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Meningeal lymphatic vessels mediate neurotropic viral drainage from the central nervous system

Nature Neuroscience volume 25, pages 577–587 (2022)Cite this article

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Abstract

Recent studies have demonstrated that brain meningeal lymphatic vessels (MLVs) act as a drainage path directly into the cervical lymph nodes (CLNs) for macromolecules contained in the cerebrospinal fluid (CSF). However, the role of MLVs during CNS viral infection remains unexplored. Here, we found that infection with several neurotropic viruses in mice promotes MLV expansion but also causes impaired MLV-mediated drainage of macromolecules. Notably, MLVs could drain virus from the CNS to CLNs. Surgical ligation of the lymph vessels or photodynamic ablation of dorsal MLVs increased neurological damage and mortality of virus-infected mice. By contrast, pretreatment with vascular endothelial growth factor C promoted expansion of functional MLVs and alleviated the effects of viral infection. Together, these data indicate that functional MLVs facilitate virus clearance, and MLVs represent a critical path for virus spreading from the CNS to the CLNs. MLV-based therapeutic strategies may thus be useful for alleviating infection-induced neurological damage.

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Fig. 1: Viral CNS infections induce dorsal meningeal lymphangiogenesis.
Fig. 2: Three-dimensional imaging of meningeal lymphangiogenesis after viral infection.
Fig. 3: Impaired MLV ability to drain biological molecules following acute viral CNS infection.
Fig. 4: Virus infection of the CNS causes MLV damage.
Fig. 5: MLVs drain neurotropic viral particles from the CNS to CLNs.
Fig. 6: Ablation of lymphatic drainage enhances, but VEGF-C pretreatment reduces, mortality of JEV-infected mice.

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Data availability

The data that support the findings of this study are available in this manuscript and the Supplementary Information. Source data are provided with this paper.

Code availability

Customized code or algorithms were not used to generate results in the present study.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81630043 to B.W., 81825011 to H.W., 81930038 to H.W. and 81961160738 to B.W.), the Ministry of Science and Technology of China (2018YFA0800702 to H.W.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19030200 to H.W.), Shanghai Municipal Science and Technology Major Project and Key National Science and Technology Programs in Shanghai (218014204 to B.W.). We thank the Core Facility and Technical Support in the Shanghai University and Wuhan Institute of Virology, CAS. We thank the Core Facility of Molecular Biology and Core Facility of Cell Biology in the Shanghai Institute of Biochemistry and Cell Biology for their technical support and help. We thank C. Gu of Huazhong Agricultural University for providing anti-JEV E. We thank M. Jin of Huazhong Agricultural University for providing VSV–GFP. We thank the laboratory of Z. Qian of Institute Pasteur of Shanghai, CAS, for providing HSV-1. We thank the laboratory of H. Wang of the Wuhan Institute of Virology, CAS, for providing ZIKV. We thank Q. Leng of the Institute Pasteur of Shanghai, CAS, for providing IFN receptor-deficient mice. We thank Q. Liang of Shanghai University of Traditional Chinese Medicine for providing the non-thermal 689-nm laser. We thank S. Cao of Huazhong Agricultural University for providing the BHK-21 cell line. We thank H. Wang of Shanghai Institute of Biochemistry and Cell Biology, CAS, for providing the 293T cell line. We thank H. Yan of Wuhan Institute of Virology, CAS, for providing the C6/36 cell line.

Author information

Author notes

  1. These authors contributed equally: Xiaojing Li, Linlin Qi. These authors jointly supervised this work: Hongyan Wang, Bin Wei.

Authors and Affiliations

  1. Institute of Geriatrics, Affiliated Nantong Hospital of Shanghai University (The Sixth People’s Hospital of Nantong), School of Medicine, Shanghai University, Nantong, China

    Xiaojing Li, Linlin Qi, ShuJie Hao, Xingguo Zhu & Bin Wei

  2. Shanghai Engineering Research Center of Organ Repair, School of Life Sciences, Shanghai University, Shanghai, China

    Xiaojing Li, Linlin Qi, Dan Yang, ShuJie Hao, Fang Zhang, Xingguo Zhu & Bin Wei

  3. Cancer Center, Shanghai Tenth People’s Hospital, School of Medicine, Tongji University, Shanghai, China

    Xiaojing Li, Fang Zhang & Bin Wei

  4. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan, China

    Xiaojing Li, Linlin Qi, Dan Yang, Fang Zhang & Bin Wei

  5. School of Life Sciences, Peking University, Beijing, China

    Yue Sun & Jing Yang

  6. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China

    Chen Chen, Jing Ye, Ling Zhao & Shengbo Cao

  7. Department of Immunology and Inflammation, Imperial College, Faculty of Medicine, Hammersmith Hospital, London, UK

    Daniel M. Altmann

  8. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China

    Hongyan Wang

  9. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China

    Hongyan Wang

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  1. Xiaojing Li
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Contributions

X.L. and L.Q. performed most of the experiments and statistical analysis. D.Y. performed the two-photon imaging. F.Z. and S.H. performed hLECs experiments. S.H. and X.Z. participated in animal experiments. Y.S. performed volume fluorescence imaging. C.C. performed RABV infection. B.W. and H.W. conceptualized and designed experiments. J. Ye., L.Z., J. Yang and S.C. helped with technical support. X.L., L.Q., B.W., D.M.A. and H.W. wrote the paper. All authors approved the final version of the manuscript.

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Correspondence to Hongyan Wang or Bin Wei.

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Nature Neuroscience thanks Robyn Klein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 JEV and VSV infections induce dorsal meningeal lymphangiogenesis.

(a-f). C57BL/6 mice were intravenously (i.v.) or intracerebrally (i.c.) infected with JEV or PBS. a. Representative images of LYVE-1 and VEGFR-3 staining and quantification of the area of LYVE-1+ MLVs in TS at 7 Dpi (n = 5). b. Representative image and quantification of the diameter of LYVE-1+ MLVs along the SSS in JEV (i.v.) infected mice (n = 7 PBS; n = 5 JEV). c. Representative images and quantification of the mean intensity of LYVE-1+ MLVs in COS after JEV (i.c.) infection (n = 7 PBS; n = 6 JEV). d. VEGF-C concentrations in the brain after JEV (i.c.) injection (n = 4 PBS; n = 5 JEV). e. The daily behavior scores of mice (n = 6) and the relative mRNA levels of JEV-C gene (n = 8 PBS; n = 4-8 JEV) in the brain at different time points after JEV (i.v.) infection. f. Representative images and quantification of the area of LYVE-1 staining in COS and TS at day 1, day 3 and day 5 after JEV (i.v.) injection (n = 5). (g-i). C57BL/6 mice were intranasally infected with VSV. g. The daily body weight of mice after VSV infection (n = 6). h. The relative mRNA levels of VSV (n = 6 PBS; n = 5 JEV) and Ccl5 (n = 4 PBS; n = 6 JEV) in the brain at 8 Dpi. i. Representative images and the quantification of meningeal LYVE-1, VEGFR-3, PDPN staining in the TS at 8 Dpi (n = 6). Data are presented as Mean ± SD. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, two-tailed, unpaired Student’s t test (a right, b right, c right, d, g, h and i right), one-way ANOVA with Holm-Sidak’s multiple comparisons test (e and f right). Data from at least 3 independent experiments. Detailed statistical information is shown in Supplementary Table 2.

Source data

Extended Data Fig. 2 HSV-1, ZIKV and RABV infections induce dorsal meningeal lymphangiogenesis.

(a-c). Mice were corneally infected with HSV-1. a. Left, the daily body weight of mice (n = 7 PBS; n = 6 HSV-1). Right, mRNA levels of HSV-1 in the brain at 8 Dpi (n = 6 PBS; n = 5 HSV-1). b. Representative meningeal LYVE-1 staining and quantification of the area and diameter of LYVE-1+ MLVs in COS or TS (n = 6). c. mRNA levels of Ccl5 in the brain (n = 4 PBS; n = 5 HSV-1). (d-f). Mice were intramuscularly infected with RABV. Representative meningeal LYVE-1 staining (d left, e left) and quantification of lymphatic branches (white arrowheads) (d right, e right) in TS (n = 6 PBS; n = 8 RABV). f. mRNA levels of RABV (n = 5 PBS; n = 9 RABV), Ccl5, Il1b and Il10 (n = 4 PBS; n = 7 RABV) in the brain at 8 Dpi. (g-j). AG6 mice were intraperitoneally infected with ZIKV. g. The daily body weight of mice (n = 5 PBS; n = 8 ZIKV). h. Viral titers were detected in the brain at 8 Dpi (n = 7). i. Representative images and quantification of the area and diameter of LYVE-1+ MLVs in COS or TS (n = 6 or 7 PBS; n = 6 or 7 ZIKV). j. mRNA levels of Il1b and Il10 in the brain at 8 dpi (n = 4 PBS; n = 5 ZIKV). k. Mice were intravenously infected with JEV or intramuscularly infected with RABV. Representative images and quantification of PDPN+ basal MLVs of JEV-infected mice at 7 dpi (n = 5). Representative images and quantification of LYVE-1+ basal MLVs of RABV-infected mice at 8 dpi (n = 4 PBS; n = 8 RABV). l. mRNA levels of PDPN, FOS, JUN and BCL-2 in hLECs treated with 0.1 MOI JEV, inactivated JEV or untreated for 72 h (n = 3). Data are presented as Mean ± SD. not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, a two-tailed, unpaired Student’s t test (a, b right, c, d right, e right, f, g, h, i right, j, k), one-way ANOVA with Holm-Sidak’s multiple comparisons test (l). Data from at least 2 independent experiments. Detailed statistical information is shown in Supplementary Table 2.

Source data

Extended Data Fig. 3 MLVs function to drain biological molecules upon virus infection.

a. Representative images of LYVE-1 staining and OVA-647 in tissue around sCLN and the superficial skin of neck at 180 min after i.c.m. injection of OVA-647. b. Representative images of LYVE-1 staining and OVA-647 in TS of meninges after i.c.m. injection of OVA-647 (2 μg/μl) at 3, 4, and 5 Dpi of JEV. c. The ventral images show the biodistribution of OVA-647 in the mice at the indicated time points after i.c.m. or intranasally injection with OVA-647. (d-f). Mice were corneally infected with HSV-1. d. The daily body weight of mice in PBS group (n = 5), the acute infection group (n = 6;) and the resolution of viral infection group (n = 4). (e, f). The ventral images show the biodistribution of OVA-647 and the retention of OVA-647 in cervical region was quantified at 10 days (e) or 28 days (f) (n = 4). g. Representative images of LYVE-1 staining and quantification of the area of LYVE-1+ MLVs in COS at 28 Dpi (n = 6 PBS; n = 4 HSV-1). (h-j). C57BL/6 mice were (i.v.) infected with JEV or PBS. 5 μl QDot655 was injected in i.c.m. of JEV or PBS-injected mice at 4 Dpi. Change of QDot655 in MLVs was monitored through a thinned skull preparation by multiphoton microscopy system. h. Schematic of QDot655 treatment. i. Representative images and the quantification of Qdot655 in MLVs were captured at different timepoints (n = 6). See also video S3. j. Representative images of Qdot655 and Lectin-488 (labeled blood vessel) were captured by multiphoton microscopy system. See also video S4. The data are presented as Mean ± SEM (d, e, f) or as Mean ± SD (g). ns, not significant (p > 0.05), **p < 0.01 and ****p < 0.0001, a two-tailed, unpaired Student’s t test (g), one-way ANOVA with Holm- Sidak’s multiple comparisons test (d) and two-way ANOVA with Holm- Sidak’s multiple comparisons test (e right, f right, i). Detailed statistical information is shown in Supplementary Table 2.

Source data

Extended Data Fig. 4 The expression of genes that regulated cell polarization of LECs.

a. The relative mRNA levels of Celsr1, Pkd1, Sema3a, Egfl7, Fat4, Nrp1 in meninges of JEV (n = 6) or PBS (n = 5 or 6) injected mice. The data are presented as Mean ± SEM. b. hLECs treated with 0.1 MOI JEV, inactivated JEV or untreated for 72 h. The relative mRNA levels of BAD and CASP9 in hLECs (n = 3). The data are presented as Mean ± SD. ns, not significant (p > 0.05), a two-tailed, unpaired Student’s t test (a) and one-way ANOVA with Holm- Sidak’s multiple comparisons test (b). Detailed statistical information is shown in Supplementary Table 2.

Source data

Extended Data Fig. 5 Meningeal lymphatic vessels drain neurotropic viral particles from CNS to CLNs.

a. Immunohistochemical analysis of JEV E protein in AxLN sections in JEV or PBS (i.c.) injected mice with severe JE cases. b. 5-week-old C57BL/6 mice were ligation of dCLVs, and 7 days later, VSV-GFP was i.c.m. injected. The viral titers of VSV-GFP in dCLNs were measured by plaque assay after 3 h injection (n = 4). c. Representative images of the colocalization of VSV-GFP (green) and LYVE-1+ MLVs in TS and COS at 3 h, 6 h, 9 h after i.c.m. injection of VSV-GFP. d. Representative images of JEV E protein staining in hLECs treated with 1 MOI JEV for 48 h. (e, f). VSV-GFP was injected in i.c.m. of C57BL/6 mice. e. Representative images of DAPI, VSV-GFP, CD11b staining in COS of meninges at 9 h after VSV-GFP injection. f. Representative images of DAPI, VSV-GFP, CD206 staining in COS of meninges at 9 h after VSV-GFP injection. (g-i). FACS analysis of the CD45+ cells, macrophages, monocytes and DCs in sCLNs and AxLNs of mice (i.c.) injected with JEV and PBS. g. Gating strategy employed to identify CD45+ cells, B cells (MHC II+ B220+), macrophages (F4/80+ CD11b+), monocytes (F4/80− Ly6c+ CD11b+) and DCs (CD11C+ MHC II+). (h, i). MFI of CD86 and CD80 in B cells, macrophages, DCs or monocytes in sCLNs (n = 5 PBS; n = 8 JEV) or AxLNs (n = 6 PBS; n = 7 JEV) of JEV-infected mice. Data are presented as mean ± SD. not significant (p > 0.05), *p < 0.05, **p < 0.01, a two-tailed, unpaired Student’s t test (b right, h and i). Data from at least 2 independent experiments. Detailed statistical information is shown in Supplementary Table 2.

Source data

Extended Data Fig. 6 Gating strategy of cells in brain of JEV-infected mice.

The gating strategy of CD45+ cells, neutrophils, macrophages, monocytes, CD8+ T cells, CD4+ T cells, NK cells, microglia and B cells in the brain of JEV-infected mice.

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Table 2

Detailed statistical information.

Supplementary Video 1

Three-dimensional reconstruction of meningeal lymphatics along the COS in PBS- or JEV-infected mice.

Supplementary Video 2

Three-dimensional projections of meningeal lymphatics in whole-mount meninges of PBS- or JEV-infected mice.

Supplementary Video 3

Flow of QD in MLVs of PBS- or JEV-infected mice.

Supplementary Video 4

Three-dimensional projections of the MLVs and blood by two-photon microscopy.

Source data

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Li, X., Qi, L., Yang, D. et al. Meningeal lymphatic vessels mediate neurotropic viral drainage from the central nervous system. Nat Neurosci 25, 577–587 (2022). https://doi.org/10.1038/s41593-022-01063-z

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