Unraveling the lymphatic system in the spinal cord meninges: a critical element in protecting the central nervous system.

The lymphatic vasculature plays a crucial role in fluid clearance and immune responses in peripheral organs by connecting them to distal lymph nodes. Recently, attention has been drawn to the lymphatic vessel network surrounding the brain's border tissue (Aspelund et al. in J Exp Med 212:991-999, 2015. https://doi.org/10.1084/jem.20142290 ; Louveau et al. in Nat Neurosci 21:1380-1391, 2018. https://doi.org/10.1038/s41593-018-0227-9 ), which guides immune cells in mediating protection against tumors (Song et al. in Nature 577:689-694, 2020. https://doi.org/10.1038/s41586-019-1912-x ) and pathogens Li et al. (Nat Neurosci 25:577-587, 2022. https://doi.org/10.1038/s41593-022-01063-z ) while also contributing to autoimmunity (Louveau et al. 2018) and neurodegeneration (Da Mesquita et al. in Nature 560:185-191, 2018. https://doi.org/10.1038/s41586-018-0368-8 ). New studies have highlighted the integral involvement of meningeal lymphatic vessels in neuropathology. However, our limited understanding of spinal cord meningeal lymphatics and immunity hinders efforts to protect and heal the spinal cord from infections, injury, and other immune-mediated diseases. This review aims to provide a comprehensive overview of the state of spinal cord meningeal immunity, highlighting its unique immunologically relevant anatomy, discussing immune cells and lymphatic vasculature, and exploring the potential impact of injuries and inflammatory disorders on this intricate environment.

Parenchymal border macrophages regulate tau pathology and tau-mediated neurodegeneration.

Parenchymal border macrophages (PBMs) reside close to the central nervous system parenchyma and regulate CSF flow dynamics. We recently demonstrated that PBMs provide a clearance pathway for amyloid-ß peptide, which accumulates in the brain in Alzheimer's disease (AD). Given the emerging role for PBMs in AD, we explored how tau pathology affects the CSF flow and the PBM populations in the PS19 mouse model of tau pathology. We demonstrated a reduction of CSF flow, and an increase in an MHCII+PBM subpopulation in PS19 mice compared with WT littermates. Consequently, we asked whether PBM dysfunction could exacerbate tau pathology and tau-mediated neurodegeneration. Pharmacological depletion of PBMs in PS19 mice led to an increase in tau pathology and tau-dependent neurodegeneration, which was independent of gliosis or aquaporin-4 depolarization, essential for the CSF-ISF exchange. Together, our results identify PBMs as novel cellular regulators of tau pathology and tau-mediated neurodegeneration.

Age-dependent immune and lymphatic responses after spinal cord injury.

Spinal cord injury (SCI) causes lifelong debilitating conditions. Previous works demonstrated the essential role of the immune system in recovery after SCI. Here, we explored the temporal changes of the response after SCI in young and aged mice in order to characterize multiple immune populations within the mammalian spinal cord. We revealed substantial infiltration of myeloid cells to the spinal cord in young animals, accompanied by changes in the activation state of microglia. In contrast, both processes were blunted in aged mice. Interestingly, we discovered the formation of meningeal lymphatic structures above the lesion site, and their role has not been examined after contusive injury. Our transcriptomic data predicted lymphangiogenic signaling between myeloid cells in the spinal cord and lymphatic endothelial cells (LECs) in the meninges after SCI. Together, our findings delineate how aging affects the immune response following SCI and highlight the participation of the spinal cord meninges in supporting vascular repair.

Microglia-mediated T cell infiltration drives neurodegeneration in tauopathy.

Extracellular deposition of amyloid-ß as neuritic plaques and intracellular accumulation of hyperphosphorylated, aggregated tau as neurofibrillary tangles are two of the characteristic hallmarks of Alzheimer's disease1,2. The regional progression of brain atrophy in Alzheimer's disease highly correlates with tau accumulation but not amyloid deposition3-5, and the mechanisms of tau-mediated neurodegeneration remain elusive. Innate immune responses represent a common pathway for the initiation and progression of some neurodegenerative diseases. So far, little is known about the extent or role of the adaptive immune response and its interaction with the innate immune response in the presence of amyloid-ß or tau pathology6. Here we systematically compared the immunological milieux in the brain of mice with amyloid deposition or tau aggregation and neurodegeneration. We found that mice with tauopathy but not those with amyloid deposition developed a unique innate and adaptive immune response and that depletion of microglia or T cells blocked tau-mediated neurodegeneration. Numbers of T cells, especially those of cytotoxic T cells, were markedly increased in areas with tau pathology in mice with tauopathy and in the Alzheimer's disease brain. T cell numbers correlated with the extent of neuronal loss, and the cells dynamically transformed their cellular characteristics from activated to exhausted states along with unique TCR clonal expansion. Inhibition of interferon-γ and PDCD1 signalling both significantly ameliorated brain atrophy. Our results thus reveal a tauopathy- and neurodegeneration-related immune hub involving activated microglia and T cell responses, which could serve as therapeutic targets for preventing neurodegeneration in Alzheimer's disease and primary tauopathies.

ApoE isoform- and microbiota-dependent progression of neurodegeneration in a mouse model of tauopathy.

Tau-mediated neurodegeneration is a hallmark of Alzheimer's disease. Primary tauopathies are characterized by pathological tau accumulation and neuronal and synaptic loss. Apolipoprotein E (ApoE)-mediated neuroinflammation is involved in the progression of tau-mediated neurodegeneration, and emerging evidence suggests that the gut microbiota regulates neuroinflammation in an APOE genotype-dependent manner. However, evidence of a causal link between the microbiota and tau-mediated neurodegeneration is lacking. In this study, we characterized a genetically engineered mouse model of tauopathy expressing human ApoE isoforms reared under germ-free conditions or after perturbation of their gut microbiota with antibiotics. Both of these manipulations reduced gliosis, tau pathology, and neurodegeneration in a sex- and ApoE isoform-dependent manner. The findings reveal mechanistic and translationally relevant interrelationships between the microbiota, neuroinflammation, and tau-mediated neurodegeneration.

Age-associated suppression of exploratory activity during sickness is linked to meningeal lymphatic dysfunction and microglia activation.

Peripheral inflammation triggers a transient, well-defined set of behavioral changes known as sickness behavior1-3, but the mechanisms by which inflammatory signals originating in the periphery alter activity in the brain remain obscure. Emerging evidence has established meningeal lymphatic vasculature as an important interface between the central nervous system (CNS) and the immune system, responsible for facilitating brain solute clearance and perfusion by cerebrospinal fluid (CSF)4,5. Here, we demonstrate that meningeal lymphatics both assist microglial activation and support the behavioral response to peripheral inflammation. Ablation of meningeal lymphatics results in a heightened behavioral response to IL-1ß-induced inflammation and a dampened transcriptional and morphological microglial phenotype. Moreover, our findings support a role for microglia in tempering the severity of sickness behavior with specific relevance to aging-related meningeal lymphatic dysfunction. Transcriptional profiling of brain myeloid cells shed light on the impact of meningeal lymphatic dysfunction on microglial activation. Furthermore, we demonstrate that experimental enhancement of meningeal lymphatic function in aged mice is sufficient to reduce the severity of exploratory abnormalities but not pleasurable consummatory behavior. Finally, we identify dysregulated genes and biological pathways, common to both experimental meningeal lymphatic ablation and aging, in microglia responding to peripheral inflammation that may result from age-related meningeal lymphatic dysfunction.

The role of protease-activated receptor 1 signaling in CD8 T cell effector functions.

CD8 T cells are essential for adaptive immunity against viral infections. Protease activated receptor 1 (PAR1) is expressed by CD8 T cells; however, its role in T cell effector function is not well defined. Here we show that in human CD8 T cells, PAR1 stimulation accelerates calcium mobilization. Furthermore, PAR1 is involved in cytotoxic T cell function by facilitating granule trafficking via actin polymerization and repositioning of the microtubule organizing center (MTOC) toward the immunological synapse. In vivo, PAR1-/- mice have reduced cytokine-producing T cells in response to a lymphocytic choriomeningitis virus (LCMV) infection and fail to efficiently control the virus. Specific deletion of PAR1 in LCMV GP33-specific CD8 T cells results in reduced expansion and diminished effector function. These data demonstrate that PAR1 plays a role in T cell activation and function, and this pathway could represent a new therapeutic strategy to modulate CD8 T cell effector function.

GABAergic neuronal IL-4R mediates T cell effect on memory.

Mechanisms governing how immune cells and their derived molecules impact homeostatic brain function are still poorly understood. Here, we elucidate neuronal mechanisms underlying T cell effects on synaptic function and episodic memory. Depletion of CD4 T cells led to memory deficits and impaired long-term potentiation. Severe combined immune-deficient mice exhibited amnesia, which was reversible by repopulation with T cells from wild-type but not from IL-4-knockout mice. Behaviors impacted by T cells were mediated via IL-4 receptors expressed on neurons. Exploration of snRNA-seq of neurons participating in memory processing provided insights into synaptic organization and plasticity-associated pathways regulated by immune cells. IL-4Rα knockout in inhibitory (but not in excitatory) neurons was sufficient to impair contextual fear memory, and snRNA-seq from these mice pointed to IL-4-driven regulation of synaptic function in promoting memory. These findings provide new insights into complex neuroimmune interactions at the transcriptional and functional levels in neurons under physiological conditions.

Skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and CNS parenchyma.

The meninges are a membranous structure enveloping the central nervous system (CNS) that host a rich repertoire of immune cells mediating CNS immune surveillance. Here, we report that the mouse meninges contain a pool of monocytes and neutrophils supplied not from the blood but by adjacent skull and vertebral bone marrow. Under pathological conditions, including spinal cord injury and neuroinflammation, CNS-infiltrating myeloid cells can originate from brain borders and display transcriptional signatures distinct from their blood-derived counterparts. Thus, CNS borders are populated by myeloid cells from adjacent bone marrow niches, strategically placed to supply innate immune cells under homeostatic and pathological conditions. These findings call for a reinterpretation of immune-cell infiltration into the CNS during injury and autoimmunity and may inform future therapeutic approaches that harness meningeal immune cells.

Aging-associated deficit in CCR7 is linked to worsened glymphatic function, cognition, neuroinflammation, and ß-amyloid pathology.

Aging leads to a progressive deterioration of meningeal lymphatics and peripheral immunity, which may accelerate cognitive decline. We hypothesized that an age-related reduction in C-C chemokine receptor type 7 (CCR7)-dependent egress of immune cells through the lymphatic vasculature mediates some aspects of brain aging and potentially exacerbates cognitive decline and Alzheimer's disease-like brain ß-amyloid (Aß) pathology. We report a reduction in CCR7 expression by meningeal T cells in old mice that is linked to increased effector and regulatory T cells. Hematopoietic CCR7 deficiency mimicked the aging-associated changes in meningeal T cells and led to reduced glymphatic influx and cognitive impairment. Deletion of CCR7 in 5xFAD transgenic mice resulted in deleterious neurovascular and microglial activation, along with increased Aß deposition in the brain. Treating old mice with anti-CD25 antibodies alleviated the exacerbated meningeal regulatory T cell response and improved cognitive function, highlighting the therapeutic potential of modulating meningeal immunity to fine-tune brain function in aging and in neurodegenerative diseases.

Meningeal lymphatics affect microglia responses and anti-Aß immunotherapy.

Alzheimer's disease (AD) is the most prevalent cause of dementia1. Although there is no effective treatment for AD, passive immunotherapy with monoclonal antibodies against amyloid beta (Aß) is a promising therapeutic strategy2,3. Meningeal lymphatic drainage has an important role in the accumulation of Aß in the brain4, but it is not known whether modulation of meningeal lymphatic function can influence the outcome of immunotherapy in AD. Here we show that ablation of meningeal lymphatic vessels in 5xFAD mice (a mouse model of amyloid deposition that expresses five mutations found in familial AD) worsened the outcome of mice treated with anti-Aß passive immunotherapy by exacerbating the deposition of Aß, microgliosis, neurovascular dysfunction, and behavioural deficits. By contrast, therapeutic delivery of vascular endothelial growth factor C improved clearance of Aß by monoclonal antibodies. Notably, there was a substantial overlap between the gene signature of microglia from 5xFAD mice with impaired meningeal lymphatic function and the transcriptional profile of activated microglia from the brains of individuals with AD. Overall, our data demonstrate that impaired meningeal lymphatic drainage exacerbates the microglial inflammatory response in AD and that enhancement of meningeal lymphatic function combined with immunotherapies could lead to better clinical outcomes.

Functional characterization of the dural sinuses as a neuroimmune interface.

Despite the established dogma of central nervous system (CNS) immune privilege, neuroimmune interactions play an active role in diverse neurological disorders. However, the precise mechanisms underlying CNS immune surveillance remain elusive; particularly, the anatomical sites where peripheral adaptive immunity can sample CNS-derived antigens and the cellular and molecular mediators orchestrating this surveillance. Here, we demonstrate that CNS-derived antigens in the cerebrospinal fluid (CSF) accumulate around the dural sinuses, are captured by local antigen-presenting cells, and are presented to patrolling T cells. This surveillance is enabled by endothelial and mural cells forming the sinus stromal niche. T cell recognition of CSF-derived antigens at this site promoted tissue resident phenotypes and effector functions within the dural meninges. These findings highlight the critical role of dural sinuses as a neuroimmune interface, where brain antigens are surveyed under steady-state conditions, and shed light on age-related dysfunction and neuroinflammatory attack in animal models of multiple sclerosis.

Meningeal γδ T cells regulate anxiety-like behavior via IL-17a signaling in neurons.

Interleukin (IL)-17a has been highly conserved during evolution of the vertebrate immune system and widely studied in contexts of infection and autoimmunity. Studies suggest that IL-17a promotes behavioral changes in experimental models of autism and aggregation behavior in worms. Here, through a cellular and molecular characterization of meningeal γδ17 T cells, we defined the nearest central nervous system-associated source of IL-17a under homeostasis. Meningeal γδ T cells express high levels of the chemokine receptor CXCR6 and seed meninges shortly after birth. Physiological release of IL-17a by these cells was correlated with anxiety-like behavior in mice and was partially dependent on T cell receptor engagement and commensal-derived signals. IL-17a receptor was expressed in cortical glutamatergic neurons under steady state and its genetic deletion decreased anxiety-like behavior in mice. Our findings suggest that IL-17a production by meningeal γδ17 T cells represents an evolutionary bridge between this conserved anti-pathogen molecule and survival behavioral traits in vertebrates.

Meningeal Lymphatics: From Anatomy to Central Nervous System Immune Surveillance.

At steady state, the CNS parenchyma has few to no lymphocytes and less potent Ag-presentation capability compared with other organs. However, the meninges surrounding the CNS host diverse populations of immune cells that influence how CNS-related immune responses develop. Interstitial and cerebrospinal fluid produced in the CNS is continuously drained, and recent advances have emphasized that this process is largely taking place through the lymphatic system. To what extent this fluid process mobilizes CNS-derived Ags toward meningeal immune cells and subsequently the peripheral immune system through the lymphatic vessel network is a question of significant clinical importance for autoimmunity, tumor immunology, and infectious disease. Recent advances in understanding the role of meningeal lymphatics as a communicator between the brain and peripheral immunity are discussed in this review.

Laboratory mice born to wild mice have natural microbiota and model human immune responses.

Laboratory mouse studies are paramount for understanding basic biological phenomena but also have limitations. These include conflicting results caused by divergent microbiota and limited translational research value. To address both shortcomings, we transferred C57BL/6 embryos into wild mice, creating "wildlings." These mice have a natural microbiota and pathogens at all body sites and the tractable genetics of C57BL/6 mice. The bacterial microbiome, mycobiome, and virome of wildlings affect the immune landscape of multiple organs. Their gut microbiota outcompete laboratory microbiota and demonstrate resilience to environmental challenges. Wildlings, but not conventional laboratory mice, phenocopied human immune responses in two preclinical studies. A combined natural microbiota- and pathogen-based model may enhance the reproducibility of biomedical studies and increase the bench-to-bedside safety and success of immunological studies.

Old T Cells Interfer(on) with Neurogenesis.

Adult neurogenesis plays an important role in brain function and declines with aging. A recent report demonstrates clonal T cell expansion within neurogenic niches of the aged brain, impairing neurogenesis through IFNγ signaling (Dulken et al.,Nature, 2019). These results highlight T cells as important contributors to and potential therapeutic targets for age-related brain dysfunction.

CNS lymphatic drainage and neuroinflammation are regulated by meningeal lymphatic vasculature.

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.

Morphological and Functional Analysis of CNS-Associated Lymphatics.

The study of meningeal lymphatic vessels of the central nervous system (CNS) has recently gathered momentum, with several papers dissecting their role in draining solutes from cerebrospinal fluid and brain (Louveau et al., Nature 523(7560):337-341, 2015; Antila et al., J Exp Med 214(12):3645-3667, 2017; Aspelund et al., J Exp Med 212(7):991-999, 2015). Methodological capabilities, however, have been limited to few laboratories due to difficulties reproducibly visualizing these rare cell subsets in the meninges. To explore meningeal lymphatics fundamental role during homeostasis and how they may contribute to human pathology, the field has begun to require purification and characterization of lymphatic endothelial cells. Here, modern cell biological techniques involving a combination of histological, flow-cytometric, and functional drainage assays are applied to brain and spinal cord meninges and detailed stepwise procedures used for successful in vivo and ex vivo characterization of meningeal lymphatic vessels.

Myeloid Cells in the Central Nervous System.

The central nervous system (CNS) and its meningeal coverings accommodate a diverse myeloid compartment that includes parenchymal microglia and perivascular macrophages, as well as choroid plexus and meningeal macrophages, dendritic cells, and granulocytes. These myeloid populations enjoy an intimate relationship with the CNS, where they play an essential role in both health and disease. Although the importance of these cells is clearly recognized, their exact function in the CNS continues to be explored. Here, we review the subsets of myeloid cells that inhabit the parenchyma, meninges, and choroid plexus and discuss their roles in CNS homeostasis. We also discuss the role of these cells in various neurological pathologies, such as autoimmunity, mechanical injury, neurodegeneration, and infection. We highlight the neuroprotective nature of certain myeloid cells by emphasizing their therapeutic potential for the treatment of neurological conditions.

Influenza A induces dysfunctional immunity and death in MeCP2-overexpressing mice.

Loss of function or overexpression of methyl-CpG-binding protein 2 (MeCP2) results in the severe neurodevelopmental disorders Rett syndrome and MeCP2 duplication syndrome, respectively. MeCP2 plays a critical role in neuronal function and the function of cells throughout the body. It has been previously demonstrated that MeCP2 regulates T cell function and macrophage response to multiple stimuli, and that immune-mediated rescue imparts significant benefit in Mecp2-null mice. Unlike Rett syndrome, MeCP2 duplication syndrome results in chronic, severe respiratory infections, which represent a significant cause of patient morbidity and mortality. Here, we demonstrate that MeCP2Tg3 mice, which overexpress MeCP2 at levels 3- to 5-fold higher than normal, are hypersensitive to influenza A/PR/8/34 infection. Prior to death, MeCP2Tg3 mice experienced a host of complications during infection, including neutrophilia, increased cytokine production, excessive corticosterone levels, defective adaptive immunity, and vascular pathology characterized by impaired perfusion and pulmonary hemorrhage. Importantly, we found that radioresistant cells are essential to infection-related death after bone marrow transplantation. In all, these results demonstrate that influenza A infection in MeCP2Tg3 mice results in pathology affecting both immune and nonhematopoietic cells, suggesting that failure to effectively respond and clear viral respiratory infection has a complex, multicompartment etiology in the context of MeCP2 overexpression.