Tim Costimulation in Liver Transplant Ischemia Injury

 描述(申请人提供)：与器官获取和冷保存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被研究的问题之一。器官IRI常导致移植物功能不全，易导致晚期慢性排斥反应，并导致可供移植器官的严重短缺。本项目将探索T细胞免疫球蛋白粘蛋白(TIM)家族细胞表面蛋白(主要由活化的CD4T细胞和巨噬细胞表达)在肝脏IRI的病理生理学中的作用，该模型是临床相关的延长冷藏(4℃，20h)后行同基因原位肝移植(OLT)的小鼠模型。总体而言，假设巨噬细胞TIM-4(固有臂)和CD4T细胞上的TIM-1(适应性臂)之间的信号调节冷藏OLT在IR应激期间的促炎(致病)和肝细胞保护(稳态)反应。目的1：明确IR应激OLTS中CD4T细胞特异性TIM-1信号的调节机制。目的1.1：探讨TIM-1信号对肝脏CD4T细胞功能的影响。假设：TIM-1阻断通过产生T细胞偏向IL-22-STAT3/c-Myc信号来减轻肝脏IRI。我们将研究在过继转移的RAG KO小鼠中，TIM-1激活如何在新的肝脏IRI模型中调节CD4T细胞的致病功能；并评估体内和体外对IL-22在肝脏细胞保护中的需求。目的：研究TIM-1-IL-22轴保肝促内稳态的作用机制。假设：TIM-1阻断可增强IL-22介导的肝细胞自噬。我们将使用改进的原位肝移植模型和良好控制的体外共培养系统来剖析TIM-1-IL-22调节下的自噬途径在肝脏保护中的需求。目的2：明确巨噬细胞特异性TIM-4信号在IR应激OLTS中的调节机制。目的：探讨TIM-4-TLR4炎症反应的机制。假设：巨噬细胞TIM-4信号缺陷通过Foxo1/？-catenin网络对TLR4激活进行自限反馈调节，从而减轻肝脏IR炎症反应。我们将利用一种新开发的CD11b-DTR小鼠肝脏IRI模型，在该模型中，有条件地消融过继转移的巨噬细胞种群允许解剖IR炎症中天然和代谢途径之间依赖TIM-4的交叉调节。目的：分析TIM-4在肝脏吞噬功能中的作用。假设：靶向TIM-4抑制局部吞噬，进一步抑制IR应激肝脏中TLR4的激活反应。由于TIM-4作为磷脂酰丝氨酸(PS)受体的功能，我们将应用一种新的吞噬实验来研究巨噬细胞TIM-4信号是否调节PS肝坏死体的结合/吞噬，从而有助于IR炎症的消退。

 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.