Alcohol and intestinal microvascular endothelium-immune axis and the role of gut derived immune nutrients

摘要 慢性酒精暴露与肠道生物失调、免疫功能受损和器官发育有关 功能障碍导致对酒精诱导的病理的器官间串扰的认识不断增加。第一次命中 摄入乙醇后，肠道和肠道微生物群在免疫系统内稳态中起着核心作用。慢性 乙醇会降低肠道细菌的丰富度和多样性，并破坏肠道上皮屏障的稳定性，从而 与鲁米那丁酸和肠道全反式维甲酸(AtRA)水平降低有关，全身内毒素， 和炎症。位于肠道固有层内的肠道微血管内皮细胞(EC) 通过重新招募和激活白细胞以及调节肠道血管灌流，对粘膜免疫功能至关重要。 乙醇诱导的上皮屏障破坏和内毒素移位后，促炎分子 与肠道微血管内皮细胞和固有层内的免疫细胞相互作用。内皮与免疫 细菌内毒素激活细胞导致细胞功能障碍。肠道微血管内皮细胞屏障 作为破坏的上皮的第二道防御屏障，提供额外的保护，防止 大分子和微生物易位。丁酸盐和全反式维甲酸是已知的肠道免疫营养素 通过T淋巴细胞调节，促进上皮屏障功能，促进免疫动态平衡。我们已经展示了 补充丁酸(三丁酸甘油酯)减轻乙醇所致的肠-肝损伤 上皮紧密连接蛋白，减少氧化应激，促进肠道微血管表达 小鼠近端结肠内皮细胞相关连接蛋白和免疫细胞。在这里，我们建议使用 假设通过乙醇对肠道微生物群的破坏，丁酸盐和全反式维甲酸的消耗 酸，以及随后肠上皮屏障的不稳定和内毒素的移位，肠 微血管内皮细胞功能紊乱和激活诱导肠道T细胞 调节失调和炎症。我们将测试两个具体目标。目的1：检验酒精诱导的假设 肠道生物失调、丁酸耗竭和上皮屏障不稳定破坏肠道微血管 内皮连接蛋白的完整性。使用体内和体外方法，我们将研究丁酸盐如何 减轻乙醇对屏障结合蛋白内皮细胞分解的影响。目标2：测试 假设乙醇激活并诱导T细胞归巢和Treg扩张的改变 丁酸盐和全反式维甲酸引起肠微血管内皮细胞失调的肠固有层 依赖的态度。我们将利用体外共培养系统，以及体内野生型和Foxp3DTR小鼠来测试 乙醇诱导肠微血管内皮细胞、T细胞归巢受体的激活以及Treg的扩张和 固有层和脾细胞的细胞因子反应。重要的是，肠微血管内皮细胞尚未 在乙醇、丁酸盐和全反式维甲酸的背景下进行研究，因此新的机理见解可能会带来新的 防止乙醇破坏肠道内环境稳定的治疗方法。

ABSTRACT Chronic ethanol exposure is associated with gut dysbiosis, impaired immunity, and the development of organ dysfunction leading to a rising appreciation for inter-organ crosstalk for ethanol-induced pathologies. First hit by ethanol ingestion, the intestine and gut microbiome play a central role in immune system homeostasis. Chronic ethanol decreases gut bacterial abundance and diversity and destabilizes the intestinal epithelial barrier, which is associated with reduced luminal butyrate and intestinal all-trans retinoic acid (atRA) levels, systemic endotoxin, and inflammation. Intestinal microvascular endothelial cells (EC), located within the intestinal lamina propria, are critical for mucosal immune function by recruiting and activating leukocytes and regulating gut vascular perfusion. Following ethanol-induced epithelial barrier disruption and endotoxin translocation, proinflammatory molecules interact with intestinal microvascular EC and immune cells within the lamina propria. Endothelial and immune cell activation by bacterial endotoxins leads to their dysfunction. The intestinal microvascular endothelial barrier serves as second defensive barrier for a disrupted epithelium, providing additional protection against macromolecule and microbe translocation. Butyrate and atRA are intestinal-generated immune nutrients known to promote epithelial barrier function, and immune homeostasis via T-lymphocyte regulation. We have shown butyrate (tributyrin) supplementation mitigates ethanol-induced gut-liver injury by preventing disassembly of epithelial tight junction proteins, reducing oxidative stress, and promoting expression of intestinal microvascular endothelial associated junctional proteins and immune cells in mouse proximal colon. Here we propose the hypothesis that through ethanol’s disruption of the gut microbiome, depletion of butyrate and all-trans retinoic acid, and subsequent destabilization of the intestinal epithelial barrier and translocation of endotoxin, intestinal microvascular endothelium become functionally disrupted and activated which induces intestinal T-cell dysregulation and inflammation. We will test two specific aims. Aim 1: Test the hypothesis that ethanol-induced gut dysbiosis, resultant butyrate depletion and epithelial barrier destabilization disrupts intestinal microvascular endothelial junctional protein integrity. Using both in vivo and ex vivo approaches, we will study how butyrate mitigates the effects of ethanol on endothelium disassembly of barrier junctional proteins. Aim 2: Test the hypothesis that ethanol activates and induces alterations in T-cell homing and Treg expansion within the intestinal lamina propria which causes intestinal microvascular endothelial dysregulation in a butyrate and atRA dependent manner. We will utilize ex vivo co-culture system, and in vivo wild-type and Foxp3DTR mice to test for ethanol-induced activation of intestinal microvascular EC, T-cell homing receptors, and Treg expansion and cytokine responses in lamina propria and splenocytes. Importantly, intestinal microvascular EC has not been studied in the context of ethanol, butyrate and atRA, and therefore new mechanistic insights may bring novel therapeutics to protect against ethanol’s disruption in intestinal homeostasis.