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Fig. 4 | Military Medical Research

Fig. 4

From: Lung epithelial cell-derived IL-25 negatively regulates LPS-induced exosome release from macrophages

Fig. 4

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

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