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.

Neuroimmune signaling at the brain borders.

The central nervous system (CNS) has been viewed as an immunologically privileged site, but emerging works are uncovering a large array of neuroimmune interactions primarily occurring at its borders. CNS barriers sites host diverse population of both innate and adaptive immune cells capable of, directly and indirectly, influence the function of the residing cells of the brain parenchyma. These structures are only starting to reveal their role in controlling brain function under normal and pathological conditions and represent an underexplored therapeutic target for the treatment of brain disorders. This review will highlight the development of the CNS barriers to host neuro-immune interactions and emphasize their newly described roles in neurodevelopmental, neurological, and neurodegenerative disorders, particularly for the meninges.

Publisher Correction: Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Change history: In this Article, Extended Data Fig. 9 was appearing as Fig. 2 in the HTML, and in Fig. 2, the panel labels 'n' and 'o' overlapped the figure; these errors have been corrected online.

Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease.

Ageing is a major risk factor for many neurological pathologies, but its mechanisms remain unclear. Unlike other tissues, the parenchyma of the central nervous system (CNS) lacks lymphatic vasculature and waste products are removed partly through a paravascular route. (Re)discovery and characterization of meningeal lymphatic vessels has prompted an assessment of their role in waste clearance from the CNS. Here we show that meningeal lymphatic vessels drain macromolecules from the CNS (cerebrospinal and interstitial fluids) into the cervical lymph nodes in mice. Impairment of meningeal lymphatic function slows paravascular influx of macromolecules into the brain and efflux of macromolecules from the interstitial fluid, and induces cognitive impairment in mice. Treatment of aged mice with vascular endothelial growth factor C enhances meningeal lymphatic drainage of macromolecules from the cerebrospinal fluid, improving brain perfusion and learning and memory performance. Disruption of meningeal lymphatic vessels in transgenic mouse models of Alzheimer's disease promotes amyloid-ß deposition in the meninges, which resembles human meningeal pathology, and aggravates parenchymal amyloid-ß accumulation. Meningeal lymphatic dysfunction may be an aggravating factor in Alzheimer's disease pathology and in age-associated cognitive decline. Thus, augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.

Meningeal Lymphatic vasculature in health and disease.

PURPOSE OF REVIEW: The recent (re)discovery of the meningeal lymphatic has brought a new player in brain neurophysiology. This review highlights the state of the current research on the meningeal lymphatic vasculature, from its specific physiology to its increasing implication in normal and pathological brain function. RECENT FINDINGS: Growing evidence are emerging about the uniqueness of the meningeal lymphatic vasculature and its implication in multiple neurological and neurotraumatic disorders. SUMMARY: These studies are highlighting a new and unexpected role for the lymphatic vasculature in brain function and a potential new therapeutic target for neurological disorders.

Structural and functional features of central nervous system lymphatic vessels.

One of the characteristics of the central nervous system is the lack of a classical lymphatic drainage system. Although it is now accepted that the central nervous system undergoes constant immune surveillance that takes place within the meningeal compartment, the mechanisms governing the entrance and exit of immune cells from the central nervous system remain poorly understood. In searching for T-cell gateways into and out of the meninges, we discovered functional lymphatic vessels lining the dural sinuses. These structures express all of the molecular hallmarks of lymphatic endothelial cells, are able to carry both fluid and immune cells from the cerebrospinal fluid, and are connected to the deep cervical lymph nodes. The unique location of these vessels may have impeded their discovery to date, thereby contributing to the long-held concept of the absence of lymphatic vasculature in the central nervous system. The discovery of the central nervous system lymphatic system may call for a reassessment of basic assumptions in neuroimmunology and sheds new light on the aetiology of neuroinflammatory and neurodegenerative diseases associated with immune system dysfunction.

Revisiting the Mechanisms of CNS Immune Privilege.

Whereas the study of the interactions between the immune system and the central nervous system (CNS) has often focused on pathological conditions, the importance of neuroimmune communication in CNS homeostasis and function has become clear over that last two decades. Here we discuss the progression of our understanding of the interaction between the peripheral immune system and the CNS. We examine the notion of immune privilege of the CNS in light of both earlier findings and recent studies revealing a functional meningeal lymphatic system that drains cerebrospinal fluid (CSF) to the deep cervical lymph nodes, and consider the implications of a revised perspective on the immune privilege of the CNS on the etiology and pathology of different neurological disorders.

Targeting the CD80/CD86 costimulatory pathway with CTLA4-Ig directs microglia toward a repair phenotype and promotes axonal outgrowth.

Among the costimulatory factors widely studied in the immune system is the CD28/cytotoxic T-lymphocyte antigen-4 (CTLA4)-CD80/CD86 pathway, which critically controls the nature and duration of the T-cell response. In the brain, up-regulated expression of CD80/CD86 during inflammation has consistently been reported in microglia. However, the role of CD80/CD86 molecules has mainly been studied in a context of microglia-T cell interactions in pathological conditions, while the function of CD80/CD86 in the regulation of intrinsic brain cells remains largely unknown. In this study, we used a transgenic pig line in which neurons express releasable CTLA4-Ig, a synthetic molecule mimicking CTLA4 and binding to CD80/CD86. The effects of CTLA4-Ig on brain cells were analyzed after intracerebral transplantation of CTLA4-Ig-expressing neurons or wild-type neurons as control. This model provided in vivo evidence that CTLA4-Ig stimulated axonal outgrowth, in correlation with a shift of the nearby microglia from a compact to a ramified morphology. In a culture system, we found that the CTLA4-Ig-induced morphological change of microglia was mediated through CD86, but not CD80. This was accompanied by microglial up-regulated expression of the anti-inflammatory molecule Arginase 1 and the neurotrophic factor BDNF, in an astrocyte-dependent manner through the purinergic P2Y1 receptor pathway. Our study identifies for the first time CD86 as a key player in the modulation of microglia phenotype and suggests that CTLA4-Ig-derived compounds might represent new tools to manipulate CNS microglia.

Impaired spatial memory in mice lacking CD3ζ is associated with altered NMDA and AMPA receptors signaling independent of T-cell deficiency.

The immunoreceptor-associated protein CD3ζ is known for its role in immunity and has also been implicated in neuronal development and synaptic plasticity. However, the mechanism by which CD3ζ regulates synaptic transmission remains unclear. In this study, we showed that mice lacking CD3ζ exhibited defects in spatial learning and memory as examined by the Barnes maze and object location memory tasks. Given that peripheral T cells have been shown to support cognitive functions and neural plasticity, we generated CD3ζ(-/-) mice in which the peripheral T cells were repopulated to a normal level by syngeneic bone marrow transplantation. Using this approach, we showed that T-cell replenishment in CD3ζ(-/-) mice did not restore spatial memory defects, suggesting that the cognitive deficits in CD3ζ(-/-) mice were most likely mediated through a T-cell-independent mechanism. In support of this idea, we showed that CD3ζ proteins were localized to glutamatergic postsynaptic sites, where they interacted with the NMDAR subunit GluN2A. Loss of CD3ζ in brain decreased GluN2A-PSD95 association and GluN2A synaptic localization. This effect was accompanied by a reduced interaction of GluN2A with the key NMDAR downstream signaling protein calcium/calmodulin-dependent protein kinase II (CaMKII). Using the glycine-induced, NMDA-dependent form of chemical long-term potentiation (LTP) in cultured cortical neurons, we showed that CD3ζ was required for activity-dependent CaMKII autophosphorylation and for the synaptic recruitment of the AMPAR subunit GluA1. Together, these results support the model that the procognitive function of CD3ζ may be mediated through its involvement in the NMDAR downstream signaling pathway leading to CaMKII-dependent LTP induction.

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis.

Meningeal Lymphatics: An Immune Gateway for the Central Nervous System.

The recent (re)discovery of the meningeal lymphatic system has opened new theories as to how immune cells traffic and interact with the central nervous system (CNS). While evidence is accumulating on the contribution of the meningeal lymphatic system in both homeostatic and disease conditions, a lot remains unknown about the mechanisms that allow for interaction between the meningeal lymphatic system and immune cells. In this review, we synthesize the knowledge about the lymphatic immune interaction in the CNS and highlight the important questions that remain to be answered.

Meningeal lymphatics, immunity and neuroinflammation.

In the past five years, the surrounding of the brain, that is the meninges (singular meninx) have evolved from being a physical barrier that protects the brain parenchyma to becoming a central player for both the maintenance of normal brain function and the modulation of neurological disorders. Indeed, the meninges are an immunologically active compartment that communicates with the periphery via the (re)discovered meningeal lymphatic system. From its ties to both the periphery and the central nervous system, the meninges are becoming a prevalent organ to understand and modulate brain homeostasis. Here we will focus on current advances in our understanding of the meningeal compartment with an emphasis on the meningeal lymphatic network as a key regulator.

Meningeal Immunity, Drainage, and Tertiary Lymphoid Structure Formation.

For decades, the brain has been considered an immune-privileged organ, meaning that the brain was mainly ignored by the immune system and that the presence of immune cells, notably of the adaptive arm, was a hallmark of pathological conditions. Over the past few decades, the definition of the immune privilege continues to be refined. There has been evidence accumulating that shows that the immune system plays a role in proper brain function. This evidence may represent an effective source of therapeutic targets for neurological disorders. In this chapter, we discuss the recent advances in understanding the immunity of the brain and describe how tertiary lymphoid structures can be generated in the central nervous system, which might represent a new avenue to treat neurological disorders.

Sex, Gut, and Microglia.

Microglia are brain-resident macrophages whose function affects a myriad of physiological processes and can in turn be affected by peripheral factors. In a recent issue of Cell, Garel, Ginhoux and colleagues describe how gender, developmental stage, and microbiome contribute to the transcriptome of microglia (Thion et al., 2018).

Meningeal whole mount preparation and characterization of neural cells by flow cytometry.

Neuroimmunologists aim to understand the interactions between the central nervous system and the immune system under both homeostatic and pathological conditions. The meninges, contrary to the brain parenchyma, are populated by numerous immune cells. Soluble factors produced by these cells are capable to diffuse into the brain parenchyma and influence the brain cells within the parenchyma, including neurons. In this unit, we will describe two protocols: analysis the meningeal compartment of rodents and the use flow cytometry to study the cells of the brain parenchyma (particularly neurons).

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.

Peripherally derived macrophages can engraft the brain independent of irradiation and maintain an identity distinct from microglia.

Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens.