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.
Hepatic injury due to trauma, surgery or induced by drugs or xenobiotics remains a major challenge in clinical practice. We have recently identified BVR, a metabolic enzyme converting biliverdin to bilirubin as a cell membrane protein (BVRsurf) that is a critical signaling molecule involved in modulation of PI3K-Akt signaling as well as functions as a zinc-finger transcription factor. We propose and will test that that fragmented DNA released from dying cells upon injury is recognized by BVRsurf, which transmits the signal into the cell and leads to activation of the macrophage and ongoing inflammation in the models of sterile inflammation in the liver.
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