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Studying the Epithelial Effects of Intestinal Inflammation <em>In Vitro</em> on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

UPMC Children’s Hospital of Pittsburgh

University of Pittsburgh School of Medicine

Plant-based Enteral Nutrition Modifies the Gut Microbiota and Improves Outcomes in Murine Models of Colitis.

The gut plays a vital role in critical illness, and alterations in the gut structure and function have been reported in endotoxemia and sepsis models. Previously, we have demonstrated a novel link between the diet-induced alteration of the gut microbiome with cellulose and improved outcomes in sepsis. As compared to mice receiving basal fiber (BF) diet, mice that were fed a non-fermentable high fiber (HF) diet demonstrated significant improvement in survival and decreased organ injury in both cecal-ligation and puncture (CLP) and endotoxin sepsis models. To understand if the benefit conferred by HF diet extends to the gut structure and function, we hypothesized that HF diet would be associated with a reduction in sepsis-induced gut epithelial loss and permeability in mice. We demonstrate that the use of dietary cellulose decreased LPS-mediated intestinal hyperpermeability and protected the gut from apoptosis. Furthermore, we noted a significant increase in epithelial cell proliferation, as evidenced by an increase in the percentage of bromodeoxyuridine-positive cells in HF fed mice as compared to BF fed mice. Thus, the use of HF diet is a simple and effective tool that confers benefit in a murine model of sepsis, and understanding the intricate relationship between the epithelial barrier, gut microbiota, and diet will open-up additional therapeutic avenues for the treatment of gut dysfunction in critical illness.

Effect of dietary cellulose supplementation on gut barrier function and apoptosis in a murine model of endotoxemia.

 Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies.  In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the  promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global  mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition,  mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria.  These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis.  NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.

Nix-Mediated Mitophagy Modulates Mitochondrial Damage During Intestinal Inflammation.

Inflammatory bowel disease (IBD) represents a set of idiopathic and chronic inflammatory diseases of the gastrointestinal tract. Central to the pathogenesis of IBD is a dysregulation of normal intestinal epithelial homeostasis. cGAS is a DNA-sensing receptor demonstrated to promote autophagy, a mechanism that removes dysfunctional cellular components. Beclin-1 is a crucial protein involved in the initiation of autophagy. We hypothesized that cGAS plays a key role in intestinal homeostasis by upregulating Beclin-1-mediated autophagy. We evaluated intestinal cGAS levels in humans with IBD and in murine colonic tissue after performing a 2% dextran sulfate sodium (DSS) colitis model. Autophagy and cell death mechanisms were studied in cGAS KO and WT mice via qPCR, WB analysis, H&E, IF, and TUNEL staining. Autophagy was measured in stimulated intestinal epithelial cells (IECs) via WB analysis. Our data demonstrates cGAS to be upregulated during human and murine colitis. Furthermore, cGAS deficiency leads to worsened colitis and decreased levels of autophagy proteins including Beclin-1 and LC3-II. Co-IP demonstrates a direct binding between cGAS and Beclin-1 in IECs. Transfection of cGAS in stimulated HCT-116 cells leads to increased autophagy. IECs isolated from cGAS KO have diminished autophagic flux. cGAS KO mice subjected to DSS have increased cell death and cleaved caspase-3. Lastly, treatment of cGAS KO mice with rapamycin decreased the severity of colitis. Our data suggest that cGAS maintains intestinal epithelial homeostasis during human IBD and murine colitis by upregulating Beclin-1-mediated autophagy and preventing IEC death. Rescue of autophagy can attenuate the severity of colitis associated with cGAS deficiency.

Article	Year
Plant-based Enteral Nutrition Modifies the Gut Microbiota and Improves Outcomes in Murine Models of Colitis. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 30716421	2019
Effect of dietary cellulose supplementation on gut barrier function and apoptosis in a murine model of endotoxemia. PloS one\| PubMed ID: 31790417	2019
Nix-Mediated Mitophagy Modulates Mitochondrial Damage During Intestinal Inflammation. Antioxidants & redox signaling\| PubMed ID: 32103677	2020
Cyclic GMP-AMP synthase contributes to epithelial homeostasis in intestinal inflammation via Beclin-1-mediated autophagy. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 35344224	2022

Elizabeth A. Novak

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