Pathological hepatocyte death results in a sterile inflammatory response (SIR) which amplifies the original liver injury. The SIR is an important component of injury in a wide range of diseases including acetaminophen (APAP) hepatotoxicity, as well as alcoholic and non-alcoholic steatohepatitis. The molecular mechanisms responsible for sterile inflammation in the liver are largely unknown. Hypothesis. There is a two signal requirement for the development of sterile inflammation in the liver: Signal 1) Activation of TLR 7 and 9 by mammalian nucleic acids resulting in up-regulation of pro-IL-1b and pro-IL-18 and signal 2) Activation of the inflammasome and caspapse-1 for cleavage of pro-IL-1b and pro-IL-18, and secretion of active cytokines. To examine these issues, we have used the mouse model of APAP hepatotoxicity and show that: i) IL- 1b and IL-18 are required for full APAP hepatotoxicity. ii) Activation of TLR7 and 9 is required for up-regulation of pro-IL-1b and pro-IL-18 transcripts in the liver and full APAP hepatotoxicity. iii) DNA from apoptotic hepatocytes can activate TLR9 on sinusoidal endothelial cells, and cause liver injury. iv) Components of the inflammasome (NLRP3, ASC, caspase-1) are required for full APAP hepatotoxicity. Based on this background we propose the following objectives: 1: Identify the interactions between mammalian nucleic acids and TLR 7 and 9 which result in up- regulation of pro-IL-1b and pro-lL-18. 1a) Test if ssRNA and nuclear and mitochondrial DNA from healthy, necrotic and apoptotic hepatocytes can up-regulate pro-IL-1b and pro-lL-18 in liver cell populations. 1b) Test if TLR 7 and 9 signaling is required for the effects seen in 1a. 1c) Identify the signaling pathways fromTLR7 and TLR9 which result in up-regulation of pro-IL-1b and pro-IL-18. 1d) Test if TLR7 and 9 are sufficient or required for inflammasome activation. 2: Identify the mechanisms of activation of the NLRP3 inflammasome. 2a) Determine the role of ATP and monosodium urate (MSU) in inflammasome activation in liver cell populations. 2b) Determine the role of the pannexin channel in-vivo in the APAP induced hepatic SIR. 2c) Determine the role of MSU in-vivo in the APAP induced hepatic SIR.
Project Narrative Chronic liver disease due to drug toxicity, alcoholic steatohepatitis (ASH), non-alcoholic hepatitis (NASH), hepatitis C and B are over-represented in the veteran population. This is due to a higher incidence of substance abuse, diabetes and exposure to needle sticks and blood products. This results in a very high degree of morbidity due to fatigue, jaundice, ankle swelling and pruritis. In addition this also results in increased mortality due to cirrhosis, hepatocellular cancer as well as liver failure. Currently there are no effective treatments these liver diseases. This is due to the fact that we have a very poor understanding of how liver inflammation and injury start in these conditions. We propose to identify the molecules in the TLR and inflammasome pathways, which are required to start the process of liver inflammation and injury. The knowledge gained from this project will therefore directly result in designing rational therapies for these conditions.