Fig 1: Systemic inflammatory response syndrome drives apoptosis and not necroptosis in intestinal epithelial cells.Co-housed 10–12 week old female C57Bl6/N wt mice were injected with 0.75 µg/g or 1 µg/g of ice-cold recombinant, LPS-free mouse TNF intravenously. Control mice received a corresponding volume of ice-cold LPS-free PBS. Mice were sacrificed 6 h post-injection. Untreated wt mice were additionally analysed (n = 3 in each group). A Representative images of small intestine and colon sections (Swiss-rolls) of wt mice as indicated were stained with H&E. Slides were digitalised in a digital slide scanner and pictures were acquired in QuPath. Scale bars: 50 µm. B Graph showing histology score in whole intestine. Data are presented as mean + SEM and each dot represents one mouse. P values were calculated via one-way Anova, Tukey’s multiple comparisons test. **P ≤ 0.01, ns not significant. C Representative images of ileum and colon sections (Swiss-rolls) of wt mice as indicated immunostained with pMLKL-S345 and c-Casp3. Slides were digitalised in a digital slide scanner and pictures were acquired in QuPath. Scale bars: 50 µm. Arrowheads indicate pMLKL-S345-positive cells and c-Casp3-positive areas. D Graph showing quantification of illustrated pictures for each immunostaining obtained via QuPath after slides were digitalised in a digital slide scanner as described in the Supplementary Methods section. Total numbers of cells were obtained to calculate the percentage of positive cells over the total amount of cells detected. Data are presented as mean + SEM and each dot represents one mouse. P values were calculated via two-way Anova, Tukey’s multiple comparisons test. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns not significant.
Fig 2: 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-γ (a), 1 μg of TDM with or without the indicated amounts of a TNF-blocking antibody (αTNF 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.
Fig 3: TREM2 deficiency exacerbates Mincle-induced inflammation.a, b WT or Trem2−/− mice (n = 4) were intraperitoneally injected with 100 μg TDM or control (vehicle) emulsion. TNF and MCP-1 in the peritoneal lavages were measured by ELISA and Nos2 mRNA levels in the peritoneal cells were measured by qRT-PCR at 24 h post injection (a). The numbers of macrophages and neutrophils in the peritoneal cavities at 72 h post injection were analyzed by flow cytometry (b). c–e WT or Trem2−/− mice (n = 5) were intravenously injected with 50 μg TDM or control emulsion, and the lungs and thymuses were collected at day 7 after the injection. LWI and TWI are shown (c). Representative hematoxylin and eosin-stained sections of lung lobes from WT and Trem2−/− mice (scale bars: upper panels, 1 mm; lower panels, 0.1 mm) are shown (d). Cytokine concentration in lung homogenates was measured by ELISA, and Nos2 mRNA levels in the lungs were measured by qRT-PCR (e). Data in a–c and e are presented as mean ± SEM and are representative of at least two independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test (a, b) or by two-tailed unpaired t test (c, e). *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.
Fig 4: Integrated Epigenetic and Transcriptional Analysis of G9a-Regulated Genes in Recurrent Tumors(A) Comparison of gene expression changes 16 h after BIX-01294 (1 μM) treatment in recurrent (x axis) and primary (y axis) tumor cells. Colored dots indicate genes whose expression was differentially regulated by BIX treatment in primary versus recurrent tumor cells (adj. p < 0.05). Sequencing was performed on three biologically independent primary cell lines, and n = 3, biologically independent recurrent cell lines in duplicate for each cell line.(B) Heatmaps showing median-centered, Z-score-normalized expression changes for differentially genes from (A).(C) A G9a gene signature was generated by overlapping genes upregulated following BIX-01294 treatment in recurrent tumor cells (adj. p < 0.05) with genes whose promoters had significantly lower H3K9ac in recurrent tumor cells (adj. p < 0.05).(D) Gene ontology analysis showing pathways enriched in the 342 gene G9a signature from (C).(E and F) GSEA plots showing enrichment of a TNF/NF-κB signature (F) and an inflammatory signature (G) in recurrent tumor cells after G9a inhibition.(G) Kaplan-Meier plots showing distant metastasis-free survival (DMFS) for all tumors (n = 1,379), HER2-enriched tumors (n = 105), and luminal B tumors (n = 225) in patients stratified by high (gray), moderate (red), or low (blue) expression of G9a signature genes.
Fig 5: Tumor necrosis factor (TNF) increases locomotor activity and exploratory behavior in pups, but has no impact on USV communication, body temperature regulation and olfactory orientation. Timeline (A). USV call rates at P6 in pups injected with TNF (20 mg/Kg) or vehicle, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (B). Change in pup body temperature during USV recording, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (C). Olfactory orientation at P9: latency to the first entry in the maternal bedding zone, presented as means ± SEM, Mann-Whitney’s U-test: p > 0.05 (D); time spent in maternal bedding zone, center zone and clean bedding zone, presented as means ± SEM, 2-way ANOVA: p(Treatment) = 0.9602, p(Time) < 0.0001, p(Interaction) = 0.7046 (E). Exploratory behavior at P13: time spent mobile in pups aged P13, by 2-min time segments, presented as means ± SEM (F). GEE estimates, 95% CI and p-values associated with the effects of treatment, time and cohort on time spent mobile: p(Treatment) = 0.0014, p(Time) = 0.1632, p(Cohort) = 0.6345 (G). Total distance traveled, presented as means ± SEM, Mann-Whitney’s U-test: p(Treatment) = 0.0157 (H). Exploratory index, presented as means ± SEM, Mann-Whitney’s U-test: p(Treatment) = 0.0266 (I). n = 29–30 Veh, n = 29–32 TNF depending on the test; *p < 0.05, **p < 0.01. Only statistically significant differences are presented.
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