Astrocytic Accumulation at the Choroid Plexus Attachment Region.

The choroid plexus (CP) plays a major role in controlling the entry of substances and immune cells into the brain as it forms the blood-cerebrospinal fluid barrier (BCSFB) in the brain ventricles. Dysregulated immune cell trafficking through the epithelial cell (EC) layer of CP is central for the pathogenesis of infectious diseases in the brain and many neurodegenerative disorders. In vitro studies elucidating the function of the CP have so far been limited to the monolayer culture of CP ECs. To mimic immune cell migration across the CP barrier, a three-dimensional model would be advantageous. Here, we present an in vitro platform for studies of the immune cell trafficking based on CP explants/organoids. The explants were generated from fragments of mouse CPs in Matrigel, where the cells formed luminal spaces and could be maintained in culture for at least 8 weeks. We demonstrate expression of the major CP markers in the explants, including transthyretin and aquaporin 1 as well as ZO1 and ICAM-1, indicating a capacity for secretion of cerebrospinal fluid (CSF) and presence of tight junctions. CP explants displayed CP-like cell polarization and formed an intact EC barrier. We also show that the expression of transthyretin, transferrin, occludin and other genes associated with various functions of CP was maintained in the explants at similar levels as in native CP. By using dendritic cells and neutrophils, we show that the migration activity of immune cells and their interactions with CP epithelium can be monitored by microscopy. Thereby, the three-dimensional CP explant model can be used to study the cellular and molecular mechanisms mediating immune cell migration through CP epithelium and other functions of choroid EC. We propose this platform can potentially be used in the search for therapeutic targets and intervention strategies to improve control of (drug) substances and (immune) cell entry into the central nervous system.

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Neuronal activity within the central nervous system (CNS) strictly depends on homeostasis and therefore does not tolerate uncontrolled entry of blood components. It has been generally believed that under normal conditions, the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) prevent immune cell entry into the CNS. This view has recently changed when it was realized that activated T cells are able to breach the BBB and the BCSFB to perform immune surveillance of the CNS. Here we propose that the immune privilege of the CNS is established by the specific morphological architecture of its borders resembling that of a medieval castle. The BBB and the BCSFB serve as the outer walls of the castle, which can be breached by activated immune cells serving as messengers for outside dangers. Having crossed the BBB or the BCSFB they reach the castle moat, namely the cerebrospinal fluid (CSF)-drained leptomeningeal and perivascular spaces of the CNS. Next to the CNS parenchyma, the castle moat is bordered by a second wall, the glia limitans, composed of astrocytic foot processes and a parenchymal basement membrane. Inside the castle, that is the CNS parenchyma proper, the royal family of sensitive neurons resides with their servants, the glial cells. Within the CSF-drained castle moat, macrophages serve as guards collecting all the information from within the castle, which they can present to the immune-surveying T cells. If in their communication with the castle moat macrophages, T cells recognize their specific antigen and see that the royal family is in danger, they will become activated and by opening doors in the outer wall of the castle allow the entry of additional immune cells into the castle moat. From there, immune cells may breach the inner castle wall with the aim to defend the castle inhabitants by eliminating the invading enemy. If the immune response by unknown mechanisms turns against self, that is the castle inhabitants, this may allow for continuous entry of immune cells into the castle and lead to the death of the castle inhabitants, and finally members of the royal family, the neurons. This review will summarize the molecular traffic signals known to allow immune cells to breach the outer and inner walls of the CNS castle moat and will highlight the importance of the CSF-drained castle moat in maintaining immune surveillance and in mounting immune responses in the CNS.

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.

The choroid plexus: a command center for brain-body communication during inflammation.

<p><b>The central nervous system was traditionally considered an immune-privileged site, defined as being immunologically inactive. However, recent studies have elucidated that a number of immune cells traffic into and out of the brain in healthy humans to conduct routine immunosurveillance. A unique immunological interface, the choroid plexus, acts as a gatekeeper for the entry of these immune cells during homeostasis. Although the mechanisms are not well described, the choroid plexus also has the capacity to regulate the responses of migrating leukocytes during inflammation.</b></p> <p>Multiple sclerosis is a complex neuroinflammatory disease characterized by demyelination in the CNS. Autoreactive immune cells invade the central nervous system and orchestrate an attack against myelin sheathes, the insulation layer that protects neurons. The disease affects nearly 1 in 1,000 New Zealanders, and currently has no cure. The most successful treatments for multiple sclerosis target the initial stages by inhibiting the entry of these cells into the central nervous system, however these are often associated with severe side and life-threatening effects and cannot prevent the progression of the disease.</p> <p>Heparanase, the ubiquitously expresses heparan sulfate degrading enzyme has been thoroughly implicated in the disease processes of multiple sclerosis, and its animal model, EAE. Autoreactive lymphocytes exploit heparanase activity to degrade the extracellular matrix and destabilize the barriers that maintain the relative immune privileged status of the central nervous system. Exogenous heparan sulfate mimetics have previously been shown to ameliorate symptoms of EAE by interfering with heparanase activity. However, the commercialization and clinical translation of these inhibitors is currently inhibited by the complexity of their synthesis. ‘HS16-35’ is a novel heparan sulfate mimetic developed by the Ferrier Institute, comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of this compound is much shorter due to its smaller size; however, it has been shown to act similarly to native heparan sulfate molecules. We proposed that HS16-35 is protective in preventing the migration of autoreactive immune cells across the choroid plexus by inhibiting lymphocyte heparanase.</p> <p>To investigate the efficacy of HS16-35 in vitro, we first established an experimental transwell model of the choroid plexus. This model incorporated core components of the choroid plexus, including fenestrated capillaries, the stromal matrix and epithelial monolayer. We first showed that the model was capable of mimicking homeostatic trafficking across the choroid plexus epithelium, which formed a selective but permeable barrier. Then, we induced T-cell specific inflammatory migration using Concanavalin A or TH1-type cytokines. This migration was found to be interferon-γ dependent and could be mitigated with anti-interferon-γ treatment.</p> <p>Once this model was established, we next investigated whether HS16-35 was effective in inhibiting inflammatory migration across this structure. To adapt HS16-35 to an in vitro dose, we performed cell viability assays. This confirmed that the compound was mildly cytotoxic to epithelial choroid plexus cells but not murine splenocytes. Further experiments found that low-dose HS16-35 did not impact monolayer permeability. Transwell migration assays showed that low-dose HS16-35 was effective in reducing ConA and interferon-γ mediated inflammatory T-cell migration to a level comparable to homeostatic trafficking. Finally, we assessed cytokine profiles of leukocytes and epithelial choroid plexus cells treated with HS16 35 and found that HS16-35 reduced the expression of key cytokines involved in MS pathogenesis.</p> <p>In summary, the work described in this thesis shows how HS16-35 may be protective during EAE by suppressing the inflammatory response of autoreactive T-cells, in addition to regulating the infiltration of immune cells into the CNS through the choroid plexus. In a broader sense, these findings show that HS16 36 may be effective in treating MS by regulating, not inhibiting lymphocyte migration into the CNS, mitigating some of the severe side effects that other migration-inhibitors face.</p>

<p><b>The central nervous system was traditionally considered an immune-privileged site, defined as being immunologically inactive. However, recent studies have elucidated that a number of immune cells traffic into and out of the brain in healthy humans to conduct routine immunosurveillance. A unique immunological interface, the choroid plexus, acts as a gatekeeper for the entry of these immune cells during homeostasis. Although the mechanisms are not well described, the choroid plexus also has the capacity to regulate the responses of migrating leukocytes during inflammation.</b></p> <p>Multiple sclerosis is a complex neuroinflammatory disease characterized by demyelination in the CNS. Autoreactive immune cells invade the central nervous system and orchestrate an attack against myelin sheathes, the insulation layer that protects neurons. The disease affects nearly 1 in 1,000 New Zealanders, and currently has no cure. The most successful treatments for multiple sclerosis target the initial stages by inhibiting the entry of these cells into the central nervous system, however these are often associated with severe side and life-threatening effects and cannot prevent the progression of the disease.</p> <p>Heparanase, the ubiquitously expresses heparan sulfate degrading enzyme has been thoroughly implicated in the disease processes of multiple sclerosis, and its animal model, EAE. Autoreactive lymphocytes exploit heparanase activity to degrade the extracellular matrix and destabilize the barriers that maintain the relative immune privileged status of the central nervous system. Exogenous heparan sulfate mimetics have previously been shown to ameliorate symptoms of EAE by interfering with heparanase activity. However, the commercialization and clinical translation of these inhibitors is currently inhibited by the complexity of their synthesis. ‘HS16-35’ is a novel heparan sulfate mimetic developed by the Ferrier Institute, comprised of a dendritic core with four heavily sulfated oligosaccharide arms. The synthesis of this compound is much shorter due to its smaller size; however, it has been shown to act similarly to native heparan sulfate molecules. We proposed that HS16-35 is protective in preventing the migration of autoreactive immune cells across the choroid plexus by inhibiting lymphocyte heparanase.</p> <p>To investigate the efficacy of HS16-35 in vitro, we first established an experimental transwell model of the choroid plexus. This model incorporated core components of the choroid plexus, including fenestrated capillaries, the stromal matrix and epithelial monolayer. We first showed that the model was capable of mimicking homeostatic trafficking across the choroid plexus epithelium, which formed a selective but permeable barrier. Then, we induced T-cell specific inflammatory migration using Concanavalin A or TH1-type cytokines. This migration was found to be interferon-γ dependent and could be mitigated with anti-interferon-γ treatment.</p> <p>Once this model was established, we next investigated whether HS16-35 was effective in inhibiting inflammatory migration across this structure. To adapt HS16-35 to an in vitro dose, we performed cell viability assays. This confirmed that the compound was mildly cytotoxic to epithelial choroid plexus cells but not murine splenocytes. Further experiments found that low-dose HS16-35 did not impact monolayer permeability. Transwell migration assays showed that low-dose HS16-35 was effective in reducing ConA and interferon-γ mediated inflammatory T-cell migration to a level comparable to homeostatic trafficking. Finally, we assessed cytokine profiles of leukocytes and epithelial choroid plexus cells treated with HS16 35 and found that HS16-35 reduced the expression of key cytokines involved in MS pathogenesis.</p> <p>In summary, the work described in this thesis shows how HS16-35 may be protective during EAE by suppressing the inflammatory response of autoreactive T-cells, in addition to regulating the infiltration of immune cells into the CNS through the choroid plexus. In a broader sense, these findings show that HS16 36 may be effective in treating MS by regulating, not inhibiting lymphocyte migration into the CNS, mitigating some of the severe side effects that other migration-inhibitors face.</p>

Before entering the central nervous system (CNS) immune cells have to penetrate any one of its barriers, namely either the endothelial blood-brain barrier, the epithelial blood-cerebrospinal fluid barrier or the tanycytic barrier around the circumventricular organs, all of which maintain homeostasis within the CNS. The presence of these barriers in combination with the lack of lymphatic vessels and the absence of classical MHC-positive antigen presenting cells characterizes the CNS as an immunologically privileged site. In multiple sclerosis a large number of inflammatory cells gains access to the CNS parenchyma. Studies performed in experimental autoimmune encephalomyelitis (EAE), a rodent model for multiple sclerosis, have enabled us to understand some of the molecular mechanisms involved in immune cell entry into the CNS. In particular, the realization that /alpha4-integrins play a predominant role in leukocyte trafficking to the CNS has led to the development of a novel drug for the treatment of relapsing-remitting multiple sclerosis, which targets /alpha4-integrin mediated immune cell migration to the CNS. At the same time, the involvement of other adhesion and signalling molecules in this process remains to be investigated and novel molecules contributing to immune cell entry into the CNS are still being identified. The entire process of immune cell trafficking into the CNS is strictly controlled by the brain barriers not only under physiological conditions but also during neuroinflammation, when some barrier properties are lost. Thus, immune cell entry into the CNS critically depends on the unique characteristics of the brain barriers maintaining CNS homeostasis.

The choroid plexus epithelium is a secretory epithelium par excellence. However, this is perhaps not the most prominent reason for the massive interest in this modest-sized tissue residing inside the brain ventricles. Most likely, the dominant reason for extensive studies of the choroid plexus is the identification of this epithelium as the source of the majority of intraventricular cerebrospinal fluid. This finding has direct relevance for studies of diseases and conditions with deranged central fluid volume or ionic balance. While the concept is supported by the vast majority of the literature, the implication of the choroid plexus in secretion of the cerebrospinal fluid was recently challenged once again. Three newer and promising areas of current choroid plexus-related investigations are as follows: 1) the choroid plexus epithelium as the source of mediators necessary for central nervous system development, 2) the choroid plexus as a route for microorganisms and immune cells into the central nervous system, and 3) the choroid plexus as a potential route for drug delivery into the central nervous system, bypassing the blood-brain barrier. Thus, the purpose of this review is to highlight current active areas of research in the choroid plexus physiology and a few matters of continuous controversy.

The choroid plexus (CP) is a highly vascularized endothelial-epithelial convolute located in the ventricular system of the brain. The CP is also the localization of the blood-cerebrospinal fluid barrier (BCSFB), which is formed by the CP epithelium. During infectious diseases of the central nervous system (CNS) the CP can play multiple roles, which will be covered by this review. To cause infection of the CNS pathogens need to cross into the brain, and the CP can present the site of entry across the BCSFB. For invading the CNS pathogens have evolved several mechanisms that can require the help of host immune cells. Infections with pathogens often also cause cell death in the CP, which can be responsible for impairment of barrier function. The CP reacts to the intruders by staging an inflammatory response involving the production of cytokines and chemokines and can function as an entry gate for host immune cells. Following entry of pathogens and host immune cells into the brain both players can cause severe damage leading to neurological sequelae. Additionally, impairment of important CP functions might be involved in further complications like hydrocephalus.

BackgroundAdenosine A2A receptor (A2AR) antagonists have been consistently demonstrated to protect against multiple sclerosis (MS) pathology, but A2AR knockout (A2AR−/−) mice exhibit exacerbated immune injury, raising concerns regarding the use of A2AR antagonists for MS treatment. Here, we revealed the critical involvement of A2AR-mediated interactions between Th1+ T cells and the choroid plexus (ChP) epithelium in the pathology of experimental autoimmune encephalomyelitis (EAE).MethodsWe assessed the effects of A2AR knockout on ChP gateway activity and the interferon gamma (IFN-γ)-secreting capacity of Th1+ T cells in an EAE model by immunofluorescence, qPCR and flow cytometry (FCM). We also investigated the effects of A2AR-mediated interactions between Th1+ T cells and the ChP epithelium on ChP gateway activity in vivo via intracerebroventricular (ICV) injection of Th1+ T cells and in vitro via coculture of ChP epithelial cells and splenic Th1+ T cells. We further knocked down IFN-γ receptor 1 (IFNGR1) specifically in the ChP of A2AR−/− mice via ICV injection of AAV2/5-shRNA (IFNGR1) to disrupt the interactions between Th1+ T cells and the ChP epithelium and thus assess the roles of these interactions in the development of EAE pathology.ResultsA2AR knockout disrupted the ChP barrier and increased T-cell infiltration across the ChP in EAE model mice. Coculture of splenic Th1+ T cells and ChP epithelial cells revealed that A2AR knockout in ChP epithelial cells strengthened the ChP barrier and attenuated T-cell migration, whereas A2AR knockout in Th1+ T cells increased the accumulation of Th1+ T cells in the ChP via the secretion of IFN-γ. Consistent with the coculture results, ICV injection of activated splenic Th1+ T cells from A2AR−/− mice increased the accumulation of T cells in the ChP to a greater extent than did injection of Th1+ T cells from A2AR+/+ mice. This effect was due to the increased secretion of IFN-γ in A2AR−/− mice compared with A2AR+/+ mice. Finally, ChP-specific knockdown of IFNGR1 attenuated A2AR knockout-induced T-cell infiltration, brain inflammation and EAE pathology.ConclusionA2AR-mediated interactions between Th1+ T cells and the ChP epithelium via the secretion of IFN-γ from CD4+ T cells and the binding IFN-γ to IFNGR1 in the ChP epithelium control immune cell invasion and the development of EAE pathology in A2AR−/− mice.

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS not to disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses are mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly regulating immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier control immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis (MS), immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of our current knowledge on the molecular mechanisms involved in immune cell entry into the CNS has been derived from studies performed in experimental autoimmune encephalomyelitis (EAE), an animal model for MS. Thus, a large part of our current knowledge on immune cell entry across the BBBs is based on the results obtained in this animal model. Similarly, knowledge on the benefits and potential risks associated with therapeutic targeting of immune cell recruitment across the BBB in human diseases are mostly derived from such treatment regimen in MS. Other mechanisms of immune cell entry into the CNS might therefore apply under different pathological conditions such as bacterial meningitis or stroke and need to be considered.

Infiltrating T cells and monocyte-derived macrophages support central nervous system repair. Although infiltration of leucocytes to the injured central nervous system has recently been shown to be orchestrated by the brain's choroid plexus, the immunological mechanism that maintains this barrier and regulates its activity as a selective gate is poorly understood. Here, we hypothesized that CD4(+) effector memory T cells, recently shown to reside at the choroid plexus stroma, regulate leucocyte trafficking through this portal through their interactions with the choroid plexus epithelium. We found that the naïve choroid plexus is populated by T helper 1, T helper 2 and regulatory T cells, but not by encephalitogenic T cells. In vitro findings revealed that the expression of immune cell trafficking determinants by the choroid plexus epithelium is specifically induced by interferon-γ. Tumour necrosis factor-α and interferon-γ reciprocally controlled the expression of their receptors by the choroid plexus epithelium, and had a synergistic effect in inducing the epithelial expression of trafficking molecules. In vivo, interferon-γ-dependent signalling controlled trafficking through the choroid plexus; interferon-γ receptor knockout mice exhibited reduced levels of T cells and monocyte entry to the cerebrospinal fluid and impaired recovery following spinal cord injury. Moreover, reduced expression of trafficking molecules by the choroid plexus was correlated with reduced CD4(+) T cells in the choroid plexus and cerebrospinal fluid of interferon-γ receptor knockout mice. Similar effect on the expression of trafficking molecules by the choroid plexus was found in bone-marrow chimeric mice lacking interferon-γ receptor in the central nervous system, or reciprocally, lacking interferon-γ in the circulation. Collectively, our findings attribute a novel immunological plasticity to the choroid plexus epithelium, allowing it to serve, through interferon-γ signalling, as a tightly regulated entry gate into the central nervous system for circulating leucocytes immune surveillance under physiological conditions, and for repair following acute injury.

BackgroundThe blood–cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis.MethodsWe compared cell line models of the mouse BCSFB derived from the Immortomouse® and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB.ResultsWe found that inducible cell line models established from the Immortomouse® or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF.ConclusionsOur study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

The choroid plexus has recently been identified as a possible migration route for peripheral immune cells into the central nervous system. For future investigation of this route, profound knowledge of the morphology of the murine choroid plexus is a prerequisite. We here present a detailed morphological description of the murine choroid plexus, its attachment regions as well as its spatial relation to the subarachnoid space. We used micro-computed tomography of immersion-contrasted fixated brains to generate three-dimensional models of the ventricle system and the choroid plexus and aligned micro-computed tomography-based sections with histological paraffin-embedded sections after immunohistochemical labeling of the basal lamina and choroid plexus epithelium marker proteins (laminin and aquaporin 1). The murine choroid plexus is located in all four ventricles and is attached to the brain parenchyma in narrow attachment regions with a specific morphology in each ventricle. While in the lateral and fourth ventricle, the attachment site is formed by thin tissue bridges, the choroid plexus attachment in the third ventricle has a more complex V-like shape. In all ventricles, the choroid plexus is in close spatial relationship with the subarachnoid space that extends from the brain surface along physiologic openings toward the choroid plexus. In summary, we here provide a description of the morphology of the murine ventricle system and choroid plexus, the attachment regions of the choroid plexus and its connection to the subarachnoid space, as well as a three-dimensional model of the ventricles, the choroid plexus, and the subarachnoid space to facilitate a spatial understanding of these complex structures.