Related Experiment Video
Updated: Jul 26, 2025
Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy
Published on: August 1, 2022
Bacterial meningitis in the early postnatal mouse studied at single-cell resolution
Jie Wang1,2, Amir Rattner1, Jeremy Nathans1,2,3,4
1Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States.
Abstract:
Bacterial meningitis is a major cause of morbidity and mortality, especially among infants and the elderly. Here, we study mice to assess the response of each of the major meningeal cell types to early postnatal E. coli infection using single nucleus RNA sequencing (snRNAseq), immunostaining, and genetic and pharamacologic perturbations of immune cells and immune signaling. Flatmounts of the dissected leptomeninges and dura were used to facilitiate high-quality confocal imaging and quantification of cell abundances and morphologies. Upon infection, the major meningeal cell types - including endothelial cells (ECs), macrophages, and fibroblasts - exhibit distinctive changes in their transcriptomes. Additionally, ECs in the leptomeninges redistribute CLDN5 and PECAM1, and leptomeningeal capillaries exhibit foci with reduced blood-brain barrier integrity. The vascular response to infection appears to be largely driven by TLR4 signaling, as determined by the nearly identical responses induced by infection and LPS administration and by the blunted response to infection in Tlr4-/- mice. Interestingly, knocking out Ccr2, encoding a major chemoattractant for monocytes, or acute depletion of leptomeningeal macrophages, following intracebroventricular injection of liposomal clodronate, had little or no effect on the response of leptomeningeal ECs to E. coli infection. Taken together, these data imply that EC responses to infection are largely driven by the intrinsic EC response to LPS.
Insights
Bacterial meningitis impacts infants and the elderly. This study shows endothelial cell responses to E. coli infection are primarily driven by Toll-like receptor 4 (TLR4) signaling, not macrophage activity.
Area of Science:
- Neuroscience
- Immunology
- Cell Biology
Background:
- Bacterial meningitis poses significant risks, particularly to vulnerable populations.
- Understanding the cellular responses within the meninges during infection is crucial for developing effective treatments.
Purpose of the Study:
- To investigate the cellular and molecular responses of meningeal cell types to early postnatal E. coli infection in mice.
- To elucidate the signaling pathways, particularly Toll-like receptor 4 (TLR4), involved in the meningeal vascular response to bacterial meningitis.
Main Methods:
- Single nucleus RNA sequencing (snRNAseq) to analyze transcriptomic changes in meningeal cells.
- Confocal imaging of leptomeninges and dura for cell quantification and morphology assessment.
- Genetic (Tlr4 knockout) and pharmacologic (liposomal clodronate) perturbations to assess immune cell and signaling roles.
Main Results:
- Distinct transcriptomic changes were observed in endothelial cells (ECs), macrophages, and fibroblasts upon infection.
- Leptomeningeal ECs showed redistribution of CLDN5 and PECAM1, with compromised blood-brain barrier integrity.
- Vascular response was largely mediated by TLR4 signaling, as evidenced by similar responses to LPS and blunted responses in Tlr4 mice.
- Depletion of macrophages or knockout of Ccr2 had minimal impact on EC responses.
Conclusions:
- Endothelial cell responses to E. coli meningitis are predominantly driven by intrinsic EC sensing of lipopolysaccharide (LPS) via TLR4.
- Macrophage activity and monocyte chemoattraction appear less critical for the early EC response in this model.
