Neuroinflammation-induced lymphangiogenesis near the cribriform plate contributes to drainage of CNS-derived antigens and immune cells

Abstract

There are no conventional lymphatic vessels within the CNS parenchyma, although it has been hypothesized that lymphatics near the cribriform plate or dura maintain fluid homeostasis and immune surveillance during steady-state conditions. However, the role of these lymphatic vessels during neuroinflammation is not well understood. We report that lymphatic vessels near the cribriform plate undergo lymphangiogenesis in a VEGFC - VEGFR3 dependent manner during experimental autoimmune encephalomyelitis (EAE) and drain both CSF and cells that were once in the CNS parenchyma. Lymphangiogenesis also contributes to the drainage of CNS derived antigens that leads to antigen specific T cell proliferation in the draining lymph nodes during EAE. In contrast, meningeal lymphatics do not undergo lymphangiogenesis during EAE, suggesting heterogeneity in CNS lymphatics. We conclude that increased lymphangiogenesis near the cribriform plate can contribute to the management of neuroinflammation-induced fluid accumulation and immune surveillance.

Fig. 1

Cribriform plate lymphatics exist dorsal to the cribriform plate. a Diagram of a coronal section through the olfactory bulbs, cribriform plate, and olfactory epithelium of a mouse. The red inset indicates the region of vessels characterized in b–j. Note that these vessels penetrate from the nasal septum through the cribriform plate and into the subarachnoid space. b–d The whole heads of healthy naive Prox1-tdTomato transgenic mice were decalcified, coronally sectioned, and immunolabeled with the lymphatic hyaluronan receptor Lyve-1 (c) and DAPI (d). Scale bars = 100 μm. e–j The whole heads of healthy naive wild-type mice were decalcified, coronally sectioned, and immunolabeled with the lymphangiogenic tyrosine kinase receptor VEGFR3, Lyve-1, and DAPI (e–g) or the lymphatic marker Podoplanin, Lyve-1, and DAPI (h–j). Scale bars = 100 μm

Fig. 2

EAE induces lymphangiogenesis near the cribriform plate. a, b Nine representative serial coronal sections of the lymphatic vessels near the cribriform plate were imaged from either a healthy (a) or an EAE Score 3.0 collected 18 days post-immunization (b) CD11c-eYFP transgenic reporter mouse were immunolabeled with Lyve-1 and DAPI. Note the increase in Lyve-1⁺ vessel area during EAE versus healthy mice. Scale bars = 100 μm. c–h Enlarged images of the red inset in a or yellow inset in b showing the section with the largest Lyve-1⁺ vessel area in healthy (c–e) versus EAE Score 3.0 (f–h) out of nine serial coronal sections. Scale bars = 100 μm. i, j Quantitation of the average Lyve-1⁺ vessel area across nine serial sections (i) (n = 4 mice per group; data are represented as mean ± SEM, ***p < 0.001, repeated two-way ANOVA using Sidaks multiple comparison test) or the average Lyve-1⁺ vessel volume (j) (n = 4 mice per group; data are represented as mean ± SEM, *p < 0.05, unpaired Student’s t-test). k–t Proliferation of lymphatic endothelial cells measured by Ki67⁺ nuclei within healthy (k–o) or EAE Score 3.0 mice (p–t). Ki67⁺ Lyve-1⁺ lymphatic endothelial cells were quantified using orthogonal views to confirm that the cells used for quantitation were in fact proliferating lymphatic endothelial cells. Yellow arrowheads indicate Ki67⁺ nuclei within Lyve-1⁺ vessels. Scale bars, 100 μm in a–t. Scale bars = 100 μm. u Quantitation of the average percent of Ki67⁺ nuclei within Lyve-1⁺ vessels between healthy and EAE Score 3.0 mice (n = 4 mice per group; data are represented as mean ± SEM, *p < 0.05, unpaired Student’s t-test

Fig. 3

Lymphangiogenic vessels can functionally carry cells and CSF during EAE. a–h Representative confocal images of the olfactory bulbs, cribriform plate, and nasal mucosa were imaged from a healthy (a–d) or EAE Score 3.0 (e–h) and immunolabeled with Lyve-1 and DAPI. b–d Magnified image of the red inset shown in a. f–h Magnified image of the red inset shown in e. Scale bars = 100 µm. i Quantitation of CD11c-eYFP⁺ cell number within Lyve-1⁺ vessels. Orthogonal views were used for quantitation to confirm that the CD11c-eYFP⁺ cells quantified were within Lyve-1⁺ vessels (n = 4 mice per group; data are represented as mean ± SEM, **p < 0.01, unpaired Student’s t-test). j, s Higher magnification images from either healthy (j–n) or EAE Score 3.0 (o–s) CD11c-eYFP transgenic reporter mice showing CD11c-eYFP⁺ cells within Lyve-1⁺ vessels. k–n Magnified image of the red inset in j. p, s Magnified image of the red inset in o. Scale bars = 100 µm for (j, o) and 20 µm for (k–n, p–s). t–E_b KikGR transgenic mice were induced with EAE, and at EAE score 3.0 were intracerebrally photoconverted to visualize if photoconverted cells originating from the CNS parenchyma can be found within lymphangiogenic vessels near the cribriform plate and the draining lymph nodes. Representative images show green-red photoconverted cells at the site of photoconversion within the brain (t–w), within lymphatic vessels near the cribriform plate (x–A_b), and within the cervical lymph nodes (CLNs) (B_b–E_b). F_b–I_b): Wild-type animals were induced with EAE, and at peak EAE 10 µl of 10% Evans blue dye was injected into the cisterna magna at a rate of 2 µl/min to visualize the drainage of CSF within cribriform plate lymphatics. Representative images show co-localization (F_b) of Evans blue dye (G_b) within Lyve-1⁺ vessels (H_b) and DAPI (I_b). Scale bars = 10 µm. J_b Representative intensity profile plot for Evans blue dye and Lyve-1 taken from a cross section of the image shown in F_b showing co-localization of Evans blue dye within Lyve-1⁺ lymphatic endothelial cells

Fig. 4

EAE increases VEGFC to promote VEGFR3-dependent lymphangiogenesis. a–d Healthy (a, c) and EAE (b, d) wild-type mice were treated I.P. with either vehicle (a, b) or the VEGFR3 tyrosine kinase inhibitor MAZ51 (c, d) beginning on day 7 post-immunization and harvested at Day 18 post-immunization. The whole heads were decalcified, and coronal sections were immunolabeled for Lyve-1 to visualize lymphatic vessels near the cribriform plate. Scale bars = 100 µm. e Quantitation of the average Lyve-1⁺ vessel area near the cribriform plate between vehicle and MAZ51-treated mice in both healthy and EAE. For each animal, nine serial sections spanning 1920 μm were analyzed and the section with the maximum Lyve-1⁺ vessel area was used for quantitation (mean ± SEM; n = 4 mice per group; ****p < 0.0001, one-way ANOVA with Tukeys post-hoc multiple comparisons test). f CNS lysates from healthy and EAE score 3.0 wild-type mice were probed for VEGFC and ß-actin as a loading control by western blot. g Quantitation of the relative VEGFC protein amount normalized to ß-actin as shown in d (mean ± SEM; n = 3–4 mice per group; **p < 0.01, unpaired Student’s t-test). h–q Representative coronal section of a healthy (h–l) or EAE Score 3.0 (m–q) CD11c-eYFP transgenic reporter mice immunolabeled for CD11b, VEGFC, Lyve-1, and DAPI to visualize VEGFC-producing cells near cribriform plate lymphatics. Scale bars = 100 µm. r–u Magnified image of the green inset taken from m showing co-localization of CD11c-eYFP⁻ CD11b⁺ macrophages with VEGFC. Yellow arrowheads indicate CD11c-eYFP⁻ CD11b⁺ macrophages that express VEGFC. v–y Magnified image of the yellow inset taken from m showing CD11c-eYFP⁺ CD11b⁺ dendritic cells expressing VEGFC. Yellow arrowheads indicate CD11c-eYFP⁺ CD11b⁺ dendritic cells that express VEGFC. Scale bars = 10 µm

Fig. 5

Lymphangiogenesis is unique to the cribriform plate during EAE. a–d Healthy (a, c) and EAE Score 3.0 (b, d) wild-type mice were treated I.P. with either vehicle (a, b) or the VEGFR3 tyrosine kinase inhibitor MAZ51 (c, d) beginning on day 7 post-immunization and harvested at day 18 post-immunization. The meninges were collected and immunolabeled for Lyve-1 to visualize meningeal lymphatics. Scale bars = 500 µm. e Quantitation of the average Lyve-1⁺ vessel area in the confluence of sinuses of the dura between vehicle and MAZ51 treated mice in both healthy and EAE (mean ± SEM; n = 4–6 mice per group; *p < 0.05, one-way ANOVA with Tukeys post hoc multiple comparisons test)

Fig. 6

Inhibition of VEGFR3 reduces CNS antigen-specific CD4 T cell proliferation. a–e ß-gal floxed ovalbumin peptides (OP) transgenic mice were crossed with CNP-Cre transgenic mice to drive ovalbumin peptide expression on oligodendrocytes (CNP-OP mice). These mice were then induced with EAE, I.P. treated with either vehicle or the VEGFR3 tyrosine kinase inhibitor MAZ51 beginning on day 7 post-immunization, and ovalbumin-specific CD8 (OT-I) or CD4 (OT-II) Thy1.1 congenic CellTrace Violet labeled T cells were adoptively transferred on day 12 post-immunization. T cell proliferation was then measured by cytofluorimetry. a Gating strategy for the adoptively transferred, CellTrace Violet labeled, Thy1.1 congenic, Ovalbumin-specific T cells. b Representative histograms of CellTrace Violet proliferation for OT-I T cells taken from the draining lymph nodes between vehicle and MAZ51 treated EAE animals. c Quantitation of the average percent of OT-I T cell proliferation in the draining lymph nodes at day 18 post-immunization (mean ± SEM; n = 4–5 mice per group; NS = non-significant, unpaired Student’s t-test). d Representative histograms of CellTrace Violet proliferation for OT-II T cells taken from the draining lymph nodes between vehicle and MAZ51 treated EAE animals. e Quantitation of the average percent of OT-II T cell proliferation in the draining lymph nodes at day 18 post-immunization (mean ± SEM; n = 4–5 mice per group; *p < 0.05, unpaired Student’s t-test)

Fig. 7

Inhibition of VEGFR3 reduces EAE severity. a–c ß-gal floxed ovalbumin peptides (OP) transgenic mice were crossed with an oligodendrocyte-specific Cre mouse (CNP-Cre) to generate CNP-OP transgenic mice. EAE was induced in CNP-OVA mice, treated with either vehicle or MAZ51, and were adoptively transferred CellTrace Violet labeled, Thy1.1 congenic, CD8 (OT-I), and CD4 (OT-II) T cells as shown in Fig. 5. EAE severity of mice receiving I.P. MAZ51 once per day beginning on day 7 post-immunization have reduced EAE severity (a), reduced average maximal EAE scores (b) (n = 5 mice per group; data are represented as mean ± SEM, *p < 0.05, unpaired Student’s t-test), and delay in the EAE onset (c) (n = 5 mice per group; data are represented as mean ± SEM, **p < 0.01, unpaired Student’s t-test). d, e The spinal cords of MAZ51 and vehicle treated animals were immunolabeled with Fluoromyelin to visualize myelin within the spinal cord (d). MAZ51-treated mice have significantly reduced percent demyelination when compared to vehicle-treated controls (e) (n = 4–5 mice per group; data are represented as mean ± SEM, *p < 0.05, ***p < 0.001, two-way ANOVA with Sidaks multiple comparisons test). f–g The spinal cords of MAZ51 and vehicle treated animals were immunolabeled with CD4 to visualize CD4⁺ T cell infiltration into the spinal cord (f). MAZ51-treated mice have significantly reduced CD4 T cell infiltration into the spinal cord when compared to vehicle-treated controls (g) (n = 4–5 mice per group; data are represented as mean ± SEM, **p < 0.01, two-way ANOVA with Sidaks multiple comparisons test)