Table 3_Short-term heavy drinking in a non-human primate model skews monocytes toward a hypo-inflammatory phenotype.xlsx

IntroductionAlcohol 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 hyper-inflammatory 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.
MethodsTo 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).
ResultsOur 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.
DiscussionOverall, these data demonstrate that 6 months of daily heavy drinking attenuates inflammatory responses in monocytes via shifts in the epigenetic landscape.

## 引言
酒精摄入在美国十分普遍，2023年数据显示，12岁以上人群中约80%有饮酒行为，其中约10%会发展为酒精使用障碍（Alcohol Use Disorder, AUD）。急性与慢性酒精摄入对免疫系统产生截然相反的影响。具体而言，急性酒精暴露（Acute Alcohol Exposure, AAE）——即体外处理3~16小时、人类单次暴饮发作或小鼠一次灌胃给药——会使单核细胞向低炎症表型倾斜，该表型伴随TNFα、IL-6及MCP-1生成减少。与之相反，慢性酒精摄入（Chronic Alcohol Consumption, CAC）——即体外处理7天、动物模型中持续摄入3~12个月或确诊酒精使用障碍的人类——会使单核细胞及其祖细胞的功能、转录组、代谢组及表观遗传谱向高炎症表型转变。尽管已有大量针对急性与慢性酒精暴露的研究，但鲜有探讨短期饮酒时长影响的相关工作。本研究旨在弥合这一研究空白，通过在恒河猴模型中开展为期6个月的乙醇摄入实验后对单核细胞进行分析——我们将该时长定义为短期饮酒。阐明酒精摄入背景下单核细胞表型与功能的纵向变化，可为识别疾病进展的诊断生物标志物奠定基础。

## 方法
为填补上述研究空白，我们在恒河猴每日乙醇摄入（每日摄入量>2.0 g/kg体重，且摄入量占比>55%）的6个月周期前后，分别分离获取其外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMC）。研究采用流式细胞术、单细胞RNA测序（Single-Cell RNA-Sequencing, scRNAseq）、酶联免疫吸附实验（ELISA）以及靶向切割与标签化（Cleavage Under Targets and Tagmentation, CUT&Tag）技术对单核细胞进行分析。

## 结果
本研究数据显示，为期6个月的乙醇摄入会将单核细胞重编程为低炎症表型，该变化可通过细胞因子生成减少得到验证。单细胞RNA测序分析显示，乙醇摄入与脂多糖（Lipopolysaccharide, LPS）刺激会使单核细胞状态/聚类发生显著变化，这与低炎症表型的转变相一致。上述变化可能由H3K4me3水平降低所介导——H3K4me3是一种组蛋白修饰，已被证实会富集于炎症与病原体应答信号通路相关基因的启动子区域。

## 讨论
综上，本研究数据证实，每日大量饮酒6个月可通过改变表观遗传谱，减弱单核细胞的炎症应答反应。