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Endothelial TLR4 signaling drives tissue inflammation, Claudin-5 internalization, and vascular barrier breakdown in a mouse model of neonatal meningitis

ABSTRACT: Neonatal bacterial meningitis is a leading cause of infant morbidity and mortality, yet the molecular and cellular basis of the leptomeningeal response to infection remains poorly defined. Here, we study a mouse model of neonatal E. coli meningitis, combining conditional gene knockouts, leptomeningeal single-nucleus RNA sequencing, and endothelial cell culture to explore the role of Toll-like receptor 4 (TLR4) signaling in the host response to infection. Deletion of Tlr4 in non-myeloid cells dramatically reduced the inflammatory response in all leptomeningeal cell types and abrogated the infection-associated increase in vascular permeability. In a brain endothelial cell line (bEnd.3 cells), exposure to E. coli triggered NF-κB activation, selective internalization of Claudin-5, and increased monolayer permeability, responses that were eliminated by Tlr4 knockout. RNA-seq showed that TLR4 controls an NF-κB–driven transcriptional program that orchestrates the endothelial response to E. coli. These findings reveal multiple TLR4-dependent host responses to neonatal Gram-negative bacterial meningitis.

ORGANISM(S): Mus musculus

PROVIDER: GSE319556 | GEO | 2026/02/20

REPOSITORIES: GEO

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Project description:Bacterial meningitis is a deadly disease most commonly caused by Streptococcus pneumoniae that leads to severe neurological sequelae including cerebral edema, seizures, stroke, and mortality when untreated. Meningitis is initiated by the transfer of S. pneumoniae from hematogenous source into the brain across the blood-brain barrier (BBB); the mechanisms however are still poorly understood. Current treatment strategies include adjuvant dexamethasone therapy for cerebral edema, the prime reason for neurological complications and mortality, which is followed up by antibiotics. The success of corticosteroids is however inconclusive, necessitating the development of new therapies for controlling cerebral edema. We have previously shown a general activation of hypoxia inducible factor (HIF-1α) in bacterial infections, we therefore hypothesized that HIF-1α, via induction of vascular endothelial growth factor (VEGF) is critically involved in the transmigration of pathogens across the BBB. In human and murine meningitis brain samples, HIF-1 activation was observed by immunohistochemistry. HIF-1α/VEGF expression and permeability was therefore analyzed in vitro in infected brain endothelial cells (ECs). Localization of S. pneumoniae in brain ECs from mouse/human sources was visualized in vitro and in vivo by confocal, super-resolution, and electron microscopy. S. pneumoniae infection in brain ECs resulted in upregulation of HIF-1α/VEGF by Western blotting and qRT-PCR that was associated with increased paracellular permeability, which was supported by bacterial localization at cell-cell junctions. RNA sequencing of brain microvessels from hematogenously infected mice with increased permeability and S. pneumoniae deposition in the brain showed upregulation of genes in HIF-1α/VEGF pathway. Inhibition of HIF-1α with echinomycin, siRNA in bEnd5 cells and by using primary brain ECs from HIF-1α KO mice revealed a reduced endothelial permeability and transmigration of S. pneumoniae. We thus demonstrate a critical role for HIF-1α/VEGF in transmigration of S. pneumoniae across the BBB and propose targeting this pathway to prevent BBB dysfunction in infections.

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Endothelial TLR4 signaling drives tissue inflammation, Claudin-5 internalization, and vascular barrier breakdown in a mouse model of neonatal meningitis

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