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HEP_24621_sm_SuppFig1.tif2908KSupplementary Figure 1 (A) Gating strategy to identify liver DC. Liver leukocytes were isolated as described in the Materials and Methods and gated on SSC/FSC, DAPI- (to exclude dead cells), single cells population (to exclude doublets), autofluorescence- (to exclude macrophage and hepatocytes), CD45+ (to exclude non hematopoietic cells), lineage markers- (to exclude contaminating subsets of NK, T and B cells that express CD11c and MHCII). Gated cells expressing PDCA-1 and CD11c were identified as plasmacytoid DC, whereas cells expressing high MHC II and CD11c levels were identified as classical DC. (B) Gating strategy to identify neutrophils and macrophages. After exclusion of death cells (DAPI negative), doublets, CD45- cells and NK cells, liver neutrophils were identified as Ly6G+CD11b+ while in the remaining Ly6G negative population liver macrophages were identified as CD11b+F480+.
HEP_24621_sm_SuppFig2.tif1640KSupplementary Figure 2 Strategies used for analysis of fibrosis regression (A), DC depletion (B), short and long term DC expansion (C and D), syngenic DC transfer (E), NK depletion using asialo-GM-1 antibody (F) and NKp46DTR chimeric mice (G), in vivo inhibition of MMP-9 during DC expansion using B16-Flt3L melanoma (H) and DC transfer (I) and DC transfer of cells isolated from MMP-9 knockout mice (J).
HEP_24621_sm_SuppFig3.tif1558KSupplementary Figure 3 (A) Less than 2% CD11b+F480+ population expressed high CD11c and MHCII levels during fibrosis regression. The intrahepatic leukocyte population analyzed by flow cytometry during fibrosis regression at 12-84 hours after the last dose of CCl4. (B) The CD11chighMHCIIhigh population significantly up-regulates CCR7 after poly I:C stimulation. Liver intrahepatic leukocytes were cultured for 8 hours with poly I:C (20 ?g/ml) and F480+CD11b+ and CD11chighMHCIIhigh were analyzed for CCR7 expression.
HEP_24621_sm_SuppFig4.tif1282KSupplementary Figure 4 Fibrosis regression is associated with a progressive decrease of liver macrophages and a delayed increased of neutrophils. Liver macrophages assessed by flow cytometry (A) are reduced during fibrosis regression while neutrophils increase (B). The influx of the neutrophils was confirmed by histochemistry staining for neutrophil specific esterase (C).
HEP_24621_sm_SuppFig5.tif1777KSupplementary Figure 5 Administration of DT in CD11c DTR mice induced depletion of cDC. (A) Liver intrahepatic cDC were analyzed at 12, 36 ad 60 hours after DT administration (4ng/g) in CD11c DTR mice. (B-D) Administration of DT in CD11c-DTR mice is not associated with changes in the pDC, NK and macrophages. DT was administered in the first day after the last dose of CCl4 and pDC, NK and macrophages were assessed by flow cytometry. DT does not induce significant changes of pDC (B), NK (C) and F480+CD11b+ populations (D). Administration of DT in CD11c DTR mice results in depletion of MHCIIhighCD11chigh cells (blue dots) that express Gfp; CD11cint/low population (red dots) does not express Gfp and respective DTR so it is not depleted by DT administration (E). The number of CD11cint/lowCD11high cells is unchanged by DT administration (F)
HEP_24621_sm_SuppFig6.tif4236KSupplementary Figure 6 Flt3L expands liver DC in vivo. C57Bl6 mice were injected subcutaneously with 5×105 B16-Flt3L cells. The number of cells MHCIIhigh, CD11chigh, and CD205+ liver DC were increased upon Flt3L treatment.
HEP_24621_sm_SuppFig7.tif1228KSupplementary Figure 7 Transfer of purified syngenic DC is associated with augmentation of liver DC population. 4×107 purified DC were CFSE labeled and retro-orbital injected. By flow cytometry, cDC (A) and pDC (B) numbers after transfer (black bars) showed significant increase in number over 12 hours period when compared with control mice without transfer (white bar). Lower histograms showed percentage of cells positive for CFSE of each population at the peak of liver DC augmentation.
HEP_24621_sm_SuppFig8.tif1359KSupplementary Figure 8 DC-induced fibrosis resolution is independent of NK cells. Two days prior to the last CCl4 injection, groups of mice were either left untreated or injected with 5×105 B16-Flt3L cells alone or in addition to anti asialo-GM1 antibody. (A) Anti asialo-GM1 antibody reduces the liver NK cell pool. Bar graph shows the number of liver NK1.1+ cells 7 days after the last CCl4 treatment in mice injected with B16 Flt3L alone or in addition to asialo-GM1 antibody. (B) DT administration during fibrosis regression depletes NK cells in chimeric mice with NKp46-DTR-eGfp bone marrow. Bar graph shows the number of NKp46+ cells 7 days after last CCl4 treatment with no treatment, B16-Flt3L treatment and combined B16-Flt3L treatment and DT. (C-D) Decreased NK cells in Flt3L treated mice does not affect fibrosis regression. Graphs show the percentage of liver area positive for Sirius red staining measured by morphometry analysis at seven days after the last CCl4 injection after antibody induced NK depletion (C) or DT administration (D). Data are presented as mean with SD of three separate experiments (n=4) (* P<0.05).
HEP_24621_sm_SuppFig9.tif1238KSupplementary Figure 9 Transfer of DC is not associated with an overall increase number of liver CD45+ cells, macrophages, and neutrophils. Liver CD45+ cells, macrophages and neutrophils numbers were assessed by flow cytometry after transfer of MMP-9 knockout or wild type purified DC. No changes in the number of CD45+ (A), macrophages (F480+CD11b+) (B) and neutrophils (C) were noted.

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