1E) and apoptosis (Fig. 1F), whereas interleukin (IL)4-stimulated (M2) conditioned medium had no effect. MK-1775 mw Altogether, these results indicate that alcohol-fed C57BL6/J mice display a predominant M1 response associated with steatosis and liver injury. In contrast, alcohol-fed BALB/c

mice are characterized by preponderant M2 KC polarization, an impairment of the M1 response, and resistance to alcohol-induced liver injury. Macrophage phenotype was further characterized by double immunohistofluorescence, combining the macrophage marker F4/80 and either the M1 marker iNOS, or the M2 marker mannose receptor CD206. F4/80+ cells that expressed neither CD206 nor iNOS were classified as M0. Control C57BL6/J and BALB/c mice both exhibited a mixed hepatic population of M0/M1/M2 polarized macrophages (Fig. 2A). However, control BALB/c mice displayed a higher proportion of M2 macrophages, as compared to control C57BL6/J mice (40% versus 20% F4/80+/CD206+ cells, respectively, Fig. 2A). Intriguingly, chronic alcohol feeding of BALB/c mice caused a marked drop in the total number of KCs, as assessed by mRNA expression

and F4/80 immunostaining (Figs. 1A, 2A), associated with a reduction in TGF-beta inhibitor both M1 and M0 KC density (Fig. 2A). Residual KCs adopted a preponderant M2 polarization (60% of F4/80+/CD206+ cells in alcohol-exposed BALB/c mice (Fig. 2A). In contrast, alcohol did not modify the density of KCs in C57BL6/J mice, but promoted predominant M1 polarization (60% F4/80+/iNOS+ cells), a decrease in M0 KCs, with no change in the proportion of M2 KCs. Differential polarization Teicoplanin adopted by alcohol-fed BALB/c and C57BL6/J KCs was confirmed by flow cytometry analysis (Fig. S2). F4/80high/CD206+ M2 cells represented 86% of total F4/80high cells in BALB/c mice but only 34% in C57BL6/J mice (Fig. S2). Chronic alcohol feeding caused a 3-fold increase in KC apoptosis in BALB/c mice, as assessed by F4/80/cleaved-caspase-3 double immunostaining (Fig. 2B). Importantly,

cleaved-caspase-3 staining was exclusively detected in F4/80+ cells (Fig. 2B), indicating that the apoptotic process selectively targets KCs in BALB/c mice, whereas there was no detectable caspase-3 signal in macrophages of alcohol-fed C57BL6/J mice (Fig. 2B). The phenotype of apoptotic KCs was further characterized by triple immunolabeling, combining F4/80, cleaved-caspase-3, iNOS, or CD206 antibodies. In alcohol-fed BALB/c mice, all cleaved-caspase-3+/ F4/80+ cells stained for iNOS, but remained CD206-, indicating selective M1 macrophage apoptosis (Fig. 2C,D). Similar results were obtained using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Fig. 2E). Thus, alcohol-fed BALB/c mice are characterized by preponderant M2 KC polarization and M1 KC apoptosis. The causal relationship between M2 KC polarization and the induction of M1 KC apoptosis was investigated in KCs isolated from C57BL6/J mice.