This proposal focuses on the role of TNFAIP8 (TIPE0) in intestinal ischemia/reperfusion injury (II/RI). Mesenteric ischemia causes 1/1000 of all hospital admissions, with a mortality rate of 60-80%. Hypoxia leads to apoptosis and loss of the protective intestinal epithelial cell (IEC) layer, resulting in bacterial translocation and often-lethal systemic inflammation. Barrier maintenance involves highly regulated interactions between IECs and the immune system, to support barrier repair and bacterial clearance, while stopping immune system hyperactivation that results in further barrier disruption, as is seen with IIRI and colitis. The TIPE proteins are phospholipid transporters that modulates both cell survival and immune function; loss of the proteins alters immune cell function and myeloid chemotaxis. Loss of TNFAIP8 (TIPE0) enhances acute colitis, likely through loss of protective IEC TNF signaling. In contrast to colitis, we recently found that TIPE0 loss protects against murine intestinal I/R injury and the mechanism is unknown; based on our preliminary data, we hypothesize that it involves loss of deleterious IEC TNF signaling and altered immune function. This proposal explores the role of TIPE0 (TNFAIP8) in II/RI in multiple cell compartments, using techniques ranging from surgery on transgenic mice to cellular assays and biochemistry.
Aim 1 investigates TIPE0 in the immune compartment during II/RI, utilizing bone marrow chimeras (BMCs). Recently, group 3 innate lymphoid cells (ILC3s) were found to help maintain IECs during small bowel injury and so the role TIPE0 in both migrating myeloid cells and ILC3s will be explored.
Aim 2 investigates TIPE0 in enterocytes, also using BMCs, and using ex vivo enteroids to study the role of TIPE0 in IEC proliferation, differentiation, and hypoxia & TNF-induced IEC cell death.
Aim 3 focuses on TIPE0 biochemistry, determining its KD for phospholipids via SPR, its ability to extract phospholipids through a novel FRET-based assay, and its ability to enhance PI3K activity. The research strategy described in this proposal combines the applicant, Dr. Goldsmith?s, expertise in II/RI, colitis, and biochemistry with new training in immunology and enteroids that are critical for him to develop a career studying the complex interactions involved in intestinal inflammation and restitution and designing new treatments to modulate this process. The research site, The University of Pennsylvania, provides comprehensive state-of-the-art physical resources and a community of experts in gastroenterology and immunology. The sponsor, Dr. Youhai Chen, is a leading expert on immunology and TIPE proteins, with expertise in the gut. His co-sponsor, Dr. Anil Rustgi, is a leading expert on IEC biology. Their collective expertise and the collaborative environment will provide ideal training to prepare Dr. Goldsmith for a career in gastrointestinal research. The research proposed here will elucidate the roles of TIPE0 in IECs, migrating myeloid cells, and ILC3s during II/RI, and more broadly during intestinal inflammation, and lay the foundation for developing therapeutics that could abrogate intestinal inflammation.
Intestinal ischemia is responsible for 1/1000 of all hospital admissions and has a mortality of 60-80%. Current treatments are limited to restoring blood flow to the intestine and supportive care; no therapies yet exist to treat the underlying intestinal injury or modulate the often-lethal inflammatory response. The aims of this research proposal are to determine the role of the TNFAIP8 protein in intestinal ischemia/reperfusion injury and biochemically prolife the protein, with the hopes of identifying novel therapeutic pathways/targets for the treatment of intestinal ischemia, focusing on the intestinal cells themselves and the deleterious inflammatory responses that occur with ischemic injury.
