However, HMGB1 levels selectively increased in the ischemic WT liver infiltrate but NVP-BEZ235 not in the ASC-deficient liver infiltrate (Supporting Fig. 1A-D). In agreement with our findings, enhanced HMGB1 activated TLR4/NF-κB and led to increased macrophage sequestration along with expression of proinflammatory cytokines (TNF-α/IL-12p40) and chemokines (MCP-1/CXCL-10). Using a well-controlled in vitro

culture system, we found that ASC deficiency decreased mRNA and protein HMGB1 expression in LPS-stimulated BMMs and that TLR4 and NF-κB expression was diminished in ASC-deficient BMMs. Furthermore, rHMGB1 increased cleaved caspase-1 and IL-1β levels in BMM cultures (Supporting Fig. 3A,B), and this suggests that HMGB1 was at least in part responsible for the activation of caspase-1/IL-1β signaling in IR-stressed livers. Interestingly, ASC deficiency resulted in inhibition of phosphorylated p38 MAPK. In agreement with the essential role of MAPKs in IL-1β–mediated inflammation,29 Syk inhibitor our data imply that HMGB1 induction in the ASC/caspase-1/IL-1β–mediated

inflammation cascade in hepatic IRI is p38 MAPK–dependent. IL-1β is an important cytokine that targets inflammatory injury due to hepatic IR. IL-1β can express its biological activity only from pro–IL-1β to mature IL-1β through proteolytic cleavage by the protease caspase-130 and induce the release of HMGB1 from monocytes and macrophages.15 Moreover, IL-1β can form complexes with HMGB1 to enhance immune responses.31, 32 Our results 上海皓元 demonstrate that IL-1β blockade reduced IR-induced hepatocellular damage and improved liver function. Blocking IL-1β decreased the expression of NF-κB and COX2. Indeed, COX2 overexpression has been associated with hypoxia/ischemia and inflammatory chronic diseases,33 whereas COX2 inhibition has improved liver transplant function.34 It is plausible that IL-1β stimulates

COX2 production through NF-κB in IR-stressed livers. Our findings support recent clinical data on the efficacy of antihuman IL-1β (canakinumab) therapy in type 2 diabetes35 and autoimmune inflammatory disorders caused by mutations in the NLRP3 nucleotide-binding domain, such as cryopyrin-associated periodic syndromes36 and Schnitzler syndrome.37 ASC, originally identified as a protein that mediates apoptosis in human leukemia cells,38 interacts with B cell lymphoma 2–associated X protein (Bax) to induce apoptosis via the p53-Bax pathway.39 In addition, HMGB1 release can occur during the process of apoptotic cell death.40 Our data demonstrate that ASC knockdown decreased HMGB1 and caspase-3 but increased antiapoptotic Bcl-2/Bcl-xL expression. These findings were further supported by the increased frequency of apoptotic cells in WT ischemic livers, whereas ASC deficiency markedly decreased hepatocellular apoptosis.