The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis.

The Gram-negative bacterium Neisseria meningitidis, which causes meningitis in humans, has been demonstrated to manipulate or alter host signalling pathways during infection of the central nervous system (CNS). However, these complex signalling networks are not completely understood. We investigate the phosphoproteome of an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with the N. meningitidis serogroup B strain MC58 in presence and absence of the bacterial capsule. Interestingly, our data demonstrates a stronger impact on the phosphoproteome of the cells by the capsule-deficient mutant of MC58. Using enrichment analyses, potential pathways, molecular processes, biological processes, cellular components and kinases were determined to be regulated as a consequence of N. meningitidis infection of the BCSFB. Our data highlight a variety of protein regulations that are altered during infection of CP epithelial cells with N. meningitidis, with the regulation of several pathways and molecular events only being detected after infection with the capsule-deficient mutant. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD038560.

The role of PLVAP in endothelial cells.

Endothelial cells play a major part in the regulation of vascular permeability and angiogenesis. According to their duty to fit the needs of the underlying tissue, endothelial cells developed different subtypes with specific endothelial microdomains as caveolae, fenestrae and transendothelial channels which regulate nutrient exchange, leukocyte migration, and permeability. These microdomains can exhibit diaphragms that are formed by the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), the only known protein component of these diaphragms. Several studies displayed an involvement of PLVAP in diseases as cancer, traumatic spinal cord injury, acute ischemic brain disease, transplant glomerulopathy, Norrie disease and diabetic retinopathy. Besides an upregulation of PLVAP expression within these diseases, pro-angiogenic or pro-inflammatory responses were observed. On the other hand, loss of PLVAP in knockout mice leads to premature mortality due to disrupted homeostasis. Generally, PLVAP is considered as a major factor influencing the permeability of endothelial cells and, finally, to be involved in the regulation of vascular permeability. Following these observations, PLVAP is debated as a novel therapeutic target with respect to the different vascular beds and tissues. In this review, we highlight the structure and functions of PLVAP in different endothelial types in health and disease.

Protocol for the setup and use of a human choroid plexus endothelial-epithelial two-cell-type in vitro model.

Choroid plexus, located in brain ventricles, is the site of blood-cerebrospinal fluid barrier that contains endothelial cells and an epithelial monolayer separated by stroma. We established a two-cell-type model of the human choroid plexus consisting of immortalized endothelial cells (iHCPEnC) and epithelial papilloma (HIBCPP) cells grown on opposite sides of filter supports. In this protocol, we describe the preparation of this model, the measurement of transepithelial electrical resistance (TEER), and immunofluorescence imaging-based analysis to determine the barrier function. For complete details on the use and execution of this protocol, please refer to Muranyi et al. (2022).

Protocol to analyze the transmigration efficiency of T. brucei using an in vitro model of the blood-cerebrospinal fluid barrier.

At present, the only approach to investigate the transmigration of Trypanosoma brucei, the causative agent of human African trypanosomiasis, from blood to cerebrospinal fluid is through animal experiments. This protocol details how to analyze the transmigration efficiency using an in vitro model of the blood-cerebrospinal fluid (blood-CSF) barrier. We describe how to grow human choroid plexus epithelial cells on cell culture filter inserts to form the barrier, followed by isolating and quantifying genomic DNA of transmigrated parasites by qPCR. For complete details on the use and execution of this protocol, please refer to Speidel et al. (2022).

Characterization of TRPV4-mediated signaling pathways in an optimized human choroid plexus epithelial cell line.

The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current (Isc) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.

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.

Invasion of the choroid plexus epithelium by Neisseria meningitidis is differently mediated by Arp2/3 signaling and possibly by dynamin dependent on the presence of the capsule.

Neisseria meningitis (Nm) is a human-specific bacterial pathogen that can cause sepsis and meningitis. To cause meningitis Nm must enter the central nervous system (CNS) across one of the barriers between the blood and the brain. We have previously shown that a capsule-depleted Serogroup B strain of Nm displays enhanced invasion into human choroid plexus (CP) epithelial papilloma (HIBCPP) cells, which represent an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB). Still, the processes involved during CNS invasion by Nm, especially the role of host cell actin cytoskeleton remodeling, are not investigated in detail. Here, we demonstrate that invasion into CP epithelial cells by encapsulated and capsule-depleted Nm is mediated by distinct host cell pathways. Whereas a Serogroup B wild-type strain enters HIBCPP cells by a possibly dynamin-independent, but actin related protein 2/3 (Arp2/3)-dependent mechanism, invasion by a capsule-depleted mutant is reduced by the dynamin inhibitor dynasore and Arp2/3-independent. Both wild-type and mutant bacteria require Src kinase activity for entry into HIBCPP cells. Our data show that Nm can employ different mechanisms for invasion into the CP epithelium dependent on the presence of a capsule.

Effect of Lipopolysaccharide-Induced Inflammatory Challenge on ß-Glucuronidase Activity and the Concentration of Quercetin and Its Metabolites in the Choroid Plexus, Blood Plasma and Cerebrospinal Fluid.

Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.

Polar Infection of Echovirus-30 Causes Differential Barrier Affection and Gene Regulation at the Blood-Cerebrospinal Fluid Barrier.

Echovirus-30 (E-30) is responsible for the extensive global outbreaks of meningitis in children. To gain access to the central nervous system, E-30 first has to cross the epithelial blood-cerebrospinal fluid barrier. Several meningitis causing bacteria preferentially infect human choroid plexus papilloma (HIBCPP) cells in a polar fashion from the basolateral cell side. Here, we investigated the polar infection of HIBCPP cells with E-30. Both apical and basolateral infections caused a significant decrease in the transepithelial electrical resistance of HIBCPP cells. However, to reach the same impact on the barrier properties, the multiplicity of infection of the apical side had to be higher than that of the basolateral infection. Furthermore, the number of infected cells at respective time-points after basolateral infection was significantly higher compared to apical infection. Cytotoxic effects of E-30 on HIBCPP cells during basolateral infection were observed following prolonged infection and appeared more drastically compared to the apical infection. Gene expression profiles determined by massive analysis of cDNA ends revealed distinct regulation of specific genes depending on the side of HIBCPP cells' infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood-cerebrospinal fluid barrier leading to central nervous system inflammation.

Non-Typeable Haemophilus influenzae Invade Choroid Plexus Epithelial Cells in a Polar Fashion.

Non-typeable Haemophilus influenzae (NTHI) is a pathogen of the human respiratory tract causing the majority of invasive H. influenzae infections. Severe invasive infections such as septicemia and meningitis occur rarely, but the lack of a protecting vaccine and the increasing antibiotic resistance of NTHI impede treatment and emphasize its relevance as a potential meningitis causing pathogen. Meningitis results from pathogens crossing blood-brain barriers and invading the immune privileged central nervous system (CNS). In this study, we addressed the potential of NTHI to enter the brain by invading cells of the choroid plexus (CP) prior to meningeal inflammation to enlighten NTHI pathophysiological mechanisms. A cell culture model of human CP epithelial cells, which form the blood-cerebrospinal fluid barrier (BCSFB) in vivo, was used to analyze adhesion and invasion by immunofluorescence and electron microscopy. NTHI invade CP cells in vitro in a polar fashion from the blood-facing side. Furthermore, NTHI invasion rates are increased compared to encapsulated HiB and HiF strains. Fimbriae occurrence attenuated adhesion and invasion. Thus, our findings underline the role of the BCSFB as a potential entry port for NTHI into the brain and provide strong evidence for a function of the CP during NTHI invasion into the CNS during the course of meningitis.

The bitter taste receptor TAS2R14 regulates resveratrol transport across the human blood-cerebrospinal fluid barrier.

The regulation of transport mechanisms at brain barriers must be thoroughly understood, so that novel strategies for improving drug delivery to the brain can be designed. The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelial cells has been poorly studied in this regard despite its relevance for the protection of the central nervous system (CNS). This study assessed the role of bitter taste receptors (TAS2Rs), TAS2R14 and TAS2R39, in the transport of resveratrol across CP epithelial cells using an in vitro model of the human BCSFB. Both receptors are expressed in human CP cells and known to bind resveratrol. First, Ca2+ imaging assays demonstrated that resveratrol specifically activates the TAS2R14 receptor, but not TAS2R39, in these human CP epithelial cells. Then, we proceeded with permeation studies that showed resveratrol can cross the human BCSFB, from the blood to the CSF side and that TAS2R14 knockdown decreased the transport of resveratrol across these cells. Conversely, inhibition of efflux transporters ABCC1, ABCC4 or ABCG2 also restrained the transport of resveratrol across these cells. Interestingly, resveratrol upregulated the expression of ABCG2 located at the apical membrane of the cells via TAS2R14, whereas ABCC1 and ABCC4 at the basolateral membrane of the cells were not affected. Altogether, our study demonstrates that the BCSFB is a gateway for resveratrol entrance into the CNS and that the receptor TAS2R14 regulates its transport by regulating the action of efflux transporters at CP epithelial cells.

Bitter taste receptors profiling in the human blood-cerebrospinal fluid-barrier.

The choroid plexus (CP) epithelial cells establish an important blood-brain interface, the blood-cerebrospinal fluid barrier (BCSFB), which constitutes a complementary gateway to the blood-brain-barrier for the entrance of several molecules into the central nervous system (CNS). However, the mechanisms that operate at the BCSFB to regulate the molecular traffic are still poorly understood. The taste signalling machinery, present in many extra-oral tissues, is involved in the chemical sensing of the composition of body fluids. We have identified this pathway in rat CP and hypothesised that it could also be present in the human BCSFB. In this study, we characterised the bitter taste receptors (TAS2Rs) expression profiling in human CP by combining data retrieved from available databases of the human CP transcriptome with its expression analysis in a human CP cell line and immunohistochemistry of human CP sections from men and women. TAS2R4, 5, 14 and 39 expression was confirmed in human CP tissue by immunohistochemistry and in HIBCPP cells by RT-PCR, immunofluorescence and Western blot. Moreover, the presence of downstream effector proteins GNAT3, PLCß2 and TRPM5 was also detected in HIBCPP cells. Then, we demonstrated that HIBCPP cells respond to chloramphenicol via TAS2R39 and to quercetin via TAS2R14. Our findings support an active role of TAS2Rs at the human BCSFB, as surveyors of the bloodstream and CSF compositions. These findings open new avenues for studies on the uptake of relevant compounds for targeted therapies of the CNS.

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.

Review of functional in vitro models of the blood-cerebrospinal fluid barrier in leukaemia research.

Acute lymphoblastic leukaemia represents the most common paediatric malignancy. Although survival rates approach up to 90% in children, investigation of leukaemic infiltration into the central nervous system (CNS) is essential due to the presence of ongoing fatal complications. Recent in vitro studies mostly employed models of the blood-brain barrier (BBB), as endothelial cells of the microvasculature represent the largest surface between the blood stream and the brain parenchyma. However, crossing the blood-cerebrospinal fluid barrier (BCSFB) within the choroid plexus (CP) has been shown to be a general capability of leukaemic blasts. Hence, in vitro models of the BCSFB to study leukaemic transmigration may be of major importance to understand the development of CNS leukaemia. This review will summarise available in vitro models of the BCSFB employed to study the cellular interactions with leukaemic blasts during cancer cell transmigration into the brain compartment across primary or immortal/immortalised BCSFB cells. It will also provide an outlook on prospective improvements in BCSFB in vitro models by developing barrier-on-a-chip models and brain organoids.

Distinct migratory pattern of naive and effector T cells through the blood-CSF barrier following Echovirus 30 infection.

BACKGROUND: Echovirus 30 (E-30) is one of the most frequently isolated pathogens in aseptic meningitis worldwide. To gain access to the central nervous system (CNS), E-30 and immune cells have to cross one of the two main barriers of the CNS, the epithelial blood-cerebrospinal fluid barrier (BCSFB) or the endothelial blood-brain barrier (BBB). In an in vitro model of the BCSFB, it has been shown that E-30 can infect human immortalized brain choroid plexus papilloma (HIBCPP) cells. METHODS: In this study we investigated the migration of different T cell subpopulations, naive and effector T cells, through HIBCPP cells during E-30 infection. Effects of E-30 infection and the migration process were evaluated via immunofluorescence and flow cytometry analysis, as well as transepithelial resistance and dextran flux measurement. RESULTS: Th1 effector cells and enterovirus-specific effector T cells migrated through HIBCPP cells more efficiently than naive CD4+ T cells following E-30 infection of HIBCPP cells. Among the different naive T cell populations, CD8+ T cells crossed the E-30-infected HIBCPP cell layer in a significantly higher number than CD4+ T cells. A large amount of effector T cells also remained attached to the basolateral side of the HIBCPP cells compared with naive T cells. Analysis of HIBCPP barrier function showed significant alteration after E-30 infection and trans- as well as paracellular migration of T cells independent of the respective subpopulation. Morphologic analysis of migrating T cells revealed that a polarized phenotype was induced by the chemokine CXCL12, but reversed to a round phenotype after E-30 infection. Further characterization of migrating Th1 effector cells revealed a downregulation of surface adhesion proteins such as LFA-1 PSGL-1, CD44, and CD49d. CONCLUSION: Taken together these results suggest that naive CD8+ and Th1 effector cells are highly efficient to migrate through the BCSFB in an inflammatory environment. The T cell phenotype is modified during the migration process through HIBCPP cells.

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Cytokine and inflammatory mediator effects on TRPV4 function in choroid plexus epithelial cells.

The choroid plexus (CP), composed of capillaries surrounded by a barrier epithelium, is the main producer of cerebrospinal fluid (CSF). The CP epithelium regulates the transport of ions and water between the blood and the ventricles, contributing to CSF production and composition. Several studies suggest a connection between the cation channel transient receptor potential vanilloid-4 (TRPV4) and transepithelial ion movement. TRPV4 is a nonselective, calcium-permeable cation channel present in CP epithelia reported to be activated by cytokines and inflammatory mediators. Utilizing the PCP-R (porcine choroid plexus-Riems) cell line, we investigated the effects of various cytokines and inflammatory mediators on TRPV4-mediated activity. Select proinflammatory cytokines (TNF-α, IL-1ß, TGF-ß1) had inhibitory effects on TRPV4-stimulated transepithelial ion flux and permeability changes, whereas anti-inflammatory cytokines (IL-10, IL-4, and IL-6) had none. Quantitative mRNA analysis showed that these cytokines had no effect on TRPV4 transcription levels. Inhibition of the transcription factor NF-κB, involved in the production and regulation of several inflammatory cytokines, inhibited TRPV4-mediated activity, suggesting a link between TRPV4 and cytokine production. Contrary to published studies, the proinflammatory mediator arachidonic acid (AA) had inhibitory rather than stimulatory effects on TRPV4-mediated responses. However, inhibition of AA metabolism also caused inhibitory effects on TRPV4, suggesting a complex interaction of AA and its metabolites in the regulation of TRPV4 activity. Together these data imply that TRPV4 activity is involved in the inflammatory response; it is negatively affected by proinflammatory mediators. Furthermore, arachidonic acid metabolites, but not arachidonic acid itself, are positive regulators of TRPV4.

Optimized cultivation of porcine choroid plexus epithelial cells, a blood-cerebrospinal fluid barrier model, for studying granulocyte transmigration.

The blood-cerebrospinal fluid barrier (BCSFB) plays important roles during the transport of substances into the brain, the pathogenesis of central nervous system (CNS) diseases, and neuro-immunological processes. Along these lines, transmigration of granulocytes across the blood-cerebrospinal fluid (CSF) barrier (BCSFB) is a hallmark of inflammatory events in the CNS. Choroid plexus (CP) epithelial cells are an important tool to generate in vitro models of the BCSFB. A porcine CP epithelial cell line (PCP-R) has been shown to present properties of the BCSFB, including a strong barrier function, when cultivated on cell culture filter inserts containing a membrane with 0.4 µm pore size. For optimal analysis of pathogen and host immune cell interactions with the basolateral side of the CP epithelium, which presents the physiologically relevant "blood side", the CP epithelial cells need to be grown on the lower face of the filter in an inverted cell culture insert model, with the supporting membrane possessing a pore size of at least 3.0 µm. Here, we demonstrate that PCP-R cells cultivated in the inverted model on filter support membranes with a pore size of 3.0 µm following a "conventional" protocol grow through the pores and cross the membrane, forming a second layer on the upper face. Therefore, we developed a cell cultivation protocol, which strongly reduces crossing of the membrane by the cells. Under these conditions, PCP-R cells retain important properties of a BCSFB model, as was observed by the formation of continuous tight junctions and a strong barrier function demonstrated by a high transepithelial electrical resistance and a low permeability for macromolecules. Importantly, compared with the conventional cultivation conditions, our optimized model allows improved investigations of porcine granulocyte transmigration across the PCP-R cell layer.

Virulence factor-dependent basolateral invasion of choroid plexus epithelial cells by pathogenic Escherichia coli in vitro.

Escherichia coli is the most common Gram-negative causative agent of neonatal meningitis and E. coli meningitis is associated with high morbidity and mortality. Previous research has been carried out with regard to the blood-brain barrier and thereby unveiled an assortment of virulence factors involved in E. coli meningitis. Little, however, is known about the role of the blood-cerebrospinal fluid (CSF) barrier (BCSFB), in spite of several studies suggesting that the choroid plexus (CP) is a possible entry point for E. coli into the CSF spaces. Here, we used a human CP papilloma (HIBCPP) cell line that was previously established as valid model for the study of the BCSFB. We show that E. coli invades HIBCPP cells in a polar fashion preferentially from the physiologically relevant basolateral side. Moreover, we demonstrate that deletion of outer membrane protein A, ibeA or neuDB genes results in decreased cell infection, while absence of fimH enhances invasion, although causing reduced adhesion to the apical side of HIBCPP cells. Our findings suggest that the BCSFB might constitute an entry point for E. coli into the central nervous system, and HIBCPP cells are a valuable tool for investigating E. coli entry of the BCSFB.

Capsule and fimbriae modulate the invasion of Haemophilus influenzae in a human blood-cerebrospinal fluid barrier model.

The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral "blood "side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.