The mechanism by which Haemophilus influenzae causes meningitis is unclear. Previously, we established murine meningitis by intranasal instillation of H. influenzae as a cell-bound organism (CBO). In this study, we aimed to identify the molecules associated with inhibiting the transmigration of cells across the blood-brain barrier (BBB). Two-dimensional difference gel electrophoresis and protein identification by mass spectrometry were used for proteomic analysis. Analysis of the membranous extract from a tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cell (HBMEC) monolayer revealed 41 differentially expressed proteins. Zyxin, which is thought to be essential for tight cell-to-cell junctions, decreased 1.8-fold in TNF-α-treated HBMECs. In addition, zyxin transcript levels decreased 1.5-fold in cells derived from TNF-α-treated HBMECs. Intranasal instillation of CBOs in zyxin-deficient mice resulted in a significant higher mortality rate than in wild-type mice. Transmigration of CBOs across a HBMEC monolayer pretreated with TNF-α (1 ng/mL), interleukin (IL)-1β (10 ng/mL), or lipopolysaccharide (LPS; 10 ng/mL) was assayed by counting CBOs that migrated from an upper chamber into a lower chamber. HBMEC pretreated with TNF-α exhibited significantly greater migration (P<0.01) than did control cells or cells treated with IL-1β or LPS. Our findings highlight that zyxin is an important protein protecting the tight junction of the BBB against cell transmigration across the BBB. Finally, TNF-α produced in respiratory infection when the primary infection reached the BBB caused decreased zyxin levels in BBB cell membranes. Furthermore, H. influenzae reaching the BBB as CBOs could transmigrate into cerebrospinal fluid across the zyxin-decreased BBB.
Keywords: TNF-α; blood-brain barrier; cell-bound organism; intracellular parasite; meningitis; secondary infection; zyxin.