Endothelial TLR4 signaling drives tissue inflammation, Claudin-5 internalization, and vascular barrier breakdown in a mouse model of neonatal meningitis
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 celltype specific 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. Endothelial-specific deletion of Tlr4 dramatically reduced the inflammatory response in all leptomeningeal cell types and abrogated the infectionassociated increase in vascular permeability. In a brain endothelial cell line (bEnd.3 cells), exposure to E. coli triggered TLR4-dependent NF-?B activation, selective internalization of Claudin-5, and increased monolayer permeability, responses that were eliminated by Tlr4 knockout. RNA-seq showed that endothelial TLR4 controls an NF-?B–driven transcriptional program that orchestrates the leptomeningeal response to infection. These findings reframe the host response in neonatal Gram-negative bacterial meningitis as an endothelial-centric process. Overall design: Here, we study a mouse neonatal meningitis model and cultured CNS ECs (bEnd.3 cells) to test whether endothelial-intrinsic TLR4 signaling (i) orchestrates the multi-cellular response of the leptomeninges to infection and (ii) regulates Cldn5 trafficking and vascular barrier function. In the present work, we (i) profiled cell-type–specific responses in the leptomeninges from WT, endothelial Tlr4 conditional knockout mice (Tlr4ECKO), and macrophage Tlr4 conditional knockouts (Tlr4MKO) using single nucleus (sn)RNA-seq; (ii) performed wholemount immunostaining to quantify ICAM-1 induction, extravascular tracer leakage, and macrophage activation in vivo; (iii) tracked Cldn5 redistribution and NF-?B nuclear translocation in bEnd.3 cells exposed to E. coli; (iv) generated CRISPR Tlr4KO bEnd.3 lines to assess the TLR4 dependence of NF-?B activation, tight-junction dynamics, and barrier permeability; (v) quantified Cldn5 colocalization with junctional, plasma-membrane, and endosomal/lysosomal markers; (vi) defined TLR4-dependent transcriptional programs in bEnd.3 cells. The results of these experiments establish a framework for understanding the pathophysiology of neonatal meningitis that establishes endothelial TLR4 signaling as a central regulator of both the multicellular leptomeningeal response to infection and Cldn5 trafficking and junctional integrity.