Interactions between the immune and central nervous systems strongly influence brain health. Although the blood–brain barrier restricts this crosstalk, we now know that meningeal gateways through brain border tissues facilitate intersystem communication. Cerebrospinal fluid (CSF), which interfaces with the glymphatic system and thereby drains the brain’s interstitial and perivascular spaces, facilitates outward signaling beyond the blood–brain barrier. In the present study, we report that CSF can exit into the skull bone marrow. Fluorescent tracers injected into the cisterna magna of mice migrate along perivascular spaces of dural blood vessels and then travel through hundreds of sub-millimeter skull channels into the calvarial marrow. During meningitis, bacteria hijack this route to invade the skull’s hematopoietic niches and initiate cranial hematopoiesis ahead of remote tibial sites. As skull channels also directly provide leukocytes to meninges, the privileged sampling of brain-derived danger signals in CSF by regional marrow may have broad implications for inflammatory neurological disorders.
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We thank J.-W. Veening for providing fluorescent bacteria and K. Joyes for editing the manuscript. This work was funded in part by US federal funds from the National Institutes of Health (grant nos. HL158040, HL142494, HL139598, HL125428, NS108419 and HL135752), the Global Research Lab program (grant no. NRF-2015K1A1A2028228) and the National Priority Research Center program (grant no. NRF-2021R1A6A1A03038865) of the Korean Research Foundation. M.H. was supported by an American Heart Association Career Development Award (no. 19CDA34490005).
M.N. has received funds or material research support from Lilly, Alnylam, Biotronik, CSL Behring, GlycoMimetics, GSK, Medtronic, Novartis and Pfizer, as well as consulting fees from Biogen, Gimv, IFM Therapeutics, Molecular Imaging, Sigilon, NovoNordisk and Verseau Therapeutics. The other authors declare no competing interests.
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a, Experimental outline. Ex-vivo z-stack (54 μm stack at 1 μm/step) of occipital, parietal and frontal skull cortex after IC and IV injection of fluorescently labeled dextran. Bone is visualized by second harmonic generation around channels b, Imaging of CSF tracer outflow through channels in different skull bones, assessed in n=2 mice. Bar graphs depict the proportion of skull channels that were positive for CSF tracer.
a, Ex vivo imaging of whole-mount skull 10 min after intracisternal (IC) injection of ovalbumin. Intravenous(IV) injection of CD31/Sca1 labeled vasculature and IV osteosense the bone. b, Ex vivo imaging of tibia 10 minutes after intracisternal injection of ovalbumin. c, Imaging 30 minutes after intracisternal injection of ovalbumin. Data is representative of 2 independent experiments.
qPCR analysis of meninges isolated from either sham controls that were intracisternally injected with artificial CSF or mice 48 hours after intracisternal infection for relative expression analysis of a, Il1β, b, Il6 and c, Tnfα (mean ± SD; n=6 mice per group; P values represent an unpaired two-tailed t-test from a single experiment). d, Raw images obtained by whole mount ex vivo imaging of the skull 48 hours after intracranial sham or S. pneumoniae injection. First representative image is the original data from Fig. 5b while the second set represents additional examples of channel morphology and bacterial propagation. Green arrow highlights bacteria (scale: 50 and 25 μm).
a, Experimental outline for calvarial hematopoietic progenitor analysis. b, Flow cytometry gating. c, Quantitation of calvarial BrdU+ common myeloid progenitors (CMP) 6 hours after intracisternal sham or S. pneumococci injection. (n=11 sham and 12 meningitis, P value represents an unpaired, two-tailed t-test). d, Quantitation of calvarial BrdU+ CMP 24 hours after intracisternal sham or S. pneumococci injection. (n=6 mice per group, P value represents an unpaired, two-tailed t-test) e, Quantitation of calvarial BrdU+ LSK 24 hours after intracisternal sham or S. pneumococci injection. (n=6 mice per group, P value represents an unpaired, two-tailed t-test).
a, Experimental outline of calvarial leukocyte analysis. b, Flow cytometry gating. c-f, Quantitation of calvarial leukocytes 6 hours after intracisternal S. pneumococci injection shows neutrophils, monocyte subsets and total lymphocytes (n=5 mice per group). g-j, Quantitation of calvarial leukocytes 12 hours after S. pneumococci injection including neutrophils (g), Ly6Chi monocytes (h), Ly6Clo monocytes (i) and total lymphocytes (j) (n=9 sham and 8 meningitis). k-n, Quantitation of calvarial leukocytes 24 hours after S. pneumococci injection including neutrophils (k), Ly6Chi monocytes (l), Ly6Clo monocytes (m) and total lymphocytes (n) (n=6 mice per group). (P values represent unpaired, two-tailed t-tests, data are mean values ± SD).
a, Experimental outline. b, flow cytometry plots of control meninges (upper panel) and meninges 48 hours after infection. c, quantification of CD11b+ myeloid cells and d, Ly6G+ neutrophils in meninges (n=4 sham and 7 meningitis mice, P values represent unpaired, two-sided t tests, data are mean values ± SD).
a, Experimental outline indicating skull marrow transplantation, followed by induction of meningitis 4 weeks later. b, flow plots and (c) quantitation of myeloid cell chimerism in irradiated skull versus lead-shielded tibia 4 weeks after transplantation (n=7 recipient mice, P value represents unpaired, two-tailed t test, data are mean values ± SD). d, flow plots and (e) quantification of myeloid cell chimerism in the meninges and in blood (n=7 recipient mice, P value represents unpaired, two-tailed t tests, data are mean values ± SD).
a, Experimental groups include wild type and Myd88−/− mice. The skull marrow was assessed by flow cytometric staining for lineage markers Sca-1 and c-kit. b, Flow cytometry plots and c, quantitation of LSK % as a total of all live lineage negative single cells in the calvarial marrow of steady-state Myd88−/− or wild-type C57/Bl6 mice (n=9 sham and 6 meningitis mice, P value represents an unpaired, two-tailed t-test, data are mean values ± SD). d, Experimental outline. Non-irradiated wild type recipient mice received a mix of 40,000 LSK from wild type donors (labeled with the membrane dye DiD) and from Myd88-/- donors (labeled with Dil). e, Flow cytometry gating and f, analysis of the skull bone marrow 3 days later showed a similar seeding of LSK irrespective of phenotype (n=4 mice per group, unpaired, two-tailed t-test).g, Experimental outline of calvarial progenitor analysis in Myd88−/− mice with and without meningitis. h, Flow cytometry gating. i, Quantitation of calvarial BrdU+ CMP 12 hours after intracisternal sham or S. pneumococci injection. (n=11 sham and 12 meningitis mice per group, unpaired two-tailed t-test, data are mean values ± SD). j, Quantitation of calvarial BrdU+ LSK 24 hours after intracisternal sham or S. pneumococci injection. (n=6 mice group, unpaired two-tailed t-test, data are mean values ± SD).
IVM of perivascular CSF tracer flow along a dural vessel. Intravenously injected dextran is shown in red, IC injected ovalbumin in green. Video depicts step-wise progression through a 127-μm z-stack acquired at 1 μm per slice.
Ex vivo acquired z-stack of the internal skull cortex showing the CSF tracer (IC injected dextran, green) traveling along channel vessels (intravenously injected dextran, red) into the marrow.
A 3D rendering of an optically cleared skull–brain specimen highlighting channels in a sham control mouse. Vasculature was labeled with intravenously injected CD31/Sca-1 antibodies and bone with osteosense.
A 3D rendering of an optically cleared skull–brain specimen from a mouse with meningitis 48 h after intracisternal injection of GFP+ S. pneumoniae showing bacterial propagation (green) in the subarachnoid space adjacent to skull channels.
A 3D rendering of a maximal intensity projection acquired by IVM of skull marrow in a sham control mouse.
A 3D rendering of a maximal intensity projection acquired by IVM of skull marrow-containing GFP+ S. pneumoniae (green) in a mouse with meningitis.
A 3D rendering of skull channels in a sham control mouse. Vasculature was labeled with intravenous injection of CD31/Sca1 antibodies and bone with osteosense.
A 3D rendering of a skull channel in a mouse with meningitis showing GFP+ S. pneumoniae (green) inside the channel. Vasculature was labeled with intravenous injection of CD31/Sca1 antibodies and bone with osteosense.
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Pulous, F.E., Cruz-Hernández, J.C., Yang, C. et al. Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis. Nat Neurosci 25, 567–576 (2022). https://doi.org/10.1038/s41593-022-01060-2
Nature Neuroscience (2022)