Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels

Abstract

It remains unclear how immune cells from skull bone marrow niches are recruited to the meninges. Here we report that cerebrospinal fluid (CSF) accesses skull bone marrow via dura-skull channels, and CSF proteins signal onto diverse cell types within the niches. After spinal cord injury, CSF-borne cues promote myelopoiesis and egress of myeloid cells into meninges. This reveals a mechanism of CNS-to-bone-marrow communication via CSF that regulates CNS immune responses.

Conflict of interest statement

Competing interests statement

J.K. is a scientific advisor, holds shares of, and has a licensing agreement with PureTech. The remaining authors declare no competing interests.

© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.

Figures

Extended Data Fig. 1.. Characterization of stem and immune cell populations in the basal skull marrow.

a, Flow cytometry gating strategy for major immune populations in the skull bone marrow. b, Absolute numbers of CD45+ cells in the dorsal and basal skull marrow. n = 3 mice. Mean ± SEM. c, Relative frequencies of immune populations in the dorsal skull, basal skull, and tibial bone marrow. n = 3 mice. Mean ± SEM.

Extended Data Fig. 2.. Characterization of differences between the skull and tibial marrow populations.

a, Dot plot demonstrating scaled gene expression and percentage of cells expressing genes for cluster phenotyping markers for bone marrow cell types from scRNA-seq analysis. b, Analysis of cluster proportions in skull and tibial bone marrow. c-f, Volcano plots of differentially expressed genes in neutrophils, monocytes, macrophages, and HSCs. Magenta dots represent upregulated transcripts, while cyan dots represent downregulated transcripts in skull populations compared to the tibia. y-axes represent adjusted log2 p value for cluster changes between skull and tibia. Dotted line represents an adjusted p value of 0.05 (general linear mixed model with Benjamini-Hochberg correction). g-j, Top 10 downregulated gene ontology pathways in skull vs. tibia for differentially expressed genes in neutrophils, monocytes, macrophages, and HSCs. k, Dot plot of receptor expression in skull bone marrow cells, scaled by gene expression and percentage of cells expressing the gene, showing expression of receptors for which there is a cognate CSF ligand.

Extended Data Fig. 3.. Effects of AMD3100 on immune cell composition of the dura and bone marrow.

a, Experimental design for injections for skull bone marrow egress experiments. AMD3100 (10 μg) or artificial cerebrospinal fluid (aCSF) was injected intra-cisterna magna (i.c.m.), and mice were left for 24 hours. The following day, tissues were processed for immunolabeling or flow cytometry. b, Representative images of Ly6b+ cells and CD3+ cells in non-sinus regions of the dura. Scale bar: 200 μm. c, d, Regional analysis of Ly6b+ myeloid and CD3+ cells in the dura following AMD3100 administration. n = 3 mice per group. Data are means ± SEM, p values represent two-way ANOVA with Sidak’s post hoc test. e, Flow cytometry gating strategy for neutrophils, Ly6Chi monocytes, macrophages, and T cells in the bone marrow following AMD3100 administration. f, g, Relative numbers of neutrophils, Ly6Chi monocytes, macrophages, and T cells in the skull and tibial bone marrow 24 hours following i.c.m. AMD3100 administration. n = 5 mice per group. Data are means ± SEM, p values represent a two-sided Student’s t test.

Extended Data Fig. 4.. Laminectomy does not affect CSF efflux to skull bone marrow.

a, Laminectomy, or sham surgery, was performed on mice and 3 hours later OVA-488 was injected into the cisterna magna. Tissues were harvested 1 hour later for flow cytometry. Representative flow plots of macrophages in skull and tibia bone marrow with either sham surgery or laminectomy. b, Quantification of i.c.m. injected OVA uptake in macrophages following sham surgery or laminectomy. n = 5 mice per group. Data are means ± SEM, p values represent a two-way ANOVA. c, Representative flow plots of i.c.m. anti-c-Kit-PE staining in LSKs in skull and tibia bone marrow with either sham surgery or laminectomy. d, Quantification of i.c.m. injected cKit-PE uptake in LSKs following sham surgery or laminectomy. n = 5 mice per group. Data are means ± SEM, p values represent a two-way ANOVA with Sidak’s post hoc test.

Extended Data Fig. 5.. Effects of spinal cord injury on vertebral bone marrow.

a, Experimental paradigm for spinal cord injury experiments. Spinal cord injury (SCI) or laminectomy (sham) was performed, and at 3 hours post-injury vertebra adjacent to the site of injury were processed for flow cytometry. b-e, Relative numbers of monocyte dendritic precursors (MDPs), common monocyte progenitors (cMoPs), Ly6Chi monocytes, and actively proliferating (Ki-67+, EdU+) monocytes in vertebral bone marrow. n = 5 mice per group. p values represent a two-sided Student’s t test. f, Multiplexed measurement of cytokines and chemokines in the CSF of sham and SCI mice using Luminex. n = 5. p values represent two-sided t tests with Holm-Sidak’s multiplicity adjustment. Data are means ± SEM.

Extended Data Fig. 6.. Intracisternal injection of LPS enhances hematopoiesis in skull bone marrow and triggers myeloid egress to the dura.

a, LPS (1.25 μg, 4 μL) was injected into the skull bone marrow. After 24 hours, skullcaps and dura were processed for flow cytometry. Representative flow plots of neutrophils in the dura in aCSF and LPS-treated mice. b, Representative flow plots of Ly6Chi monocytes in the dura in aCSF and LPS-treated mice. c, Representative flow plots of LSKs in the skull BM of aCSF and LPS-treated mice. d, Quantification of the proportion of CD45+ immune cells and absolute number of neutrophils and Ly6Chi monocytes in the dura of aCSF and LPS-treated mice. n = 5 mice per group. Mean ± SEM. p values represent a two-sided Student’s t test. e, Quantification of the proportion of live cells and the proportion of actively proliferating Ki67+ stem/progenitor (LSK, MDP, cMoP, GMP, GP) and myeloid (neutrophils, Ly6Chi monocytes) cells in the skull bone marrow of aCSF and LPS-treated mice. n = 5 mice per group. Mean ± SEM. p values represent a two-sided Student’s t test.

Extended Data Fig. 7.. Summary schematic for proposed mechanism.

Brain interstitial fluid and cerebrospinal fluid can efflux to skull bone marrow during healthy conditions. During CNS insults—for example pathogenic infections or spinal cord injury—CSF-derived cues can promote skull bone marrow hematopoiesis and egress of myeloid cells to underlying dura. HSC; hematopoietic stem cell, CSF; cerebrospinal fluid, ISF; interstitial fluid, SAS; subarachnoid space, BM; bone marrow, CNS, central nervous system.

Fig. 1.. CSF accesses skull bone marrow niches.

a, Representative maximum intensity projection of a decalcified and cleared skull cap-dura whole mount 1 hour after an intra-cisterna magna (i.c.m.) injection of OVA-A594. Scale bar: 2 mm. b, Z-sections through the dura, cortical skull bone, and trabecular bone marrow in a region of interest from the skull cap-dura whole mount. Arrowheads highlight perivascular OVA accumulation. Arrows denote OVA-positive cells within the bone marrow cavity. Scale bar: 100 μm. c, Anatomy of skull bone marrow niches in the dorsal and basal skull. d, Sagittal sections through the dorsal and basal skull. Scale bar: 3 mm. e, High-magnification images of tracer accumulation in the dorsal (left) and basal (right) bone marrow of the skull 1 hour after an i.c.m. injection of OVA-A594. Scale bars: 50 μm. f, Representative two-photon image of skull bone marrow in a live mouse 30 min following administration of 70 kDa FITC-dextran (i.v.) and OVA-594 (i.c.m.). Scale bar: 100 μm. g, Gating strategy and representative plots of i.c.m. OVA-A488 labelling in bone marrow macrophages. h, Gating strategy and representative plots of i.c.m. c-Kit-PE labelling in bone marrow HSCs. i-k, Experimental design, percentage of HSCs positive for i.c.m. c-Kit-PE, and percentage of macrophages positive for i.c.m. OVA-A488 1 hour post injection. n = 4 or 5 mice. Data are means ± SEM, p values represent a one-way ANOVA with Tukey’s post-hoc test. l-n, Experimental design, percentage of HSCs positive for i.c. c-Kit-PE, and percentage of macrophages positive for i.c. OVA-A594 1 hour post injection. n = 4 mice. Data are means ± SEM, p values represent a one-way ANOVA with Tukey’s post-hoc test. o, p, Experimental design and percentage of bone marrow HSCs positive for c-Kit-PE 1 hour after i.c.m. injection in postnatal day 7, 14, 21, adult (2–3 months old), and aged (20–24 months old) mice. n = 4 mice. Data are means ± SEM, p values represent a two-way ANOVA with Dunnett’s post-hoc test versus 2–3 months.

Fig. 2.. Functional interactions between CSF and the skull bone marrow niche.

a, b, t-distributed stochastic neighbor embedding (tSNE) visualizations of single-cell RNAseq from dorsal skull and tibial bone marrow from 2 month old mice colored by cell type a, or sample b. c, Chord plot detailing between CSF ligands identified by unlabeled liquid chromatography-mass spectrometry (LC-MS) and receptors on skull bone marrow hematopoietic stem cells (HSCs), macrophages, monocytes, and neutrophils identified by sc-RNA-seq. d, e, Gene ontology pathway analysis on receptor genes with at least one CSF ligand in monocytes and neutrophils. f, Representative immunohistochemistry of CD3+ and Ly6b+ cells at the superior sagittal sinus (S.S. Sinus) and transverse sinus (T. Sinus) of the dura mater, 24 hours after an intra-cisterna magna (i.c.m.) injection of 10 μg AMD3100 or artificial cerebrospinal fluid (aCSF). Scale bar: 200 μm. g-j, Flow cytometry gating strategy and frequency of Ly6Chi monocytes, neutrophils, and T cells proportions following an i.c.m. injection of 10 μg AMD3100 or aCSF. n = 5 mice per group. Data are means ± SEM, p values represent a two-sided Student’s t-test.

Fig. 3.. CSF-contained cues mobilize the skull bone marrow in response to CNS injury.

a, Experimental design for spinal cord injury experiments. Spinal cord injury was performed in 2 month old mice at T7 following laminectomy (sham group included laminectomy). EdU (10 mg/kg) was injected one hour prior to harvest. Three hours later, skull bone marrow was processed for flow cytometry. b, Gating strategy for monocyte dendritic precursors (MDP), common monocyte progenitors (cMoP), and actively proliferating Ki67+ EdU+ Ly6Chi monocytes. c, Schematic detailing differentiation of the monocyte lineage from common myeloid progenitors (CMPs). d-g, Frequency of MDPs, cMoPs, Ly6Chi monocytes, and actively proliferating Ki67+ EdU+ Ly6Chi monocytes in the skull bone marrow of sham and spinal cord injury animals. n = 5 mice per group. Data are means ± SEM, p values represent a two-sided Student’s t-test. h, Experimental design for CSF transfer experiments post spinal cord injury. CSF was harvested from the cisterna magna of sham or spinal cord injury mice 3 hours post injury, and 10 μL was transferred to naive mice via intra-cisterna magna (i.c.m.) injection. After 6 hours, dural meninges from mice with CSF transferred were harvested. i, Flow cytometry gating strategy for dural Ly6Chi monocytes following transfer of sham or spinal cord injury CSF. j, k, Absolute numbers and frequency of monocytes in the dura following transfer of CSF from sham and spinal cord injury mice. n = 5 mice per group. Data are means ± SEM, p values represent a two-sided Student’s t-test.