Choroid plexus immune cell response in murine hydrocephalus induced by intraventricular hemorrhage.

BACKGROUND: Intraventricular hemorrhage (IVH) and associated hydrocephalus are significant complications of intracerebral and subarachnoid hemorrhage. Despite proximity to IVH, the immune cell response at the choroid plexus (ChP) has been relatively understudied. This study employs CX3CR-1GFP mice, which marks multiple immune cell populations, and immunohistochemistry to outline that response. METHODS: This study had four parts all examining male adult CX3CR-1GFP mice. Part 1 examined naïve mice. In part 2, mice received an injection 30 µl of autologous blood into right ventricle and were euthanized at 24 h. In part 3, mice underwent intraventricular injection of saline, iron or peroxiredoxin 2 (Prx-2) and were euthanized at 24 h. In part 4, mice received intraventricular iron injection and were treated with either control or clodronate liposomes and were euthanized at 24 h. All mice underwent magnetic resonance imaging to quantify ventricular volume. The ChP immune cell response was examined by combining analysis of GFP(+) immune cells and immunofluorescence staining. RESULTS: IVH and intraventricular iron or Prx-2 injection in CX3CR-1GFP mice all induced ventriculomegaly and activation of ChP immune cells. There were very marked increases in the numbers of ChP epiplexus macrophages, T lymphocytes and neutrophils. Co-injection of clodronate liposomes with iron reduced the ventriculomegaly which was associated with fewer epiplexus and stromal macrophages but not reduced T lymphocytes and neutrophils. CONCLUSION: There is a marked immune cell response at the ChP in IVH involving epiplexus cells, T lymphocytes and neutrophils. The blood components iron and Prx-2 may play a role in eliciting that response. Reduction of ChP macrophages with clodronate liposomes reduced iron-induced ventriculomegaly suggesting that ChP macrophages may be a promising therapeutic target for managing IVH-induced hydrocephalus.

Ectopic CD4+ T cells in choroid plexus mediate neuropsychiatric lupus symptoms in mice via interferon-γ induced microglia activation.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a disabling and potentially life-threatening complication of SLE. This study aims to investigate whether ectopic CD4+ T cells in the choroid plexus mediate NPSLE in mice. Intracerebroventricular (ICV) injection of anti-CD4 antibody effectively depleted CP-resident CD4+ T cells and alleviated NPSLE-like symptoms in MRL/lpr mice. Following ICV injection, the majority of isolated lupus CD4+ T cells from donor MRL/lpr mice predominantly stayed in the CP for at least 28 days in recipient C57BL/6 mice, while nearly all isolated CD4+ T cells from MRL/MpJ mice disappeared within 7 days. ICV injection of lupus CD4+ T cells resulted in NPSLE-like symptoms, including impaired behavioral performances, increased microglial activation, and abnormal microstructure changes. Flow cytometry analysis revealed that the majority of isolated lupus CD4+ T cells were positive for IFN-γ. Neutralizing intracerebral IFN-γ alleviated NPSLE-like symptoms in MRL/lpr mice. Moreover, ICV injection of anti-IFN-γ antibody or microglial depletion by PLX3397 benefited most NPSLE-like symptoms in lupus CD4+ T-treated mice, while ICV injection of IFN-γ mimicked most NPSLE-like symptoms. In conclusion, CP-resident lupus CD4+ T cells contribute to NPSLE-like symptoms in mice via Interferon-γ induced microglia activation. Depleting CP-resident lupus CD4+ T cells, interferon-γ, or activated microglia may be potential therapeutic targets for NPSLE.

Monocytes promote acute neuroinflammation and become pathological microglia in neonatal hypoxic-ischemic brain injury.

Rationale: Monocytes belong to the mononuclear phagocyte system and are immune responders to tissue injury and infection. There were also reports of monocytes transforming to microglia-like cells. Here we explore the roles of monocytes in microglia ontogeny and the pathogenesis of neonatal cerebral hypoxic-ischemic (HI) brain injury in mice. Methods: We used three genetic methods to track the development of monocytes, including CX3CR1GFP/+; CCR2RFP/+ reporter mice, adoptive transfer of GFP+ monocytes, and fate-mapping with CCR2-CreER mice, in neonatal mouse brains with or without lipopolysaccharide (LPS, 0.3 mg/kg)-sensitized Vannucci HI. We also used genetic (CCR2RFP/ RFP, CCR2 knockout) and pharmacological methods (RS102895, a CCR2 antagonist) to test the roles of monocytic influx in LPS/HI brain injury. Results: CCR2+ monocytes entered the late-embryonic brains via choroid plexus, but rapidly became CX3CR1+ amoeboid microglial cells (AMCs). The influx of CCR2+ monocytes declined after birth, but recurred after HI or LPS-sensitized HI (LPS/HI) brain injury, particularly in the hippocampus. The CCR2-CreER-based fate-mapping showed that CCR2+ monocytes became CD68+ TNFα+ macrophages within 4 d after LPS/HI, and maintained as TNFα+ MHCII+ macrophages or persisted as Tmem119+ Sall1+ P2RY12+ ramified microglia for at least five months after injury. Genetic deletion of the chemokine receptor CCR2 markedly diminished monocytic influx, the expression of pro- and anti-inflammatory cytokines, and brain damage. Post-LPS/HI application of RS102895 also reduced inflammatory responses and brain damage, leading to better cognitive functions. Conclusion: These results suggest that monocytes promote acute inflammatory responses and may become pathological microglia long after the neonatal LPS/HI insult. Further, blocking the influx of monocytes may be a potential therapy for neonatal brain injury.

Capsule-dependent impact of MAPK signalling on host cell invasion and immune response during infection of the choroid plexus epithelium by Neisseria meningitidis.

BACKGROUND: The Gram-negative bacterium Neisseria meningitidis (Nm) can cause meningitis in humans, but the host signalling pathways manipulated by Nm during central nervous system (CNS) entry are not completely understood. METHODS: We investigate the role of the mitogen-activated protein kinases (MAPK) Erk1/2 and p38 in an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with Nm serogroup B (NmB) and serogroup C (NmC) strains. A transcriptome analysis of HIBCPP cells following infection with Nm by massive analysis of cDNA ends (MACE) was done to further characterize the cellular response to infection of the barrier. RESULTS: Interestingly, whereas NmB and NmC wild type strains required active Erk1/2 and p38 pathways for infection, invasion by capsule-deficient mutants was independent of Erk1/2 and, in case of the NmB strain, of p38 activity. The transcriptome analysis of HIBCPP cells following infection with Nm demonstrated specific regulation of genes involved in the immune response dependent on Erk1/2 signalling. Gene ontology (GO) analysis confirmed loss of MAPK signalling after Erk1/2 inhibition and revealed an additional reduction of cellular responses including NFκB and JAK-STAT signalling. Interestingly, GO terms related to TNF signalling and production of IL6 were lost specifically following Erk1/2 inhibition during infection with wild type Nm, which correlated with the reduced infection rates by the wild type in absence of Erk1/2 signalling. CONCLUSION: Our data point towards a role of MAPK signalling during infection of the CP epithelium by Nm, which is strongly influenced by capsule expression, and affects infection rates as well as the host cell response.

Elevated CSF inflammatory markers in patients with idiopathic normal pressure hydrocephalus do not promote NKCC1 hyperactivity in rat choroid plexus.

BACKGROUND: Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological condition of unresolved etiology characterized by a clinical triad of symptoms; gait disturbances, urinary incontinence, and cognitive deterioration. In the present study, we aimed to elucidate the molecular coupling between inflammatory markers and development of iNPH and determine whether inflammation-induced hyperactivity of the choroidal Na+/K+/2Cl- cotransporter (NKCC1) that is involved in cerebrospinal fluid (CSF) secretion could contribute to the iNPH pathogenesis. METHODS: Lumbar CSF samples from 20 iNPH patients (10 with clinical improvement upon CSF shunting, 10 without clinical improvement) and 20 elderly control subjects were analyzed with the novel proximity extension assay technique for presence of 92 different inflammatory markers. RNA-sequencing was employed to delineate choroidal abundance of the receptors for the inflammatory markers found elevated in the CSF from iNPH patients. The ability of the elevated inflammatory markers to modulate choroidal NKCC1 activity was determined by addition of combinations of rat version of these in ex vivo experiments on rat choroid plexus. RESULTS: 11 inflammatory markers were significantly elevated in the CSF from iNPH patients compared to elderly control subjects: CCL28, CCL23, CCL3, OPG, CXCL1, IL-18, IL-8, OSM, 4E-BP1, CXCL6, and Flt3L. One inflammatory marker, CDCP1, was significantly decreased in iNPH patients compared to control subjects. None of the inflammatory markers differed significantly when comparing iNPH patients with and without clinical improvement upon CSF shunting. All receptors for the elevated inflammatory markers were expressed in the rat and human choroid plexus, except CCR4 and CXCR1, which were absent from the rat choroid plexus. None of the elevated inflammatory markers found in the CSF from iNPH patients modulated the choroidal NKCC1 activity in ex vivo experiments on rat choroid plexus. CONCLUSION: The CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. Although the corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, their activation did not modulate the NKCC1-mediated fraction of choroidal CSF secretion ex vivo. The molecular mechanisms underlying ventriculomegaly in iNPH, and the possible connection to inflammation, therefore remains to be elucidated.

Acute Effect of Caffeine on the Synthesis of Pro-Inflammatory Cytokines in the Hypothalamus and Choroid Plexus during Endotoxin-Induced Inflammation in a Female Sheep Model.

This study was designed to determine the effect of acute caffeine (CAF) administration, which exerts a broad spectrum of anti-inflammatory activity, on the synthesis of pro-inflammatory cytokines and their receptors in the hypothalamus and choroid plexus (ChP) during acute inflammation caused by the injection of bacterial endotoxin-lipopolysaccharide (LPS). The experiment was performed on 24 female sheep randomly divided into four groups: control; LPS treated (iv.; 400 ng/kg of body mass (bm.)); CAF treated (iv.; 30 mg/kg of bm.); and LPS and CAF treated. The animals were euthanized 3 h after the treatment. It was found that acute administration of CAF suppressed the synthesis of interleukin (IL-1ß) and tumor necrosis factor (TNF)α, but did not influence IL-6, in the hypothalamus during LPS-induced inflammation. The injection of CAF reduced the LPS-induced expression of TNF mRNA in the ChP. CAF lowered the gene expression of IL-6 cytokine family signal transducer (IL6ST) and TNF receptor superfamily member 1A (TNFRSF1) in the hypothalamus and IL-1 type II receptor (IL1R2) in the ChP. Our study on the sheep model suggests that CAF may attenuate the inflammatory response at the hypothalamic level and partly influence the inflammatory signal generated by the ChP cells. This suggests the potential of CAF to suppress neuroinflammatory processes induced by peripheral immune/inflammatory challenges.

Translational value of choroid plexus imaging for tracking neuroinflammation in mice and humans.

Neuroinflammation is a pathophysiological hallmark of multiple sclerosis and has a close mechanistic link to neurodegeneration. Although this link is potentially targetable, robust translatable models to reliably quantify and track neuroinflammation in both mice and humans are lacking. The choroid plexus (ChP) plays a pivotal role in regulating the trafficking of immune cells from the brain parenchyma into the cerebrospinal fluid (CSF) and has recently attracted attention as a key structure in the initiation of inflammatory brain responses. In a translational framework, we here address the integrity and multidimensional characteristics of the ChP under inflammatory conditions and question whether ChP volumes could act as an interspecies marker of neuroinflammation that closely interrelates with functional impairment. Therefore, we explore ChP characteristics in neuroinflammation in patients with multiple sclerosis and in two experimental mouse models, cuprizone diet-related demyelination and experimental autoimmune encephalomyelitis. We demonstrate that ChP enlargement-reconstructed from MRI-is highly associated with acute disease activity, both in the studied mouse models and in humans. A close dependency of ChP integrity and molecular signatures of neuroinflammation is shown in the performed transcriptomic analyses. Moreover, pharmacological modulation of the blood-CSF barrier with natalizumab prevents an increase of the ChP volume. ChP enlargement is strongly linked to emerging functional impairment as depicted in the mouse models and in multiple sclerosis patients. Our findings identify ChP characteristics as robust and translatable hallmarks of acute and ongoing neuroinflammatory activity in mice and humans that could serve as a promising interspecies marker for translational and reverse-translational approaches.

Hydrocephalus Induced by Intraventricular Peroxiredoxin-2: The Role of Macrophages in the Choroid Plexus.

The choroid plexus (CP) is the primary source of cerebrospinal fluid in the central nervous system. Recent evidence indicates that inflammatory pathways at the CP may be involved in hydrocephalus development. Peroxiredoxin 2 (Prx2) is a major component of red blood cells. Extracellular Prx2 is proinflammatory, and its release after red blood cell lysis may contribute to hydrocephalus after intraventricular hemorrhage. This study aimed to identify alterations in CP macrophages and dendritic cells following intracerebroventricular Prx2 injection and investigate the relationship between macrophages/dendritic cells and hydrocephalus. There were two parts to this study. In the first part, adult male Sprague-Dawley rats received an intracerebroventricular injection of Prx2 or saline. In the second part, Prx2 was co-injected with clodronate liposomes or control liposomes. All animals were euthanized at 24 h after magnetic resonance imaging. Immunohistochemistry was used to evaluate macrophages in CP, magnetic resonance imaging to quantify hydrocephalus, and histology to assess ventricular wall damage. The intracerebroventricular injection of Prx2 not only increased the OX-6 positive cells, but it also altered their location in the CP and immunophenotype. Co-injecting clodronate liposomes with Prx2 decreased the number of macrophages and simultaneously attenuated Prx2-induced hydrocephalus and ventricular wall damage. These results suggest that CP macrophages play an essential role in CP inflammation-induced hydrocephalus. These macrophages may be a potential therapeutic target in post-hemorrhagic hydrocephalus.

Intrathecal Inflammation in Progressive Multiple Sclerosis.

Progressive forms of multiple sclerosis (MS) are associated with chronic demyelination, axonal loss, neurodegeneration, cortical and deep gray matter damage, and atrophy. These changes are strictly associated with compartmentalized sustained inflammation within the brain parenchyma, the leptomeninges, and the cerebrospinal fluid. In progressive MS, molecular mechanisms underlying active demyelination differ from processes that drive neurodegeneration at cortical and subcortical locations. The widespread pattern of neurodegeneration is consistent with mechanisms associated with the inflammatory molecular load of the cerebrospinal fluid. This is at variance with gray matter demyelination that typically occurs at focal subpial sites, in the proximity of ectopic meningeal lymphoid follicles. Accordingly, it is possible that variations in the extent and location of neurodegeneration may be accounted for by individual differences in CSF flow, and by the composition of soluble inflammatory factors and their clearance. In addition, "double hit" damage may occur at sites allowing a bidirectional exchange between interstitial fluid and CSF, such as the Virchow-Robin spaces and the periventricular ependymal barrier. An important aspect of CSF inflammation and deep gray matter damage in MS involves dysfunction of the blood-cerebrospinal fluid barrier and inflammation in the choroid plexus. Here, we provide a comprehensive review on the role of intrathecal inflammation compartmentalized to CNS and non-neural tissues in progressive MS.

Inflammation of the Embryonic Choroid Plexus Barrier following Maternal Immune Activation.

The choroid plexus (ChP) regulates brain development by secreting instructive cues and providing a protective brain barrier. Here, we show that polyI:C-mediated maternal immune activation leads to an inflammatory response in the developing embryonic mouse brain that manifests as pro-inflammatory cerebrospinal fluid (CSF) and accumulation of ChP macrophages. Elevation of CSF-CCL2 was sufficient to drive ChP immune cell recruitment, activation, and proliferation. In addition, ChP macrophages abandoned their regular tiling pattern and relocated to the ChP-free margin where they breached the weakened epithelial barrier. We further found that these immune cells entered from the ChP into the brain via anatomically specialized "hotspots" at the distal tips of ChP villi. In vivo two-photon imaging demonstrated that surveillance behaviors in ChP macrophages had already emerged at this early stage of embryogenesis. Thus, the embryonic ChP forms a functional brain barrier that can mount an inflammatory response to external insults.

Tracking Calcium Dynamics and Immune Surveillance at the Choroid Plexus Blood-Cerebrospinal Fluid Interface.

The choroid plexus (ChP) epithelium is a source of secreted signaling factors in cerebrospinal fluid (CSF) and a key barrier between blood and brain. Here, we develop imaging tools to interrogate these functions in adult lateral ventricle ChP in whole-mount explants and in awake mice. By imaging epithelial cells in intact ChP explants, we observed calcium activity and secretory events that increased in frequency following delivery of serotonergic agonists. Using chronic two-photon imaging in awake mice, we observed spontaneous subcellular calcium events as well as strong agonist-evoked calcium activation and cytoplasmic secretion into CSF. Three-dimensional imaging of motility and mobility of multiple types of ChP immune cells at baseline and following immune challenge or focal injury revealed a range of surveillance and defensive behaviors. Together, these tools should help illuminate the diverse functions of this understudied body-brain interface.

The T Cell Receptor Repertoire in Neuropsychiatric Systemic Lupus Erythematosus.

Objective: In systemic lupus erythematosus (SLE), widespread T cell infiltration into target organs contributes to inflammation and organ damage. Autoreactive T cells become aberrantly activated in this disease due to dysfunctional T cell receptor signaling that lowers the activation threshold. Characterizing the T cell repertoire can provide further insight into the specific homing and proliferation of these T cells into lupus target organs. In the spontaneous lupus model, MRL/lpr, the TCR repertoire has not been fully elucidated, especially for T cells infiltrating the brain. Our aim was to investigate and compare the TCR repertoire between MRL/lpr mice and its congenic controls, MRL/MpJ, and within MRL/lpr tissues. Methods: Spleen, salivary gland, and brain choroid plexus were isolated from female MRL/lpr mice and MRL/MpJ mice. The TCRß CDR3 region was analyzed by multiplex PCRs and sequencing. Results: Significant differences were seen not only between the MRL/lpr and MRL/MpJ spleens, but also between MRL/lpr tissues. The TCR repertoire in MRL/lpr choroid plexus tissues had significantly increased clonality and sequence homology compared to MRL/lpr spleen and salivary gland. The consensus sequence, CASSQDWGGYEQYFF, was identified in the MRL/lpr choroid plexus repertoire. Conclusions: The TCR repertoire in lupus prone mice is not uniform between target organs, and suggests that T cells are specifically recruited into the choroid plexus of MRL/lpr mice. Further studies are needed to determine the antigen specificities for these infiltrating T cells in target organs of lupus mice, and their possible contribution to the pathogenesis of neuropsychiatric disease and other lupus manifestations.

The molecular anatomy and functions of the choroid plexus in healthy and diseased brain.

The choroid plexus (CP) is located in the ventricular system of the brain (one in each ventricle), and the CP epithelial cells form an important barrier between the blood and the cerebrospinal fluid (CSF). Their main function comprises CSF secretion, maintenance of brain homeostasis, signalling, and forming a neuroprotective barrier against harmful external and internal compounds. The CPs mature early and demonstrate expressional changes of barrier-specific genes and proteins related to location and developmental stage of the CP. Important proteins for the barrier function include tight junction proteins, numerous transporters and enzymes. Natural senescence leads to structural changes in the CP cells and reduced or loss of function, while further loss of CP function and changes in immune status may be relevant in neurodegenerative diseases such as Alzheimer's disease and Multiple Sclerosis. Neuroprotective genes expressed at CPs may be unexplored targets for new therapies for neurodegenerative diseases.

The Lyme disease bacterium, Borrelia burgdorferi, stimulates an inflammatory response in human choroid plexus epithelial cells.

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.

Global alterations to the choroid plexus blood-CSF barrier in amyotrophic lateral sclerosis.

The choroid plexus (CP) is a highly vascularized structure located in the ventricles that forms the blood-CSF barrier (BCSFB) and separates the blood from the cerebrospinal fluid (CSF). In addition to its role as a physical barrier, the CP functions in CSF secretion, transport of nutrients into the central nervous system (CNS) and a gated point of entry of circulating immune cells into the CNS. Aging and neurodegeneration have been reported to affect CP morphology and function and increase protein leakage from blood to the CSF. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease associated with both upper and lower motor neuron loss, as well as altered proteomic and metabolomic signatures in the CSF. The role of the BCSFB and the CP in ALS is unknown. Here we describe a transcriptomic and ultrastructural analysis of BCSFB and CP alterations in human postmortem tissues from ALS and non-neurologic disease controls. ALS-CP exhibited widespread disruptions in tight junctional components of the CP epithelial layer and vascular integrity. In addition, we detected loss of pericytes around ALS blood vessels, accompanied by activation of platelet aggregation markers vWF and Fibrinogen, reminiscent of vascular injury. To investigate the immune component of ALS-CP, we conducted a comprehensive analysis of cytokines and chemokine panels in CP lysates and found a significant down-regulation of M-CSF and V-CAM1 in ALS, as well as up-regulation of VEGF-A protein. This phenotype was accompanied by an infiltration of MERTK positive macrophages into the parenchyma of the ALS-CP when compared to controls. Taken together, we demonstrate widespread structural and functional disruptions of the BCSFB in human ALS increasing our understanding of the disease pathology and identifying potential new targets for ALS therapeutic development.

Neonatal Stroke and TLR1/2 Ligand Recruit Myeloid Cells through the Choroid Plexus in a CX3CR1-CCR2- and Context-Specific Manner.

Neonatal stroke is as frequent as stroke in the elderly, but many pathophysiological injury aspects are distinct in neonates, including immune signaling. While myeloid cells can traffic into the brain via multiple routes, the choroid plexus (CP) has been identified as a uniquely educated gate for immune cell traffic during health and disease. To understand the mechanisms of myeloid cell trafficking via the CP and their influence on neonatal stroke, we characterized the phenotypes of CP-infiltrating myeloid cells after transient middle cerebral artery occlusion (tMCAO) in neonatal mice of both sexes in relation to blood-brain barrier permeability, injury, microglial activation, and CX3CR1-CCR2 signaling, focusing on the dynamics early after reperfusion. We demonstrate rapid recruitment of multiple myeloid phenotypes in the CP ipsilateral to the injury, including inflammatory CD45+CD11b+Ly6chighCD86+, beneficial CD45+CD11b+Ly6clowCD206+, and CD45+CD11b+Ly6clowLy6ghigh cells, but only minor leukocyte infiltration into acutely ischemic-reperfused cortex and negligible vascular albumin leakage. We report that CX3CR1-CCR2-mediated myeloid cell recruitment contributes to stroke injury. Considering the complexity of inflammatory cascades triggered by stroke and a role for TLR2 in injury, we also used direct TLR2 stimulation as an independent injury model. TLR2 agonist rapidly recruited myeloid cells to the CP, increased leukocytosis in the CSF and blood, but infiltration into the cortex remained low over time. While the magnitude and the phenotypes of myeloid cells diverged between tMCAO and TLR2 stimulation, in both models, disruption of CX3CR1-CCR2 signaling attenuated both monocyte and neutrophil trafficking to the CP and cortex.SIGNIFICANCE STATEMENT Stroke during the neonatal period leads to long-term disabilities. The mechanisms of ischemic injury and inflammatory response differ greatly between the immature and adult brain. We examined leukocyte trafficking via the choroid plexus (CP) following neonatal stroke in relation to blood-brain barrier integrity, injury, microglial activation, and signaling via CX3CR1 and CCR2 receptors, or following direct TLR2 stimulation. Ischemia-reperfusion triggered marked unilateral CX3CR1-CCR2 dependent accumulation of diverse leukocyte subpopulations in the CP without inducing extravascular albumin leakage or major leukocyte infiltration into the brain. Disrupted CX3CR1-CCR2 signaling was neuroprotective in part by attenuating monocyte and neutrophil trafficking. Understanding the migratory patterns of CP-infiltrating myeloid cells with intact and disrupted CX3CR1-CCR2 signaling could identify novel therapeutic targets to protect the neonatal brain.

Human CD4+ T cell subsets differ in their abilities to cross endothelial and epithelial brain barriers in vitro.

BACKGROUND: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. METHODS: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

Inflammation of the choroid plexus in progressive multiple sclerosis: accumulation of granulocytes and T cells.

The choroid plexus (CP) is strategically located between the peripheral blood and the cerebrospinal fluid, and is involved in the regulation of central nervous system (CNS) homeostasis. In multiple sclerosis (MS), demyelination and inflammation occur in the CNS. While experimental animal models of MS pointed to the CP as a key route for immune cell invasion of the CNS, little is known about the distribution of immune cells in the human CP during progressive phases of MS. Here, we use immunohistochemistry and confocal microscopy to explore the main immune cell populations in the CP of progressive MS patients and non-neuroinflammatory controls, in terms of abundance and location within the distinct CP compartments. We show for the first time that the CP stromal density of granulocytes and CD8+ T cells is higher in progressive MS patients compared to controls. In line with previous studies, the CP of both controls and progressive MS patients contains relatively high numbers of macrophages and dendritic cells. Moreover, we found virtually no B cells or plasma cells in the CP. MHCII+ antigen-presenting cells were often found in close proximity to T cells, suggesting constitutive CNS immune monitoring functions of the CP. Together, our data highlights the role of the CP in immune homeostasis and indicates the occurrence of mild inflammatory processes in the CP of progressive MS patients. However, our findings suggest that the CP is only marginally involved in immune cell migration into the CNS in chronic MS.

N-acetylcysteine inhibits bacterial lipopeptide-mediated neutrophil transmigration through the choroid plexus in the developing brain.

The etiology of neurological impairments associated with prematurity and other perinatal complications often involves an infectious or pro-inflammatory component. The use of antioxidant molecules have proved useful to protect the neonatal brain from injury. The choroid plexuses-CSF system shapes the central nervous system response to inflammation at the adult stage, but little is known on the neuroimmune interactions that take place at the choroidal blood-CSF barrier during development. We previously described that peripheral administration to neonatal mice of the TLR2 ligand PAM3CSK4 (P3C), a prototypic Gram-positive bacterial lipopeptide, induces the migration of innate immune cells to the CSF. Here we showed in neonatal rats exposed to P3C that the migration of neutrophils into the CSF, which occurred through the choroid plexuses, is abolished following administration of the antioxidant drug N-acetylcysteine. Combining light sheet microscopy imaging of choroid plexus, a differentiated model of the blood-CSF barrier, and multiplex cytokine assays, we showed that the choroidal epithelium responds to the bacterial insult by a specific pattern of cytokine secretion, leading to a selective accumulation of neutrophils in the choroid plexus and to their trafficking into CSF. N-acetylcysteine acted by blocking neutrophil migration across both the endothelium of choroidal stromal vessels and the epithelium forming the blood-CSF barrier, without interfering with neutrophil blood count, neutrophil tropism for choroid plexus, and choroidal chemokine-driven chemotaxis. N-acetylcysteine reduced the injury induced by hypoxia-ischemia in P3C-sensitized neonatal rats. Overall, the data show that a double endothelial and epithelial check point controls the transchoroidal migration of neutrophils into the developing brain. They also point to the efficacy of N-acetylcysteine in reducing the deleterious effects of inflammation-associated perinatal injuries by a previously undescribed mechanism, i.e. the inhibition of innate immune cell migration across the choroid plexuses, without interfering with the systemic inflammatory response to infection.

The Choroid Plexus Is Permissive for a Preactivated Antigen-Experienced Memory B-Cell Subset in Multiple Sclerosis.

The role of B cells in multiple sclerosis (MS) is increasingly recognized. B cells undergo compartmentalized redistribution in blood and cerebrospinal fluid (CSF) during active MS, whereby memory B cells accumulate in the CSF. While B-cell trafficking across the blood-brain barrier has been intensely investigated, cellular diapedesis through the blood-CSF barrier (BCSFB) is incompletely understood. To investigate how B cells interact with the choroid plexus to transmigrate into the CSF we isolated circulating B cells from healthy donors (HC) and MS patients, utilized an inverted cell culture filter system of human choroid plexus papilloma (HIBCPP) cells to determine transmigration rates of B-cell subsets, immunofluorescence, and electron microscopy to analyze migration routes, and qRT-PCR to determine cytokines/chemokines mediating B-cell diapedesis. We also screened the transcriptome of intrathecal B cells from MS patients. We found, that spontaneous transmigration of HC- and MS-derived B cells was scant, yet increased significantly in response to B-cell specific chemokines CXCL-12/CXCL-13, was further boosted upon pre-activation and occurred via paracellular and transcellular pathways. Migrating cells exhibited upregulation of several genes involved in B-cell activation/migration and enhanced expression of chemokine receptors CXCR4/CXCR5, and were predominantly of isotype class switched memory phenotype. This antigen-experienced migratory subset displayed more pronounced chemotactic activities in MS than in HC and was retrieved in intrathecal B cells from patients with active MS. Trafficking of class-switched memory B cells was downscaled in a small cohort of natalizumab-exposed MS patients and the proportions of these phenotypes were reduced in peripheral blood yet were enriched intrathecally in patients who experienced recurrence of disease activity after withdrawal of natalizumab. Our findings highlight the relevance of the BCSFB as important gate for the entry of potentially harmful activated B cells into the CSF.