Fig. 1

TNF-Exo induces lung injury by affecting M1 macrophage activation and pyroptosis in vivo. WT C57BL/6 mice were administered with PBS-Exo/TNF-Exo (300 μg/mouse) intraperitoneally for 24 h. An equal volume of PBS was used as negative control. a Ex vivo fluorescence signals in the lungs of mice injected i.p. with Dil-labeled exosomes. b Evaluation of lung histology by H&E staining (magnification × 400). Green arrows indicate neutrophils in the alveolar and interstitial space, red arrows indicate alveolar macrophages, yellow arrows indicate hyaline membranes, and black arrows indicate thickening of the alveolar walls. Scale bar, 50 μm. c Detection of inflammatory cytokine mRNA (IL-1β, TNF-α) and iNOS mRNA expression in the lung tissues by RT-qPCR. d Representative images of direct immunofluorescence staining of DNA (blue), F4/80 (red) and iNOS (green) in the lung sections, and white arrows indicate iNOS positive macrophages. Negative control represents immunofluorescence images stained with species-specific secondary antibodies coupled with Alexa Fluor Dyes alone. Scale bar, 50 μm. e Flow cytometry detection of CD11c and CD206 expression on peritoneal macrophage (PMϕ) after being gated with macrophage marker F4/80. f Representative flow cytometry plots of Annexin V/PI staining of PMϕ, and analysis of Annexin V/PI double-stained cells by dying. g Representative flow cytometry plots and quantitation of PMϕ pyroptosis (Caspase-1/TUNEL double-positive cells). One-way analysis of variance with Tukey's multiple comparisons test was used for the analysis. Graphs represent means ± SEM, n ≥ 3; *P < 0.05, **P < 0.01 compared within two groups