Ethanol effects on pulmonary innate immunity in a murine model of aging

项目摘要 老年人(65岁的≥)越来越普遍的行为是饮酒，其中40%的人饮酒 平均一周三天每天喝1-2杯酒，被认为是“适度”的摄入量。即使老年人通常 饮酒比年轻人少，酒精对健康的负面影响在年长的饮酒者中可能更强 由于新陈代谢和药物使用较慢。饮酒与全身性疾病相关 炎症和细胞免疫功能降低，类似于“炎症老化”的基础升高 炎症状态，即使是健康的老年人也存在这种状态。此外，饮酒和高龄 与感染的发病率和严重性增加有关，否则是无害的病原体，如 肺炎链球菌；然而，酒精对老年人免疫功能的影响尚未得到证实 调查过了。肺泡巨噬细胞(AM)是驻留在肺部的先天免疫细胞，调节体内平衡。 并成为关键的效应细胞，以启动炎症反应，以应对肺部病原体。巨噬细胞 病原体刺激导致丝裂原活化蛋白激酶(MAPK)在细胞内激活，并 NF-κB途径，以及产生趋化物质以招募和激活额外的免疫细胞 感染部位。我们实验室以前的研究表明，高龄和急性体内乙醇 单独暴露可减少巨噬细胞产生促炎细胞因子 体外被Toll样受体(TLR)激动剂刺激，与抑制p38MAPK激活平行。 此外，初步的体内数据显示，适度的、多天的酒精暴露会导致减少 白介素6、肿瘤坏死因子α和单核细胞趋化因子C-C基序趋化因子配体 2(CCL2)基因在老龄小鼠肺组织匀浆中的表达 不考虑酒精暴露的小鼠。根据这些发现，我们假设高龄和酒精 暴露协同作用抑制小鼠肺泡巨噬细胞中p38 MAPK的激活，导致 产生促炎介质、吞噬功能受损和/或免疫细胞调节失调 在感染后重新招募。为了测试这一点，在目标1中，我们将检查乙醇和高级 AGE对MAPK活化和巨噬细胞功能的影响。在经过多天的车辆或乙醇处理后，我们将 体外调节肺泡巨噬细胞p38MAPK的表达并测定其吞噬功能和产物 促炎细胞因子和趋化因子。在目标2中，我们将评估CCL2在肺免疫中的作用 CCL2重组蛋白处理后的细胞募集和体内肺炎链球菌感染。在.期间 在感染过程中，我们将用体积描记仪测量呼吸功能，并对肺部细菌进行量化 负担通过定量聚合酶链式反应。总的来说，这些研究将扩大我们对免疫学的了解 老年人饮酒的后果，并可能确定改善的治疗目标 饮酒老年人中与感染相关的健康后果。

Project Summary An increasingly common behavior in the elderly ( ≥ 65 years old) is alcohol consumption, with 40% consuming an average of 1-2 drinks/day 3 days a week, considered “moderate” intake. Even though the elderly typically drink less than younger adults do, the negative health effects of alcohol may be more potent in older drinkers due to slower metabolism and medication use. Alcohol consumption correlates with heightened systemic inflammation and reduced cell-mediated immunity, similar to the effects of “inflamm-aging,” the elevated basal inflammatory state which is present even in healthy elderly individuals. Further, alcohol use and advanced age are associated with increased incidence and severity of infection with otherwise innocuous pathogens such as Streptococcus pneumoniae; however, the effects of alcohol on immunity in the elderly have yet to be investigated. Alveolar macrophages (AMs), the resident innate immune cells in the lung, regulate homeostasis and become critical effector cells to initiate inflammation in response to pulmonary pathogens. Macrophage stimulation by pathogens results in intracellular activation of the mitogen-activated protein kinase (MAPK) and NF-κB pathways, and the production of chemoattractants to recruit and activate additional immune cells at the site of infection. Previous studies from our laboratory have shown that advanced age and acute in vivo ethanol exposure independently contribute to decreased production of pro-inflammatory cytokines by macrophages stimulated with Toll-like receptor (TLR) agonists ex vivo, which paralleled suppression of p38 MAPK activation. In addition, preliminary in vivo data show that moderate, multi-day ethanol exposure leads to decreased Interleukin- (Il-) 6, Tumor necrosis factor- (Tnf-) α, and monocyte chemoattractant C-C motif chemokine ligand 2 (Ccl2) gene expression in lung homogenates of aged mice, compared to aged mice given vehicle, and young mice regardless of ethanol exposure. Given these findings, we hypothesize that advanced age and ethanol exposure act synergistically to suppress p38 MAPK activation in murine AMs, leading to decreased production of pro-inflammatory mediators, impaired phagocytosis, and/or dysregulated immune cell recruitment following infection. To test this, in Aim 1, we will examine the impact of ethanol and advanced age on MAPK activation and macrophage function. Following multi-day vehicle or ethanol treatment, we will modulate p38 MAPK expression in alveolar macrophages ex vivo and measure phagocytosis and production of pro-inflammatory cytokines and chemokines. In Aim 2, we will assess the role of CCL2 in pulmonary immune cell recruitment after treatment with CCL2 recombinant protein and in vivo S. pneumoniae infection. During the course of infection, we will measure respiratory function by plethysmography and quantify lung bacterial burden by quantitative PCR. Collectively, these studies will expand our knowledge of the immunological consequences of alcohol consumption in the elderly, and potentially identify therapeutic targets to improve infection-related health outcomes in older adults who consume alcohol.