Figure 4

Alcohol and HFD increased hepatic inflammation. (a) Increase in number of leukocytes and macrophages in the livers of mice with treatments compared with Chow. Representative images of immunofluorescent stains. CD45-positive leukocyte clusters increased in number and size (arrows) in all groups compared with Chow. Both Alc+HFD and HFD treatment groups showed more CD45+ cells compared with Alcohol group. × 40 Magnification. F4/80-positive Kupffer cells increased in numbers with Alc+HFD, followed by HFD and Alcohol treatment compared with Chow. HFD treatment showed more large size clusters of F4/80-positive cells compared with Alc+HFD (small arrows). Alcohol alone did not show F4/80+ cell clustering. × 40 Magnification. CD68-positive macrophages: CD68+ cells increased with all treatments compared with Chow but did not show clustering of cells. × 10 Magnification. Isotype (IgG) controls (bottom panel) for specific antibodies did not show positive staining. × 10 Magnification. Nuclei were stained with DAPI (blue). (b) Alcohol and HFD increased expression of inflammation-related mRNAs (Q-PCR). All treatments induced expression of F4/80 mRNA compared with Chow, but only reached significance with Alc+HFD. HFD significantly induced TNF-α mRNA expression from Chow. Alc+HFD also increased TNF-α, but this was not significant, and Alcohol alone had no effect. mRNA expression of target genes was normalised using housekeeper Hsp90ab1 (n= 5–6). Data are mean±s.d.; *P<0.05 compared with Chow; ^#P<0.05 compared with Alcohol.