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Alcohol use in persons living with HIV (PLWH) worsens the severity of bacterial pneumonia. However, the exact mechanism(s) by which this occurs remain ill-defined. We hypothesized that alcohol in the setting of HIV infection decreases Streptococcus pneumoniae clearance from the lung through mechanisms mediated by the gut microbiota. Humanized BLT (bone marrow, liver, thymus) mice were infected with 1 × 104 TCID50 of HIV (BAL and JRCSF strains) via intraperitoneal (i.p.) injection. One week post-HIV infection, animals were switched to a Lieber-DeCarli 5% ethanol diet or an isocaloric control diet for 10 days. Alcohol-fed animals were also given two binges of 2 g/kg ethanol on days 5 and 10. Feces were also collected, banked, and the community structures were analyzed. Mice were then infected with 1 × 105 CFU (colony-forming units) of S. pneumoniae and were sacrificed 48 h later. HIV-infected mice had viral loads of ∼2 × 104 copies/mL of blood 1 week post-infection, and exhibited an ∼57% decrease in the number of circulating CD4+ T cells at the time of sacrifice. Fecal microbial community structure was significantly different in each of the feeding groups, as well as with HIV infection. Alcohol-fed mice had a significantly higher burden of S. pneumoniae 48 h post-infection, regardless of HIV status. In follow-up experiments, female C57BL/6 mice were treated with a cocktail of antibiotics daily for 2 weeks and recolonized by gavage with intestinal microbiota from HIV+ ethanol-fed, HIV+ pair-fed, HIV- ethanol-fed, or HIV- pair-fed mice. Recolonized mice were then infected with S. pneumoniae and were sacrificed 48 h later. The intestinal microbiota from alcohol-fed mice (regardless of HIV status) significantly impaired clearance of S. pneumoniae. Collectively, these data indicate that alcohol feeding, as well as alcohol-associated intestinal dysbiosis, compromise pulmonary host defenses against pneumococcal pneumonia. Determining whether HIV infection acts synergistically with alcohol use in impairing pulmonary host defenses will require additional study.

AbstractBackgroundChronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa‐associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol‐associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear.MethodsTo investigate the impact of alcohol on MAIT cells, mice were treated with binge‐on‐chronic alcohol for 10 days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol‐induced MAIT cell changes: (1) Cecal microbiota from alcohol‐fed (AF) mice were adoptive transferred into antibiotic‐pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry.ResultsBinge‐on‐chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue‐associated MAIT cells was increased in AF mice compared with pair‐fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals.ConclusionMAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol‐associated dysbiosis. MAIT cell dysfunction may contribute to alcohol‐induced innate and adaptive immunity and consequently end‐organ pathophysiology.

AbstractThe intestinal microbiota generates many different metabolites which are critical for the regulation of host signaling pathways. In fact, a wide-range of diseases are associated with increased levels of local or systemic microbe-derived metabolites. In contrast, certain bacterial metabolites, such as tryptophan metabolites, are known to contribute to both local and systemic homeostasis. Chronic alcohol consumption is accompanied by alterations to intestinal microbial communities, and their functional capacities. However, little is known about the role of alcohol-associated dysbiosis on host defense against bacterial pneumonia. Our previous work using fecal transplantation demonstrated that alcohol-associated intestinal dysbiosis, independent of ethanol consumption, increased susceptibility toKlebsiellapneumonia. Here, we demonstrate that intestinal microbiota treatments mitigate the increased risk of alcohol-associated pneumonia. Treatment with the microbial metabolite indole or with probiotics reduced pulmonary and extrapulmonary bacterial burden, restored immune responses, and improved cellular trafficking required for host defense. Protective effects were, in part, mediated by aryl hydrocarbon receptors (AhR), as inhibition of AhR diminished the protective effects. Thus, alcohol appears to impair the production/processing of tryptophan catabolites resulting in immune dysregulation and impaired cellular trafficking. These data support microbiota therapeutics as novel strategies to mitigate the increased risk for alcohol-associated bacterial pneumonia.

BackgroundAlcohol use causes significant disruption of intestinal microbial communities, yet exactly how these dysbiotic communities interact with the host is unclear. We sought to understand the role of microbial products associated with alcohol dysbiosis in mice on intestinal permeability and immune activation in an in vitro model system.MethodsMicrobiota samples from binge‐on‐chronic alcohol‐fed and pair‐fed male and female mice were cultured in Gifu Anaerobic Broth for 24 hours under anaerobic conditions. Live/whole organisms were removed, and microbial products were collected and added to human peripheral blood mononuclear cells (PBMCs) or polarized C2BBe1 intestinal epithelial monolayers. Following stimulation, transepithelial electrical resistance (TEER) was measured using a volt/ohm meter and immune activation of PBMC was assessed via flow cytometry.ResultsMicrobial products from male and female alcohol‐fed mice significantly decreased TEER (mean percentage change from baseline alcohol‐fed 0.86 Ω/cm2 vs. pair‐fed 1.10 Ω/cm2) compared to microbial products from control mice. Following ex vivo stimulation, immune activation of PBMC was assessed via flow cytometry. We found that microbial products from alcohol‐fed mice significantly increased the percentage of CD38+ CD4+ (mean alcohol‐fed 17.32% ± 0.683% standard deviation (SD) vs. mean pair‐fed 14.2% ± 1.21% SD, p < 0.05) and CD8+ (mean alcohol‐fed 20.28% ± 0.88% SD vs. mean pair‐fed 12.58% ± 3.59% SD, p < 0.05) T cells.ConclusionsCollectively, these data suggest that microbial products contribute to immune activation and intestinal permeability associated with alcohol dysbiosis. Further, utilization of these ex vivo microbial product assays will allow us to rapidly assess the impact of microbial products on intestinal permeability and immune activation and to identify probiotic therapies to ameliorate these defects.