy. Acetaldehyde, a metabolite of alcohol, circulates systemically and directly impacts alveolar macrophages (AMs), the primary reservoir of HIV in the lungs. Acetaldehyde promotes AM HIV replication and triggers IL-1β release. We explored the mechanisms by which alcohol-derived acetaldehyde drives HIV replication and IL-1β release in AMs. Furthermore, we tested if the transcription factor peroxisome proliferator-activated receptor (PPAR)γ agonist, pioglitazone, attenuates AM HIV replication and IL-1β release. Primary mouse AMs, MH-S cells (an AM cell line), and THP-1 (human monocyte cell line)-derived macrophages were treated with alcohol-derived acetaldehyde (acetaldehyde-generating system [AGS]), HIV 1ADA, and EcoHIV, a chimeric HIV that infects murine cells. HIV expression was confirmed by HIV gag RNA (qRT-PCR) and p24 release (ELISA). IL-1β was measured by qRT-PCR and ELISA. Extracellular hydrogen peroxide (H2O2) release was quantified by Amplex Red assay. Furthermore, immunoblot analysis of ERK1/2, PPARγ, and NF-ĸB/p65 (p65) was used to identify how acetaldehyde potentiates HIV replication and IL-1β activation in AMs. AGS increased H2O2, leading to ERK1/2 phosphorylation, which deactivated PPARγ. AGS drove nuclear p65 translocation in HIV-infected cells, which enhanced HIV replication and IL-1β release. Treatment with pioglitazone decreased nuclear p65, attenuating AGS-induced HIV replication and IL-1β activation in AMs. We identified mechanisms underlying acetaldehyde-induced inflammatory activation and potentiation of HIV replication in AMs, which could be therapeutically targeted with pioglitazone to decrease HIV-related respiratory comorbidities among people living with HIV who misuse alcohol.

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

Post-acute sequelae of SARS-CoV-2 (SARS2) infection (PASC) is a heterogeneous condition, but the main viral drivers are unknown. Here, we use MENSA, Media Enriched with Newly Synthesized Antibodies, secreted exclusively from circulating human plasmablasts, to provide an immune snapshot that defines the underlying viral triggers. We provide proof-of-concept testing that the MENSA technology can capture the new host immune response to accurately diagnose acute primary and breakthrough infections when known SARS2 virus or proteins are present. It is also positive after vaccination when spike proteins elicit an acute immune response. Applying the same principles for long-COVID patients, MENSA is positive for SARS2 in 40% of PASC vs none of the COVID recovered (CR) patients without any sequelae demonstrating ongoing SARS2 viral inflammation only in PASC. Additionally, in PASC patients, MENSAs are also positive for Epstein-Barr Virus (EBV) in 37%, Human Cytomegalovirus (CMV) in 23%, and herpes simplex virus 2 (HSV2) in 15% compared to 17%, 4%, and 4% in CR controls respectively. Combined, a total of 60% of PASC patients have a positive MENSA for SARS2, EBV, CMV, and/or HSV2. MENSA offers a unique antibody snapshot to reveal the underlying viral drivers in long-COVID thus demonstrating the persistence of SARS2 and reactivation of viral herpes in 60% of PASC patients.

People with alcohol use disorder (AUD) have an increased risk of developing pneumonia and pulmonary diseases. Alveolar macrophages (AMs) are immune cells of the lower respiratory tract that are necessary for clearance of pathogens. However, alcohol causes AM oxidative stress, mitochondrial damage and dysfunction, and diminished phagocytic capacity, leading to lung injury and immune suppression. AMs were isolated by bronchoalveolar lavage from people with AUD and male and female C57BL/6J mice given chronic ethanol (20% w/v, 12 weeks) in drinking water. The peroxisome proliferator-activated receptor γ ligand, pioglitazone, was used to treat human AMs ex vivo (10 μM, 24 h) and mice in vivo by oral gavage (10 mg/kg/day). Levels of AM mitochondrial superoxide and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA, a marker of oxidative stress, were measured by fluorescence microscopy and RT-qPCR, respectively. Mouse AM phagocytic ability was determined by internalized Staphylococcus aureus, and mitochondrial capacity, dependency, and flexibility for glucose, long-chain fatty acid, and glutamine oxidation were measured using an extracellular flux analyzer. In vitro studies used a murine AM cell line, MH-S (±0.08% ethanol, 72 h) to investigate mitochondrial fuel oxidation and ATP-linked respiration. Pioglitazone treatment decreased mitochondrial superoxide in AMs from people with AUD and ethanol-fed mice and HIF-1α mRNA in ethanol-fed mouse lungs. Pioglitazone also reversed mouse AM glutamine oxidation and glucose or long-chain fatty acid flexibility to meet basal oxidation needs. In vitro, ethanol decreased the rate of AM mitochondrial and total ATP production, and pioglitazone improved changes in glucose and glutamine oxidation. Pioglitazone reversed chronic alcohol-induced oxidative stress in human AM and mitochondrial substrate oxidation flexibility and superoxide levels in mouse AM. Decreased ethanol-induced AM HIF-1α mRNA with pioglitazone suggests that this pathway may be a focus for metabolic-targeted therapeutics to improve morbidity and mortality in people with AUD.

This matched case–control study aimed to provide epidemiologic evidence of increased burden of respiratory symptoms and pulmonary function decline among people living with human immunodeficiency virus (HIV) and a history of heavy alcohol consumption. Cases were participants with HIV (PWH; n = 75, 33%), and controls were participants without HIV (PWoH; n = 150, 67%). PWH were matched to PWoH by age and sex in the ratio of 1:2. Eligible participants responded to the respiratory health National Health and Nutrition Examination Survey questionnaire [prolonged coughs (≥3 months), bringing up of phlegm (≥3 months), and a history of wheezing or whistling in the chest (past year)]. The effects of both alcohol and HIV on participants’ pulmonary function were determined using linear regression analysis. History of heavy alcohol consumption was more prevalent among PWH (40%) compared to PWoH (27%). PWH who had a history of heavy alcohol consumption had a higher prevalence of coughing most days (45% vs. 4%, P = .0010), bringing up phlegm most days (31% vs. 0%, P = .0012), and wheezing or whistling in the chest (40% vs. 20%, P = .058) compared to participants who did not heavily consume alcohol. Furthermore, a history of heavy alcohol consumption was associated with decreased forced expiratory volume (ml) in 1 s/forced vital capacity among PWH (β = − 0.098 95% C.I. −0.16, −0.04, P = .03) after adjusting for having smoked at least 100 cigarettes in life. A history of heavy alcohol use increased respiratory symptoms and suppressed pulmonary function among people living with HIV. This study provides epidemiological evidence of the respiratory symptom burden of people living with HIV who have a history of heavy alcohol consumption.