The TRPM2 Ion Channel in Hepatic Innate Immunity

描述（由申请方提供）：本提案旨在探索TRPM 2离子通道在肝脏先天免疫中的作用，重点关注肝细胞。我们以前已经表明，TRPM 2是小鼠成功克服单核细胞增生李斯特菌（Lm）感染所必需的。基于TRPM 2在髓系细胞中的强代表性和已知作用，我们最初假设免疫细胞中TRPM 2的缺乏是Lm易感性高的唯一原因。然而，对骨髓嵌合体动物的分析显示，与脾脏相反， 是在造血和实质隔室中组合TRPM 2依赖性功能的结果。引人注目的是，我们发现早期肝细胞死亡是Lm感染小鼠的TRPM 2-/-表型的标志，其仅依赖于TRPM 2在肝实质中的存在。Lm是一种兼性细胞内细菌病原体，当静脉注射时靶向肝细胞，并且主要在这些细胞中复制。因此，我们假设TRPM 2-/-肝细胞对Lm感染有效应答的能力受损。为了研究TRPM 2功能的这一新方面，我们提出了以下具体目标：1）确定TRPM 2在肝细胞中响应李斯特菌和相关刺激的作用，以及2）确定TRPM 2在肝脏先天免疫中功能的分子决定因素。在第一个目标下，我们将在肝细胞系中建立TRPM 2的过表达和抑制。我们将在该细胞系以及野生型与TRPM 2-/-原代小鼠肝细胞中评估响应Lm和病原体识别受体激动剂的TRPM 2活性水平变化对各种参数（如细胞因子产生）的影响。在目标2下，我们将利用TRPM 2突变体在体外和体内肝脏抗病毒防御的背景下进行TRPM 2的结构-功能关系研究。我们期望获得关于TRPM 2作为肝实质和免疫细胞中先前未被怀疑的信号通路的见解，该信号通路可能与导致肝损伤的许多疾病相关。这项研究的长期目标将是操纵TRPM 2功能来影响肝脏先天免疫和炎症，并治疗或预防肝纤维化和肝硬化等临床疾病。

DESCRIPTION (provided by applicant): The purpose of this proposal is to explore the role of the TRPM2 ion channel in hepatic innate immunity, with a focus on hepatocytes. We have previously shown that TRPM2 is essential for mice to successfully overcome infections with Listeria monocytogenes (Lm). Based on the strong representation and known role of TRPM2 in cells of the myeloid lineage, we originally assumed that the absence of TRPM2 in immunocytes was the sole cause for high Lm susceptibility. However, the analysis of bone marrow chimeric animals revealed that the highly elevated listerial burdens in the liver, as opposed to the spleen, are the result of combined TRPM2-dependent functions both in the hematopoietic and parenchymal compartments. Strikingly, the early hepatocytic death that we found to be a hallmark of the TRPM2-/- phenotype of Lm-infected mice is solely dependent upon TRPM2's presence in the liver parenchyma. Lm is a facultative intracellular bacterial pathogen that target hepatocytes when injected intravenously, and mostly replicates in these cells. We thus hypothesize that TRPM2-/- hepatocytes are impaired in their ability to efficiently respond to Lm infection. To investigate this novel aspect of TRPM2 function, we propose to pursue following specific aims: 1) To define the role of TRPM2 in response to Listeria and related stimuli in hepatocytes, and 2) To define molecular determinants of TRPM2 function in hepatic innate immunity. Under the first aim, we will establish overexpression and inhibition of TRPM2 in a hepatocyte cell line. We will evaluate in this cell line, as well as in wildtype versus TRPM2-/- primary mouse hepatocytes, the impact of changing TRPM2 activity levels in response to Lm and pathogen recognition receptor agonists on various parameters such as cytokine production. Under aim 2, we will utilize TRPM2 mutants to perform a structure-function relationship study of TRPM2 in the context of hepatic anti-listerial defense, both in vitro and in vivo. We expect to gain insights about TRPM2 as a previously unsuspected signaling pathway in hepatic parenchymal and immune cells that could have relevance for many conditions leading to liver injury. The long-term goal of this research will be to manipulate TRPM2 function to influence hepatic innate immunity and inflammation, and treat or prevent clinical conditions such as liver fibrosis and cirrhosis.