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Abstract

Neuroinflammatory diseases, such as multiple sclerosis, are characterized by invasion of the brain by autoreactive T cells. The mechanism for how T cells acquire their encephalitogenic phenotype and trigger disease remains, however, unclear. The existence of lymphatic vessels in the meninges indicates a relevant link between the CNS and peripheral immune system, perhaps affecting autoimmunity. Here we demonstrate that meningeal lymphatics fulfill two critical criteria: they assist in the drainage of cerebrospinal fluid components and enable immune cells to enter draining lymph nodes in a CCR7-dependent manner. Unlike other tissues, meningeal lymphatic endothelial cells do not undergo expansion during inflammation, and they express a unique transcriptional signature. Notably, the ablation of meningeal lymphatics diminishes pathology and reduces the inflammatory response of brain-reactive T cells during an animal model of multiple sclerosis. Our findings demonstrate that meningeal lymphatics govern inflammatory processes and immune surveillance of the CNS and pose a valuable target for therapeutic intervention.

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Data availability

All RNA-seq datasets are available at GEO: LEC experiment, GSE99743; T cell lymphatic ablation experiment, GSE99764. The data that supports the finding of the study are available from the corresponding authors upon reasonable request.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

We thank S. Smith for editing the manuscript. We also thank all the members of the Kipnis lab and the members of the Center for Brain Immunology and Glia (BIG) for their valuable comments during multiple discussions of this work. This work was supported by grants from the National Institutes of Health (AG034113 and NS096967), National Multiple Sclerosis Society (NMSS), and the German Research Council (DFG; CRC-TR-128, B11) to J.K.; National Institutes of Health HL073402 to G.O.; Howard Hughes Medical Institute Medical Research Fellow Program to M.Q.D. and by Henry M. Weitzner, Edna K. Weitzner, Dorothea M. Weitzner, Morton L. Weitzner and Lymphatic Education & Research Network postdoctoral fellowship to A.L.

Author information

Author notes

  1. These authors contributed equally: Antoine Louveau, Jasmin Herz.

Affiliations

  1. Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA

    • Antoine Louveau
    • , Jasmin Herz
    • , Maria Nordheim Alme
    • , Andrea Francesca Salvador
    • , Michael Q. Dong
    • , Kenneth E. Viar
    • , S. Grace Herod
    • , James Knopp
    • , Joshua C. Setliff
    • , Alexander L. Lupi
    • , Sandro Da Mesquita
    • , Elizabeth L. Frost
    • , Alban Gaultier
    • , Tajie H. Harris
    • , John R. Lukens
    • , Igor Smirnov
    • , Christopher C. Overall
    •  & Jonathan Kipnis
  2. Department of Neuroscience, University of Virginia, Charlottesville, VA, USA

    • Antoine Louveau
    • , Jasmin Herz
    • , Maria Nordheim Alme
    • , Andrea Francesca Salvador
    • , Michael Q. Dong
    • , Kenneth E. Viar
    • , S. Grace Herod
    • , James Knopp
    • , Joshua C. Setliff
    • , Alexander L. Lupi
    • , Sandro Da Mesquita
    • , Elizabeth L. Frost
    • , Alban Gaultier
    • , Tajie H. Harris
    • , John R. Lukens
    • , Igor Smirnov
    • , Christopher C. Overall
    •  & Jonathan Kipnis
  3. Department of Clinical Medicine, University of Bergen, Bergen, Norway

    • Maria Nordheim Alme
  4. Department of Neurology, Haukeland University Hospital, Bergen, Norway

    • Maria Nordheim Alme
  5. Neuroscience Graduate Program, University of Virginia, Charlottesville, VA, USA

    • Andrea Francesca Salvador
    •  & Jonathan Kipnis
  6. Department of Biomedical Engineering, University of Virginia, Charlottesville, VA, USA

    • Rui Cao
    •  & Song Hu
  7. Northwestern University, Feinberg School of Medicine, Chicago, IL, USA

    • Guillermo Oliver
  8. Gutenberg Research Fellowship Group of Neuroimmunology, Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine Main Neuroscience Network (rmn²), University Medical Center of the Johannes Gutenberg University Mainz, Mainz, Germany

    • Jonathan Kipnis

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Contributions

A.L. designed and performed most of the experiments and wrote the manuscript; J.H. designed and performed the experiments related to the spinal cord lymphatics and CCR7-dependent cellular migration; M.N.A. initiated experiments related to cribriform plate and nasal lymphatics; A.F.S. assisted A.L. with experimental procedures and analysis; M.Q.D. assisted J.H. with spinal cord lymphatic related experiments; K.E.V. assisted A.L. with experimental procedures; G.H. assisted A.L. with experimental procedures; J. Knopp assisted A.L. with experimental procedures; J.S. assisted A.L. with experimental procedures; A.L.L. assisted J.H. with experimental procedures; S.D.M. helped with experimental procedures; E.L.F. performed the multiplex experiment on CSF; I.S. performed ligation surgeries, intracranial pressure measurements, and harvested CSF; R.C. performed the experiments related to photoacoustic microscopy; S.H. designed the experiments related to photoacoustic microscopy; A.G. helped with initial EAE induction and scoring; T.H.H. contributed intellectually to experimental design through multiple discussions; J.R.L. designed the multiplex experiment; C.C.O. provided bioinfomatic analysis of the data; G.O. provided Prox1het mice and helped with experiments design related to these mice and writing; J. Kipnis designed experiments, supervised the work, and wrote the manuscript.

Competing interests

J. Kipnis is an Advisor to PureTech Health/Ariya.

Corresponding authors

Correspondence to Antoine Louveau or Jonathan Kipnis.

Integrated supplementary information

  1. Supplementary Figure 1 Lack of dural leakage and characterization of hot spots of the meningeal lymphatic vessels.

    a, Representative images of the lymphatic vessels immunostained by both an i.c.v. injected anti-Lyve-1A488 and exogenously applied anti-Lyve-1A660 at different time points after i.c.v. injection. Inset at 5 and 15 min illustrate the initial points where the i.c.v. injected anti-Lyve-1A488 labelled the meningeal lymphatics. Inset at 60 min illustrate the lack of staining of the middle meningeal artery lymphatics. Scale bar = 1000 µm, 75 µm (insets). Representative of 2-3 independent mice per group. b, Representative images of the meningeal beads around the meningeal lymphatics of the transverse sinuses after i.c.m., i.c.v. or i.n. injection of 2µl of fluorescently conjugated beads. Scale bar = 300 µm, 75 µm (insets). Arrowheads in the i.n. injection inset points towards individual beads. Representative of 3 independent mice. c, Representative images of whole mount meninges immunostained for macrophages (Iba1 – green) one hour after i.c.m. injection or ex-vivo exposure to fluorescent ovalbumin. Scale bar = 500 µm, 120 µm (insets). Representative of 2-3 independent mice per group. d, Quantification of the ovalbumin uptake by meningeal macrophages in different meningeal regions one hour after i.c.m. injection or ex-vivo exposure to ovalbumin (mean ± s.e.m.) ei, Representative images of Ova diffusion in the skullcap meninges (dura) and at the surface of the brain (pia). Scale bar = 1000 µm. eii, Representative images of the diffusion of Ova (both perivascular diffusion and macrophage uptake) on different region of the cerebellum 10 min after i.c.m. injection. Scale bar = 300 or 200 µm. Images in e, are representative of 3 independent mice. f, Representative images of VE-Cadherin and Claudin-5 expression on lymphatic sprouts of the meninges (upper panel) and diaphragm (lower panel) of adult wild-type mice. Scale bar = 20 µm (meninges) and 30 µm (Diaphragm). Representative images of sprouts from 2 mice. g, Representative side view of the meningeal lymphatics along the upper region of the superior sagittal sinus by multiphoton microscopy. The meningeal lymphatics (Prox1GFP – green) are detected below the skull and the dura matter (secondary harmonic – blue). Yellow arrow points to sub-arachnoid lymphatic extensions. Scale bar = 40 µm. Representative of 2 independent animals. h, Prox1GFP mice were injected intravenously (i.v.) with 5µl of Qdot655 (diluted in 95µl of saline). The transverse sinus was imaged through a thinned skull ~ 5min after injection. Representative images of the complex meningeal lymphatics associated with the transverse sinuses at the lymphatic extension-rich area (arrows point to lymphatic extensions). Arrow on the 3D reconstruction points to a descending lymphatic extension directed towards the SAS. Scale bar = 60 µm. Representative of 2 independent animals.

  2. Supplementary Figure 2 Spinal cord lymphatic and hot spots.

    a, Scheme of the extension of the meningeal lymphatic vasculature in the brain and spinal cord meninges. b, Representative images of the entire CNS dorsal meninges dissected from a Prox1CreERT2XRosatdTOMATO mice (Red) after 3 weeks on tamoxifen. Scale bar = 2 mm. c, Representative images of spinal cord lymphatic organization (Lyve-1 – red) around the nerve roots (green) of the spinal cord. Scale bar = 300 µm, 10 µm (inset). d, Representative images of Prox1CreERT2XRosatdTOMATO mice (Red) after 3 weeks on tamoxifen and Lyve-1 (grey) expression by the lymphatics of the nerve roots. Scale bar = 30 µm. e, Representative images Podoplanin (green) and VEGFR3 (pink) expression by the lymphatics (Lyve-1 – grey) of the nerve roots. Scale bar = 40 µm. Images from b-e are representative of 3 independent mice. f, Representative images of the cerebellar ring lymphatics (Lyve-1 – grey). Yellow arrowheads points to button-like structures along the cerebellar lymphatics. Scale bar = 1000 µm. Representative of 5 independent mice. g, Representative images of beads distribution around the nerve roots 2h after i.c.m. or i.c.v. injection of beads. Scale bar = 2 mm. Representative of 3 independent mice per group. h-i, Representative images of the meningeal lymphatic network on the side (h) and at the base (i) of the skull. Arrows point to valve like structures characteristic of collecting lymphatics. Scale bar = 1500 µm, 400 µm (insets). Representative of 2 independent mice. j, Representative image of beads distribution at the bottom of the skull, around lymphatic vessels (Lyve-1 – red). Scale bar = 1500 µm, 400 µm (insets). Representative of 2 independent mice.

  3. Supplementary Figure 3 Drainage of T cells and dendritic cells into the cervical lymph nodes in a CCR7-dependent manner.

    a, Representative multiphoton images of endogenous T cells (LcktdTOMATO - red) localized within lymphatic extension (Prox1GFP - green) along the transverse sinus. Scale bar = 20 µm. b, Representative dot plots of GFP + CD4 + T cells in the dCLNs, sCLNs and lumbar lymph nodes at different time points after injection. c, Quantification of the percentage of GFP + CD4 + T cells in the dCLNs, sCLNs, Lumbar and ILN at 2, 6 and 12h post i.c.m. injection (mean ± s.e.m.; n = 10 mice/group; F(3,78) = 20.58; two-way ANOVA with Tukey’s multiple comparisons test). d, Quantification of the number of GFP + CD4 + T cells per dCLNs, sCLNs, Lumbar and ILN at 2, 6 and 12h post i.c.m. injection (mean ± s.e.m.; n = 10 mice/group; F(3,78) = 28.35; two-way ANOVA with Tukey’s multiple comparison test). e, CD4 T cells (isolated by negative selection from wild-type spleen and lymph nodes) were activated in vitro by CD3/CD28 stimulation followed by sustained IL-2 supplementation every other day for 9 days. Cells were then labeled with CFSE and mixed with Cell Tracer Violet-labeled, freshly isolated CD4 T cells. f, Representative image of i.c.m. injected in vitro activated T cells in the dCLNs 12h post injection. Scale bar = 100 µm, 45 µm (inset). g, Quantification of the density of activated T cells/mm2 of dCLNs at different time points post injection (mean ± s.e.m). h, Representative dot plots of naïve and activated T cells in the dCLNs 12h post injection. Both naïve and activated T cells are found draining into the dCLN from the subarachnoid space. The draining activated CD4 T cells bear an effector memory phenotype (CD44+CD62Llow/+). Representative of 3 independent mice. i, Representative images of control and PTX-treated T cells in the dCLNs of wild-type mice 12h after i.c.m. injection. Scale bar = 200 µm, 60 µm (inset). j, Quantification of the density of T cells/mm2 of dCLN (mean ± s.e.m.; pooled from 2 independent experiments; t = 3.901 df = 16; two-tailed unpaired t-test). k, Gating of the CCR7-WT and CCR7-KO DCs after LPS stimulation. l, Representative histograms of expression of CCR7, CD40, CD80 and CD103 by CCR7-WT and CCR7-KO DCs after LPS stimulation. Representative of 2 independent experiment. m, Representative images of the exogenously injected DCs (TAMRA – red) located within the meningeal lymphatics (Lyve-1 – white) of the transverse sinus. Scale bar = 20 µm. Representative of 3 independent mice. n, Scheme of the experiment illustrated in panels. (O-R). Meninges of KiKGR mice were exposed for 2 min to a violet light every 12h for photo-conversion. After 2 days, mice were injected i.c.m. with Poly(I:C) and peptide. Tissues were harvested 24h after injection. o, Representative gating strategy to identify dendritic cells. p, Representative dot plots of dendritic cells in B6 control and KiKGR control mice. Representative of 3 independent mice. q, Representative dot plots of photoconverted KiKR + dendritic cells in the dCLN, sCLN and ILN 24h after Poly(I:C) injection in converted mice. Representative of 4 independent mice. r, Quantification of the percentage of KiKR + dendritic cells in the dCLNs, sCLNs and ILNs of control and converted mice 24h after Poly(I:C) injection (mean ± s.e.m.; representative of 2 independent experiments; F(3,8) = 14.96; one-way ANOVA with Tukey’s multiple comparisons test). s, Representative images of i.c.m. injected DCs (CFSE – green) in dCLNs of wild-type mice 1, 2, and 4 days after i.c.m. injection. Scale bar = 200 µm, 50 µm (insets). Representative of 5 independent mice. t, Quantification of the density of the injected DCs/mm2 of dCLN and ILN at different time points after injection (mean ± s.e.m.; n = 4-5 mice/group; representative of 2 independent experiments). u, Representative image of dCLNs 24h after i.c.m. injection of CCR7-WT (CFSE – green) and CCR7-KO (TAMRA – red) DCs at a 1:1 ratio. Scale bar = 200 µm. v, Quantification of the density of CCR7-WT and CCR7-KO DCs (per mm2) in the dCLN of wild-type mice 24 and 48h post i.c.m. injection (mean ± s.e.m.; n = 4-7 mice/group; F(1,18) = 17.47; two-way ANOVA with Sidak’s multiple comparison test).

  4. Supplementary Figure 4 Drainage in the Prox1het mice, characterization of the cribriform plate lymphatics, and lethality of i.c.m. injection of tamoxifen and diphtheria toxin.

    a, Representative images of exogenously injected T cells (CFSE – green) in the dCLN of Prox1-WT and Prox1-Het mice 12h after i.c.m. injection. Scale bar = 200 µm, 100 µm (insets). b, Quantification of the density of T cells per mm2 of dCLN of Prox1-WT and Prox1-Het mice (mean ± s.e.m.; t = 10.72 df = 5; two-tailed unpaired t-test). c, Representative images of exogenously injected fluorescent microbeads (0.5 µm in diameter – red, 1 µm in diameter - green) in the dCLN of Prox1-WT and Prox1-Het mice 2h after i.c.m. injection. Scale bar = 200 µm, 80 µm (insets). d, Quantification of the percentage of beads coverage in the dCLN of Prox1-WT and Prox1-Het mice ((0.25µlof beads in a total of 2µl were injected) mean ± s.e.m.; F(1,9) = 3.038; two-way ANOVA with Sidak’s multiple comparisons test). e, Quantification of the average dCLN section size in Prox1-WT and Prox1-Het mice (mean ± s.e.m.; pooled from 2 independent experiments). f, Representative contour plots of T cells in the meninges of Prox1-WT and Prox1-Het mice. g, Quantification of the number of T cells (TCRb + ) in the meninges of Prox1-WT and Prox1-Het mice (mean ± s.e.m.; pooled from 2 independent experiment; Two-tailed unpaired t-test). h, Representative images of exogenously i.c.m. injected DCs (CFSE – green) in the dCLN of sham and ligated mice. Scale bar = 100 µm. i, Quantification of the density (per mm2) of exogenously i.c.m. injected DCs in the dCLN of sham and ligated mice (mean ± s.e.m.; t = 2.627 df = 7; two-tailed unpaired t test). j, Representative photographs of the mouse cribriform plate. k, Representative photograph of cribriform lymphatics (i) and representative images (Lyve-1 – red) on different sections of the mouse nose (ii). White arrowhead points to a cribriform lymphatic crossing the cribriform plate along the olfactory nerve (laminin – green). Laminin stains the vasculature but also the meninges surrounding the olfactory bulbs and the olfactory nerves. Scale bar = 300 µm. Note that the large majority of the cribriform plate lymphatics are localized on the CNS side of the bone, with some examples of crossing into the nasal side. Representative of 3 independent mice. l, Representative images of VEGFR3 (grey) expression by the cribriform plate lymphatics (Prox1GFP – green, Lyve-1 – red). Arrowheads point to area of colocalization of Prox1, Lyve-1 and VEGFR3. Scale bar = 100 µm. Representative of 2 independent mice. m, Representative images of podoplanin expression (PDPN – grey) by the cribriform plate lymphatics (Prox1GFP – green, Lyve-1 – red). Arrowheads point to area of colocalization of Prox1, Lyve-1 and PDPN). Scale bar = 100 µm. Representative of 2 independent mice. n, Representative images of CCL21 (green) expression by the cribriform lymphatics (Lyve-1 – red). Arrowheads point to CCL21+ vesicles inside the cribriform LEC. Scale bar = 100 µm. Representative of 2 independent mice. o, Representative images of a sagittal section of Prox1GFP (green) nose region immunostained with Lyve-1 (red). Inset illustrates the large lymphatic bundle localized at the base of the nose draining the lymphatic vasculature of the nasal region. Scale bar = 1000 µm. Representative of 2 independent mice. p, Representative images of the endogenous T cells (CD3e – red) around the lymphatics (Lyve-1 – green) of the cribriform plate of naïve mice. Arrowheads point to T cells. Scale bar = 100 µm. q, Quantification of the number of T cells (CD3e + ) on the CNS, nasal side or within the cribriform plate per section of wild-type mouse cribriform region (mean ± s.e.m.; F(2,12) = 57.22; one way ANOVA with Tukey’s multiple comparison test.) r, Representative images of fluorescently labeled T cells (CFSE – red) around the cribriform plate 12h after low (0.5µl/min) or high (4µl/min) speed of injection. Scale bar = 100 µm. s, Quantification of the number of T cells on the CNS and the nasal mucosa sides of the cribriform plate 12h after i.c.m. injection at 0.5, 1 or 4 µl/min (mean ± s.e.m.; F(2,8) = 11.98; two-way ANOVA with Sidak’s multiple comparison test). t-x, Adult Prox1CreERT2::DTAfl/fl and Prox1CreERT2 mice were injected i.c.m. with 2µl of 4-hydroxi-tamoxifen (TAM - 0.1mg/ml). t, Survival curve of Prox1CreERT2::DTAfl/fl and Prox1CreERT2 mice after i.c.m. injection of TAM (representative of 2 independent experiments). u, Representative images of the lymphatics (Lyve-1 – green) of the superior sagittal sinus of Prox1CreERT2::DTAfl/fl after injection of low-dose (100µg) of TAM. Scale bar = 150 µm. Representative of 2 independent mice. v, Survival curve of Prox1CreERT2::DTRfl/fl and Prox1CreERT2 mice after repeated i.c.m. injection of diphtheria toxin (DT) at different concentrations (250ng to 0.25ng; representative of 2 independent experiments). w, Representative images of the meningeal lymphatics (Lyve-1 – green) in Prox1CreERT2::DTRfl/fl 10 days after repeated i.c.m. injection of PBS or low dose of DT (25ng). Scale bar = 1000 µm. x, Quantification of the ratio of total lymphatic length over the length of the intracranial sinuses in mice injected i.c.m. with PBS or low dose of DT (mean ± s.e.m.).

  5. Supplementary Figure 5 Analysis of potential side effects of Visudyne treatment on CNS homeostasis.

    a, Representative images of the meningeal lymphatics (Lyve-1 – red) of the transverse sinuses 7 days after laser or Visudyne (i.c.m.) + laser treatment. Scale bar = 300 µm, 80 µm. b, Quantification of the ratio between the total lymphatic length and the sinus length of the superior sagittal sinus (SSS) and transverse sinuses (TS) 1 and 7 days after laser and Visudyne (i.c.m.) + laser treatment (mean ± s.e.m.; F(1,23) = 37.68; two-way ANOVA). c, Quantification of the average intracranial pressure (over 2 minutes of recording) of naïve mice, mice treated with Acetazolamide (20mg/kg), mice with bilateral jugular vein ligation, laser (PBS), Visudyne (i.c.m.) and Visudyne (i.c.m.) + laser treated mice (analysis 3 days after treatment (mean ± s.e.m.; pooled from 3 independent experiments; F(5,56) = 16.60; one-way ANOVA with Tukey’s multiple comparison tests). d, Representative intracranial pressure curves of a laser (PBS) and Visudyne (i.c.m.) + laser treated mice. Representative of 10 independent mice per group. e, Representative images of the CD31 (red) and Claudin-5 (green) expression on and around the transverse sinus of laser and Visudyne (i.c.m.) + laser treated mice (analysis 4 days after treatment). Scale bar = 300 µm, 75 µm (insets). Representative of 3 independent mice. f, Representative images of αSMA expression on the transverse sinus of laser and Visudyne (i.c.m.) + laser treated mice (analysis 4 days after treatment). Scale bar = 200 µm. Representative of 3 independent mice. g, Representative density plot of blood oxygenation of laser and Visudyne (i.c.m.) + laser treated mice recorded by photoacoustic microscopy. Scale bar = 500 µm. h, Quantification of arterial and venous oxygenation of the meningeal vasculature of laser and Visudyne (i.c.m.) + laser treated mice (mean ± s.e.m.). i, Representative density plot of meningeal blood flow speed in laser and Visudyne (i.c.m.) + laser treated mice by photoacoustic imaging. Scale bar = 500 µm. j, Quantification of meningeal blood flow speed in laser and Visudyne (i.c.m.) + laser treated mice (4 days after treatment; mean ± s.e.m.). k, Representative images of glia limitans integrity (GFAP – red) on the brain of laser and Visudyne (i.c.m.) + laser treated mice. Scale bar = 100 µm, 50 µm (insets). l, Representative images of pial integrity (Laminin – green) in the brain of laser and Visudyne (i.c.m.) + laser treated mice. Scale bar = 50 µm. Images in k and l are representative of 3 independent mice. m-n, Multiplex based measurements of inflammatory cytokines (m) and chemokines (n) in the CSF of control, laser and Visudyne (i.c.m.) + laser treated mice at 1 and 4 days after treatment. (mean ± s.e.m.; each biological samples is composed of pooled CSF from 3 independent mice). o, Representative images of macrophages (Iba1 – green) along the transverse sinus of laser, or Visudyne (i.c.m.) + laser treated mice 4 days after ablation. Scale bar = 100 µm. p, Quantification of the size of macrophages (Iba-1 + ) on the transverse sinus of laser and Visudyne (i.c.m.) + laser treated mice (mean ± s.e.m.). q, Quantification of the mean MHCII intensity (arbitrary units) by meningeal macrophages (Iba1+) around the transverse sinus of laser and Visudyne (i.c.m.) + laser treated mice (mean ± s.e.m.). r, Representative histograms of ICAM-1 and VCAM-1 expression by the endothelial cells (CD45- CD31 + ) of the brain in control, laser and Visudyne (i.c.m.) + laser treated mice at D1 and D4 post treatment. s, Quantification of the geometric mean fluorescence intensity of ICAM1 and VCAM1 expression by the brain and spinal cord endothelial cells of control, laser and Visudyne (i.c.m.) + laser treated mice at D1 and D4 post treatment (mean ± s.e.m.; F(2,19) = 11.65; two-way ANOVA with Sidak’s multiple comparison test). t, Representative plots of neutrophils (Ly6Ghigh), monocytes (Ly6CHigh) and macrophages (Ly6Glow/negLy6Cneg) in the meninges of laser and Visudyne (i.c.m.) + laser treated mice, at 4 days post treatment. u, Quantification of the number of neutrophils, monocytes and macrophages in the meninges of laser and Visudyne (i.c.m.) + laser treated mice (mean ± s.e.m, pooled from 2 independent experiments). v, Representative histogram of CD11c and MHCII expression by the meningeal macrophages of laser and Visudyne (i.c.m.) + laser treated mice. w, Quantification of the geometric mean fluorescence intensity (gMFI) for CD11c and MHCII expression by the meningeal macrophages of laser and Visudyne (i.c.m.) + laser treated mice at day 4 post treatment (mean ± s.e.m.). x, Representative images of the cribriform plate lymphatics (Lyve-1 – grey) of laser and Visudyne (i.c.m.) + laser treated mice. Laminin stains the vasculature, the meninges surrounding the olfactory bulbs and the olfactory nerves. Scale bar = 100 µm. y, Quantification of the number of T cells on the nasal side of the cribriform plate in laser and Visudyne (i.c.m.) + laser treated mice at 12h post i.c.m. injection. (mean ± s.e.m.). z, Adult mice were injected i.c.m. with Visudyne. Fifteen to thirty minutes after injection, Visudyne was converted using a non-thermal 689nm laser applied on the intact skull. In the targeted group, the laser was aimed at 5 different spots localized above the meningeal lymphatics. In the non-targeted group, the laser was aimed away of the sinuses. Quantification of the density of exogenously injected T cells/mm2 in dCLN of targeted and non-targeted Visudyne treated mice (expressed as percentage of the control condition). The dotted blue line corresponds to the average drainage in the laser treated group (mean ± s.e.m.; t = 3.575 df = 8; two-tailed unpaired student’s t-test).

  6. Supplementary Figure 6 Restricted CNS lymphangiogenesis during neuroinflammatory conditions.

    a, Representative images of the meningeal lymphatics (Lyve1 – red) adjacent to the transverse sinuses of MOG35-55– immunized mice at D0 (prior to immunization) and D21 post immunization. Scale bar = 400 µm, 100 µm (insets). b, Quantification of the total (i) length, (ii) number of (iii) sprouts and width of the lymphatics along the transverse sinus at D0, D3, D8, D13 and D21 after MOG35-55– immunization. (mean ± s.e.m.). c, Representative images of the meningeal lymphatics (Lyve1 – red) of the spinal cord of MOG35-55–immunized mice at D0 (prior to immunization) and D21 post immunization. Scale bar = 1000 µm, 200 µm (insets). d, Quantification of the (i) area and (ii) width of the lymphatics (Lyve-1 – red) around the nerve roots of the thoracic/lumbar region of the MOG35-55– immunized mice at D0, D3, D8, D13 and D21 after immunization (mean ± s.e.m.). e, Representative images of the lymphatics (Lyve-1 – red) around the cribriform plate of MOG35-55– immunized mice at D0 (prior to immunization) and D21 post immunization. Scale bar = 150 µm. f, Quantification of the lymphatic coverage (area) around the cribriform plate of MOG35-55– immunized mice at D0, D3, D8, D13 and D21 after immunization (mean ± s.e.m.; Kruskal-Wallis test with Dunn’s multiple comparisons test). g, Representative images of T cells (CD3e – grey) in and around the meningeal lymphatics (Lyve1 – red) of the superior sagittal sinus of CFA– and MOG35-55– immunized mice at D13 after immunization. Scale bar = 500 µm. h, Quantification of the density of T cells on the (i) superior sagittal and (ii) transverse sinuses of naïve, CFA–, and MOG35-55– immunized mice at different times after immunization (mean ± s.e.m, n = 2-5 mice per group). i, Quantification of the density of T cells (i) outside and (ii) inside the meningeal lymphatic vessels of the superior sagittal sinus at different time points after immunization (mean ± s.e.m, n = 2-5 mice/group).

  7. Supplementary Figure 7 EAE development upon ligation and characterization of peripheral immunity during EAE.

    a, EAE clinical symptom development in dCLN resected and sham operated mice (3 weeks prior to EAE induction; mean ± s.e.m.; F(1,9) = 6.057; repeated measures two-way ANOVA). b, Development of EAE clinical symptoms in dCLN-ligated, brachial lymph node-ligated, and sham operated mice (on the day of EAE induction; mean ± s.e.m.; representative of 2 independent experiments; F(2,20) = 2.119; repeated measures two-way ANOVA with Tukey’s multiple comparisons test). c, Incidence of EAE development (day mice reach a score of 1 or above) in dCLN-ligated, brachial lymph node-ligated or sham operated mice (representative of 2 independent experiments: Log-rank (Mantel-Cox) test). di, Representative contour plots of T cell activation/phenotype (CD44+CD62L) in the spleen and dCLN of laser and Visudyne (i.c.m.) + laser treated mice during early onset EAE (D8). dii, Representative histograms of CD69 expression by splenic and dCLN activated T cells from laser and Visudyne (i.c.m.) + laser treated mice during early onset EAE (D8). Representative of 5 independent mice. e, Representative histograms of splenic T cell proliferation from laser or Visudyne (i.c.m.) + laser treated mice (3 days after CD3/CD28 stimulation) extracted during early onset EAE (D8). f, Quantification of the percentage of proliferating splenic T cells from laser and Visudyne (i.c.m.) + laser treated mice at D8 post EAE induction (mean ± s.e.m.). g, Quantification of the percentage of proliferating splenic T cells from laser and Visudyne (i.c.m.) + laser treated mice after stimulation with increase MOG35-55 concentration (mean ± s.e.m.). h, Representative dot plots of IL-17 and IFNγ production by splenic T cells (upon PMA/ionomycin re-stimulation) isolated from laser and Visudyne (i.c.m.) + laser treated mice during early onset EAE (D8). Representative of 5 independent mice. i, Quantification of the percentage coverage of CD11c and MHCII in the dCLNs of laser and Visudyne (i.c.m.) + laser treated mice at day 8 post EAE induction (mean ± s.e.m.).

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–7

  2. Reporting Summary

  3. Supplementary Video 1

    Lack of dural leakage after i.c.m. injection of Qdot655. Adult CX3CR1GFP mice were injected i.v. with furamidine (to label the nuclei of the cells of the bones/dura and blood endothelial cells) and i.c.m. with 3µl of Qdot655 through a catheter implanted above the cisterna magna. Qdot655 was injected at t=0 of the movie. The meninges covering the cerebellum were imaged through a thin skull. Part 1: Top down view of the cerebellum covering meninges. Part 2: Side view of the movie presented in part 1.

  4. Supplementary Video 2

    Qdot655 uptake by the meningeal lymphatics along the transverse sinus. Adult Prox1GFP mice were injected with 3µl of Qdot655 through a catheter implanted into the CSF above the cisterna magna. Qdot655 was injected at t=0 of the movie. The meningeal lymphatics along the transverse sinuses were imaged though a thin skull. Part 1: Imaging of the lymphatics in the region of the transverse sinus proximal to the pineal gland (associated with lymphatic extensions, hot-spots). Representative of 2 independent mice. Part 2: Imaging of the lymphatics in the region of the transverse sinus distal to the pineal gland (devoid of lymphatic extensions, hot-spots). Representative of 2 independent mice.

  5. Supplementary Video 3

    Lymphatic sprouts of the meningeal lymphatics. Part 1: The upper region of the superior sagittal sinus of a Prox1GFP (green) mice was imaged through a thin skull. Secondary harmonic signal was used to separate the bone and the dura matter (Blue). Representative of 2 independent mice. Part 2: Adult Prox1GFP (green) mice were injected i.v. with 5µl of Qdot655 (diluted in 95µl of saline) to label the vasculature (red). The “hot spots” of the transverse sinus lymphatics were imaged through the thinned skull.

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https://doi.org/10.1038/s41593-018-0227-9