Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion

Extended Data Fig. 1 Nociceptors suppress meninges-mediated protection of CNS to infection.

a, Whole-mount confocal images of mouse meninges (dura mater) showing extravascular localization of S. pneumoniae 24 h post-injection of CMTPX-labeled bacteria. Scale bar = 100 µm. b, Bacterial load in samples collected from Nav1.8-DTA and control mice 48h after injection of S. pneumoniae (n = 6/group). c, Bacterial load in samples collected from Nav1.8-DTA and control mice 24 h after injection of S. pneumoniae (n = 4/group). d, Left, Illustration created with BioRender.com (https://biorender.com). Right, Imaging and quantification of cleaved caspase-3 staining in brain samples collected from Nav1.8-DTA and control mice 24 h after injection of CMTPX-labeled S. pneumoniae. Results are presented as fold-change relative to cCasp-3 staining of brain samples from uninfected mice (n = 4/group). Scale bar = 200 μm. e, Left, Illustration created with BioRender.com (https://biorender.com). Hematoxylin and eosin staining of brain sections, and blinded histopathology scores of brain samples from Nav1.8-DTA and control mice 24 h after injection of S. pneumoniae. n = 48/group (12 fields/sample, 4 samples/group). Scale bar = 50 μm. f, g, Meningeal innervation by CGRP⁺ neurons (green = CGRP staining; white = skeletonization) in (f) Nav1.8-DTA and control mice, and in (g) WT mice treated with systemic injection of resiniferatoxin (RTX) or vehicle (n = 3/group). Scale bar = 300 μm. h, Bacterial load in samples collected 24 h after injection of S. pneumoniae from mice treated with RTX or vehicle (n = 5/group). i, Left, illustration created with BioRender.com (https://biorender.com). Right, Bacterial load in samples collected from Nav1.8-DTA and control mice 24 h after intracisternal injection of S. pneumoniae (n = 4/group). Statistical analysis: (b—i) Unpaired t-tests. *p < 0.05, **p < 0.01, ****p < 0.0001. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Mean ±SEM. Exact p-values in Supplementary Table 1.

Extended Data Fig. 2 Local depletion of meningeal nociceptors reduces CNS infection by bacteria.

a, b, Back skin innervation by CGRP⁺ neurons quantified 3 weeks after either local injection of (a) resiniferatoxin (RTX) or vehicle into WT mice (n = 4/group), or (b) diphtheria toxin (DTX) into Nav1.8-Calca-DTR (Nav1.8-cre^+/−Calca-DTR^+/−) mice (n = 4) or into littermate control (Nav1.8-cre^+/−Calca-DTR^−/−) mice (n = 3). Scale bar = 100 μm. c, Meningeal innervation by CGRP⁺ neurons after local application of DTX in Nav1.8-Calca-DTR mice or control littermates (n = 3/group). Scale bar = 300 μm. d, Bacterial load in samples collected 24h after injection of S. pneumoniae from Nav1.8-Calca-DTR mice or control littermates treated locally with DTX (n = 3/group). a, b, c, green = CGRP staining; white = skeletonization. Statistical analysis: (a, b, c, d) Unpaired two-sided t-tests. *p < 0.05, **p < 0.01. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Mean ±SEM. Exact p-values in Supplementary Table 1.

Extended Data Fig. 3 Nociceptors regulate meningeal immunity against bacterial infection.

a, Representative flow cytometry plots and quantification of total leukocytes (CD45⁺ gate), neutrophils (CD11b⁺Ly6G⁺ gates), and monocytes (CD11b⁺Ly6G⁻Ly6C^hi gates) in the meninges at baseline and different time points after injection of S. pneumoniae (n = 4/group). b, Representative flow cytometry plots and quantification of total leukocytes (CD45⁺ gate), monocytes (CD11b⁺Ly6G⁻Ly6C^hi gates), B cells (CD11b⁻CD19⁺ gates), and T cells (CD11b⁻CD3⁺) in the meninges of Nav1.8-DTA mice or control littermates 24 h after S. pneumoniae injection (n = 5/group). c, Flow cytometric quantification of myeloid and lymphoid immune cells in the blood and spleen of Nav1.8-DTA mice and littermate controls (n = 4/group). d, Quantification of immune cells in the meninges from Nav1.8-DTA mice and wt controls by flow cytometry (n = 5/group for monocytes, B cells, and T cells; n = 6/group for macrophages, neutrophils, and DCs). Statistical analysis: (a) One-way ANOVA with Tukey post-tests. (b, c, d) Unpaired two-sided t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Box plots = median, IQR, min/max. Exact p-values in Supplementary Table 1.

Extended Data Fig. 4 S. pneumoniae and S. agalactiae induce pain and act on trigeminal ganglion neurons.

a, Left, Representative pictures of mice at baseline (uninfected), 1 day, or 2 days after S. pneumoniae infection. Right, Grimace scores of mice at baseline (uninfected), 1 day, and 2 days after injection of S. pneumoniae (n = 4), S. agalactiae (n = 4), or saline (n = 5). Orbital tightening (white arrowhead), nose bulge (black arrowhead), cheek bulge (white arrow), and ear position (black arrow) were scored. b, Grimace scores of mice injected with CGRP (2 µg, i.p.) or vehicle (n = 4/group). c, Trigeminal ganglion from Calca-GFP (Green) mice 24 h after injection of dye-labelled S. pneumoniae (red). Scale bar = 100 µm. d, Whole-mount staining of meninges from Calca-GFP mice (CGRP) collected 24 h after injection of dye-labelled S. pneumoniae (red) and co-stained with anti-Pneumolysin antibody (cyan). Scale bar = 10 µm. e, f, Trigeminal ganglion neurons plated in microfluidic chambers with cell bodies on left and axons growingto right chamber. Vehicle or PLY was added to right chamber during Fura-2 calcium imaging. (e) Left, illustration created with BioRender.com (https://biorender.com). Right, representative fields of neurons; (f) Traces and quantification (f) of intracellular calcium levels in individual axons stimulated with vehicle or S. pneumoniae toxin pneumolysin (n = 4/group). Scale bar = 50 µm. g, Left, Representative Fura-2 calcium traces of individual trigeminal ganglion neurons stimulated with wildtype S. agalactiae or the isogenic ΔcylE toxin-deficient mutant bacteria (2×10⁷ c.f.u.), followed by capsaicin (1uM) and KCl (40 mM). Right, proportions of capsaicin non-responsive and capsaicin-responsive neurons that responded to wild-type or ΔcylE S. agalactiae (n = 6/group). h, Blood CGRP levels from Nav1.8-DTA mice and control littermates 24 h after injection of S. pneumoniae (3×10⁷ c.f.u.) (n = 3/group). Statistical analysis: (b, f, g, h) Unpaired two-sided t-tests. (a) One-way ANOVA with Tukey post-tests. *p < 0.05, **p < 0.01, ***p < 0.001. n = individual mice (a, b) and biologically independent samples from mouse primary cells (f–g) and tissues (h). Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Mean ±SEM. Exact p-values in Supplementary Table 1.

Extended Data Fig. 5 CGRP and RAMP1 signaling impair host response against bacterial meningitis.

a, Representative flow cytometry plots and quantification of total leukocytes (CD45⁺ gate) and monocytes (CD11b⁺Ly6G⁻Ly6C^hi gates) in the meninges of Ramp1 knockout (Ramp1^−/−) and control (Ramp1^+/+) mice 24 h after S. pneumoniae injection (n = 4/group). b, Representative flow cytometry plots and quantification of total leukocytes (CD45⁺ gate) and monocytes (CD11b⁺Ly6G⁻Ly6C^hi gates) 24h after S. pneumoniae injection in the meninges of mice treated with CGRP or vehicle (n = 10/group). c, Bacterial load 24h after injection of S. agalactiae in samples from mice treated with CGRP or vehicle (n = 5/group). d, Bacterial load in samples collected from mice treated with vehicle (n = 4 for blood; n = 5 for meninges and brain) or CGRP (CGRP 2 µg i.p., daily) and either isotype control (n = 5) or neutralizing anti-IL-10 (200 µg i.p., daily) (n = 5). Statistical analysis: (a, b, c) Unpaired two-sided t-tests. (d) One-way ANOVA with Tukey post-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns = not ANOVA with Tukey post-tests. *p < 0.05, **p < 0.01, ***p < 0.001. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Error bars = mean ±SEM. Box plots = median, IQR, min/max. Exact p-values in Supplementary Table 1.

Extended Data Fig. 6 Blockade of CGRP signaling affects bacterial meningitis.

a, Analysis of disease progression in mice treated with BIBN (300 µg/kg, i.p.) or vehicle control, starting at 6 h post injection with S. pneumoniae (n = 6/group). b, Bacterial load recovery from samples at 24 h post infection in mice treated with BIBN (300 µg/kg, i.p.) or vehicle control, starting at 6 h post injection with S. pneumoniae (n = 5/group). c, Bacterial load in samples collected from peripheral tissues 24 h after injection of S. pneumoniae in mice treated with BIBN (300 µg/kg, i.p.) or vehicle (n = 5/group). Statistical analysis: (a, b, c) Unpaired two-sided t-tests. (a, weight loss) One-way ANOVA with Tukey post-tests. (a, survival) Kaplan Meier with Mantel-Cox comparison. *p < 0.05, **p < 0.01, ****p < 0.0001. n = individual mice (a) and biologically independent samples from mouse tissues (b–c). Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Error bars = mean±SEM. Box plots = median, IQR, min/max. Exact p-values in Supplementary Table 1.

Extended Data Fig. 7 Single-cell RNA-sequencing analysis of meningeal cells.

a, Single-cell RNA-sequencing analysis of meningeal immune cells (CD45-positive cells). Left, heatmap showing normalized expression of 100 top cluster marker genes in meningeal immune cells of the meninges, with key marker genes highlighted. Right, UMAP visualization of the expression of key marker genes for each immune cell cluster (n = 10 pooled mouse meninges). b, Left, Illustration created with BioRender.com (https://biorender.com). showing single-cell RNA-sequencing analysis of meningeal nonimmune cells (CD45-negative cells). Center, Uniform Manifold Approximation and Projection (UMAP) visualizations of CD45-negative cell types in the meninges at baseline. Right, heatmap showing normalized expression of 100 top cluster marker genes in nonimmune cells of the meninges, with key marker genes highlighted (n = 10 pooled mouse meninges).

Extended Data Fig. 8 Transcriptional responses of meningeal immune cells to bacterial meningitis.

a, Single-cell RNA-sequencing analysis of meningeal immune responses to bacterial infection. Left, Uniform Manifold Approximation and Projection (UMAP) visualizations of CD45-positive cell types in the meninges at baseline and 24 h after injection of S. pneumoniae (meningitis). Right, heatmap showing normalized expression of 100 top cluster marker genes with key immune marker genes highlighted. b, Number of genes that were differentially expressed in each immune cell population during infection (baseline vs meningitis). c, Annotated GO biological processes of genes differentially expressed by the cluster of macrophages in response to infection (baseline vs meningitis), highlighting the enrichment of processes related to chemotaxis. d, Annotated GO biological processes and volcano plot of genes differentially expressed by the cluster of neutrophils in response to infection (baseline vs meningitis), highlighting upregulation of processes and genes related to antimicrobial activity. e, Annotated GO biological processes and volcano plot of genes differentially expressed by the cluster of monocytes in response to infection (baseline vs meningitis). (n = 10 pooled mouse meninges/group). Statistical analysis: (b, c, d, e) Fisher’s Exact score (enriched biological processes) and Wilcoxon rank-sum test (volcano plots of DEG), dashed purple line = p < 0.01.

Extended Data Fig. 9 Meningeal macrophages engulf bacteria and regulate immune responses against bacterial invasion.

a, Whole-mount confocal images of mouse meninges (dura mater) showing meningeal macrophages (Mrc1⁺ cells) associated with S. pneumoniae 24 h post-injection of CMTPX-labeled bacteria. Scale bar = 500 µm (left) and 50 µm (right). b, c, d, Tissue-specific impact of depletion of meningeal Mrc1⁺ macrophages by intracisternal injection of clodronate liposomes (CLL). b, Flow cytometric quantification of meningeal macrophages, monocytes, neutrophils, and dendritic cells 3 days after intracisternal injection with clodronate liposomes (CLL, 5 mL) or vehicle (5 mL) (n = 4/group). c, Representative flow panels and quantification of neutrophils (CD11b⁺Ly6G⁺ gates), monocytes (CD11b⁺Ly6G⁻Ly6C^hi gates), B cells (CD11b^–CD19⁺ gates), and T cells (CD11b^–CD3⁺ gates) 24 h after injection of S. pneumoniae in mice treated with CLL (5 μL) or vehicle. d, Representative flow panels and quantification of macrophages (CD11b⁺Mrc1⁺ gates), neutrophils (CD11b⁺Ly6G⁺ gates), and monocytes (Cd11b⁺Ly6G⁻Ly6C^hi gates) in the liver of mice treated with CLL (5 μL) or vehicle. (n = 4/group). Statistical analysis: (b, c, d) Unpaired two-sided t-tests. *p < 0.05, ***p < 0.001, ****p < 0.0001. n = biologically independent samples from mouse tissues. Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Box plots = median, IQR, min/max. Exact p-values in Supplementary Table 1.

Extended Data Fig. 10 CGRP and RAMP1 polarization of macrophage responses.

a, Phagocytic killing assay showing amount of S. pneumoniae recovered after incubation with macrophages (BMDM) in presence of CGRP or vehicle (n = 4/group). b, Concentration of chemotaxis-related mediators in macrophage supernatants after 24 h of incubation with vehicle (n = 3), S. pneumoniae (n = 5), or S. pneumoniae+CGRP (n = 5). c, Crem and Jdp2 expression determined by qPCR in macrophages after 4 h incubation with S. pneumoniae together with CGRP, PKA inhibitor (PKAi), or vehicle. Results are normalized to Beta-actin gene expression (n = 6/group). d, Crem, Jdp2, Ramp1, and Ccl7 transcript levels determined by qPCR in FACS purified macrophages from meninges of Pf4^ΔRamp1 mice (n = 5 for Crem and Jdp2; n = 4 for Ramp1 and Ccl7) or control mice (n = 4 for Crem, Jdp2, Ramp1, Ccl7) 24 h after injection of S. pneumoniae. Results are normalized to Beta-actin expression. e, Expression of chemotactic mediators determined by qPCR in meningeal macrophages from Nav1.8-DTA (n = 5) and control mice (n = 6). f, Flow cytometric quantification of total leukocytes (CD45⁺) and macrophages (Cd11b⁺Mrc1⁺) in meninges of Pf4^ΔRamp1 and control mice 24 h after S. pneumoniae injection (n = 5/group). g, Illustration created with BioRender.com (https://biorender.com). Bacterial pathogens S. pneumoniae and S. agalactiae invade the meninges, activating trigeminal nociceptors to induce release of CGRP. CGRP acts through its receptor RAMP1 on Pf4⁺Mrc⁺ meningeal macrophages, downregulating expression of chemokines, suppressing leukocyte recruitment and antimicrobial defenses. Nociceptor ablation or blockade of CGRP signaling enhances host defense against meningitis. Statistical analysis: (a, b, c) One-way ANOVA with Tukey post-tests. (d, e, f) Unpaired two-sided t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = biologically independent samples from mouse tissues (d–f) and cells (a–c). Each experiment was performed at least twice, and results presented are representative of 2 or more replicates. ns = not significant. Error bars = mean ±SEM. Box plots = median, IQR, min/max. Exact p-values in Supplementary Table 1.

Supplementary Figures

Supplementary Fig. 1. Gating strategy for flow cytometry. a, Gating strategy used in Figs. 1j, 3b,d, 5d and 6i–j, and in Extended Data Figs. 3a,b, 5a,b, 9c,d and 10f. Cells were separated from debris by FSC-A versus SSC-A. Singlets were gated in SSC-A versus SSC-H. Live cells were separated by DAPI exclusion (DAPI^– population). Immune cells were identified by positive staining for CD45. Myeloid subpopulations were identified as neutrophils (CD11b⁺Ly6G⁺ population), macrophages (Ly6G^–CD11b⁺MRC1⁺ population) and monocytes (Ly6G^–MRC1^–CD11b⁺Ly6C⁺ population). Lymphoid subpopulations were identified as B cells (CD11b^–CD19⁺ population) and T cells (CD11b^–CD3⁺ population). b, The gating strategy used to quantify meningeal immune cells in Extended Data Fig. 3d and meningeal phagocytes in Extended Data Fig. 9b included additional gating for dendritic cells (b, green dashed line) before quantification of monocytes. This gating strategy was used to quantify neutrophils (CD11b⁺Ly6G⁺ population), macrophages (Ly6G^–CD11b⁺MRC1⁺ population), dendritic cells (Ly6G^–MRC1^–CD11c⁺ population) and monocytes (Ly6G^–MRC1^–CD11c^–Ly6C⁺ population). Lymphoid subpopulations were identified as B cells (CD11b^–CD19⁺ population) and T cells (CD11b^–CD3⁺ population). Supplementary Fig. 2. Gating strategy for flow cytometry of blood and spleen. a–d, Gating strategy used in Extended Data Fig. 3c. a, Gating strategies for detection of myeloid cells in the blood. b, Gating strategies for detection of myeloid cells in the spleen. c, Gating strategies for detection of lymphocytes in the blood. d, Gating strategies for detection of lymphocytes in the spleen.

Supplementary Table 1

Table listing specific information on figure panels, type of sample measured, groups compared and P values.

Cite this article

Pinho-Ribeiro, F.A., Deng, L., Neel, D.V. et al. Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion.
Nature (2023). https://doi.org/10.1038/s41586-023-05753-x

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• Received: 22 March 2022

• Accepted: 23 January 2023

• Published: 01 March 2023

• DOI: https://doi.org/10.1038/s41586-023-05753-x