To explore the inflammatory effect of exosomes derived from alveolar epithelial cells stimulated by lipopolysaccharide (LPS) on the alveolar macrophages (NR8383).

The alveolar epithelial cells disposed with different treatments were co-cultured with alveolar macrophages by using a Transwell system separately. Alveolar epithelial cells (RLE-6TN) were randomly divided into 4 groups: normal group, LPS-stimulated group, exosome inhibitor group, and exosome inhibitor pretreatment + LPS stimulation group. NR8383 cultured alone was considered as a blank control. After the 12-h co-culture, the real-time PCR (qPCR) was performed to examine the mRNA relative expression of IL-6, TNF-α, and IL-1β in NR8383 cells. To further explore the role of exosomes derived from RLE-6TN on alveolar macrophages mediated inflammationary response, the experimental exosomes (exosomes derived from LPS-induced RLE-6TN) and control exosomes exosomes derived from normal RLE-6TN were extracted by gradient ultracentrifugation. Transmission electron microscopy and Western blotting analyses was performed to identify the exosomes, and qNano particle diameter analyzer was conducted to measure the particle diameter of exosomes. In vitro, NR8383 cells were divided into 3 groups which were cultured with exosomes derived from LPS-stimulated RLE-6TN at a concentration of 10 μg/mL (experimental group), exosomes derived from untreated RLE-6TN at the same concentration of 10 μg/mL (control group), and the PBS at the same volume with experimental group (PBS group), respectively for 12 h. After the treatment, the phagocytosis of NR8383 cells was observed by laser confocal microscope and the release of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in supernatants of NR8383 was detected by enzyme-linked immunosorbent assay ELISA

(1)In the co-culture experiment, the mRNA relative expression of pro-inflammatory cytokine in the LPS group was significantly increased compared with the blank control group (P<0.01), however comparing the exosome inhibitor pretreatment+LPS group with the LPS group, the expression of pro-inflammatory cytokine was decreased (P<0.01). (2) The extracted exosomes were observed as circular or elliptical vesicles with a diameter of 40-100 nm under the transmission electron microscopy. Western blotting analyses showed that the extracted exosomes express the protein marker, such as CD63 and CD9; After incubation with NR8383 cells for 5 h, laser scanning confocal microscope showed that the exosomes labeled with red fluorescent were uptaken by NR8383 cells. (3)After the exosomes derived from the LPS-disposed RLE-6TN and the normal RLE-6TN cells were incubated with NR8383 cells respectively. The ELISA test showed that treated the alveolar macrophages with LPS induced alveolar epithelial secreted exosomes led to a robustly increased release of pro-inflammatory cytokine (P<0.01), but there was no significant difference between the control group and PBS group (P>0.05).

Exosomes derived from LPS-disposed alveolar epithelial cells activate the alveolar macrophage-mediated inflammatory response.

Lipopolysaccharide; Exosomes; Alveolar epithelial cells; Alveolar macrophages; Inflammation