studying-epithelial-effects-intestinal-inflammation-vitro-on

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

 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.

Ferroptosis and necroptosis are two pro-inflammatory cell death programs contributing to major pathologies and their inhibition has gained attention to treat a wide range of disease states. Necroptosis relies on activation of RIP1 and RIP3 kinases. Ferroptosis is triggered by oxidation of polyunsaturated phosphatidylethanolamines (PUFA-PE) by complexes of 15-Lipoxygenase (15LOX) with phosphatidylethanolamine-binding protein 1 (PEBP1). The latter, also known as RAF kinase inhibitory protein, displays promiscuity towards multiple proteins. In this study we show that RIP3 K51A kinase inactive mice have increased ferroptotic burden and worse outcome after irradiation and brain trauma rescued by anti-ferroptotic compounds Liproxstatin-1 and Ferrostatin 16-86. Given structural homology between RAF and RIP3, we hypothesized that PEBP1 acts as a necroptosis-to-ferroptosis switch interacting with either RIP3 or 15LOX. Using genetic, biochemical, redox lipidomics and computational approaches, we uncovered that PEBP1 complexes with RIP3 and inhibits necroptosis. Elevated expression combined with higher affinity enables 15LOX to pilfer PEBP1 from RIP3, thereby promoting PUFA-PE oxidation and ferroptosis which sensitizes Rip3 kinase-deficient mice to total body irradiation and brain trauma. This newly unearthed PEBP1/15LOX-driven mechanism, along with previously established switch between necroptosis and apoptosis, can serve multiple and diverse cell death regulatory functions across various human disease states.

Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death.

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.

Cyclic GMP-AMP synthase contributes to epithelial homeostasis in intestinal inflammation via Beclin-1-mediated autophagy.

The colonic epithelium requires continuous renewal by crypt resident intestinal stem cells (ISCs) and transit amplifying (TA) cells to maintain barrier integrity, especially after inflammatory damage. The diet of high-income countries contains increasing amounts of sugar, such as sucrose. ISCs and TA cells are sensitive to dietary metabolites, but whether excess sugar affects their function directly is unknown.

Article	Year
Nix-Mediated Mitophagy Modulates Mitochondrial Damage During Intestinal Inflammation. Antioxidants & redox signaling\| PubMed ID: 32103677	2020
Inactivation of RIP3 kinase sensitizes to 15LOX/PEBP1-mediated ferroptotic death. Redox biology\| PubMed ID: 35101798	2022
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
Excess dietary sugar alters colonocyte metabolism and impairs the proliferative response to damage. Cellular and molecular gastroenterology and hepatology\| PubMed ID: 37172822	2023

Heather L. Mentrup

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