Role of biliverdin reductase during sterile inflammation in the liver.

 描述（由申请人提供）：无菌性炎症是在无病原体的情况下由炎症细胞的损伤、浸润和活化引起的，是细胞坏死后对器官损伤的急性反应，但如果不加控制，可能导致器官衰竭和死亡。从坏死细胞释放的危险相关分子模式（DAMP）中包括线粒体（mt）和核DNA，它们被Toll样受体9（TLR 9）识别。响应于细胞坏死的无菌炎症模拟微生物炎症，并且由多种信号传导途径调节，包括血红素降解酶：血红素加氧酶-1（HO-1）和胆绿素还原酶（BVR）。 胆绿素还原酶（BVR）是一种代谢酶，其主要功能是将胆绿素（BV）转化为胆红素（BR），然而BVR也在细胞表面（BVRsurf）上表达，在细胞表面它可以作为受体发挥作用并激活巨噬细胞中的PI 3 K-Akt信号传导。在这个提议中，我们假设BVRsurf也作为一种新的模式识别受体样蛋白，可以与细胞外mtDNA片段相互作用，介导和放大巨噬细胞中的促炎信号。胆色素已被证明可以防止休克诱导的肝损伤，以及对乙酰氨基酚（APAP）损伤的保护。由于缺乏可用的基因敲除小鼠，尚未在体内测试BVR对肝损伤的免疫调节功能。我们现在已经产生了BVRflfl条件性敲除小鼠，并证明巨噬细胞中BVR的缺失导致巨噬细胞中DNA介导的信号传导的抑制。我们的部分假设是BVR结合细胞外mtDNA并将其递送至细胞内TLR 9。 本申请的主要重点是阐明BVRsurf作为细胞外mtDNA片段受体的能力，从而介导无菌炎症期间巨噬细胞中的部分反应。具体而言，在本提案中，我们打算：1。使用新的BVRflfl菌株，用条件性免疫抑制剂研究BVR在肝脏无菌性炎症模型中的作用。 巨噬细胞中BVR的缺失（CreLyz：BVRflfl）。 2.评价胆色素在无菌炎症模型中通过直接影响BVRsurf活性抑制炎症的潜在治疗作用。 3.探讨巨噬细胞BVR和胆色素的具体作用机制。我们将测试mtDNA-BVR相互作用是否导致其下游信号转导的激活。我们将测试BVRsurf是否独立于胞外酶活性结合mtDNA片段，以及BVRsurf表达、BVRsurf：DNA相互作用、运输和信号传导是否以及如何受到胞外胆色素的影响。 总之，本研究将揭示巨噬细胞中BVR和胆色素的新机制，这些机制有助于肝脏中无病原体相关炎症期间的先天免疫应答。

 DESCRIPTION (provided by applicant): Sterile inflammation is caused by injury, infiltration and activation of inflammatory cells in the absence of a pathogen and is an acute response to organ injury after cell necrosis, however if uncontrolled can lead to organ failure and death. Among the Danger Associated Molecular Patterns (DAMPs) that are released from the necrotic cells is mitochondrial (mt) and nuclear DNA, which are recognized by toll like receptor 9 (TLR9). Sterile inflammation in response to cellular necrosis mimics microbial inflammation and is regulated by multiple signaling pathways including heme degradation enzymes: heme oxygenase-1 (HO-1) and biliverdin reductase (BVR). Biliverdin reductase (BVR) is a metabolic enzyme whose principal function is to convert biliverdin (BV) to bilirubin (BR), however BVR is also expressed on the cell surface (BVRsurf) where it can function as a receptor and activate PI3K-Akt signaling in macrophages. In this proposal, we hypothesize that BVRsurf also serves as a novel pattern recognition receptor-like protein, and can interact with extracellular mtDNA fragments to mediate and amplify pro-inflammatory signaling in macrophages. Bile pigments have been shown to prevent shock-induced liver injury as well as protected against acetaminophen (APAP) injury in rats. The immunomodulatory function of BVR has not been tested in vivo in response to liver injury due to lack of available knockout mice. We have now generated BVRflfl conditional knockout mice and demonstrated that deletion of BVR in macrophages results in inhibition of DNA-mediated signaling in macrophages. Part of our hypothesis is that BVR binds extracellular mtDNA and delivers it to TLR9 intracellular. The major focus of this application is to elucidate the ability of BVRsurf to acts as a receptor for extracellular mtDNA fragments and therefore mediate part of the responses in macrophages during sterile inflammation. Specifically, in this proposal we intend to: 1. Investigate a role of BVR in models of sterile inflammation in the liver using a novel strain of BVRflfl with conditional deletion of BVR in macrophages (CreLyz:BVRflfl). 2. Evaluate the potential therapeutic role of bile pigments in sterile inflammation models to suppress inflammation by directly impacting BVRsurf activity. 3. Study a detail mechanism of BVR and bile pigments in macrophages. We will test whether mtDNA-BVR interaction results in activation of its downstream signaling. We will test whether BVRsurf binds mtDNA fragments independent of the ecto-enzymatic activity and whether and how BVRsurf expression, BVRsurf:DNA interaction, trafficking and signaling are influenced by extracellular bile pigments. In summary, this study will unravel the novel mechanisms of BVR and bile pigments in macrophages that contribute to innate immune responses during pathogen-free associated inflammation in the liver.