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hep26607-sup-0001-suppfig1.tif3151KSupporting Information Figure S1: Chronic alcohol feeding of C57BL6/J and BALB/c mice is not associated with significant recruitment of Gr-1/CCR2 positive cells in the liver. (A) RT PCR analysis of CCR2 mRNA expression (n=12 per CD group and n=30 per ETOH group from two independent experiments). (B) Flow cytometry analysis of Gr-1 expression in hepatic immune cell-enriched fractions; bone marrow cells were used as positive control; typical cytograms representative of three experiments.
hep26607-sup-0002-suppfig2.tif1604KSupporting Information Figure S2: EtOH-fed BALB/c display high M2 KC polarization as compared to EtOH-fed C57BL6/J mice. F4/80high cells are quantified by flow cytometry analysis of hepatic KC-enriched fraction as CD206-or CD206+ (typical cytogram representative of three experiments).
hep26607-sup-0003-suppfig3.tif5277KSupporting Information Figure S3: M2 macrophages trigger selective M1 macrophage apoptosis via IL10 secretion and arginase activation. (A and C) Impact of conditioned medium from M2 Raw264.7 macrophages (IL4 CM) and (B) impact of 10 ng/ml IL10, on apoptosis (caspase 3 positive cells: red arrows) and morphology (spindle-shaped cells: black arrows) of LPS-stimulated macrophages counterstained with hematoxylin eosin. (Scale bar: 50μm). When indicated, the arginase inhibitor NOR-NOHA (100 μg/ml) (A and B) or a neutralizing anti-IL10 antibody (10 μg/ml) (C), was preincubated with LPS-stimulated Raw264.7 cells prior to addition of IL4 CM (A and C) or IL10 (B). Assays were done in quadruplicates and data are representative of three independent experiments. * P< 0.05 vs control LPS-stimulated Raw264.7 cells. * P< 0.05 vs corresponding control.
hep26607-sup-0004-suppfig4.tif8636KSupporting Information Figure S4: M2 peritoneal macrophages trigger selective M1 peritoneal macrophage apoptosis via IL10 secretion and arginase activation. (A) Conditioned medium from IL4-treated peritoneal macrophages (IL4 CM) or IL10 induce apoptosis and decrease cell survival of LPS-stimulated peritoneal macrophages. (Scale bar: 20μM). When indicated, the arginase inhibitor NOR-NOHA (100 μg/ml) or a neutralizing anti-IL10 antibody (10 μg/ml) were preincubated with LPS-stimulated macrophages, prior to addition of IL10 or IL4 CM. Data are representative of two experiments. * P< 0.05 vs control LPS-stimulated macrophages. (B) iNOS+/Arg1+ phenotype of apoptotic peritoneal macrophages of LPS-stimulated macrophages incubated with IL4 CM. Scale bar: 5μm.
hep26607-sup-0005-suppfig5.tif5961KSupporting Information Figure S5: Early response to alcohol is characterized by M2 KC polarization in C57BL6/J and is associated with a decrease in total KC number. KC polarization was characterized in C57BL6/J mice exposed to intragastric gavage with a single dose of alcohol (ETOH) or isocaloric maltodextrin by (A) confocal microscopy (animals were sacrificed 6h after gavage, n=6 mice/condition, typical results obtained from two experiments); M1 or M2 KCs were identified using a combination of F4/80 and either iNOS (M1) or CD206 (M2) antibodies; F4/80+-CD206--iNOS- cells were classified as M0. In representative images, double positive cells are indicated by white arrows. Scale bar: 5 μm. (B and C) RT PCR analysis of M1 and M2 marker genes (animals were sacrificed 3 or 6h after gavage, n=8 mice per condition from two independent experiments). * P< 0.05 for EtOH vs isocaloric.
hep26607-sup-0006-suppfig6.tif4532KSupporting Information Figure S6: Preponderant M2 polarization is not associated with enhancement of fibrogenic gene expression in alcohol- or high fat-fed mice. RT-PCR analysis of Col1 and TGFß mRNA expression in (A) BALB/c and C57BL6/J mice chronically fed control diet (n=12 mice/CD group) or alcohol (n=30 mice/EtOH group), (B) C57BL6/J mice chronically fed control diet or alcohol with or without resveratrol (400mg/kg/day) (n=6 for CD, n=15 for EtOH, in each group) and (C) C57BL6/J mice fed normal diet or high fat diet for 27 weeks, with or without resveratrol (400mg/kg/day) during the last 3 weeks (n=6 and n=10 mice for ND and HFD condition, respectively). * P< 0.05.
hep26607-sup-0007-suppfig7.tif1030KSupporting Information Figure S7: Chronic alcohol feeding in BALB/c mice is not associated with proliferation of macrophages in the liver. (A) BALB/c mice chronically fed alcohol were injected with BrdU 90 min before sacrifice. Immunohistofluorescence using F4/80 and BrdU antibodies shows only few BrdU-positive cells that do not stain F4/80. Scale bar: 20μm. Typical image representative of three experiments.
hep26607-sup-0008-supptable1.doc40KSupporting Information Table S1: General characteristics of mice fed EtOH or high fat diet.
hep26607-sup-0009-supptable2.doc38KSupporting Information Table S2: primer sequences for RT-PCR
hep26607-sup-0010-suppinfo.doc120KSupporting Information

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