Data from: Meningeal inflammation and arachnoid barrier breakdown in a mouse model of neonatal bacterial meningitis
Data files
Abstract
Newborns are especially susceptible to bacterial meningitis, primarily caused by Group B Streptococcus (GBS), due to incomplete maturation of immune and barrier systems. While meningitis is well known to break down the blood-brain barrier, its effect on the meningeal arachnoid barrier remains poorly understood. Using a neonatal bacterial meningitis model, we demonstrate that GBS infection significantly increases arachnoid barrier permeability, disrupts tight junctions, and elevates meningeal production of proinflammatory cytokines. CD206+/LYVE-1+ border-associated macrophages undergo morphological and molecular activation post-infection, but their depletion prior to infection does not attenuate arachnoid barrier leakage or cytokine levels. We show that meningeal fibroblasts are a source of proinflammatory cytokines in response to GBS infection and that a systemic exposure of TNF-α alone is sufficient to induce barrier breakdown. These results support that neonatal arachnoid barrier is vulnerable to cytokine-induced breakdown and highlight the role of non-immune meningeal cells like fibroblasts during bacterial infection.
Dataset DOI: 10.5061/dryad.s7h44j1qg
Description of the data and file structure
Each subfolder is titled by corresponding figures and contains raw files for each panel represented in the manuscript. Images (IF/RNAscope/ICC) are czi extension and can be opened using ImageJ/Fiji, ZenBlue, or IMARIS.
Files and variables
File Description:
fig1.zip: Neonatal meningitis model using Group B Streptococcus (GBS) and arachnoid barrier (AB) pathology. Contains raw images of arachnoid barrier integrity assay (coronal brain sections) and E-cadherin/Claudin-11 junctions (leptomeninges whole mount). GBS refers to GBS-infected animals and Mock refers to PBS-injected animals.
fig3.zip: Leptomeningeal immune cell profiles in GBS-infected leptomeninges (LPM). Contains raw images of CD206+/Lyve1+ border-associated macrophages (BAMs) in the leptomeninges whole mount. GBS refers to GBS-infected animals and Mock refers to PBS-injected animals.
fig4.zip: Proinflammatory cytokines (Il6, Tnfa) in dural and leptomeningeal BAMs and the role of BAMs in arachnoid barrier breakdown during infection. Contains raw RNAscope images of cytokine transcripts in BAMs and arachnoid barrier integrity assay with and without clodronate-treatment. Leptomeninges (LPM) line the border of the cortex and dura is a thin membranous structure detached from the cortex (refer to Fig. 5A in manuscript). GBS refers to GBS-infected animals, Mock refers to PBS-injected animals, Clodronate refers to Clodronate-treated animals, Vehicle refers to liposome-treated animals, Clo+GBS refers to GBS-infected animals that received Clodronate, and Veh+GBS refers to GBS-infected animals that received liposome.
fig5.zip: Proinflammatory cytokines (Il6, Tnfa) in dural and leptomeningeal fibroblasts. Contains raw RNAscope images of cytokine transcripts in meningeal fibroblasts. Leptomeninges (LPM) line the border of the cortex and dura is a thin membranous structure detached from the cortex (refer to Fig. 5A in manuscript). GBS refers to GBS-infected animals and Mock refers to PBS-injected animals. Dura and LPM panels in Fig. 5b are from the same image file and dura and LPM panels in Fig. 5c are from different image files.
fig6.zip: Meningeal fibroblast response to GBS in vitro. Contains raw images of dural and leptomeningeal (LPM) fibroblast cultures with GBS.
fig7.zip: The role of TNFa in arachnoid barrier breakdown. Contains raw images of the arachnoid barrier integrity assay (coronal brain sections) and E-cadherin/Claudin-11 junctions (leptomeninges whole mount) after systemic TNFa injection. TNFa refers to TNFa-treated animals and Vehicle refers to PBS-injected animals.
supp3.zip: Contains raw images of the brains and dura from animals treated with clodronate or vehicle. Shows CD206+ BAM staining. GBS_Clodronate refers to GBS-infected animals that received Clodronate, GBS_Vehicle refers to GBS-infected animals that received liposome, Mock_Clodronate refers to PBS-injected animals that received Clodronate, and Mock_Vehicle refers to PBS-injected animals that received liposome.
supp4.zip: Contains raw RNAscope images of the leptomeninges (LPM) and cortex from GBS-infected animals. Pdgfra expressed in all fibroblasts and AB cells, Aldh1a2 expressed in arachnoid fibroblasts, Cxcl12 enriched in pial fibroblasts, and Pecam expressed in endothelial cells.
supp5.zip: Contains raw images of leptomeningeal (LPM) and dural fibroblast cultures stained with CD45 and PDGFRa.
supp6.zip: The role of IL-6 in arachnoid barrier breakdown. Contains raw images of the arachnoid barrier integrity assay (coronal brain sections) and E-cadherin/Claudin-11 junctions (leptomeninges whole mount) after systemic IL-6 injection. IL6 refers to IL-6-treated animals and Vehicle refers to PBS-injected animals.
Code/software
ZenBlue, FIJI/ImageJ, IMARIS (v10.2)
Access information
Other publicly accessible locations of the data:
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Data was derived from the following sources:
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Procedures for LPM whole mount dissection, fixation, and immunostaining are detailed at length in our protocol paper (Jones et al. 2022). For cryosections of whole head used for RNAscope and brains prepared for vibratome sectioning, mice were euthanized by injecting a fatal dose of pentobarbital followed by transcardiac perfusion with 1X PBS followed by 4% paraformaldehyde (PFA). Heads (skin and lower jaw removed) were post-fixed in 4% PFA for 24 hours, then in 20% sucrose solution for 24 hours at 4°C. Prior to cryosectioning (12µm), the heads were embedded in Optimal Cutting Temperature (OCT) compound and flash-frozen in pre-cooled isopentane. For thick sections (100-500µm), brains were post-fixed in 4% PFA for 24h and stored in PBS until embedded in 3% agarose gel (1.5% low-melt agarose and 1.5% agarose) prior to vibratome sectioning. Incubation in the primary antibodies (1:200 dilution) was conducted overnight at 4°C in appropriate solution (Jones et al. 2022). Following incubation with primary antibodies, tissue sections were incubated for 60 min at room temperature with appropriate AlexaFluor-conjugated secondary antibodies (1:500; Invitrogen) and DAPI (1:1000; Invitrogen). For RNAscope in situ hybridization, cryosectioned tissues were processed following the manufacturer’s protocol (ACD Bio) with appropriate probes.