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

摘要 慢性乙醇暴露与肠道生态失调、免疫力受损和器官发育有关。 功能障碍导致对乙醇诱导的病理学的器官间串扰的日益重视。第一次被 在乙醇摄入后，肠和肠道微生物组在免疫系统体内平衡中发挥核心作用。慢性 乙醇降低了肠道细菌的丰度和多样性，并使肠上皮屏障不稳定， 与降低的鲁米那丁酸和肠全反式维甲酸（atRA）水平，全身内毒素， 和炎症。肠道微血管内皮细胞（EC）位于肠道固有层内， 通过募集和激活白细胞以及调节肠道血管灌注，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 与肠微血管EC和固有层内的免疫细胞相互作用。内皮和免疫 细菌内毒素引起的细胞活化导致其功能障碍。肠微血管内皮屏障 作为受损上皮的第二道防御屏障，提供额外保护， 大分子和微生物易位。丁酸盐和atRA是已知的脑内产生的免疫营养素 通过T淋巴细胞调节促进上皮屏障功能和免疫稳态。我们已经表明 丁酸（三丁酸甘油酯）补充剂通过防止分解乙醇诱导的肠-肝损伤 上皮紧密连接蛋白，减少氧化应激，促进肠微血管表达 内皮相关连接蛋白和免疫细胞在小鼠近端结肠。在这里，我们建议 假设通过乙醇破坏肠道微生物组，丁酸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 微血管内皮功能被破坏和激活，诱导肠道T细胞 失调和炎症。我们将测试两个具体目标。目的1：检验乙醇诱导的 肠道生态失调，导致丁酸消耗和上皮屏障不稳定，破坏肠道微血管 内皮连接蛋白完整性。使用体内和体外方法，我们将研究丁酸盐 减轻乙醇对内皮细胞屏障连接蛋白分解的影响。目标2：测试 假设乙醇激活并诱导T细胞归巢和Treg扩增的改变， 肠固有层引起丁酸盐和atRA中肠微血管内皮调节异常 依赖的方式。我们将利用离体共培养系统，以及体内野生型和Foxp 3DTR小鼠来测试 乙醇诱导的肠微血管EC、T细胞归巢受体和Treg扩增的活化， 固有层和脾细胞中的细胞因子应答。重要的是，肠微血管EC尚未被 研究的背景下，乙醇，丁酸和atRA，因此，新的机制的见解可能会带来新的 治疗，以防止乙醇的破坏肠道内稳态。

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.