Non-Typeable Haemophilus influenzae Induces Expression of 14-3-3ε to Negatively Regulate Pulmonary Inflammatory Response

This paper aimed to investigate the effect of 14-3-3ε on the secretion of proinflammatory cytokines by human bronchial epithelial cells induced by NTHi (non-typeable Haemophilus influenzae) and its mechanism. BEAS-2B cells were infected with NTHi with MOI (multiplicity of infection) of 5, 10, and 20, respectively. Real-time quantitative PCR or Western blot were used to detect the expression of 14-3-3ε in the cells and the role of tyrosine kinase c-Src before and after infection. The binding levels of 14-3-3ε and TLR2 were detected by co-immunoprecipitation. The activation of NF-κB and the concentration of TNF-α and IL-6 in the culture supernatant after RNA interference or BEAS-2B cell overexpression of 14-3-3ε were measured by immunofluorescence and ELISA. Finally, the effects of 14-3-3ε inhibitor treatment on pulmonary inflammation after NTHi infection were observed in animal experiments. The results showed that following the infection of epithelial cells with NTHi, there was an observed elevation in the protein and mRNA levels of 14-3-3ε concomitant with increasing MOI. However, upon pre-treatment of cells with ActD and CHX, the expression levels of 14-3- 3ε decreased by 71.05% and 59.21% respectively, in comparison to the control group. At 15 minutes post NTHi infection, the phosphorylation levels of c-Src in BEAS-2B cells surged by 3.63-fold. However, upon inhibition of c-Src activity, the expression of 14-3-3ε decreased by 50.60%. Immunoprecipitation results also revealed that following infection with varying MOI of NTHi, the binding of 14-3-3ε to TLR2 was augmented by 1.78, 4.33, and 6.89-fold respectively. Silencing of 14-3-3ε expression led to a decrease in the inhibitor of NF-κB, IκB, by 75.24% and an intranuclear increase of the p65 subunit by 36.9%. The secretion levels of TNF-α and IL-6 were elevated from (269.24±16.71) pg/mL and (116.08±5.61) pg/mL to (332.27±20.57) pg/mL and (172.32±9.78) pg/mL respectively. Conversely, cellular overexpression of 14-3-3ε resulted in reduced levels of TNF-α and IL-1β to (145.34±22.16) pg/mL and (65.22±11.74) pg/mL respectively. All aforementioned differences were statistically significant (P<0.05). Additionally, following NTHi infection in C57BL/6 mice, there was a noticeable increase in the mRNA expression levels of 14-3-3ε in lung tissues. However, prior intraperitoneal injection with the 14-3-3ε inhibitor R18 led to an escalation in the pathological scoring of mouse lung tissue from 6.42±1.52 to 11.86±1.63. The levels of TNF-α and IL-6 in bronchoalveolar lavage fluid were increased from (186.39±16.71) pg/mL and (76.21±12.63) pg/mL to (258.91±32.05) pg/mL and (151.25±23.87) pg/mL respectively. These results indicated that infection with NTHi leads to the upregulation of 14-3-3ε expression in airway epithelial cells. Subsequently, 14-3-3ε acts to downregulate the activity of the TLR2/NF-κB signaling pathway, culminating in the negative modulation of pulmonary inflammatory responses

CSTR: 32200.14.cjcb.2023.09.0004