The TRPM2 Ion Channel in Hepatic Innate Immunity

描述(申请人提供)：这项建议的目的是探索TRPM2离子通道在肝脏先天性免疫中的作用，重点是肝细胞。我们之前已经证明，TRPM2对于小鼠成功克服单核细胞增多性李斯特菌(Lm)感染是必不可少的。基于TRPM2在髓系细胞中的强表达和已知作用，我们最初认为免疫细胞中TRPM2的缺失是导致高度LM敏感性的唯一原因。然而，对骨髓嵌合动物的分析表明，与脾相反，肝脏的李斯特菌负担高度升高， 是在造血室和实质室结合TRPM2依赖功能的结果。值得注意的是，我们发现的早期肝细胞死亡是LM感染小鼠TRPM2-/-表型的一个标志，这完全依赖于TRPM2的S在肝实质中的存在。LM是一种兼性的细胞内细菌病原体，静脉注射时以肝细胞为靶标，主要在这些细胞中复制。因此，我们假设TRPM2-/-肝细胞对LM感染的有效反应能力受损。为了研究TRPM2功能的这一新方面，我们提出了以下具体目标：1)确定TRPM2在肝细胞对李斯特菌和相关刺激的反应中的作用；2)确定TRPM2功能在肝脏天然免疫中的分子决定因素。在第一个目标下，我们将在一个肝细胞系中建立TRPM2的过表达和抑制。我们将在该细胞系以及野生型与TRPM2-/-原代小鼠肝细胞中评估因LM和病原体识别受体激动剂而改变TRPM2活性水平对细胞因子产生等各种参数的影响。在目标2下，我们将在体外和体内利用TRPM2突变体在肝脏抗李斯特菌防御的背景下进行TRPM2的结构-功能关系研究。我们希望深入了解TRPM2是肝实质和免疫细胞中一种先前未被怀疑的信号通路，可能与导致肝损伤的许多情况相关。这项研究的长期目标将是操纵TRPM2的功能，以影响肝脏的先天免疫和炎症，并治疗或预防肝纤维化和肝硬变等临床疾病。

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.