Fig 2: Differential Regulation of IL-17 and IL-22 in CD18-/- Colon after C. rodentium Infection(A and B) Eight-week-old CD18-/- mice and CD18-sufficient littermate controls (CD18+/- and/or CD18+/+) were orally inoculated with C. rodentium. On day 5, colonic tissue was harvested, and expression of the indicated cytokines was determined (A) at the mRNA level by quantitative real-time PCR and (B) at the protein level by ex vivo colon culture ELISA.(C and D) On day 5 post-infection, LPMCs were isolated from the large intestine of CD18-/- or CD18+/- mice and analyzed by flow cytometry for IL-22+ ILC3 cells (shown are representative fluorescence-activated cell sorting [FACS] plots [left] and absolute numbers [right] of IL-22-expressing ROR?t+ ILCs [gated on CD3-CD5-B220-NK1.1-F4/80-Gr-1-CD127+CD90.2+ROR?t+]) (C) and for total number of ILC3 cells (D).Data are means ± SDs and are pooled from 2 independent experiments with 3 mice/group in each experiment, for a total of 6 mice/group (A and B); are from 2 independent experiments with 3 mice/group in one experiment and 4 mice/group in the second experiment, for a total of 7 mice/group (C); or are from 3 independent experiments with 3 mice/group in each experiment, for a total of 9 mice/group (D). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (A, C, and D: 2-tailed Student’s t test; B: or 1-way ANOVA with Tukey’s multiple-comparisons test). NS, non-significant.

Fig 3: MGLL deficiency in tumor-associated macrophages results in lipid accumulation. a FACS gating strategy for tissue macrophages and lipid measurement. Debris and doublets were removed, and tissue macrophages were then assessed as CD3-CD45R-Gr1-CD45+F4/80+. The average fluorescence degree of macrophages stained by Bodipy (GFP) was measured. b The percentage of TAMs in inoculated tumors. Six-week-old WT mice were subcutaneously inoculated with CT-26, MC-38 or 4T1 cells and the TAMs were quantified at 1st and 3rd week. Each tested sample was pooled from five individual ones. c Lipid staining of macrophages from spleens (TSMs) or tumors (TAMs). Six-week-old mice were subcutaneously inoculated with MC-38 tumors and sacrificed two weeks later. Tissue macrophages were isolated and stained with Bodipy (Green). The nucleus was visualized by DAPI staining (Blue). This experiment was repeated four times. Representative images are displayed. Scale bars, 10 µm. d The lipid levels in TSMs and TAMs. Six-week-old mice were subcutaneously injected with indicated cells. Two weeks later, TSMs and TAMs were isolated for lipid staining with bodipy. The Geometric mean fluorescence intensity (MFI) of Bodipy in each group was measured. Each tested sample was pooled from five individual ones. e A diagram of glycerolipid metabolism. The blue arrow indicates up-regulation, and the orange arrow indicates down-regulation. A gene-microarray analysis was performed on the peritoneal macrophages, which were treated with regular or conditioned medium from CT-26 cells for 24 h. TG triglyceride, DG diglyceride, MG monoglyceride. f Relative mRNA expression of MGLL in TSMs and TAMs. Six-week-old mice were subcutaneously inoculated with indicated tumors and sacrificed two weeks later. TSMs and TAMs were isolated for real-time PCR assays. g Cellular lipid levels were measured in TSMs and TAMs from the MC-38 tumor-bearing mice as described in c. h Relative mRNA expression of MGLL in TSMs and TAMs. Six-week-old WT or myeloid MGLL transgenic (TgMGLL) mice were subcutaneously inoculated with MC-38 tumors for 2 weeks. TSMs and TAMs were then isolated for mRNA assays of MGLL. i Cellular lipid levels in TSMs and TAMs from the MC-38 tumor-bearing mice as described in h. Data in f–i represent the means ± s.e.ms (n = 5, *P < 0.05, **P < 0.01, ***P < 0.005; student’s t-test; ns not significant)

Fig 4: Systemic inflammation affects the polarization of macrophages in bone marrow cells in vivo. (A–B) Quantitative data of the proportions (A) and FCM photographs of MDSCs (B) in the bone marrow cells from mice with indicated treatments. (C–D) Quantitative data of the proportions (C) and FCM photographs (D) of subgroups of MDSCs in the bone marrow cells from mice with indicated treatments. (E–G) Quantitative data of the proportions of macrophages (E), subgroups of macrophages (F), and CD4+ and CD8+ T cells (G) in the bone marrow cells from mice with indicated treatments. *P < 0.01, †P < 0.05 vs. control. FCM: Flow cytometry; G-MDSCs: Granulocyte-myeloid-derived suppressor cells; Gr-1: Glutathione reductase-1; iNOS: Inducible nitric oxide synthase; LPS: Lipopolysaccharides; M-MDSCs: Monocytic-myeloid-derived suppressor cells.

Fig 5: M1 type macrophages infiltrates into tumor microenvironments under local inflammation in vivo. (A–B) Quantitative data of the proportions (A) and FCM photographs of MDSCs (B) in the tumor tissues from mice with indicated treatments. (C–D) Quantitative data of the proportions (C) and FCM photographs (D) of subgroups of MDSCs in the tumor tissues from mice with indicated treatments. (E–G) Quantitative data of the proportions of macrophages (E), subgroups of macrophages (F), and CD4+ and CD8+ T cells (G) in the tumor tissues from mice with indicated treatments. (H) Single cells were extracted from the tumor tissues and used for qRT-PCR to determine the expression of targeted genes. *P < 0.01, †P < 0.05 vs. control. FCM: Flow cytometry; G-MDSCs: Granulocyte-myeloid-derived suppressor cells; Gr-1: Glutathione reductase-1; iNOS: Inducible nitric oxide synthase; MDSCs: Myeloid-derived suppressor cells; M-MDSCs: Monocytic-myeloid-derived suppressor cells; qRT-PCR: Quantitative real time-polymerase chain reaction; TLR: Toll-like receptor.