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.
Alcohol use disorder, a leading cause of morbidity and mortality worldwide, is associated with gut dysbiosis, intestinal permeability and increased endotoxin into circulation, which induces inflammatory responses that drive organ injury, but the target and driver of ethanol-induced inflammation is uncertain. We recently showed the gut fermentation byproduct butyrate is protective against ethanol-induced gut permeability and associated liver inflammation and injury, and that this protection was linked with preserved markers for intestinal endothelium and reduced oxidative stress in mice. Here we will determine the mechanisms for protective effects of immune nutrients, butyrate and all trans retinoic acid, in intestinal microvascular endothelium and T-lymphocytes, with the long-term goal of this project aimed at identifying therapeutic agents that will protect against ethanol-induced injury to intestine and subsequently other organs.
