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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).

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.

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&ndash, 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&rsquo, infection. Altogether, our data highlights the polar effects of E-30 infection in a human in vitro model of the blood&ndash, cerebrospinal fluid barrier leading to central nervous system inflammation.

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.

Background Echovirus (E) 30 (E-30) meningitis is characterized by neuroinflammation involving immune cell pleocytosis at the protective barriers of the central nervous system (CNS). In this context, infection of the blood-cerebrospinal fluid barrier (BCSFB), which has been demonstrated to be involved in enteroviral CNS pathogenesis, may affect the tight junction (TJ) and adherens junction (AJ) function and morphology. Methods We used an in vitro human choroid plexus epithelial (HIBCPP) cell model to investigate the effect of three clinical outbreak strains (13-311, 13-759, and 14-397) isolated in Germany in 2013, and compared them to E-30 Bastianni. Conducting transepithelial electrical resistance (TEER), paracellular dextran flux measurement, quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence analysis, we investigated TJ and AJ function and morphology as well as strain-specific E-30 infection patterns. Additionally, transmission electron and focused ion beam microscopy electron microscopy (FIB-SEM) was used to evaluate the mode of leukocyte transmigration. Genome sequencing and phylogenetic analyses were performed to discriminate potential genetic differences among the outbreak strains. Results We observed a significant strain-dependent decrease in TEER with strains E-30 Bastianni and 13-311, whereas paracellular dextran flux was only affected by E-30 Bastianni. Despite strong similarities among the outbreak strains in replication characteristics and particle distribution, strain 13-311 was the only outbreak isolate revealing comparable disruptive effects on TJ (Zonula Occludens (ZO) 1 and occludin) and AJ (E-cadherin) morphology to E-30 Bastianni. Notwithstanding significant junctional alterations upon E-30 infection, we observed both para- and transcellular leukocyte migration across HIBCPP cells. Complete genome sequencing revealed differences between the strains analyzed, but no explicit correlation with the observed strain-dependent effects on HIBCPP cells was possible. Conclusion The findings revealed distinct E-30 strain-specific effects on barrier integrity and junctional morphology. Despite E-30-induced barrier alterations leukocyte trafficking did not exclusively occur via the paracellular route. Electronic supplementary material The online version of this article (10.1186/s12974-018-1061-4) contains supplementary material, which is available to authorized users.

BackgroundEchovirus 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.MethodsIn 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.ResultsTh1 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.ConclusionTaken 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.

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

Listeria monocytogenes, a Gram-positive bacterium, can cause meningitis after invading the human central nervous system. The blood-cerebrospinal fluid barrier (BCSFB), located at the epithelium of the choroid plexus, is a possible entry site for L. monocytogenes into the brain, and in vitro L. monocytogenes invades human choroid plexus epithelial papilloma (HIBCPP) cells. Although host cell signal transduction subsequent to infection by L. monocytogenes has been investigated, the role of mitogen-activated protein kinases (MAPK) is not clarified yet. We show that infection with L. monocytogenes causes activation of the MAPKs Erk1/2 and p38 preferentially when bacteria are added to the physiologically more relevant basolateral side of HIBCPP cells. Deletion of the listerial virulence factors Internalin (InlA) and InlB reduces MAPK activation. Whereas inhibition of either Erk1/2 or p38 signaling significantly attenuates infection of HIBCPP cells with L. monocytogenes, simultaneous inhibition of both MAPK pathways shows an additive effect, and Erk1/2 and p38 are involved in regulation of cytokine and chemokine expression following infection. Blocking of endocytosis with the synthetic dynamin inhibitor dynasore strongly abrogates infection of HIBCPP cells with L. monocytogenes. Concurrent inhibition of MAPK signaling further reduces infection, suggesting MAPKs mediate infection with L. monocytogenes during inhibition of dynamin-mediated endocytosis.

Background The human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens. Methods Using a recently established in vitro model of the human BCSFB based on human malignant CP papilloma (HIBCPP) cells we investigated the cellular response of HIBCPP cells challenged with the meningitis-causing Nm strain, MC58, employing transcriptome and RT-PCR analysis, cytokine bead array, and enzyme-linked immunosorbent assay (ELISA). In comparison, we analyzed the answer to the closely related unencapsulated carrier isolate Nm α14. The presence of TLRs in HIBCPP and their role during signal transduction caused by Nm was studied by RT-PCR and the use of specific agonists and mutant bacteria. Results We observed a stronger transcriptional response after infection with strain MC58, in particular with its capsule-deficient mutant MC58siaD−, which correlated with bacterial invasion levels. Expression evaluation and Gene Set Enrichment Analysis pointed to a NFκB-mediated pro-inflammatory immune response involving up-regulation of the transcription factor IκBζ. Infected cells secreted significant levels of pro-inflammatory chemokines and cytokines, including, among others, IL8, CXCL1-3, and the IκBζ target gene product IL6. The expression profile of pattern recognition receptors in HIBCPP cells and the response to specific agonists indicates that TLR2/TLR6, rather than TLR4 or TLR2/TLR1, is involved in the cellular reaction following Nm infection. Conclusions Our data show that Nm can initiate a pro-inflammatory response in human CP epithelial cells probably involving TLR2/TLR6 signaling and the transcriptional regulator IκBζ. Electronic supplementary material The online version of this article (doi:10.1186/s12974-014-0163-x) contains supplementary material, which is available to authorized users.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.