High-mobility box 1 (HMGB1) was quantified using an ELISA kit (IBL International GmbH, Hamburg, Germany). Serum cytokine quantification

was performed using the Cytometric Bead Array Mouse Inflammation Kit (BD Biosciences). A western blotting assay was performed using whole cell lysates from either liver tissue or HCs, as previously GDC-0449 in vivo described.13 Membranes were incubated overnight using the following antibodies (Abs): TLR4 (Imgenex Corp., San Diego, CA), HMGB1, and heme oxygenase 1 (HO-1; Abcam, Cambridge, MA); mouse monoclonal HMGB1 Ab and β-actin (Sigma-Aldrich); and phospho-p38, p38, phospho-c-Jun, c-Jun, phospho-JNK, JNK, extracellular signal-regulated kinase (ERK), phospho-ERK, p65, and phospho-p65 (Cell Signaling Technology, Inc., Danvers, MA). Immunofluorescent (IF) staining was performed using HMGB1 Ab (1:1,000; Abcam), as previously described.14 Immunohistochemistry (IHC) for neutrophil infiltration was accomplished using Anti-Neutrophil Ab [7/4] (Abcam). An E1- and E3-deleted adenoviral vector carrying AdTLR4 and AdLacZ cDNA was constructed and utilized in vivo as previously described.15 SYBR green polymerase chain reaction (PCR) was performed as previously described using β-actin as endogenous control.14 Specific primers were as follows: IL-10, forward 5′-TACCTGGTAGAAGTGATGCC-3′ and reverse 5′-CATCATGTATGCTTCTATGC-3′, and HO-1, which

is commercially available from Qiagen. Results are expressed as either mean ± standard error of the mean (SEM) or mean ± standard

deviation (SD). Group comparisons were performed using analysis of variance and Student Wnt inhibitor t test. A probability value selleck chemicals of P ≤ 0.05 was considered statistically significant. To investigate the role of TLR4 on an individual cellular population, we generated HC-, myeloid-cell–, and DC-specific TLR4 KO (Alb-TLR4−/−, Lyz-TLR4−/−, and CD11c-TLR4−/−, respectively) mice using Cre-loxP technology. Mice with loxP sites flanking exon 2 of TLR4 were interbred with mice that had Cre recombinase linked to the desired promoter. WT mice used had loxP inserted without expression of Cre recombinase, and TLR4−/− mice were globally lacking the loxP flanked exon 2. Both WT and TLR4−/− mice were born healthy and fertile, without any grossly apparent phenotypic differences. Verification of specificity of TLR4 KO in Alb-TLR4−/− mice was accomplished by isolating both HCs and NPCs as well as analyzing these cells for the presence of TLR4 mRNA transcription using reverse-transcriptase (RT)-PCR with primers specific for exon 2 of TLR4 (Fig. 1A). TLR4 was present in both HCs and NPCs of WT mice, whereas Alb-TLR4−/− mice had TLR4 expressed only in NPCs. Global TLR4−/− had no detectable TLR4 in either cell population. Western blotting analysis was performed to confirm that HCs isolated from Alb-TLR4−/− mice had TLR4 protein levels that were undetectable (Fig. 1B).