Ckcross the IRAK-M2/2 mice. SNP test showed 
that our IRAK-M2/2 mice were fully back-crossed to B6 genetic background (Figure 1).Results Mouse Genome AnalysisOne pitfall of using genetically engineered mice is the purity of the mouse strain, as genomic contamination could affect the data interpretation. The genetic purity of the IRAK-M2/2 mice used in this study was analyzed by mouse genome SNP analysis (www. dartmouse.org). The genomic DNA IRAK-M2/2 mice wereAlcohol Induced Worse Liver Damage in IRAK-M2/2 MiceTo study the role of innate immunity, in particular IRAK-M, in alcohol-induced liver damage, we treated wild type (WT) and IRAK-M2/2 
B6 mice with alcohol as described in the Materials and Methods. 10 alcohol in drinking water was administered to mimic a daily light alcohol consumption and the single gavageFigure 4. Inflammatory cytokine in LMNCs. Ex vivo LMNCs were stained with intracellular cytokines and different surface markers as described in Materials and Methods. (A) Representative FACS plots showing IFNc+ cells after gating CD8+ T cells in alcohol treated mice. (B) Summary of percentage of IFNc producing CD8 T cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M2/2 mice (red). (C) Representative FACS plots showing IL-6 producing CD11b+ Fruquintinib chemical information 1113-59-3 web Kupffer cells after gating CD11b+ LMNCs in alcohol treated mice. (D) Summary of percentage of IL-6 producing CD11b+ Kupffer cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M2/2 mice (red). Error bars represent the SD of samples within a group. Experiments were performed 4 times and n = 2? in each group of each experiment. The data presented are from two pooled experiments. *P,0.05, (Two-way ANOVA test). doi:10.1371/journal.pone.0057085.gIRAK-M Regulates Liver InjuryFigure 5. Phagocytic activity of Kupffer cells in liver after alcohol treatment. (A) Representative histogram of FITC-dextran intake LMNCs in wild type B6 mice (blue line) and IRAK-M2/2 mice (red line, 2 ). (B) FITC-dextran uptake by LMNCs in wild type B6 (blue) and IRAK-M2/2 mice (red, 23 ). (C) FITC-dextran uptake by CD11b+ Kupffer cells in wild type B6 (blue) and IRAK-M2/2 mice (red). (D) FITC-dextran uptake by CD68+ Kupffer cells in wild type B6 (blue) and IRAK-M2/2 mice (red). Experiments were performed 3 times. N = 3? in each group of each experiment. The data presented are from one of the 3 experiments. Error bars represent the SD of samples within a group. *P,0.05, **P,0.01, ***P,0.001, Two way ANOVA test. doi:10.1371/journal.pone.0057085.gwith a larger amount of alcohol (60 alcohol in 200 ml, ,6 g/kg) was to mimic an alcoholic binge, which has been reported to be one of the main triggers of alcoholic liver damage in human [29]. There was very mild liver damage induced by daily 10 alcohol water consumption in both WT and IRAK-M2/2 mice, indicated by serum ALT levels (Figure 2A) and liver histology (Figure 2C and 2E, without binge). However, the difference between WT and IRAK-M2/2 was negligible (Figure 2A) although it appeared that IRAK-M2/2 mice showed more liver damage (Figure 2E). In contrast, a single episode of heavy alcohol consumption triggered liver inflammation and injury as evidenced by increased serum ALT levels in both WT and IRAK-M2/2 mice (Figure 2B) and LMNC infiltration in the liver of IRAKM2/2 mice (Figure 2D and 2F). We also examined the absolutenumber of LMNC infiltration per gram liver tissue analyzed, and the results were consistent (Figure 2G). W.Ckcross the IRAK-M2/2 mice. SNP test showed that our IRAK-M2/2 mice were fully back-crossed to B6 genetic background (Figure 1).Results Mouse Genome AnalysisOne pitfall of using genetically engineered mice is the purity of the mouse strain, as genomic contamination could affect the data interpretation. The genetic purity of the IRAK-M2/2 mice used in this study was analyzed by mouse genome SNP analysis (www. dartmouse.org). The genomic DNA IRAK-M2/2 mice wereAlcohol Induced Worse Liver Damage in IRAK-M2/2 MiceTo study the role of innate immunity, in particular IRAK-M, in alcohol-induced liver damage, we treated wild type (WT) and IRAK-M2/2 B6 mice with alcohol as described in the Materials and Methods. 10 alcohol in drinking water was administered to mimic a daily light alcohol consumption and the single gavageFigure 4. Inflammatory cytokine in LMNCs. Ex vivo LMNCs were stained with intracellular cytokines and different surface markers as described in Materials and Methods. (A) Representative FACS plots showing IFNc+ cells after gating CD8+ T cells in alcohol treated mice. (B) Summary of percentage of IFNc producing CD8 T cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M2/2 mice (red). (C) Representative FACS plots showing IL-6 producing CD11b+ Kupffer cells after gating CD11b+ LMNCs in alcohol treated mice. (D) Summary of percentage of IL-6 producing CD11b+ Kupffer cells in LMNCs of control (CTL) and alcohol treated (ALC) B6 (blue) and IRAK-M2/2 mice (red). Error bars represent the SD of samples within a group. Experiments were performed 4 times and n = 2? in each group of each experiment. The data presented are from two pooled experiments. *P,0.05, (Two-way ANOVA test). doi:10.1371/journal.pone.0057085.gIRAK-M Regulates Liver InjuryFigure 5. Phagocytic activity of Kupffer cells in liver after alcohol treatment. (A) Representative histogram of FITC-dextran intake LMNCs in wild type B6 mice (blue line) and IRAK-M2/2 mice (red line, 2 ). (B) FITC-dextran uptake by LMNCs in wild type B6 (blue) and IRAK-M2/2 mice (red, 23 ). (C) FITC-dextran uptake by CD11b+ Kupffer cells in wild type B6 (blue) and IRAK-M2/2 mice (red). (D) FITC-dextran uptake by CD68+ Kupffer cells in wild type B6 (blue) and IRAK-M2/2 mice (red). Experiments were performed 3 times. N = 3? in each group of each experiment. The data presented are from one of the 3 experiments. Error bars represent the SD of samples within a group. *P,0.05, **P,0.01, ***P,0.001, Two way ANOVA test. doi:10.1371/journal.pone.0057085.gwith a larger amount of alcohol (60 alcohol in 200 ml, ,6 g/kg) was to mimic an alcoholic binge, which has been reported to be one of the main triggers of alcoholic liver damage in human [29]. There was very mild liver damage induced by daily 10 alcohol water consumption in both WT and IRAK-M2/2 mice, indicated by serum ALT levels (Figure 2A) and liver histology (Figure 2C and 2E, without binge). However, the difference between WT and IRAK-M2/2 was negligible (Figure 2A) although it appeared that IRAK-M2/2 mice showed more liver damage (Figure 2E). In contrast, a single episode of heavy alcohol consumption triggered liver inflammation and injury as evidenced by increased serum ALT levels in both WT and IRAK-M2/2 mice (Figure 2B) and LMNC infiltration in the liver of IRAKM2/2 mice (Figure 2D and 2F). We also examined the absolutenumber of LMNC infiltration per gram liver tissue analyzed, and the results were consistent (Figure 2G). W.
