Role of biliverdin reductase during sterile inflammation in the liver.

 描述(申请人提供)：无菌炎症是在没有病原体的情况下，炎性细胞的损伤、渗透和激活引起的，是细胞坏死后对器官损伤的急性反应，但如果不加以控制，可能会导致器官衰竭和死亡。坏死细胞释放的危险相关分子模式(DAMP)包括线粒体(MT)和核DNA，它们被Toll样受体9(TLR9)识别。细胞坏死后的无菌炎症模拟微生物炎症，受多种信号通路调节，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)，但BVR也表达于细胞表面(BVRsurf)，在巨噬细胞中可作为受体激活PI3K-Akt信号。在这个提议中，我们假设BVRsurf还可以作为一种新的模式识别受体样蛋白，并可以与细胞外mtDNA片段相互作用，介导和放大巨噬细胞中的促炎信号。胆汁色素已被证明可以预防休克诱导的大鼠肝损伤，并对对乙酰氨基酚(APAP)损伤具有保护作用。由于缺乏可用的基因敲除小鼠，BVR的免疫调节功能尚未在体内进行测试，以应对肝损伤。我们现在已经建立了BVRflfl条件基因敲除小鼠，并证明了巨噬细胞中BVR的缺失会导致巨噬细胞中DNA介导的信号抑制。我们的部分假设是BVR与细胞外的mtDNA结合，并将其运送到TLR9细胞内。这一应用的主要焦点是阐明BVRsurf作为细胞外mtDNA片段的受体的能力，从而在无菌炎症期间介导巨噬细胞的部分反应。具体地说，在这项建议中，我们打算：1.使用一种新的BVRflfl株和条件性条件感染来研究BVR在肝脏无菌炎症模型中的作用 巨噬细胞中BVR的缺失(CreLyz：BVRflfl)。2.评价胆汁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.研究BVR和巨噬细胞胆汁色素的作用机制。我们将测试mtDNA-BVR相互作用是否导致其下游信号的激活。我们将测试BVRsurf是否独立于胞外酶活性与mtDNA片段结合，以及BVRsurf的表达、BVRsurf：DNA相互作用、转运和信号是否以及如何受到细胞外胆汁色素的影响。综上所述，这项研究将揭开BVR和巨噬细胞中胆汁色素在肝脏无病原体相关炎症过程中促进先天性免疫反应的新机制。

 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.