Neuroinflammation creates an immune regulatory niche at the meningeal lymphatic vasculature near the cribriform plate.

Meningeal lymphatics near the cribriform plate undergo lymphangiogenesis during neuroinflammation to drain excess fluid. Here, we hypothesized that lymphangiogenic vessels may acquire an altered phenotype to regulate immunity. Using single-cell RNA sequencing of meningeal lymphatics near the cribriform plate from healthy and experimental autoimmune encephalomyelitis in the C57BL/6 model, we report that neuroinflammation induces the upregulation of genes involved in antigen presentation such as major histocompatibility complex class II, adhesion molecules including vascular cell adhesion protein 1 and immunoregulatory molecules such as programmed cell death 1 ligand 1, where many of these changes are mediated by interferon-γ. The inflamed lymphatics retain CD11c+ cells and CD4 T cells where they capture and present antigen, creating an immunoregulatory niche that represents an underappreciated interface in the regulation of neuroinflammation. We also found discontinuity of the arachnoid membrane near the cribriform plate, which provides unrestricted access to the cerebrospinal fluid. These findings highlight a previously unknown function of local meningeal lymphatics in regulating immunity that has only previously been characterized in draining lymph nodes.

An antioxidant and anti-ER stress combo therapy decreases inflammation, secondary brain damage and promotes neurological recovery following traumatic brain injury in mice.

The complex pathophysiology of post-traumatic brain damage might need a polypharmacological strategy with a combination of drugs that target multiple, synergistic mechanisms. We currently tested a combination of apocynin (curtails formation of reactive oxygen species; ROS), tert-butylhydroquinone (promotes disposal of ROS), and salubrinal (prevents endoplasmic reticulum stress) following a moderate traumatic brain injury (TBI) induced by controlled cortical impact in adult mice. Adult mice of both sexes treated with the above tri-combo showed alleviated motor and cognitive deficits, attenuated secondary lesion volume, and decreased oxidative DNA damage. Concomitantly, tri-combo treatment regulated post-TBI inflammatory response by decreasing the infiltration of T cells and neutrophils and activation of microglia in both sexes. Interestingly, sexual dimorphism was seen in the case of TBI-induced microgliosis and infiltration of macrophages in the tri-combo treated mice. Moreover, the tri-combo treatment prevented TBI-induced white matter volume loss in both sexes. The beneficial effects of tri-combo treatment were long-lasting and were also seen in aged mice. Thus, the present study supports the tri-combo treatment to curtail oxidative stress and endoplasmic reticulum stress concomitantly as a therapeutic strategy to improve TBI outcomes.SIGNIFICANCE STATEMENTOf the several mechanisms that contribute to TBI pathophysiology, oxidative stress, endoplasmic reticulum (ER) stress, and inflammation play a major role. The present study shows the therapeutic potential of a combination of apocynin, tert-butylhydroquinone, and salubrinal to prevent oxidative stress and ER stress and the interrelated inflammatory response in mice subjected to TBI. The beneficial effects of the tri-combo include alleviation of TBI-induced motor and cognitive deficits and lesion volume. The neuroprotective effects of the tri-combo are also linked to its ability to prevent TBI-induced white matter damage. Importantly, neuroprotection by the tri-combo treatment was observed to be not dependent on sex or age. Our data demonstrate that a polypharmacological strategy is efficacious after TBI.

CXCL13 expressed on inflamed cerebral blood vessels recruit IL-21 producing TFH cells to damage neurons following stroke.

BACKGROUND: Ischemic stroke is a leading cause of mortality worldwide, largely due to the inflammatory response to brain ischemia during post-stroke reperfusion. Despite ongoing intensive research, there have not been any clinically approved drugs targeting the inflammatory component to stroke. Preclinical studies have identified T cells as pro-inflammatory mediators of ischemic brain damage, yet mechanisms that regulate the infiltration and phenotype of these cells are lacking. Further understanding of how T cells migrate to the ischemic brain and facilitate neuronal death during brain ischemia can reveal novel targets for post-stroke intervention. METHODS: To identify the population of T cells that produce IL-21 and contribute to stroke, we performed transient middle cerebral artery occlusion (tMCAO) in mice and performed flow cytometry on brain tissue. We also utilized immunohistochemistry in both mouse and human brain sections to identify cell types and inflammatory mediators related to stroke-induced IL-21 signaling. To mechanistically demonstrate our findings, we employed pharmacological inhibitor anti-CXCL13 and performed histological analyses to evaluate its effects on brain infarct damage. Finally, to evaluate cellular mechanisms of stroke, we exposed mouse primary neurons to oxygen glucose deprivation (OGD) conditions with or without IL-21 and measured cell viability, caspase activity and JAK/STAT signaling. RESULTS: Flow cytometry on brains from mice following tMCAO identified a novel population of cells IL-21 producing CXCR5+ CD4+ ICOS-1+ T follicular helper cells (TFH) in the ischemic brain early after injury. We observed augmented expression of CXCL13 on inflamed brain vascular cells and demonstrated that inhibition of CXCL13 protects mice from tMCAO by restricting the migration and influence of IL-21 producing TFH cells in the ischemic brain. We also illustrate that neurons express IL-21R in the peri-infarct regions of both mice and human stroke tissue in vivo. Lastly, we found that IL-21 acts on mouse primary ischemic neurons to activate the JAK/STAT pathway and induce caspase 3/7-mediated apoptosis in vitro. CONCLUSION: These findings identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and provide a potential new therapeutic target for stroke.

Molecular Mechanisms of Neuroimmune Crosstalk in the Pathogenesis of Stroke.

Stroke disrupts the homeostatic balance within the brain and is associated with a significant accumulation of necrotic cellular debris, fluid, and peripheral immune cells in the central nervous system (CNS). Additionally, cells, antigens, and other factors exit the brain into the periphery via damaged blood-brain barrier cells, glymphatic transport mechanisms, and lymphatic vessels, which dramatically influence the systemic immune response and lead to complex neuroimmune communication. As a result, the immunological response after stroke is a highly dynamic event that involves communication between multiple organ systems and cell types, with significant consequences on not only the initial stroke tissue injury but long-term recovery in the CNS. In this review, we discuss the complex immunological and physiological interactions that occur after stroke with a focus on how the peripheral immune system and CNS communicate to regulate post-stroke brain homeostasis. First, we discuss the post-stroke immune cascade across different contexts as well as homeostatic regulation within the brain. Then, we focus on the lymphatic vessels surrounding the brain and their ability to coordinate both immune response and fluid homeostasis within the brain after stroke. Finally, we discuss how therapeutic manipulation of peripheral systems may provide new mechanisms to treat stroke injury.

Regional Distribution of CNS Antigens Differentially Determines T-Cell Mediated Neuroinflammation in a CX3CR1-Dependent Manner.

T cells continuously sample CNS-derived antigens in the periphery, yet it is unknown how they sample and respond to CNS antigens derived from distinct brain areas. We expressed ovalbumin (OVA) neoepitopes in regionally distinct CNS areas (Cnp-OVA and Nes-OVA mice) to test peripheral antigen sampling by OVA-specific T cells under homeostatic and neuroinflammatory conditions. We show that antigen sampling in the periphery is independent of regional origin of CNS antigens in both male and female mice. However, experimental autoimmune encephalomyelitis (EAE) is differentially influenced in Cnp-OVA and Nes-OVA female mice. Although there is the same frequency of CD45high CD11b+ CD11c+ CX3CL1+ myeloid cell-T-cell clusters in neoepitope-expressing areas, EAE is inhibited in Nes-OVA female mice and accelerated in CNP-OVA female mice. Accumulation of OVA-specific T cells and their immunomodulatory effects on EAE are CX3C chemokine receptor 1 (CX3CR1) dependent. These data show that despite similar levels of peripheral antigen sampling, CNS antigen-specific T cells differentially influence neuroinflammatory disease depending on the location of cognate antigens and the presence of CX3CL1/CX3CR1 signaling.SIGNIFICANCE STATEMENT Our data show that peripheral T cells similarly recognize neoepitopes independent of their origin within the CNS under homeostatic conditions. Contrastingly, during ongoing autoimmune neuroinflammation, neoepitope-specific T cells differentially influence clinical score and pathology based on the CNS regional location of the neoepitopes in a CX3CR1-dependent manner. Altogether, we propose a novel mechanism for how T cells respond to regionally distinct CNS derived antigens and contribute to CNS autoimmune pathology.

Immune responses in stroke: how the immune system contributes to damage and healing after stroke and how this knowledge could be translated to better cures?

Stroke is one of the leading causes of death and disability worldwide. The long-standing dogma that stroke is exclusively a vascular disease has been questioned by extensive clinical findings of immune factors that are associated mostly with inflammation after stroke. These have been confirmed in preclinical studies using experimental animal models. It is now accepted that inflammation and immune mediators are critical in acute and long-term neuronal tissue damage and healing following thrombotic and ischaemic stroke. Despite mounting information delineating the role of the immune system in stroke, the mechanisms of how inflammatory cells and their mediators are involved in stroke-induced neuroinflammation are still not fully understood. Currently, there is no available treatment for targeting the acute immune response that develops in the brain during cerebral ischaemia. No new treatment has been introduced to stroke therapy since the discovery of tissue plasminogen activator therapy in 1996. Here, we review current knowledge of the immunity of stroke and identify critical gaps that hinder current therapies. We will discuss advances in the understanding of the complex innate and adaptive immune responses in stroke; mechanisms of immune cell-mediated and factor-mediated vascular and tissue injury; immunity-induced tissue repair; and the importance of modulating immunity in stroke.

Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

Dual role of Vascular Endothelial Growth Factor-C (VEGF-C) in post-stroke recovery.

Using a mouse model of ischemic stroke, this study characterizes stroke-induced lymphangiogenesis at the cribriform plate (CP). While blocking CP lymphangiogenesis with a VEGFR-3 inhibitor improves stroke outcome, administration of VEGF-C induced larger brain infarcts. Abstract: Cerebrospinal fluid (CSF), antigens, and antigen-presenting cells drain from the central nervous system (CNS) into lymphatic vessels near the cribriform plate and dural meningeal lymphatics. However, the pathological roles of these lymphatic vessels surrounding the CNS during stroke are not well understood. Using a mouse model of ischemic stroke, transient middle cerebral artery occlusion (tMCAO), we show that stroke induces lymphangiogenesis near the cribriform plate. Interestingly, lymphangiogenesis is restricted to lymphatic vessels at the cribriform plate and downstream cervical lymph nodes, without affecting the conserved network of lymphatic vessels in the dura. Cribriform plate lymphangiogenesis peaks at day 7 and regresses by day 14 following tMCAO and is regulated by VEGF-C/VEGFR-3. These newly developed lymphangiogenic vessels transport CSF and immune cells to the cervical lymph nodes. Inhibition of VEGF-C/VEGFR-3 signaling using a blocker of VEGFR-3 prevented lymphangiogenesis and led to improved stroke outcomes at earlier time points but had no effects at later time points following stroke. Administration of VEGF-C after tMCAO did not further increase post-stroke lymphangiogenesis, but instead induced larger brain infarcts. The differential roles for VEGFR-3 inhibition and VEGF-C in regulating stroke pathology call into question recent suggestions to use VEGF-C therapeutically for stroke.

Neuroinflammation-Driven Lymphangiogenesis in CNS Diseases.

The central nervous system (CNS) undergoes immunosurveillance despite the lack of conventional antigen presenting cells and lymphatic vessels in the CNS parenchyma. Additionally, the CNS is bathed in a cerebrospinal fluid (CSF). CSF is continuously produced, and consequently must continuously clear to maintain fluid homeostasis despite the lack of conventional lymphatics. During neuroinflammation, there is often an accumulation of fluid, antigens, and immune cells to affected areas of the brain parenchyma. Failure to effectively drain these factors may result in edema, prolonged immune response, and adverse clinical outcome as observed in conditions including traumatic brain injury, ischemic and hypoxic brain injury, CNS infection, multiple sclerosis (MS), and brain cancer. Consequently, there has been renewed interest surrounding the expansion of lymphatic vessels adjacent to the CNS which are now thought to be central in regulating the drainage of fluid, cells, and waste out of the CNS. These lymphatic vessels, found at the cribriform plate, dorsal dural meninges, base of the brain, and around the spinal cord have each been implicated to have important roles in various CNS diseases. In this review, we discuss the contribution of meningeal lymphatics to these processes during both steady-state conditions and neuroinflammation, as well as discuss some of the many still unknown aspects regarding the role of meningeal lymphatics in neuroinflammation. Specifically, we focus on the observed phenomenon of lymphangiogenesis by a subset of meningeal lymphatics near the cribriform plate during neuroinflammation, and discuss their potential roles in immunosurveillance, fluid clearance, and access to the CSF and CNS compartments. We propose that manipulating CNS lymphatics may be a new therapeutic way to treat CNS infections, stroke, and autoimmunity.

Current concepts on communication between the central nervous system and peripheral immunity via lymphatics: what roles do lymphatics play in brain and spinal cord disease pathogenesis?

The central nervous system (CNS) lacks conventional lymphatics within the CNS parenchyma, yet still maintains fluid homeostasis and immunosurveillance. How the CNS communicates with systemic immunity has thus been a topic of interest for scientists in the past century, which has led to several theories of CNS drainage routes. In addition to perineural routes, rediscoveries of lymphatics surrounding the CNS in the meninges revealed an extensive network of lymphatics, which we now know play a significant role in fluid homeostasis and immunosurveillance. These meningeal lymphatic networks exist along the superior sagittal sinus and transverse sinus dorsal to the brain, near the cribriform plate below the olfactory bulbs, at the base of the brain, and surrounding the spinal cord. Inhibition of one or all of these lymphatic networks can reduce CNS autoimmunity in a mouse model of multiple sclerosis (MS), while augmenting these lymphatic networks can improve immunosurveillance, immunotherapy, and clearance in glioblastoma, Alzheimer's disease, traumatic brain injury, and cerebrovascular injury. In this review, we will provide historical context of how CNS drainage contributes to immune surveillance, how more recently published studies fit meningeal lymphatics into the context of CNS homeostasis and neuroinflammation, identify the complex dualities of lymphatic function during neuroinflammation and how therapeutics targeting lymphatic function may be more complicated than currently appreciated, and conclude by identifying some unresolved questions and controversies that may guide future research.

Experimental Autoimmune Encephalomyelitis in the Mouse.

This article details the materials and methods required for both active induction and adoptive transfer of experimental autoimmune encephalomyelitis (EAE) in the SJL mouse strain using intact proteins or peptides from the two major myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). Additionally, active induction of EAE in the C57BL/6 strain using myelin oligodendrocyte glycoprotein (MOG) peptide is also discussed. Detailed materials and methods required for the purification of both PLP and MBP are described, and a protocol for isolating CNS-infiltrating lymphocytes in EAE mice is included. Modifications of the specified protocols may be necessary for efficient induction of active or adoptive EAE in other mouse strains. © 2021 Wiley Periodicals LLC. Basic Protocol: Active induction of EAE with PLP, MBP, and MOG protein or peptide Alternate Protocol: Adoptive induction of EAE with PLP-, MBP-, or MOG-specific lymphocytes Support Protocol 1: Purification of proteolipid protein Support Protocol 2: Purification of myelin basic protein Support Protocol 3: Isolation of CNS-infiltrating lymphocytes.

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

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.

CCR7 deficient inflammatory Dendritic Cells are retained in the Central Nervous System.

Dendritic cells (DC) accumulate in the CNS during neuroinflammation, yet, how these cells contribute to CNS antigen drainage is still unknown. We have previously shown that after intracerebral injection, antigen-loaded bone marrow DC migrate to deep cervical lymph nodes where they prime antigen-specific T cells and exacerbate experimental autoimmune encephalomyelitis (EAE) in mice. Here, we report that DC migration from brain parenchyma is dependent upon the chemokine receptor CCR7. During EAE, both wild type and CCR7-/- CD11c-eYFP cells infiltrated into the CNS but cells that lacked CCR7 were retained in brain and spinal cord while wild type DC migrated to cervical lymph nodes. Retention of CCR7-deficient CD11c-eYFP cells in the CNS exacerbated EAE. These data are the first to show that CD11chigh DC use CCR7 for migration out of the CNS, and in the absence of this receptor they remain in the CNS in situ and exacerbate EAE.

Contrasting roles of immune cells in tissue injury and repair in stroke: The dark and bright side of immunity in the brain.

Despite considerable efforts in research and clinical studies, stroke is still one of the leading causes of death and disability worldwide. Originally, stroke was considered a vascular thrombotic disease without significant immune involvement. However, over the last few decades it has become increasingly obvious that the immune responses can significantly contribute to both tissue injury and protection following stroke. Recently, much research has been focused on the immune system's role in stroke pathology and trying to elucidate the mechanism used by immune cells in tissue injury and protection. Since the discovery of tissue plasminogen activator therapy in 1996, there have been no new treatments for stroke. For this reason, research into understanding how the immune system contributes to stroke pathology may lead to better therapies or enhance the efficacy of current treatments. Here, we discuss the contrasting roles of immune cells to stroke pathology while emphasizing myeloid cells and T cells. We propose that focusing future research on balancing the beneficial-versus-detrimental roles of immunity may lead to the discovery of better and novel stroke therapies.

Group A Streptococcus intranasal infection promotes CNS infiltration by streptococcal-specific Th17 cells.

Group A streptococcal (GAS) infection induces the production of Abs that cross-react with host neuronal proteins, and these anti-GAS mimetic Abs are associated with autoimmune diseases of the CNS. However, the mechanisms that allow these Abs to cross the blood-brain barrier (BBB) and induce neuropathology remain unresolved. We have previously shown that GAS infection in mouse models induces a robust Th17 response in nasal-associated lymphoid tissue (NALT). Here, we identified GAS-specific Th17 cells in tonsils of humans naturally exposed to GAS, prompting us to explore whether GAS-specific CD4+ T cells home to mouse brains following i.n. infection. Intranasal challenge of repeatedly GAS-inoculated mice promoted migration of GAS-specific Th17 cells from NALT into the brain, BBB breakdown, serum IgG deposition, microglial activation, and loss of excitatory synaptic proteins under conditions in which no viable bacteria were detected in CNS tissue. CD4+ T cells were predominantly located in the olfactory bulb (OB) and in other brain regions that receive direct input from the OB. Together, these findings provide insight into the immunopathology of neuropsychiatric complications that are associated with GAS infections and suggest that crosstalk between the CNS and cellular immunity may be a general mechanism by which infectious agents exacerbate symptoms associated with other CNS autoimmune disorders.

Stepwise recruitment of transcellular and paracellular pathways underlies blood-brain barrier breakdown in stroke.

Brain endothelial cells form a paracellular and transcellular barrier to many blood-borne solutes via tight junctions (TJs) and scarce endocytotic vesicles. The blood-brain barrier (BBB) plays a pivotal role in the healthy and diseased CNS. BBB damage after ischemic stroke contributes to increased mortality, yet the contributions of paracellular and transcellular mechanisms to this process in vivo are unknown. We have created a transgenic mouse strain whose endothelial TJs are labeled with eGFP and have imaged dynamic TJ changes and fluorescent tracer leakage across the BBB in vivo, using two-photon microscopy in the t-MCAO stroke model. Although barrier function is impaired as early as 6 hr after stroke, TJs display profound structural defects only after 2 days. Conversely, the number of endothelial caveolae and transcytosis rate increase as early as 6 hr after stroke. Therefore, stepwise impairment of transcellular followed by paracellular barrier mechanisms accounts for the BBB deficits in stroke.