8-1.0, 1.0-1.2, 0.2-0.4, and 0.5-0.7 AU, respectively). We further analyzed messenger RNA (mRNA)–level coding for HMGB1 by qRT-PCR (Fig. 2B). ASC KO mice showed decreased hepatic expression of HMGB1 versus controls (P < 0.05). In contrast to ASC-proficient (WT) controls, the baseline LY294002 solubility dmso HMGB1 levels were decreased in ASC-deficient mice, as evidenced both in vitro (BMM cultures) and in vivo (normal livers; Supporting Fig. 4A,B). To determine whether ASC signaling may have influenced macrophage and neutrophil

trafficking patterns, we performed immunohistochemical staining in IR-stressed livers. ASC-deficient livers were devoid of both CD11b+ macrophages (Fig. 2Ca-c) and Ly6G+ neutrophils (Fig. 2Cd-f) in comparison with WT controls (7.4 ± 4.4 versus 38.1 ± 18.6 CD11b+ macrophages per HPF, P < 0.005; 10.7 ± 5.6 versus 42.5 ± 18.5 LY6G+ neutrophils per HPF, P < 0.0001). In agreement selleck chemicals with these immunostaining data, the qRT-PCR–assisted detection of mRNA coding for TNF-α/IL-12p40 (P < 0.01), CXCL-10/MCP-1 (P < 0.05), and CXCL-1 (P < 0.0005) was reduced in ASC-deficient

livers versus WT controls (Fig. 2D). To determine whether ASC affects IR-induced apoptosis, we performed western blots to detect antiapoptotic genes. The expression of Bcl-2 and Bcl-xL was up-regulated in ASC KO livers (1.8-2.0 and 1.5-1.7 AU; Fig. 3A) versus WT livers (0.2-0.4 AU). Moreover, ASC deficiency inhibited the expression of cleaved caspase-3 (0.3-0.5 AU) in comparison with controls (1.9-2.1 AU). In agreement with

the western analysis, the frequency of TUNEL+ cells per HPF in the ischemic liver lobes was diminished in ASC KO mice versus their WT counterparts [7.9 ± 15.22 (Fig. 3Bc) versus 75.4 ± 15.12 TUNEL+ cells per HPF (Fig. 3Bb), P < 0.001]. To clarify the function of HMGB1 in the ASC-mediated inflammatory response, we administered rHMGB1 to ASC KO mice immediately at reperfusion after 90 minutes of warm ischemia. As shown in Fig. 3C, an rHMGB1 infusion increased sALT levels at 6 hours of reperfusion in comparison with untreated ASC-deficient mice (29,354.3 ± 2971 versus 12,506.8 ± 12,717 IU/L, P < 0.05). These data correlated with Suzuki's grading of histological liver damage (Fig. 3D). Hence, unlike ASC-deficient but otherwise untreated livers (Suzuki's score = 1.5 ± 0.7), those conditioned with adjunctive rHMGB1 revealed moderate Selleck Baf-A1 to severe edema, sinusoidal congestion, and hepatocellular necrosis (Suzuki’s score = 3.6 ± 0.51, P < 0.0001). A similar effect was displayed in rHMBG1-treated WT livers subjected to 90 minutes of ischemia and 6 hours of reperfusion (Supporting Fig. 5A,B). In contrast, rHMGB1 did not affect well-preserved histological architecture in sham controls. Furthermore, adjunctive rHMGB1 significantly increased the expression of mRNA coding for TNF-α (P < 0.05) and IL-1β (P < 0.005) in ASC KO livers versus otherwise untreated ASC-deficient livers (Fig. 3E).