Developmental Dysfunction of the Central Nervous System Lymphatics Modulates the Adaptive Neuro-Immune Response in the Perilesional Cortex in a Mouse Model of Traumatic Brain Injury

Rationale The recently discovered meningeal lymphatic vessels (mLVs) have been proposed to be the missing link between the immune and the central nervous system. The role of mLVs in modulating the neuro-immune response following a traumatic brain injury (TBI), however, has not been analyzed. Parenchymal T lymphocyte infiltration has been previously reported as part of secondary events after TBI, suggestive of an adaptive neuro-immune response. The phenotype of these cells has remained mostly uncharacterized. In this study, we identified subpopulations of T cells infiltrating the perilesional areas 30 days post-injury (an early-chronic time point). Furthermore, we analyzed how the lack of mLVs affects the magnitude and the type of T cell response in the brain after TBI. Methods TBI was induced in K14-VEGFR3-Ig transgenic (TG) mice or in their littermate controls (WT; wild type), applying a controlled cortical impact (CCI). One month after TBI, T cells were isolated from cortical areas ipsilateral or contralateral to the trauma and from the spleen, then characterized by flow cytometry. Lesion size in each animal was evaluated by MRI. Results In both WT and TG-CCI mice, we found a prominent T cell infiltration in the brain confined to the perilesional cortex and hippocampus. The majority of infiltrating T cells were cytotoxic CD8+ expressing a CD44(hi)CD69+ phenotype, suggesting that these are effector resident memory T cells. K14-VEGFR3-Ig mice showed a significant reduction of infiltrating CD4+ T lymphocytes, suggesting that mLVs could be involved in establishing a proper neuro-immune response. Extension of the lesion (measured as lesion volume from MRI) did not differ between the genotypes. Finally, TBI did not relate to alterations in peripheral circulating T cells, as assessed one month after injury. Conclusions Our results are consistent with the hypothesis that mLVs are involved in the neuro-immune response after TBI. We also defined the resident memory CD8+ T cells as one of the main population activated within the brain after a traumatic injury.Peer reviewe

Significance of developmental meningeal lymphatic dysfunction in experimental post-traumatic injury

Publisher Copyright: © 2022 The AuthorsPeer reviewe

A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules

The central nervous system (CNS) is considered an organ devoid of lymphatic vasculature. Yet, part of the cerebrospinal fluid (CSF) drains into the cervical lymph nodes (LNs). The mechanism of CSF entry into the LNs has been unclear. Here we report the surprising finding of a lymphatic vessel network in the dura mater of the mouse brain. We show that dural lymphatic vessels absorb CSF from the adjacent subarachnoid space and brain interstitial fluid (ISF) via the glymphatic system. Dural lymphatic vessels transport fluid into deep cervical LNs (dcLNs) via foramina at the base of the skull. In a transgenic mouse model expressing a VEGF-C/D trap and displaying complete aplasia of the dural lymphatic vessels, macromolecule clearance from the brain was attenuated and transport from the subarachnoid space into dcLNs was abrogated. Surprisingly, brain ISF pressure and water content were unaffected. Overall, these findings indicate that the mechanism of CSF flow into the dcLNs is directly via an adjacent dural lymphatic network, which may be important for the clearance of macromolecules from the brain. Importantly, these results call for a reexamination of the role of the lymphatic system in CNS physiology and disease.Peer reviewe

Anatomy and function of the vertebral column lymphatic network in mice

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO(+)-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.Peer reviewe

Molecular anatomy of adult mouse leptomeninges.

Leptomeninges, consisting of the pia mater and arachnoid, form a connective tissue investment and barrier enclosure of the brain. The exact nature of leptomeningeal cells has long been debated. In this study, we identify five molecularly distinct fibroblast-like transcriptomes in cerebral leptomeninges; link them to anatomically distinct cell types of the pia, inner arachnoid, outer arachnoid barrier, and dural border layer; and contrast them to a sixth fibroblast-like transcriptome present in the choroid plexus and median eminence. Newly identified transcriptional markers enabled molecular characterization of cell types responsible for adherence of arachnoid layers to one another and for the arachnoid barrier. These markers also proved useful in identifying the molecular features of leptomeningeal development, injury, and repair that were preserved or changed after traumatic brain injury. Together, the findings highlight the value of identifying fibroblast transcriptional subsets and their cellular locations toward advancing the understanding of leptomeningeal physiology and pathology

VEGF-C/VEGFR3 signalointijärjestelmä silmän ja kovakalvon imusuoniston kehityksessä ja taudeissa

Lymphatic vessels (LVs) are found in almost all tissues of the vertebrate body and contribute to the regulation of interstitial fluid (ISF) homeostasis and immune surveillance by draining excess fluids, macromolecules, and immune cells into lymph nodes (LNs) and systemic circulation. Despite a few studies reporting isolated LVs inside the skull, the mammalian central nervous system (CNS) and its meningeal linings have been though to lack a significant lymphatic network. Together with another PhD student, we serendipitously discovered the lymphatic-like nature of the Schlemm’s canal (SC) in the eye. During this work, when observing eyes and, later, skulls of transgenic mice that allowed direct visualization of lymphatic vasculature under fluorescent light, we also discovered the lymphatic vasculature in the dura mater. The subsequent aims of the studies I-IV included in my thesis were to elucidate the basic anatomy, development, and function of the lymphatic-like ocular vasculature and the lymphatic vasculature in the dura mater. Furthermore, we sought to characterize the function of the newly discovered dural lymphatic vascular system in different neuropathological processes, with my thesis work focusing on Alzheimer’s disease (AD) In study I, we explored the lymphatic-like anatomy and development of the SC in the eye, as well as the translational therapeutic implications of the possibility that the SC would function as a lymphatic vessel. We characterized the evolutionary conservation of the lymphatic-like phenotype of the SC and discovered VEGF-C growth factor and VEGFR3 growth factor receptor as critical regulators of SC growth from pre-existing lymphatic structures, a process known as lymphangiogenesis. Our findings provide a basis for further studies related to therapeutic manipulation of the SC using VEGF-C in glaucoma treatment. In study II, we reported the surprising finding of extensive and functional network of fully differentiated LVs in the outermost meningeal layer, the dura mater, surrounding the brain. We described the unique morphology of the dural lymphatic vessels (dLVs) localized in both the dorsal and basal cranium. Furthermore, we identified that dLVs function in the absorption and transportation of macromolecular brain ISF and cerebrospinal fluid (CSF) into deep cervical LNs. These results open new avenues for research and call for a reexamination of the role of the lymphatic system in CNS physiology and disease. In study III, we studied the development and plasticity of dLVs using a range of genetic models and modulators that affect lymphangiogenic signaling. We characterized an extensive LV network also in the meninges surrounding the spinal cord, within the dura mater and the epidural space overlying the dura mater. These spinal meningeal LVs (mLVs) were interconnected with cranial dural LVs, collectively referred to as mLVs. Cranial and spinal mLVs developed during the first postnatal month along the blood vessels and nerves through lymphangiogenic sprouting and fusion of isolated lymphatic endothelial cell clusters. We discovered that the extent and functionality of mLVs could be surprisingly selectively modulated by manipulating the VEGF-C/VEGFR3 signaling axis. The plasticity and regenerative potential of mLVs should provide improved methods for assessing their importance in CSF drainage and neuropathological processes within the CNS. The discovery of the significant meningeal lymphatic system prompted a reevaluation of some fundamental assumptions in neurobiology, particularly regarding CNS immune privilege and macromolecule clearance. In study IV, we set out to characterize the function of cranial dLVs in AD-related amyloid beta (Aβ) pathology by blocking or overexpressing VEGF-C in two widely used mouse models of AD. Our results indicated that sustained functional manipulation of dLV does not affect overall Aβ deposition in the brain and that compensatory mechanisms promote CSF clearance. Overall, the findings in my thesis establish the foundations for future research into the lymphatic-like Schlemm’s canal, as well as the cranial and spinal mLVs. Furthermore, our findings highlight the need for further comparison of different methods to manipulate mLVs and for better mechanistic understanding of CNS-related fluid and particle production, circulation, and outflow under both physiological and pathological conditions. Importantly, our findings may open entirely new therapeutic avenues in ocular and CNS-associated diseases.Imusuonia löytyy lähes kaikista selkärankaisten kudoksista. Imusuoniston tärkeimpiä tehtäviä on kudosnesteen tasapainon säätely sekä immuunijärjestelmän valvonta, joihin se osallistuu kuljettamalla ylimääräistä nestettä, suuria molekyylejä ja immuunisoluja imusolmukkeisiin sekä systeemiseen verenkiertoon. Vaikka muutamat aiemmat tutkimukset ovat raportoineet yksittäisiä imusuonia kallon sisällä, merkittävän imusuoniverkoston on ajateltu puuttuvan nisäkkäiden keskushermostosta ja sitä ympäröivistä aivokalvoista. Yhdessä toisen tohtoriopiskelijan kanssa kuvasimme silmässä sijaitsevan Schlemmin kanavan olevan imusuonen kaltainen suonirakenne. Kyseisen tutkimuksen aikana tarkastelimme pään aluetta muuntogeenisillä hiirillä, joiden imusuonisto näkyi suoraan fluoresoivan valon avulla, ja huomasimme, että myös aivoja ympäröivä kovakalvo sisältää laajan imusuoniverkoston. Väitöskirjani osat I-IV keskittyivät kartoittamaan imusuonen kaltaisen Schlemmin kanavan sekä kovakalvon imusuonten perustavanlaatuista anatomiaa, kehitystä ja toimintaa fysiologisessa tilassa. Lisäksi pyrimme selvittämään kovakalvon imusuoniston toimintaa erilaisissa neuropatologisissa tiloissa, joista väitöskirjatyö keskittyi erityisesti Alzheimerin tautiin. Tutkimuksessa I tutkimme Schlemmin kanavan anatomiaa, kehitystä ja mahdollisuutta vaikuttaa sen imusuonen kaltaiseen luonteeseen terapeuttisten vaikutusten aikaansaamiseksi. Havaitsimme, että Schlemmin kanavan imusuonen kaltaisuus on säilynyt evoluutiossa ja totesimme VEGF-C-kasvutekijän sekä VEGFR3-kasvutekijäreseptorin kriittisiksi säätelijöiksi rakenteen imusuoniuudiskasvussa olemassa olevista imusuonirakenteista; tätä prosessia kutsutaan myös nimellä lymfangiogeneesi. Tuloksemme avaavat mahdollisuuksia tutkia Schlemmin kanavan terapeuttista muokkausta esimerkiksi glaukooman hoidossa VEGF-C-kasvutekijän avulla. Tutkimuksessa II raportoimme yllättävän havainnon täysin erilaistuneista imusuonista, jotka muodostavat laajan ja toiminnallisen verkoston aivoja ympäröivässä uloimmassa aivokalvossa, kovakalvossa. Tutkimuksessa kuvasimme kovakalvon imusuonten ainutlaatuista rakennetta kallon eri osissa. Osoitimme myös, että kovakalvon imusuonet osallistuvat aivojen kudosvälinesteen ja aivoselkäydinnesteen kiertoon kuljettamalla nestettä ja suuria molekyylejä kallon ulkopuolelle kaulan syviin imusolmukkeisiin. Nämä tulokset avaavat uusia tutkimusmahdollisuuksia ja korostavat tarvetta tarkastella imusuonten merkitystä keskushermoston normaalissa toiminnassa ja poikkeavissa tiloissa. Tutkimuksessa III tutkimme kovakalvon imusuonten kehitystä ja muokkautuvuutta käyttäen erilaisia geneettisiä malleja ja säätelijöitä, jotka vaikuttavat imusuonten signalointiin. Kuvasimme laajan imusuoniverkoston myös selkäydintä ympäröivissä aivokalvoissa, jotka olivat yhteydessä kallonsisäisiin kovakalvon imusuoniin. Kallon ja selkäydinkanavan sisäiset aivokalvojen imusuonet kehittyivät ensimmäisen syntymänjälkeisen kuukauden aikana. Kehitys tapahtui verisuonten ja hermojen vierustilassa lymfangiogeneesin sekä erillisten imusuoniendoteelisaarekkeiden yhdistymisen kautta. Havaitsimme myös, että vaikuttamalla VEGF-C/VEGFR3-signalointijärjestelmään, pystyimme yllättävän selektiivisesti muokkaamaan aivokalvojen imusuonten määrää ja toiminnallisuutta. Aivokalvojen imusuonten erityislaatuinen muokkautuvuus ja uudelleenkasvupotentiaali tarjoavat uusia mahdollisuuksia niiden merkityksen tutkimiseen keskushermoston fysiologiassa ja sairauksissa. Merkittävän aivokalvojen imusuonijärjestelmän löytyminen herätti monia kysymyksiä erityisesti keskushermoston immuunijärjestelmän toiminnasta ja molekyylien poistumisesta. Tutkimuksessa IV pyrimme ymmärtämään kovakalvon imusuonten toimintaa Alzheimerin taudissa ja erityisesti siihen liittyvässä beeta-amyloidin (Aβ) patologiassa. Tutkimuksessa muokkasimme aivokalvojen imusuonia estämällä ja tehostamalla VEGF-C-kasvutekijän vaikutusta kahdessa laajasti käytetyssä Alzheimerin taudin hiirimallissa. Tuloksemme osoittavat, että aivokalvojen imusuonten pitkäkestoinen toiminnallinen muokkaaminen ei vaikuta Aβ-plakkien kertymiseen aivoihin ja että kompensoivat mekanismit ylläpitävät aivoselkäydinnesteen ulosvirtausta kallon ulkopuolelle. Kaiken kaikkiaan väitöskirjani tulokset luovat perustan Schlemmin kanavan imusuonen kaltaisuuteen sekä kallon ja selkäydinkanavan aivokalvoihin liittyvälle tutkimukselle. Lisäksi tuloksemme korostavat tarvetta vertailla erilaisia menetelmiä aivokalvojen imusuonten muokkaamiseksi sekä tarvetta saada syvempi mekanistinen ymmärrys keskushermostoon liittyvien nesteiden, solujen, ja molekyylien tuotannosta, kierrosta ja ulosvirtauksesta niin fysiologisissa kuin patologisissa olosuhteissa. Erityisen tärkeää on, että tuloksemme saattavat avata aivan uusia hoitomahdollisuuksia silmään ja keskushermoston sairauksille

Blockade of VEGFR3 signaling leads to functional impairment of dural lymphatic vessels without affecting autoimmune neuroinflammation

This is the author's version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science Immunology 8, (2023-04-14), doi: 10.1126/sciimmunol.abq0375.The recent discovery of lymphatic vessels (LVs) in the dura mater, the outermost layer of meninges around the central nervous system (CNS), has opened a possibility for the development of alternative therapeutics for CNS disorders. The vascular endothelial growth factor C (VEGF-C)/VEGF receptor 3 (VEGFR3) signaling pathway is essential for the development and maintenance of dural LVs. However, its significance in mediating dural lymphatic function in CNS autoimmunity is unclear. We show that inhibition of the VEGF-C/VEGFR3 signaling pathway using a monoclonal VEGFR3-blocking antibody, a soluble VEGF-C/D trap, or deletion of the Vegfr3 gene in adult lymphatic endothelium causes notable regression and functional impairment of dural LVs but has no effect on the development of CNS autoimmunity in mice. During autoimmune neuroinflammation, the dura mater was only minimally affected, and neuroinflammation-induced helper T (TH) cell recruitment, activation, and polarization were significantly less pronounced in the dura mater than in the CNS. In support of this notion, during autoimmune neuroinflammation, blood vascular endothelial cells in the cranial and spinal dura expressed lower levels of cell adhesion molecules and chemokines, and antigen-presenting cells (i.e., macrophages and dendritic cells) had lower expression of chemokines, MHC class II–associated molecules, and costimulatory molecules than their counterparts in the brain and spinal cord, respectively. The significantly weaker TH cell responses in the dura mater may explain why dural LVs do not contribute directly to CNS autoimmunity.Peer reviewe

Angiopoietin-2 blockade ameliorates autoimmune neuroinflammation by inhibiting leukocyte recruitment into the CNS

Angiopoietin-2 (Ang2), a ligand of the endothelial Tie2 tyrosine kinase, is involved in vascular inflammation and leakage in critically ill patients. However, the role of Ang2 in demyelinating central nervous system (CNS) autoimmune diseases is unknown. Here, we report that Ang2 is critically involved in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis. Ang2 expression was induced in CNS autoimmunity, and transgenic mice overexpressing Ang2 specifically in endothelial cells (ECs) developed a significantly more severe EAE. In contrast, treatment with Ang2-blocking Abs ameliorated neuroinflammation and decreased spinal cord demyelination and leukocyte infiltration into the CNS. Similarly, Ang2-binding and Tie2-activating Ab attenuated the development of CNS autoimmune disease. Ang2 blockade inhibited expression of EC adhesion molecules, improved blood-brain barrier integrity, and decreased expression of genes involved in antigen presentation and proinflammatory responses of microglia and macrophages, which was accompanied by inhibition of α5β1 integrin activation in microglia. Taken together, our data suggest that Ang2 provides a target for increasing Tie2 activation in ECs and inhibiting proinflammatory polarization of CNS myeloid cells via α5β1 integrin in neuroinflammation. Thus, Ang2 targeting may serve as a therapeutic option for the treatment of CNS autoimmune disease.Peer reviewe