Bacterial meningitis in the early postnatal mouse studied at single-cell resolution
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eLife assessment
This study presents valuable findings on the changes of immune cell populations and stromal cells occurring at the CNS borders in a neonatal bacterial meningitis model, focusing on fibroblasts, macrophages and endothelial cells. The study provides solid snRNA-seq dataset and high quality immune fluorescence images of dissected brain border regions, that will be useful for the community. These observations and datasets will be of interest to the neuro-immunology community.
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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.
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Author Response
Reviewer #2 (Public Review):
In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.
The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall …
Author Response
Reviewer #2 (Public Review):
In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.
The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall changes in the periphery, survival curves or bacterial counts (in the KO models) in the meninges/brain are not mentioned.
Thank you for those comments. We hope that the text is now more readable. We have added a separate section to describe the meninges model and added data on survival and E coli counts (Supplemental Figure 3).
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eLife assessment
This study presents valuable findings on the changes of immune cell populations and stromal cells occurring at the CNS borders in a neonatal bacterial meningitis model, focusing on fibroblasts, macrophages and endothelial cells. The study provides solid snRNA-seq dataset and high quality immune fluorescence images of dissected brain border regions, that will be useful for the community. These observations and datasets will be of interest to the neuro-immunology community.
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Reviewer #1 (Public Review):
In their manuscript, Wang et al. investigate the changes occurring at the CNS borders upon neonatal bacterial meningitis. Both the dural meninges and the leptomeninges display changes. Using single nuc RNAseq and imaging approaches, they show that fibroblasts, endothelial cells and macrophages get inflamed, with an increase vascular leakage. Mechanistically, TLR4 KO but not CCR2 KO or liposome treatment (to deplete leptomeningeal macrophages) was able to rescue the vascular impairment. This is an interesting study that provides useful datasets for the community. However, we recommend several additions regarding data analysis (definitions, single cell, imaging) as well as additional studies (bacterial load, protein validation).
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Reviewer #2 (Public Review):
In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.
The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall changes in the …
Reviewer #2 (Public Review):
In a neonatal model of bacterial meningitis induced by s.c. injection of E. coli, transcriptional changes were found across all major cell types including endothelial cells, fibroblasts and macrophages. Among macrophages, they describe 2 resident subsets and 2 inflammatory subsets. By immunohistochemistry of arachnoid and dura flatmounts, they show vascular changes upon infection, including clustering of CLDN5 and PECAM1, and disorganized capillary morphology, which was dependent on Tlr4 signaling but independent of arachnoid macrophages.
The manuscript would benefit from rewriting, it is not written in a concise manner and the rationale for experiments, time points for analyses and their conclusions are not clear. The model of s.c. bacterial infection is not well introduced and overall changes in the periphery, survival curves or bacterial counts (in the KO models) in the meninges/brain are not mentioned.
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