Fig 2: Suppression of exosome release from Mϕ attenuates the secretion of TNFα from Mϕ. a and b BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) for 24 h. Then, TNF-α and IL-6 mRNA levels in the BMDM and protein levels in the supernatants were measured by RT-qPCR and ELISA, respectively. c BMDM were treated with LPS (1 μg/ml) with or without dimethyl amiloride (DMA, 25 μmol/L) for 24 h, and then the TNF-α and IL-6 levels in the supernatants were measured by ELISA. d BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of recombinant IL-25 (200 ng/ml), anti-IL-25 neutralizing antibody (10 μg/ml), or non-specific IgG (10 μg/ml) for 24 h. The level of TNF-α in the supernatants was measured by ELISA. e Immunofluorescence images show Mϕ internalization of exosomes. BMDM were incubated with DiI-labeled exosomes (red) that were isolated from the culture media of untreated or LPS-treated BMDM (1 μl/ml) for 2 h. Nuclei were counterstained with Hoechst (blue). The fold changes in DiI fluorescence intensity were calculated by ImageJ. f Exosomes were isolated from the culture media of untreated or LPS-treated BMDM (1 μl/ml) for 24 h and were then added to WT or TLR4−/− BMDM and incubated for 6 h. The level of TNF-α in the supernatants was measured by ELISA. All results are representative of three independent experiments. Three random fields in the images were counted. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups or with the control. NS, no significant difference

Fig 3: Expansion of ILC2s in AF mice is dependent on IL-25. Single-cell suspensions of the siLP mononuclear cells isolated from indicated mice were analyzed by flow cytometry. (A and B) Dot plots show IL-17RB and CD25 expressions in CD45+ Lin− Thy1.2+ cells in the indicated mice (A). Graphs show statistical analysis (N=3~5) (B). Data are representative of three independent experiments. (C) GF mice were administered with either PBS (vehicle) or recombinant mouse (rm) IL-25 plus IL-2 daily for three consecutive days. Dot plots show CD69, and Ki-67 expressions in ILC2s, and the graph shows statistical analysis of CD69 expressions. (D–F) Contour plots show frequencies of ILC2s and their CD69 expressions in AF mice administrated with 250µg anti-IL-25 or anti-IL-17RA/RB-blocking antibodies for five consecutive days (D). Graphs show statistical analysis of the frequencies of ILC2s (E) and CD69 MFIs (F). Results were pooled from 3 independent experiments (N=2~11). *p<0.05, **p<0.01. ***p<0.001. NS, not significant (unpaired two-tailed student t-test). GF, germ-free; AF, antigen free; Lin, Lineage; MFI, mean fluorescence intensity.

Fig 4: IL-25 mediates LEPC-mediated suppression of exosome release from Mϕ. a BMDM were treated with LPS (1 μg/ml) with or without recombinant IL-25 (200 ng/ml), IL-33 (200 ng/ml), or TSLP (200 ng/ml) for 24 h followed by isolation of exosomes from the cell culture supernatants, and CD63+ exosomes were identified by flow cytometry. b BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of anti-IL-25 neutralizing antibody (10 μg/ml) or control non-specific IgG (10 μg/ml). CD63+ exosomes were detected by flow cytometry. c and d LEPCs were treated with LPS (1 μg/ml) for 0, 6, 12, or 24 h, and the IL-25 mRNA expression in the LEPCs and IL-25 protein concentration in the supernatants were measured by RT-qPCR (c) and ELISA (d), respectively. e BMDM were treated with LPS (1 μg/ml) for 24 h, and the cell surface expression of IL-25R was measured by flow cytometry. All results are representative of three independent experiments. The graphs show the mean ± SEM, n = 3; *P < 0.05 or **P < 0.01, compared with the indicated groups or with the control

Fig 5: IL-25 downregulates LPS-induced Rab27a and Rab27b to suppress exosome release from macrophages. a BMDM were transfected with siRab27a, siRab27b, or siNC (control) for 36 h and were then treated with LPS (1 μg/ml) for 24 h. Exosomes were isolated from the culture media, and CD63 staining was detected by flow cytometry. b BMDM were treated with LPS (1 μg/ml) for 0, 6, 12, and 24 h. Rab27a and Rab27b expression were detected by Western blotting. c BMDM cultured alone or cocultured with LEPCs were treated with LPS (1 μg/ml) in the presence or absence of recombinant IL-25 (200 ng/ml) for 24 h. The expression of Rab27a and Rab27b were detected by Western blotting. All results are representative of three independent experiments. The graphs show the mean ± SEM, n = 3; * or # P < 0.05, ** or ## P < 0.01, compared with the LPS groups. * and ** indicate Rab27a, # and ## indicate Rab27b