Fig 2: Chitinase 3-like-1 (Chi3l1)/CD44 signaling in M?s upregulates podoplanin expression and platelet adhesion.(A) Male WT, Chil1-/-, Cd44-/- mice were treated with acetaminophen (APAP) (n = 4 mice/group). After 3 hr, mice were sacrificed and M?s were isolated to measure mRNA levels of various adhesion molecules, including selectin P ligand (Selplg), Cd40, melanoma cell adhesion molecule (Mcam), Fc receptor (Fcr), intercellular adhesion molecule 1 (Icam1), lymphocyte function-associated antigen 1 (Lfa1), von Willebrand factor (Vwf), and podoplanin (Pdpn). (B, C) Wild-type (WT) mice were treated with APAP. Chil1-/- and Cd44-/- mice were treated with PBS or rmChi3l1 followed by APAP challenge simultaneously and mice were sacrificed 3 hr after APAP (n = 3 mice/group). (B) M?s were isolated and mRNA levels of Pdpn in M?s were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (C) Immunofluorescence (IF) staining of liver sections for podoplanin and F4/80 is shown and the proportions of M?s that express Pdpn were quantified, Scale bar, 25 µm. (D–F) Chil1-/- mice reconstituted with rmChi3l1 were treated with either Ctrl IgG or a-podoplanin Ab for 16 hr and subsequently challenged with APAP. (D) Serum levels of ALT and (E) liver histology were evaluated 24 hr after APAP treatment (n = 6 mice/group). Scale bar, 250 µm. (F) IF staining for intrahepatic platelets (CD41+) and M?s (F4/80+) was performed 3 hr after APAP (n = 3 mice/group). Scale bar, 25 µm. One-way ANOVA were performed in A–C. Two-tailed, unpaired Student’s t-test was performed in D.

Fig 3: Loss of Ccr2 Prevents Accumulation of C-LAMs with NAFLD(A) Image of a macrophage aggregate stained for F4/80, CLEC4F, and Ccr2-GFP.(B) Image of macrophage clusters from mice co-expressing Ccr2-RFP and Cx3cr1-GFP.(C–F) Body weight (C), liver TAG (D), and serum ALT (E) from WT and KO mice fed a STD/HFD for 20 weeks. (F) ITT from WT or KO mice fed a HFD.(G) Ly6Chi monocyte counts in the liver.(H) Flow plot of F4/80hi, CD11bint macrophages where red and green text indicate two phenotypes observed in Ccr2 KO mice.(I) Quantification of indicated macrophage populations in WT and KO mice. The red and green symbols indicate the phenotype of the mice from (H).(J and K) Quantification of C-LAM number as an absolute value (J) and as a percentage of the Tim4neg macrophages (K). Each dot represents an individual mouse, and the bar reflects the mean.The data represent three independent experiments with n = 3–17 mice/group. The p values are shown. Scale bars, 30 μm.

Fig 4: IFN-I signaling in differentiating monocytes unexpectedly leads to strong induction of arginase-1. (A–C) The MACS-purified BM monocytes were cultured in M-CSF ± IFN for 60 h. Microarray analyses were performed. Heatmap of differentially expressed genes is presented in (A). (B) Genes with the greatest fold-changes are presented (Arg1 highlighted). (C) Protein samples were analyzed by Western blot (WB). Average quantifications of normalized ARG1 levels (± range) from two independent experiments are marked. (D-F) Mouse BM mononuclear cells (D), mature BMDMs (E) or human PBMCs were treated w/M-CSF ± IFNβ for indicated times (PBMC with IFNα for 5 days). RNA samples were analyzed by qPCR. Average data (± range) from two independent experiments are presented. (G and H) Relative levels of Arg1 mRNA as normalized to its liver levels (G) or ARG1 protein (H) in control and treated tumors (day 14) are presented. In (G), data were from 3 independent experiments (±SEM, P value marked). In (H), some normal tissue samples were included. Quantifications of normalized tumor ARG1 levels were averaged (± range, n = 2). (I) BM mononuclear cells were cultured within the control or poly(I:C) group of supernatants from minced tumor tissues for 60 h in two independent experiments. The poly(I:C) group was also added with neutralizing antibody against IFN receptor (αR1). The mRNA levels of Arg1 were determined (± range). (J and K) BM mononuclear cells were cultured in M-CSF ± IFNβ for 60 h. After cytospin, the slides were then co-stained with indicated antibodies for immunofluorescence analyses ((J), scale: 50 μm). Arrowheads point at positively stained cells. In (K), the cells positive for ARG1 (A+), F4/80 (F+) or both markers (F + A+) were quantitated (±SEM, 3 fields, P values between given groups marked). (L and M) In (L), sections of tumors (day 14) were immunostained as in (J). In (M), tumor associated monocytes or macrophages were sorted by flow cytometry in two independent experiments. Samples from sorted cells were subjected to WB analysis. Average quantifications of normalized ARG1 levels are marked (± range). (N and O) Tumor-burden mice were treated ± poly(I:C) ± GW2580 and tumors were harvested (day 14). The samples were subjected to qPCR (N) or WB analyses (O), respectively. Average quantifications from two independent experiments are presented (± range). (P) Tumor-burden mice were treated with ± poly(I:C) ± CCR2 antagonist RS504393 (CCR2-A) on day 6 after tumor implantation. Tumors were harvested on day 14 and subjected to qPCR analyses (± range, n = 2).

Fig 5: Immunofluorescence observation of the expression of GATA6 in the peritoneal macrophages after CPT-11 treatment and syngeneic adoptive transfer with peritoneal exudate cells (PECs) and bone marrow cells (BMCs). C57BL/6 mice were intraperitoneally injected with CPT-11 (200 mg/kg body weight, in phosphate-buffered saline containing 2% Tween-80) or vehicle. Seven days later, the CPT-11-treated mice were transferred with syngeneic PECs (1 × 106/mouse) or BMCs (1 × 107/mouse). The mice were bred for additional 21 days, and then their PECs were collected. The expression of CD11b (green), F4/80 (magenta), and GATA6 (red) was observed by immunofluorescence microscopy with the nuclei (blue) being revealed by Hoechst 33342 staining. (A) A representative set of immunofluorescence images. Scale bar: 10 mm. (B) Statistical analysis of the percentages of CD11b+GATA6+ macrophages. n = 6; ***P < 0.001; ns, not significant.