Tim Costimulation in Liver Transplant Ischemia Injury

 描述（由申请人提供）：与器官获取和冷藏相关的缺血再灌注损伤（IRI）是临床肝移植中最具挑战性但尚未得到充分研究的问题之一。器官 IRI 常常导致原发性移植物无功能，可能导致晚期慢性排斥反应，并导致可用于移植的器官严重短缺。该项目将在临床相关小鼠模型中探索 T 细胞免疫球蛋白粘蛋白 (TIM) 细胞表面蛋白家族（主要由激活的 CD4 T 细胞和巨噬细胞表达）在肝 IRI 病理生理学中的新兴功能（4C 下 20 小时），然后进行同基因原位肝移植 (OLT)。总体假设表明，CD4 T 细胞（适应性臂）上的巨噬细胞 TIM-4（先天臂）和 TIM-1 之间的信号传导在冷储存 OLT 的 IR 应激期间调节促炎（致病）和肝细胞细胞保护（稳态）反应。目标 1：定义 IR 应激 OLT 中 CD4 T 细胞特异性 TIM-1 信号传导的调节机制。目标 1.1：研究 TIM-1 信号传导是否极化肝 CD4 T 细胞功能。假设：TIM-1 阻断通过产生对 IL-22-STAT3/c-Myc 信号传导的 T 细胞偏向来减轻肝脏 IRI。我们将在过继移植的 RAG KO 小鼠的新肝脏 IRI 模型中研究 TIM-1 激活如何调节 CD4 T 细胞致病功能；并评估体内和体外肝细胞保护对 IL-22 的需求。目标1.2：研究TIM-1-IL-22轴发挥保肝作用并导致体内平衡的机制。假设：TIM-1 阻断增强 IL-22 介导的肝细胞自噬。我们将采用精细的OLT模型和良好控制的体外共培养系统来剖析TIM-1 - IL-22调控下保肝作用中自噬途径的需求。目标 2：定义 IR 应激 OLT 中巨噬细胞特异性 TIM-4 信号传导的调节机制。目标2.1：评估TIM-4-TLR4炎症反应的机制。假设：有缺陷的巨噬细胞 TIM-4 信号传导通过 Foxo1/ß-catenin 网络对 TLR4 激活进行自我限制反馈调节，从而减轻肝脏 IR 炎症。我们将在 CD11b-DTR 小鼠中利用新开发的肝脏 IRI 模型，其中过继转移的巨噬细胞群的条件消融允许剖析 IR 炎症中先天和代谢途径之间的 TIM-4 依赖性交叉调节。目标2.2：分析TIM-4在肝吞噬作用中的作用。假设：靶向 TIM-4 会抑制局部吞噬作用，从而进一步抑制 IR 应激肝脏中的 TLR4 激活反应。由于 TIM-4 作为磷脂酰丝氨酸 (PS) 受体发挥作用，我们将应用新开发的吞噬试验来研究巨噬细胞 TIM-4 信号传导是否调节 PS 肝坏死体的结合/吞噬，并有助于解决 IR 炎症。

 DESCRIPTION (provided by applicant): Ischemia-reperfusion injury (IRI) related to organ procurement and cold preservation represents one of the most challenging yet understudied problems in clinical liver transplantation. Organ IRI often leads to primary graft non-function, may predispose to late chronic rejection, and contributes to acute shortage of organs available for transplantation. This project will explore the emerging function of T cell Immunoglobulin Mucin (TIM) family of cell surface proteins (expressed primarily by activate CD4+ T cells and macrophages) in the pathophysiology of hepatic IRI in a clinically relevant mouse model of extended cold storage (20h at 4C) followed by syngeneic orthotopic liver transplantation (OLT). Overall hypothesis states that signaling between macrophage TIM-4 (innate arm) and TIM-1 on CD4+ T cells (adaptive arm) regulates pro-inflammatory (pathogenic) and hepatocyte cytoprotective (homeostatic) responses during IR-stress in cold-stored OLTs. Aim 1: Define regulatory mechanisms of CD4+ T cell-specific TIM-1 signaling in IR-stressed OLTs. Objective 1.1: To investigate whether TIM-1 signaling polarizes hepatic CD4+ T cell function. Hypothesis: TIM- 1 blockade mitigates liver IRI by producing a T cell bias towards IL-22-STAT3/c-Myc signaling. We will study how TIM-1 activation regulates CD4+ T cell pathogenic functions in a new model of liver IRI in adoptively transferred RAG KO mice; and assess the requirement for IL-22 in hepatic cytoprotection both in vivo and in vitro. Objective 1.2: To study the mechanism by which TIM-1 - IL-22 axis exerts hepatoprotection leading to homeostasis. Hypothesis: TIM-1 blockade enhances IL-22-mediated hepatocyte autophagy. We will employ refined OLT models and well-controlled in vitro co-culture systems to dissect the requirement for autophagy pathway in hepatoprotection under TIM-1 - IL-22 regulation. Aim 2: Define regulatory mechanisms of macrophage-specific TIM-4 signaling in IR-stressed OLTs. Objective 2.1: To assess the mechanism of TIM-4-TLR4 inflammation response. Hypothesis: Defective macrophage TIM-4 signaling mitigates liver IR-inflammation by self-limiting feedback regulation of TLR4 activation via Foxo1/ß-catenin network. We will utilize a newly developed model of liver IRI in CD11b-DTR mice in which conditional ablation of adoptively transferred macrophage populations allows dissecting TIM-4- dependent cross-regulation between innate and metabolic pathways in IR-inflammation. Objective 2.2: To analyze the role of TIM-4 in hepatic phagocytosis. Hypothesis: Targeting TIM-4 inhibits local phagocytosis, to further suppress TLR4-activation response in IR-stressed livers. As TIM-4 functions as a phosphatidylserine (PS) receptor, we will apply a newly developed phagocytosis assays to study whether macrophage TIM-4 signaling regulates binding/engulfment of PS+ hepatic necrotic bodies, and contributes to the resolution of IR- inflammation.