The role and mechanism of TNFAIP8 in intestinal inflammation and wound healing

项目摘要 本研究旨在探讨TNFAIP8(TIPE0)在肠缺血/再灌注损伤(II/RI)中的作用。肠系膜 在所有住院病人中，有1/1000是由缺血引起的，死亡率为60%-80%。低氧导致细胞凋亡 和失去保护性的肠道上皮细胞(IEC)层，导致细菌易位，通常是致命的 全身炎症。屏障维护涉及IECS和IECS之间高度规范的交互 免疫系统，支持屏障修复和细菌清除，同时阻止免疫系统过度激活 这会导致进一步的屏障破坏，就像IIRI和结肠炎一样。TIPE蛋白是磷脂 同时调节细胞存活和免疫功能的转运体；蛋白质的丢失会改变免疫细胞 功能和髓系趋化。丢失TNFAIP8(TIPE0)可增强急性结肠炎，可能是通过丢失 保护性的IEC肿瘤坏死因子信号。与结肠炎相反，我们最近发现TIPE0丢失对小鼠有保护作用 肠I/R损伤及其机制尚不清楚；根据我们的初步数据，我们假设它 包括有害的IEC肿瘤坏死因子信号的丢失和免疫功能的改变。这项提案探讨了 TIPE0(TNFAIP8)在多个细胞室的II/RI中，使用从转基因手术到 对小鼠进行细胞分析和生化检测。目的1研究II/RI期间免疫隔膜中的TIPE0， 利用骨髓嵌合体(BMC)。最近，第三组先天淋巴样细胞(ILC3)被发现有助于 在小肠损伤期间维持IECS，因此TIPE0在迁移的髓系细胞和ILC3中的作用将是 探索过了。目的2研究TIPE0在肠上皮细胞中的作用，也使用骨髓细胞，并使用体外肠样组织来研究 TIPE0在IEC增殖、分化及缺氧和肿瘤坏死因子诱导的IEC细胞死亡中的作用目标3侧重于 TIPE0生物化学，通过SPR测定其对磷脂的Kd值，它通过 新的基于FRET的检测及其增强PI3K活性的能力。本文件中描述的研究策略 建议将申请者，戈德史密斯博士在II/RI、结肠炎和生化方面的专业知识与新的培训相结合 在免疫学和肠系膜方面对他发展研究复杂相互作用的职业至关重要 参与肠道炎症和修复，并设计新的治疗方法来调节这一过程。这个 研究网站，宾夕法尼亚大学，提供全面的最先进的物理资源和 胃肠病学和免疫学方面的专家社区。发起人陈友海博士是一位领先的专家 在免疫学和TIPE蛋白方面，拥有肠道方面的专业知识。他的共同赞助人Anil Rustgi博士是一位领先的专家 关于IEC生物学。他们的集体专业知识和协作环境将为准备工作提供理想的培训 戈德史密斯博士的胃肠研究生涯。这里提出的研究将阐明 IECS、移行髓系细胞和ILC3在II/RI期间的TIPE0，以及更广泛的肠道炎症期间的TIPE0， 并为开发可消除肠道炎症的治疗方法奠定了基础。

PROJECT ABSTRACT 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.