Bouhlel, M.A. , Derudas, B. , Rigamonti, E. , Dievart, R. , Brozek, J. , Haulon, S. , Zawadzki, C. , Jude, B. , Torpier, G. , Marx, N. , Staels, B. , Chinetti\Gbaguidi, G. (2007) PPARgamma activation primes human monocytes into alternative M2 macrophages with anti\inflammatory properties. CHK1-IN-3 evaluate the molecular mechanisms that govern the M1/M2 polarization during ALD. Transcription factors play a major role in regulation of macrophage polarization. The M1 macrophage signals, IFN\and LPS, control gene expression via transcription factors, including STAT1, JAK2, IFN regulatory factors, NF\coactivator 1comparative threshold values by use of the ratio of the SLC2A2 fold change in target gene expression versus the fold change in reference gene expression (18 s). Western blot Tissue lysates and cell lysates were analyzed on a 10% polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane overnight and then blocked for 2 h in blocking buffer containing TBS, 0.1% Tween 20, and 5% nonfat milk. Primary antibodies against mouse KLF4 and 0.05 was considered statistically significant. Prism software (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. RESULTS M1 and M2 macrophages are present in the liver after chronic alcohol feeding in mice Chronic alcohol feeding in mice is characterized by inflammation, immune cell activation, cellular injury, and steatosis in the liver. The serum ALT levels were increased in the EtFed mice, indicating liver injury and steatosis was present on H&E staining of the liver compared with PF controls (Supplemental Fig. 1A and B). Livers from EtFed mice showed a 2\ to 4\fold increase in macrophage activation markers TNF\compared with PF controls ( Fig. 1A ). We also observed a significant increase (2\ to 8\fold) in the expression of M2 genes (Arg1, Mrc1, and IL\10; Fig. 1B). We also found an increase in the levels of CD163 mRNA (Fig. 1C). Open in a separate window Figure 1 Chronic alcohol feeding induced a mixed M1/M2 macrophage phenotype in vivo. C57Bl/6 female mice received Lieber\DeCarli alcohol CHK1-IN-3 (EtFed) or PF diet for 5 wk. Liver samples and cells were collected from the mice. (A) mRNA expression of proinflammatory genes TNF\was quantified by qRT\PCR. (B) M2 macrophage markers; Arg1, Mrc1, and IL\10 were quantified by qRT\PCR. (C) CD163 mRNA levels were also evaluated by qRT\PCR. (D) The percentage of CD45+CD11b+F4/80+ population in the LMNCs was assessed by flow cytometry. (E) Expression of F4/80+ macrophages expressing CD206 and CD163 in liver was determined by flow cytometry. (F) mRNA expression of KLF4 was determined by qRT\PCR. (G) Western blot analysis shows the expression of KLF4 and = 6C8 mice/group. * 0.05. We evaluated further the levels of M1 and M2 markers in the KCs in vivo. We observed that TNF\= 6/group), and total RNA was isolated and analyzed for (A and B) mRNA expression of M1 and M2 genes in the KCs and (C) mRNA levels of KLF4 by use of specific primers by qRT\PCR. Values of relative mRNA expression CHK1-IN-3 normalized for housekeeping gene 18s are shown as mean sem. Statistically significant differences are shown (* 0.05 vs. PF control cells). We performed flow cytometric analysis of the liver immune cell population and observed a significant increase in the frequency of CD45+CD11b+F4/80+ macrophages in the EtFed mice compared with PF controls (Fig. 1D). We observed further a 3\fold increase in the frequency of CD163+CD206+ macrophages in the EtFed mice compared with PF controls (Fig. 1E). KLF4 expression is increased in vivo in ALD in mice KLF4 has been identified as a critical regulator of M2 macrophage polarization . As we observed a significant increase in M2 gene expression in the liver of the chronic EtFed mice, we hypothesized that KLF4 may mediate alcohol\induced M2 polarization. CHK1-IN-3 We found up\regulation of KLF4 mRNA and protein levels in the livers of chronic EtFed compared with PF mice (Fig. 1F and G). Furthermore, we studied the expression levels of KLF4 in the KCs and observed a significant increase in EtFed compared with control diet\fed mice (Fig. 2C). Altogether, these results demonstrated that chronic alcohol feeding in mice led to KLF4 up\regulation, macrophage activation, and induction of M2 genes in the liver. Alcohol increases KLF4 expression and transcriptional activity in macrophages in vitro CHK1-IN-3 To test the hypothesis that KLF4 is induced by alcohol, we treated the macrophage RAW264.7 cells with 50 mM EtOH or with well\established M1.