Fig. 4. Macrophage-derived exosomal APN/CD13 promotes lung epithelial cell necroptosis.

a Protein quantification of exosomes secreted by BMDMs exposed to LPS (1 µg/mL) for 24 h using BCA, PBS was used as the control. b ELISA was used to determine the levels of APN expression in exosomes. c Representative fluorescence images showed LPS-treated BEAS-2B cells incubated with PKH67-labeled exosomes (green) for 6 h. The nuclei were stained with DAPI (blue), and the cytoskeleton with phalloidin (red). Scale bar, 15 μm. d–l BEAS-2B cells treated with LPS were cocultured for 24 h with exosomes isolated from the supernatant of BMDM stimulated with PBS (PBS-exo) or LPS (LPS-exo). d Western blotting was used to detect APN expression in recipient BEAS-2B cells. As an internal reference, GAPDH was used. e Effects of BMDM exosomes on the mRNA expression of IL-6, TNF-α and IL-1β in LPS-stimulated BEAS-2B cells. RNA was isolated and analyzed using qPCR. f Representative images and quantified data showing BEAS-2B cell apoptosis determined by a percentage of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei/total nuclei in the indicated group (n = 3). Scale bar: 20 μm. g Flow cytometric analysis shows the proportion of apoptotic cells (Annexin V/PI-positive), after treatment with BMDM-derived exosomes. Bar graphs depict the quantitative analysis. h Western blotting analysis of the protein levels of NF-κB signaling genes shows that the levels of P-p65, RelB, p50, and p100 were significantly elevated in the recipient BEAS-2B cells treated with BMDM-derived exosomes in the presence of LPS. The BEAS-2B cells were co-treated with exosomes and LPS (1 μg/mL) for 30 min or 6 h. i Immunofluorescence was used to examine the translocation of the NF-κB p65 subunit from the cytoplasm to the nucleus. The blue spots are cell nuclei, and the red spots are NF-κB p65 staining. j Flow cytometry analysis of intracellular ROS level. k The increase in RIPK1-RIPK3 complex in BEAS-2B cells after co-treatment with LPS-exo, indicated by immunofluorescence and (l) immune-precipitation. m Endogenous RIP1 interacts with RIP3 indicate necrosomes formation by treatment with LPS-exo or PBS-exo. Cells were fixed and proximity ligation assay (PLA) was performed using α-RIP1 and either α-RIP3, as indicated. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. Representative confocal microscopy images are shown for each condition, which was repeated at least three times. Scale bar, 10 μm. PLA signals per cell were quantified by image software-assisted analysis. n Western blotting analysis of the protein levels of caspase-3 cleavage and p-MLKL. Ctrl, LPS-treated BEAS-2B cells without exosomes. All of the results are based on three separate experiments. The data are presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01.