Lipopolysaccharide Administration Alters Extracellular Vesicles in Cell Lines and Mice

Abstract

Extracellular vesicles (EVs) play a fundamental role in cell and infection biology and have the potential to act as biomarkers for novel diagnostic tools. In this study, we explored the in vitro impact of bacterial lipopolysaccharide administration on cell lines that represents a target for bacterial infection in the host. Administration of lipopolysaccharide at varying concentrations to A549 and BV-2 cell lines caused only modest changes in cell death, but EV numbers were significantly changed. After treatment with the highest concentration of lipopolysaccharide, EVs derived from A549 cells packaged significantly less interleukin-6 and lysosomal-associated membrane protein 1. EVs derived from BV-2 cells packaged significantly less tumor necrosis factor after administration of lipopolysaccharide concentrations of 0.1 µg/mL and 1 µg/mL. We also examined the impact of lipopolysaccharide administration on exosome biogenesis and cargo composition in BALB/c mice. Serum-isolated EVs from lipopolysaccharide-treated mice showed significantly increased lysosomal-associated membrane protein 1 and toll-like receptor 4 levels compared with EVs from control mice. In summary, this study demonstrated that EV numbers and cargo were altered using these in vitro and in vivo models of bacterial infection.

Fig. 1

LPS treatment alters A549 cell viability. Cell viability following LPS treatment (0.1 µg/mL, 1 µg/mL, and 10 µg/mL) or without treatment was determined using the MTT assay at 48 h. Mean ± SEM data are from five experiments

Fig. 2

TEM anlaysis of EVs after LPS treatment. TEM analaysis was performed on EVs derived from BV-2 cells (a–f) and A549 cells (g–i) in the abscene or prescence of LPS treatment at 1 µg/mL and 10 µg/mL for 24 or 48 h. TEM analysis was performed in duplicate

Fig. 3

LPS treatment alters EVs from BV-2 and A549 cells. a, b Mean sizes and c, d particle concentrations (per mL) were determined for BV-2-derived and A549-derived EVs following LPS treatment using Nanosight Tracking Analysis. ELISAs of BV-2 derived EVs and A549-derived EVs were used to examine expressions of e, f LAMP-1, and g, h RRP44/DIS3 proteins. Mean fold change ± SEM data are from a total of five experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001

Fig. 4

Immunomodulator responses in EVs from LPS-treated cells. The expressions of immunomodulators a IL-6, b IL-1β, c TLR4, and d TNFα in EVs from LPS-treated (0.1 µg/mL, 1 µg/mL, and 10 µg/mL) BV-2-derived EVS and untreated EVs were determined by ELISA. The expressions of immunomodulators e IL-6, f IL-1β, g TLR4, h TNFα, and i iNOS in EVs from LPS-treated (0.1 µg/mL, 1 µg/mL, and 10 µg/mL) A549-derived EVs and untreated EVs were also determined by ELISA. Mean fold change ± SEM data are from a total of 6–8 experiments. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001

Fig. 5

EV characterization from LPS-treated mice in vivo. a Mean sizes and b particle concentrations (per mL) were determined for mouse-derived exosomes following LPS treatment using Nanosight Tracking Analysis. Mean ± SEM data are from a total of eight control mice and six experimental mice

Fig. 6

EV-associated protein expression from LPS-treated BALB/c mice. The expressions of a CD9, b CD81, c CD63, and d LAMP-1 were determined by ELISA in EVs isolated from mice. Mean fold change ± SEM data from a total of five experiments. **P ≤ 0.01

Fig. 7

Expression of immunomodulators after LPS treatment in vivo. The expressions of a IL-6, b TLR4, and c TLR7 were determined by ELISA in EVs isolated from mice. Mean fold change ± SEM data from a total of five experiments. ****P ≤ 0.0001