Short-term heavy drinking in a non-human primate model skews monocytes toward a hypo-inflammatory phenotype

Madison B Blanton¹Hami Hemati²Qi Qiao³Rupak Khadka⁴Greg Hawk³Kathleen A. Grant⁴Ilhem Messaoudi^2*

• ¹Pharmaceutical Sciences, University of Kentucky, Lexington, United States
• ²Microbiology, Immunology and Molecular Genetics, University of Kentucky, Lexington, United States
• ³Dr. Bing Zhang Department of Statistics, University of Kentucky, Lexington, United States
• ⁴Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, Oregon, United States

Introduction: Alcohol use is prevalent in the United States (US), with ~80% of persons over 12 years old reporting alcohol consumption in 2023 and ~10% of those individuals developing alcohol use disorder (AUD). Acute and chronic alcohol consumption exert opposite effects on the immune system. Specifically, acute alcohol exposure (AAE), (3-16 hours of in-vitro treatment, one binge episode in humans, or one gavage feeding in mice) skews monocytes towards a hypo-inflammatory phenotype associated with reduced TNFα, IL-6, and MCP-1 production. In contrast, chronic alcohol consumption (CAC) (7 days of in-vitro treatment, 3-12 months of consumption in animal models, or humans with confirmed AUD diagnosis), shifts the functional, transcriptional, metabolic, and epigenetic landscapes of monocytes and their progenitors towards a hyperinflammatory profile. Despite the extensive work investigating AAE and CAC, few studies have examined short-term drinking durations. We sought to bridge this gap by assessing monocytes after 6 months of ethanol consumption in a rhesus macaque model, which we considered short-term drinking. Understanding the longitudinal changes in monocytes' phenotype and function in the context of alcohol consumption could pave the way to identifying diagnostic biomarkers for disease progression. Methods: To bridge this gap, we obtained peripheral blood mononucleated cells (PBMC) isolated from rhesus macaques before and after 6 months of daily ethanol consumption (>55% of intakes over 2.0 g/kg/day). Monocytes were analyzed using a combination of flow cytometry, single-cell RNA-sequencing (scRNAseq), ELISAs, and Cleavage Under Targets and Tagmentation (CUT&Tag). Results: Our data show that 6 months of ethanol consumption rewires monocytes towards a hypo-inflammatory profile as evidenced by reduced cytokine production. scRNAseq analysis revealed distinct shifts in monocyte states/clusters with ethanol consumption and LPS stimulation in line with a shift to a hypo-inflammatory state. These changes may be driven by reduced levels of H3k4me3, a histone modification shown to be deposited at promoter regions of genes involved in inflammation and pathogen response signaling. Discussion: Overall, these data demonstrate that 6 months of daily heavy drinking attenuates inflammatory responses in monocytes via shifts in the epigenetic landscape.