Fig 1: Effects of low- and high-dose LPS priming on ROS production and MCP-1 release in bone marrow neutrophils. Cells were primed with different doses of LPS (low-dose: 10 pg/mL; high-dose: 100 ng/mL) and re-challenged on day 2 with a fixed dose LPS (100 ng/mL) as described previously. (A) ROS levels (n = 6) were assessed by the 2',7'–dichlorofluorescin diacetate (DCFDA) assay, whereas the production of (B) MCP-1 (n = 7) was measured using ELISA (normalized to total protein concentration). Data are shown as scatter dot plots, mean + SEM, # p < 0.05, # significant differences vs. unprimed condition (UP, gray column).
Fig 2: 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 3: Distinct lipid recognition and macrophage activation through Mincle and TREM2.a–c Peritoneal macrophages from wild-type (WT), Trem2-/-, or Clec4e-/- mice (a), WT, Tyrobp-/-, or Fcer1g-/- mice (b), or WT or Card9-/- mice (c) were stimulated with the indicated amounts of TDM, GMM, GroMM, or fMA coated on the plates or with LPS (100 ng/ml) for 24 h. WT cells were stimulated in the presence or absence of the Syk inhibitor BAY-613606 (BAY) (c). MCP-1 and TNF production in the culture supernatant was measured by ELISA. Data are presented as the mean ± SEM of triplicate assays and are representative of three independent experiments. The statistical significance was calculated by two-way ANOVA followed by Bonferroni’s test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.
Fig 4: 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.
Fig 5: Role of microbiota-derived small EVs altering pro- and anti-inflammatory mediators in bone marrow neutrophils. Murine bone marrow neutrophils were primed for 45 min by microbiota-derived small EVs (low concentration: 1 ng/mL; high concentration: 28,1 µg/mL) and re-challenged with 100 ng/mL LPS for 4 h on day 2. Production of (A) ROS (n = 7) was determined by DCFDA assay, whereas (B) MCP-1 (n = 7) and (C) IL-10 (n = 7) were measured by ELISA (normalized to total protein concentration). Real-time qPCR was used to analyze the gene expression of (D) IL-10 (n = 3; unprimed state assigned as 1.0). Data are presented as scatter dot plots, mean + SEM, # p < 0.05, # versus unprimed condition (UP, gray bar).
Supplier Page from BioLegend for ELISA MAX(TM) Standard Set Mouse MCP-1