Fig 2: OrfV-stimulated neutrophils exhibited enhanced tumor necrosis factor (TNF)-?-mediated cytotoxicity against tumor cells: (a) naïve seven- to nine-week-old female C57BL/6 mice (n = 8 per group) were injected intraperitoneally with phosphate-buffered saline (PBS) or 5 × 107 PFU of OrfV. Two hours following treatment, bone marrow was flushed from femurs to isolate viable neutrophils (defined by a CD45+ Ly6Ghi CD11bhi phenotype) by flow cytometric cell sorting. Neutrophils were co-cultured in vitro with B16F10 melanoma cells at increasing tumor-to-neutrophil ratios for 16 h. The viability of tumor cells was quantified via flow cytometry; (b) twenty-four hours after intravenous treatment with OrfV or PBS, B16F10 metastasis-bearing lungs were collected from the mice, and the cytokine response of immature and mature neutrophils (defined by CD101 expression) was quantified with flow cytometry after intracellular cytokine staining. TNF-a, tumor necrosis factor-alpha; IL-10, interleukin 10; IL-17A, interleukin 17A, IFN?, interferon-gamma; PD-1, programmed cell death protein 1; IL-12p40, interleukin 12p40; IL-6, interleukin 6; (c) two hours after treating mice with OrfV, bone marrow-derived neutrophils (CD45+ Ly6Ghi CD11bhi) were flow-sorted. The isolated neutrophils were incubated with TNF-?-neutralizing antibodies and co-cultured in vitro with B16F10 melanoma cells at increasing tumor-to-neutrophil ratios for 16 h. The cell viability of tumor cells was quantified with flow cytometry. Statistical analysis was performed using two-way analysis of variance. Statistical significance was designated as * p = 0.05, *** p = 0.001, and **** p = 0.0001.

Fig 3: Autocrine TNF-a secretion and impact on MDSC suppressive activity. (A) TNF-a concentration in the supernatant of NUP or NUP-MDSC cells. (B,C) CFSE-labelled CD8 T cells were stimulated alone or in the presence of NUP-MDSCCas9 transduced with non-targeting gRNAs or gRNAs targeting Nos2, Hif1a or Tnf. (B) Histograms show CFSE dilution on CD8 T cells after 3 days of stimulation. (C) IFN? concentration in the supernatant after 3 days of stimulation. Summary of 2 independent experiments (3 gRNAs/gene). (D) TNF-a concentration in the supernatant of NUP-MDSCCas9 cells transduced with non-targeting gRNAs (NT) or gRNAs specific for Tnf. Summary of 2 independent experiments (3 gRNAs/gene). (E and F) CFSE-labeled CD8 T cells were stimulated alone or in the presence of NUP-MDSC together with a neutralizing antibody against TNF-a or an isotype control. (E) Histograms show CFSE level on CD8 T cells after 3 days of stimulation. (F) IFN-? concentration in the supernatant after 3 days of stimulation. Summary of 2 independent experiments. (G) Relative Nos2 expression in NUP cells, NUP-MDSCCas9 and NUP-MDSCCas9 transduced with non-targeting gRNAs or gRNAs targeting Tnf. Summary of 3 independent experiments (3 gRNAs/gene). ***P < 0.001, Student T-test.

Fig 4: Inflammatory signal upregulates Gp2 expression in pancreatic acinar cells.a Cultured pancreatic acinar cells are shown. b Pancreas of control (upper) or Ptf1aCre-ERTMCre-tdTomato (bottom) mice received tamoxifen are shown. c Cultured pancreatic acini from control of Ptf1aCre-ERTM-tdTomato mice stimulated with 4OHT are shown. Data are representing for three independent experiments. d Gp2 expression in no stimulation (None) or stimulated by 200 ng/mL LPS, 20 ng/mL TNF, 20 ng/mL IL-6 (n = 10 /group), 100 ng/mL RANKL (n = 4) pancreatic acini were examined. Relative expressions mRNA levels were determined by quantitative RT-PCR and normalized to Gapdh. *p = 0.040 (one-way ANOVA). Data are presented as mean values ± SEM., N.S. indicates not significant. e Pancreas of mice systemically administered PBS (control) or TNF (100 ng, two times). GP2 (red), DAPI (blue). Representative data of three independent experiments are shown. f GP2 concentration in pancreatic juice. *p = 0.04 (two-tailed Mann–Whitney U test). Data are presented as mean values ± SEM. g TNF concentration in serum *p = 0.021 (two-tailed unpaired t-test). Data are presented as mean values ± SEM. h Representative photograph showing the abdominal-organ matrices surrounding the pancreas in Ptf1aCre-ERTM-tdTomato mice that received tamoxifen. i TNF concentration in abdominal-organ matrices, as determined by enzyme-linked immunosorbent assay. Pancreas (intact n = 15, colitis n = 13), Omentum (intact n = 10, colitis n = 11) mesenteric fat (intact n = 15, colitis n = 13), Retroperitoneal fat and Epididymis fat (intact n = 3, colitis n = 3) *p < 0.05 (two-tailed unpaired t-test). Data are presented as mean values ± SEM. j 100 µg of anti-TNF antibody (Biolegend, MP6-XT22, #506332) or Isotype Rat IgG2a (Biolegend, RTK2758, #400503) was intraperitoneal administered three times (on days 1, 3, and 5) during DSS treatment, and pancreases were collected on day 6. GP2 (red), DAPI (blue). Representative data of three independent experiments are shown. Source data are provided as a Source Data file.

Fig 5: Triggering of TREM2 by MA induces the permissive macrophages.a, b BMDMs were stimulated for 24 h with the indicated amount of TDM or fMA in the presence of 10 ng/ml IFN-? (a), 1 µg of TDM with or without the indicated amounts of a TNF-blocking antibody (aTNF Ab) or an isotype-matched control antibody (Control Ab) (b), or 1 µg of fMA with or without recombinant TNF (rTNF) (b). NO production was determined by Griess assay. Data represent as the mean ± SEM of triplicate assays from three independent experiments. The statistical significance was calculated by one-way ANOVA followed by Bonferroni’s test. **p < 0.01, ***p < 0.001, n.s. not significant. c Representative images of the hematoxylin and eosin-stained lungs from WT, Clec4e-/-, or Card9-/- mice at day 7 after intravenous injection of 50 µg TDM or 250 µg fMA emulsion. The images are representative of two independent experiments. Scale bars: 0.1 mm. d–f Peritoneal lavages were collected at 4, 24, and 72 h after intraperitoneally injection of 500 µg fMA or 100 µg TDM emulsion to WT mice (n = 4). MCP-1 and TNF concentrations in the lavages were measured by ELISA, and Nos2 mRNA levels were measured by quantitative RT-PCR. e Flow cytometric analysis of peritoneal neutrophils (CD11b+Ly6G+F4/80-) and monocyte-derived macrophages (CD11b+Ly6G-F4/80low SPMs) from WT mice (n = 4) at 48 and 72 h post injection of MA or TDM emulsions as in d. f, g WT or Trem2-/- mice were injected intraperitoneally with fMA or control (vehicle) emulsion. The concentrations of MCP-1 (control, n = 4; fMA, n = 6; at 4 h) and TNF (n = 4; at 72 h) in the peritoneal lavages was measured by ELISA (f). Peritoneal exudate cells (n = 4; at 72 h) was analyzed as in e and g. h–k WT mice were injected intraperitoneally with TDM, fMA, or control (vehicle) emulsion, and infiltrated macrophages at days 1, 2, and 3 were analyzed by flow cytometry for the expression of CD38 and iNOS (h). The numbers of total recruited monocyte-derived macrophages (CD11b+Ly6C+F4/80+) (i), the M1 macrophages (CD11b+Ly6C+F4/80+CD38highNOS+) (j), and permissive macrophages (CD11b+Ly6C+F4/80+CD38dullNOS-) (k) at day 3 are shown. Data in d–k represent as the mean ± SEM from at least three independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test (f–g) or by two-tailed unpaired t test (i–k). *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant. Source data are provided as a Source data file.