Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

BACKGROUND: Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. METHODS: Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. RESULTS: After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87 ± 4.95 nm and 96.23 ± 2.16 nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67 ± 26.3 nm and 325 ± 6.06 nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35 mg, 41.90 mg, 16.44 mg, and 12.65 mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750 mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. CONCLUSIONS: Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Activating Mas receptor protects human pulmonary microvascular endothelial cells against LPS-induced apoptosis via the NF-kB p65/P53 feedback pathways.

The balance between Ang II/AT1R and Ang-(1-7)/Mas plays a pivotal role in the development of lipopolysaccharides (LPS)-induced acute respiratory distress syndrome. However, the mechanisms underlying the balancing process still remain unclear. Here we investigated the roles of nuclear factor (NF)-κB and p53 in regulating AT1R and Mas expression. The results demonstrated that Ang II pretreatment resulted in downregulation of Mas and upregulation of AT1R, phosphorylated p65, and apoptosis in LPS-treated Human pulmonary microvascular endothelial cells (HPMVECs), but had no effect on p53 expression. Lentiviral vector-mediated P65 knockdown, but not a P53 knockdown, reversed all these effects of Ang II. On the other hand, Ang-(1-7) pretreatment lead to an increased in Mas expression and a decrease in AT1R, p53, and phosphorylated p65 expressions with suppressed apoptosis in LPS-treated cells. P65 knockdown promoted the protein expression of both AT1R and Mas while inhibiting p53 expression. P53 knockdown, but not a p65 knockdown, reversed all these effects of Ang-(1-7). Interestingly, p65 overexpression upregulated p53 and AT1R but downregulated Mas. P53 knockdown activated p65. These results suggest that there is a two-way feedback regulation between AT1R and Mas receptor via the NF-kB p65/P53 pathway, which may play a key role in LPS-induced HPMVECs apoptosis.