https://www.nature.com/articles/s41586-023-05753-x Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Advertisement Nature * View all journals * Search * Log in * Explore content * About the journal * Publish with us * Subscribe * Sign up for alerts * RSS feed 1. nature 2. articles 3. article * Article * Published: 01 March 2023 Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion * Felipe A. Pinho-Ribeiro^1,2, * Liwen Deng^1, * Dylan V. Neel ORCID: orcid.org/0000-0001-6964-9842^1, * Ozge Erdogan^3, * Himanish Basu ORCID: orcid.org/0000-0001-8236-1157^1, * Daping Yang^1, * Samantha Choi^1, * Alec J. Walker^4,5,6, * Simone Carneiro-Nascimento ORCID: orcid.org/0000-0002-6141-6080^ 7, * Kathleen He^1, * Glendon Wu^1, * Beth Stevens ORCID: orcid.org/0000-0003-4226-1201^4,5,6,8, * Kelly S. Doran^9, * Dan Levy^5,7 & * ... * Isaac M. Chiu ORCID: orcid.org/0000-0002-0723-4841^1 Show authors Nature (2023)Cite this article * 8043 Accesses * 1 Citations * 317 Altmetric * Metrics details Subjects * Antimicrobial responses * Meningitis * Neuroimmunology * Pain Abstract The meninges are densely innervated by nociceptive sensory neurons that mediate pain and headache^1,2. Bacterial meningitis causes life-threatening infections of the meninges and central nervous system, affecting more than 2.5 million people a year^3,4,5. How pain and neuroimmune interactions impact meningeal antibacterial host defences are unclear. Here we show that Nav1.8^+ nociceptors signal to immune cells in the meninges through the neuropeptide calcitonin gene-related peptide (CGRP) during infection. This neuroimmune axis inhibits host defences and exacerbates bacterial meningitis. Nociceptor neuron ablation reduced meningeal and brain invasion by two bacterial pathogens: Streptococcus pneumoniae and Streptococcus agalactiae. S. pneumoniae activated nociceptors through its pore-forming toxin pneumolysin to release CGRP from nerve terminals. CGRP acted through receptor activity modifying protein 1 (RAMP1) on meningeal macrophages to polarize their transcriptional responses, suppressing macrophage chemokine expression, neutrophil recruitment and dural antimicrobial defences. Macrophage-specific RAMP1 deficiency or pharmacological blockade of RAMP1 enhanced immune responses and bacterial clearance in the meninges and brain. Therefore, bacteria hijack CGRP-RAMP1 signalling in meningeal macrophages to facilitate brain invasion. Targeting this neuroimmune axis in the meninges can enhance host defences and potentially produce treatments for bacterial meningitis. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution Access options Access through your institution Access through your institution Change institution Buy or subscribe Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $29.99 per month cancel any time Learn more Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Rent or buy this article Get just this article for as long as you need it $39.95 Learn more Prices may be subject to local taxes which are calculated during checkout Additional access options: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support Fig. 1: Nociceptors regulate bacterial CNS invasion by S. pneumoniae and S. agalactiae. [41586_2023_5753_Fig1_HTML] Fig. 2: Bacteria activate nociceptors that release CGRP in meninges. [41586_2023_5753_Fig2_HTML] Fig. 3: CGRP and RAMP1 signalling contribute to bacterial meningitis. [41586_2023_5753_Fig3_HTML] Fig. 4: Loss of myeloid Ramp1 expression improves the meningeal response against infection. [41586_2023_5753_Fig4_HTML] Fig. 5: Meningeal macrophages are required for host defence against S. pneumoniae infection. [41586_2023_5753_Fig5_HTML] Fig. 6: CGRP suppresses meningeal macrophage immunity. [41586_2023_5753_Fig6_HTML] Data availability All scRNA-seq datasets and bulk RNA-seq datasets generated and analysed during this study have been deposited into the NCBI Gene Expression Omnibus database under accession number GSE221681. The reference mouse genome mm10 v.2020-A can be accessed under the assembly number GRCm38. Raw imaging or other datasets from this paper will be made available upon request to the corresponding author. References 1. Levy, D., Labastida-Ramirez, A. & MaassenVanDenBrink, A. 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Sannajust, E. Erhard, O. Clancy and A. Prystupa for technical help and analysis; A. Frey for manuscript feedback; and J. L. Gibbs for mentorship. This work was supported by National Institutes of Health grants R01AI130019 and R01DK127257 to I.M.C.; Burroughs Wellcome fund, the Kenneth Rainin Foundation, the Food Allergy Science Initiative, Fairbairn Lyme Initiative to I.M.C.; 2R01NS078263 and 5R01NS115972 to D.L.; P50MH112491 to the Conte Center; R01NS116716 to K.S.D; and T32GM007753 to D.V.N. K.H. was supported by the Harvard Medical School Undergraduate Immunology Summer Program. Author information Authors and Affiliations 1. Department of Immunology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA Felipe A. Pinho-Ribeiro, Liwen Deng, Dylan V. Neel, Himanish Basu, Daping Yang, Samantha Choi, Kathleen He, Glendon Wu & Isaac M. Chiu 2. Division of Dermatology, John T. Milliken Department of Medicine, Washington University School of Medicine in St Louis, St Louis, MO, USA Felipe A. Pinho-Ribeiro 3. Department of Restorative Dentistry and Biomaterial Sciences, Harvard School of Dental Medicine, Boston, MA, USA Ozge Erdogan 4. F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, USA Alec J. Walker & Beth Stevens 5. Harvard Medical School, Boston, MA, USA Alec J. Walker, Beth Stevens & Dan Levy 6. Stanley Center for Psychiatric Research, The Broad Institute of MIT and Harvard, Cambridge, MA, USA Alec J. Walker & Beth Stevens 7. Departments of Anesthesia, Critical Care and Pain Medicine, Beth Israel Deaconess Medical Center, Boston, MA, USA Simone Carneiro-Nascimento & Dan Levy 8. Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA, USA Beth Stevens 9. Department of Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA Kelly S. Doran Authors 1. Felipe A. Pinho-Ribeiro View author publications You can also search for this author in PubMed Google Scholar 2. Liwen Deng View author publications You can also search for this author in PubMed Google Scholar 3. Dylan V. Neel View author publications You can also search for this author in PubMed Google Scholar 4. Ozge Erdogan View author publications You can also search for this author in PubMed Google Scholar 5. Himanish Basu View author publications You can also search for this author in PubMed Google Scholar 6. Daping Yang View author publications You can also search for this author in PubMed Google Scholar 7. Samantha Choi View author publications You can also search for this author in PubMed Google Scholar 8. Alec J. Walker View author publications You can also search for this author in PubMed Google Scholar 9. Simone Carneiro-Nascimento View author publications You can also search for this author in PubMed Google Scholar 10. Kathleen He View author publications You can also search for this author in PubMed Google Scholar 11. Glendon Wu View author publications You can also search for this author in PubMed Google Scholar 12. Beth Stevens View author publications You can also search for this author in PubMed Google Scholar 13. Kelly S. Doran View author publications You can also search for this author in PubMed Google Scholar 14. Dan Levy View author publications You can also search for this author in PubMed Google Scholar 15. Isaac M. Chiu View author publications You can also search for this author in PubMed Google Scholar Contributions Conceptualization: F.A.P.-R. and I.M.C. Resources and bacterial strains: D.L., K.S.D. and B.S. Experimentation and acquisition of data: F.A.P.-R., L.D., O.E., S.C.-N., S.C., D.Y., G.W. and K.H. Data analysis: F.A.P.-R., D.V.N., H.B., D.Y., O.E., K.H. and A.J.W. Writing the manuscript: F.A.P.-R. and I.M.C., with input from all authors. Funding acquisition: I.M.C. Corresponding author Correspondence to Isaac M. Chiu. Ethics declarations Competing interests I.M.C. and F.A.P-R. are named inventors on US patent application 2021 /0145937A1, 'Methods and compositions for treating a microbial infection', which includes targeting CGRP and its receptors to treat infections. The Chiu Lab receives research support from Abbvie/ Allergan and Moderna. Peer review Peer review information Nature thanks Victor Nizet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Additional information Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Extended data figures and tables 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 um. 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 mm. 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 mm. 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 mm. 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 mm. 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 mm. 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 ug, 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 um. 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 um. 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 um. g, Left, Representative Fura-2 calcium traces of individual trigeminal ganglion neurons stimulated with wildtype S. agalactiae or the isogenic DcylE toxin-deficient mutant bacteria (2x10^7 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 DcylE S. agalactiae (n = 6/group). h, Blood CGRP levels from Nav1.8-DTA mice and control littermates 24 h after injection of S. pneumoniae (3x10^7 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 ug i.p., daily) and either isotype control (n = 5) or neutralizing anti-IL-10 (200 ug 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 ug/ 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 ug/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 ug/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 um (left) and 50 um (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 mL) 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 mL) 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^DRamp1 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^DRamp1 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 information 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. Reporting Summary Supplementary Table 1 Table listing specific information on figure panels, type of sample measured, groups compared and P values. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and Permissions About this article Verify currency and authenticity via CrossMark 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 Download citation * Received: 22 March 2022 * Accepted: 23 January 2023 * Published: 01 March 2023 * DOI: https://doi.org/10.1038/s41586-023-05753-x Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. 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