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Choroid plexus organoids reveal mechanisms of Streptococcus suis translocation at the blood-cerebrospinal fluid barrier

Fluids and Barriers of the CNS volume 22, Article number: 14 (2025) Cite this article

Abstract

Streptococcus suis is a globally emerging zoonotic pathogen that can cause invasive disease commonly associated with meningitis in pigs and humans. To cause meningitis, S. suis must invade the central nervous system (CNS) by crossing the neurovascular unit, also known as the blood-brain barrier (BBB), or vascularized choroid plexus (ChP) epithelium known as the blood-cerebrospinal fluid barrier (BCSFB). Recently developed ChP organoids have been shown to accurately replicate the cytoarchitecture and physiological functions of the ChP epithelium in vivo. Here, we used human induced pluripotent stem cells (iPSC)-derived ChP organoids as an in vitro model to investigate S. suis interaction and infection at the BCSFB. Our study revealed that S. suis is capable of translocating across the epithelium of ChP organoids without causing significant cell death or compromising the barrier integrity. Plasminogen (Plg) binding to S. suis in the presence of tissue plasminogen activator (tPA), which converts immobilized Plg to plasmin (Pln), significantly increased the basolateral to apical translocation across ChP organoids into the CSF-like fluid in the lumen. S. suis was able to replicate at the same rate in CSF and laboratory S. suis culture medium but reached a lower final density. The analysis of transcriptomes in ChP organoids after S. suis infection indicated inflammatory responses, while the addition of Plg further suggested extracellular matrix (ECM) remodeling. To our knowledge, this is the first study using ChP organoids to investigate bacterial infection of the BCSFB. Our findings highlight the potential of ChP organoids as a valuable tool for studying the mechanisms of bacterial interaction and infection of the human ChP in vitro.

Introduction

Streptococcus suis, a Gram-positive bacterium, is an important swine pathogen causing a wide range of infections such as meningitis and arthritis, resulting in significant economic losses to the swine industry worldwide [1]. S. suis is also an emerging zoonotic pathogen causing sepsis and meningitis in humans [2]. S. suis serotype 2 strains are the most common cause of bacterial meningitis in pigs and also humans in southern Vietnam and Thailand [3, 4]. Despite advancements in antimicrobial therapy, bacterial meningitis is a severe and potentially life-threatening condition that may lead to complications, including brain damage, hearing loss, and learning disabilities, if not promptly and effectively treated [5]. Understanding the mechanisms of bacterial meningitis could lead to the development of more effective treatments and preventive measures, such as targeting vaccines to virulence factors required for crossing the blood-brain barrier (BBB) or blood-cerebrospinal fluid barrier (BCSFB), or developing drugs that inhibit entry.

The brain microvascular endothelial cells (BMEC) are the primary components of the BBB, protecting the central nervous system (CNS) from bacterial entry. Additionally, BMEC express pattern recognition receptors to detect microorganisms, leading to the local production of defensins and immune mediators that recruit leukocytes. The association of BMEC with pericytes and astrocytes supports the barrier function and repair processes following injury [6].

Studies of S. suis interaction with brain endothelial cell lines suggest that the BBB might be a route of entry into the brain [7,8,9]. A recent study used monolayers of endothelial cells generated from human induced pluripotent stem cells (iPSC) to investigate translocation of S. suis (data not shown, manuscript in preparation submitted). The endothelial monolayers mimic the high barrier integrity in vivo and show that S. suis can translocate without affecting trans-endothelial electrical resistance (TEER), possibly via the paracellular route within 3 h after a challenge with 10 bacteria per cell.

The BCSFB is another potential route for pathogens to enter the brain [10]. The BCSFB is primarily located at the base of the brain ventricles where the choroid plexus (ChP) epithelium produces cerebrospinal fluid (CSF) from fenestrated blood vessels surrounding the ChP [11, 12]. In S. suis-associated cases of porcine meningitis, extracellular identification of S. suis in histological sections of the ChP indicates that S. suis might translocate across the ChP into the CSF [4]. In addition, S. suis has been detected in CSF samples from human meningitis patients, suggesting the possibility of S. suis invasion of the CNS [13, 14].

Previous studies on the interaction between S. suis and the BCSFB have used primary porcine ChP epithelial cells (PCPEC) [15,16,17,18] and human choroid plexus epithelial papilloma (HIBCPP) cancer cells [19]. In vitro invasion and translocation of S. suis across PCPEC were shown, supporting a possible mechanism of S. suis translocation via the physiologically relevant basal side of the epithelium (blood side) to the apical CSF side without the loss of barrier function [20]. Similar results were obtained using HIBCPP cells grown on the underside of an “inverted” Transwell system [21, 22].

Both PCPEC and HIBCPP monolayers display typical features of a functional BCSFB in the “inverted” Transwell system, including formation of tight junctions and development of high TEER values up to 3000 Ω × cm2 [20] and 800 Ω × cm2 [21], respectively. Transformed cell lines can provide valuable insights into host-pathogen interaction in vitro but are known to display genetic and transcriptional evolution in culture, leading to biological heterogeneity over time [23]. Furthermore, TEER values of ChP epithelial cell line monolayers are dependent on various factors, such as the number of cells seeded, serum concentration in medium, and culture conditions [21, 24], which can lead to issues with reproducing similar TEER values across laboratories [21, 25, 26].

Recently, Pellegrini et al. developed a pluripotent stem cell (PSC)-derived ChP organoid model, which recapitulates the critical properties of human ChP tissue including the formation of a polarized barrier with apical tight junctions, the secretion of a CSF-like fluid within the self-contained compartment, and the presence of different epithelial cell types [27]. This advanced ChP organoid model has been employed for studying the SARS-Cov-2 infection at the BCSFB in vitro [28], but no studies have been published on its use to investigate bacterial infection.

Recently, we reported that host plasminogen (Plg) binding to S. suis surface-expressed enolase and its subsequent conversion to proteolytic Plasmin (Pln) facilitates translocation of S. suis across the BBB [7], but its effect on S. suis translocation across the BCSFB has not yet been investigated. Enolase is not only an abundantly expressed cytosolic enzyme that catalyzes the reversible conversion of 2-phospho-D-glycerate (2-PG) to phosphoenolpyruvate (PEP), but is also present on the cell surface of streptococci that binds host Plg, including S. suis [29, 30], S. pneumoniae [31,32,33], and group A streptococci [34]. Streptococcal surface-bound Plg can be activated by host activators such as tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), converting it to proteolytic Pln [30,31,32,33,34], which degrades fibrin networks in blood clots, extracellular matrix (ECM) proteins, and some cell-cell junctional proteins [35,36,37,38,39]. The enolase-Plg interaction facilitates S. suis translocation across the BBB is most likely due to the degradation of ECM proteins in endothelial barriers.

In this study, we used iPSC-derived ChP organoids to investigate the ability of S. suis to translocate across the basal side of ChP epithelium and study the responses of ChP organoids to S. suis using transcriptomics. We also investigated whether the known binding of Plg to S. suis surface enolase and its conversion to Pln by tPA would facilitate S. suis translocation across the epithelium of ChP organoids and alter the ChP response to infection.

Materials and methods

Bacterial culture

S. suis strain P1/7 was used in this study. A green fluorescent protein (GFP)-expressing derivative was provided by Dr. Manouk Vrieling at the Wageningen Bioveterinary Research (WBVR) of Wageningen University and Research [40]. S. suis strains were cultured on agar media containing Todd Hewitt Broth (THB; Difco Laboratories, Detroit, MI, The United States) with 0.2% Yeast extract (THY), and incubated overnight at 37 ˚C with 5% CO2. The following day, liquid cultures were prepared from overnight colonies grown in THY medium.

Generation of ChP organoids from human iPSC

The 6-well plates (Corning, 07-200-83) were pre-coated with Vitronectin (STEMCELL, 07180) in CellAdhere™ Dilution Buffer (STEMCELL, 07183) at room temperature for 1 h. Human iPSC line EDi002-A (EBiSC™) were maintained on vitronectin-coated 6-well plates in mTeSR1 (STEMCELL, 85857). Media was changed daily, and cells were passaged once a week. ChP organoids were generated using the STEMdiff™ Choroid Plexus Organoid Differentiation Kit (STEMCELL, 100–0824) and the STEMdiff™ Choroid Plexus Organoid Maturation Kit (STEMCELL, 100–0825), following previous protocols [27] with minor modifications. Briefly, iPSC were dissociated into single-cell suspensions using Accutase (STEMCELL, 07920). On day 1, 1 × 105 cells were seeded into a well of the Corning® 96-well round-bottom ultra-low attachment microplate (Corning, 7007) in 100 µL of embryoid body (EB) Formation Medium and 10 µM Y-27,632 (ROCK inhibitor; STEMCELL, 72302). On day 2 and day 4, fresh 100 µL of EB Formation Medium was added to each well. On day 5, we assessed whether EB display clear and smooth edges and reach a diameter ranging between 400 and 600 μm using light field microscopy. Afterwards, EB Formation Medium was replaced with 200 µL/well of Induction Medium in the same plate. On day 7, each EB was embedded in 15 µL of Matrigel® (Corning, 734–1101) dropwise on sheets of parafilm and incubated at 37 ˚C for 30 min to polymerize Matrigel® (16 EB per sheet of parafilm). The sheet of parafilm was positioned above one well of a 6-well ultra-low adherent plate (STEMCELL, 100 − 0083) using sterile forceps. All 16 Matrigel® droplets were gently washed off the sheet and into one well by using 3 mL of Expansion Medium. The plate was shaken back and forth three times to ensure even distribution of EB before being incubated at 37 ˚C for 3 days. On day 10, Expansion Medium was carefully replaced with 3 mL/well of Choroid Plexus Differentiation Medium, and the plate was placed on the platform rotator (Fisherbrand™, 15504080) in the incubator. On day 13, Choroid Plexus Differentiation Medium was refreshed. From day 15, Choroid Plexus Differentiation Medium was replaced with 3 mL/well Maturation Medium and renewed every 3 days. By day 30, ChP organoids epithelia resemble cyst-like structures filled with CSF-like fluid. ChP organoids between day 30 to day 40 were used for experiments.

Embedding and cryosectioning of ChP organoids

ChP organoids were fixed in 4% paraformaldehyde (PFA) at 4 ˚C for 20 min, washed three times in PBS, and moved to 30% sucrose buffer at 4 ˚C for overnight incubation. Organoids were then embedded in gelatin/sucrose solution (7.5% gelatin in 10% sucrose in PBS) and sectioned as previously described [41]. Briefly, organoids were pre-incubated in warm gelatin/sucrose solution at 37 ˚C for 15 min, while the bottom of the embedding mold (Polysciences, 18646D-1) was covered by a layer of gelatin/sucrose and placed at 4 ˚C to harden. Organoids were put on top of the hardened gelatin, immersed with warm gelatin/sucrose, and placed at 4 ˚C for 1 h. Each gelatin block containing one organoid was withdrawn from the embedding mold, cut into appropriately sized cube, and glued to a small piece of rough cardboard by using Tissue-Tek O.C.T. compound. The gelatin block was immersed in the cold 2-Methylbutane (Sigma-Aldrich, 277258) bath containing dry ice (until the temperature reached -35 ˚C to -50 ˚C) to freeze for the maximum of 1 min. Frozen gelatin blocks were wrapped in aluminum foil and stored at -80 ˚C. For cryosectioning, the Cryostat (Leica, CM3050 S) was set at -14 ˚C to -18 ˚C ambient temperature/blade temperature and − 13 ˚C to -18 ˚C block temperature. The cardboard side of the ChP organoid block was attached to the metal disk by using Tissue-Tek O.C.T. compound. Sections were cut at 10-µm thickness and collected on Epredia™ SuperFrost Plus™ Adhesion slides (Fisher, J1800AMNZ) keeping track of the order of sections. Slides containing organoid sections were left at room temperature for several hours to dry and then stored at -80 ˚C.

Immunostaining and imaging

Prior to immunostaining, frozen slides with organoid sections were transferred from the − 80 ˚C freezer to the − 20 ˚C freezer overnight, then thawed overnight at 4 ˚C to maintain cellular morphology. A PAP-pen (Abcam, ab2601) was used to draw hydrophobic borders around the sections on the slide, then rinsed with PBS and allowed to dry. After blocking and permeabilizing with 4% of bovine serum albumin (BSA) and 0.25% Triton-X in PBS at room temperature for 1 h, sections were incubated overnight with primary antibodies: mouse anti-TTR (1:50; R&D Systems, MAB7505), rabbit anti-CLIC6 (1:200; Abcam, ab204567), rabbit anti-ZO1 (1:50; Invitrogen, 61-7300), and goat anti-MSX1 (1:20; R&D Systems, AF5045) at 4 ˚C. After three washes with PBS, slides were incubated in 0.1% Triton-X and 4% of BSA in PBS containing secondary antibodies labelled with Alexafluor 555 and 647 (1:500) and DAPI (1:1000) at room temperature for 1 h. Following three washes with PBS again, slides were mounted with Prolong Diamond mounting media and covered with coverslip. Slides were imaged using a Leica DM6b microscope equipped with epifluorescence illumination. Captured images were adjusted using Leica LAS X software version 4.5.0.25531 (Leica) and Fiji (NIH), using identical fluorescence intensity settings for each channel.

S. suis growth in CSF collected from the lumen of ChP organoids

CSF in the lumen of ChP organoids has previously been shown to be highly similar to in vivo CSF [27]. One day before collecting CSF from the lumen of ChP organoids, the culture medium was replaced with fresh Maturation Medium. On the day of collection, the culture medium was changed at least 1 h before removing CSF with a 1 mL syringe and a 0.30 × 12 mm BL/LB needle. Fresh CSF was dispensed into a 96-well plate at a volume of 200 µL/well, or stored at -20 ˚C for future use. The overnight S. suis culture was centrifuged and resuspended in PBS to reach a concentration of 3 × 108 CFU/mL. Then, S. suis were added to 96-well plate wells containing 200 µL of CSF to a final concentration of 5 × 106 CFU/mL. The 96-well plate was incubated at 37 ˚C in presence of 5% CO2. 10 µL of CSF was withdrawn from each well and serially diluted in PBS to enumerate the CFU of S. suis at one-hour interval for a total duration of 8 hours. For comparison, S. suis was cultured in THY medium under same conditions as described above.

S. suis translocation assay of ChP organoids

Experiments were performed by inoculating S. suis into the ChP organoid culture medium at a multiplicity of infection (MOI) of 10 bacteria per cell (MOI of 10) and incubating at 37 ˚C with 5% CO2 for 6 hours. In each independent experiment, we distributed three ChP organoids to three wells of a Costar® 24-well ultra-low attachment plate (Corning, 3473) as technical triplicates. Because the size of ChP organoids varies, we estimated cell numbers per organoid by dissociating three organoids (small, medium, and large) into single cells using pre-warmed TrypLE™ Express Enzyme (Gibco, 12604013), then calculating the average cell number among the three organoids, resulting in an estimated 4.5 × 106 cells per organoid. 700 µL of Maturation Medium was added to each well to submerge the organoid. In the case of ChP organoids challenged with S. suis + Plg, 10 µL of Plg protein solution (10 mg/mL) and 5.6 µL of tPA solution (42.8 KIU/mL) were added to the S. suis inoculum. After 6-hour incubation, S. suis challenged organoids were washed three times with warm PBS and subsequently incubated with 700 µL fresh warm Maturation Medium containing 100 µg/mL of gentamicin and 5 µg/mL of penicillin G for 1 h to inactivate extracellular and adhered S. suis. After washing three times with PBS, CSF was collected from the organoid lumen using a 0.30 × 12 mm BL/LB needle attached to a 1 mL syringe and serially diluted to enumerate S. suis numbers that translocated across the epithelium of ChP organoids.

Cytotoxicity assay

The potential cytotoxic effect of S. suis on ChP organoids was assessed by measuring the lactate dehydrogenase (LDH) release of the organoid in the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780) as previously described [42]. It has been shown that LDH release in challenged cells is associated with the cell death [43]. After 6-hour incubation with S. suis, ChP organoids culture medium was pipetted into a 96-well plate at a volume of 50 µL/well. 50 µL of the CytoTox 96® Reagent was added to each well. The plate was covered with aluminum foil and incubated at room temperature for 30 min. As a positive control, 10 × Lysis Solution was added to wells containing unchallenged ChP organoids for 45 min before adding CytoTox 96® Reagent. The absorbance at 490 nm (OD490) was measured within 1 h after adding 50 µL of Stop Solution to each well. Background LDH release from unchallenged organoids was subtracted from all measured values and the corrected values were used to calculate cytotoxicity (%) via the formula:

$${\text{Cytotoxicity}}\left( \% \right){\text{ = }}\frac{{{\text{Experimental}}\:{\text{LDH}}\:{\text{Release}}\:({\text{O}}{{\text{D}}_{{\text{490}}}}{\text{)}}}}{{{\text{Maximum}}\:{\text{LDH}}\:{\text{Release}}\:({\text{O}}{{\text{D}}_{{\text{490}}}}{\text{)}}}} \times {\text{100}}\% $$

ChP organoids permeability assay

The permeability assay of ChP organoids challenged with S. suis (or S. suis + Plg) was determined by the influx of 4 kDa fluorescein isothiocyanate-dextran (FITC-dextran) (Sigma-Aldrich 46944). After 6-hour incubation with S. suis, ChP organoids were gently washed three times with warm PBS and then incubated with 1 mg/mL FITC-dextran solution for 2 h, after which CSF was collected from ChP organoids to measure the fluorescence intensity (FI) (excitation 490 nm, emission 515 nm) using a spectrophotometer (Molecular Devices, CA). The FITC intensity (%) was determined by calculating the FI of CSF divided by the FI of 1 mg/mL FITC-dextran solution.

RNA isolation and DNA removal

The total RNA was isolated from ChP organoids (challenged with S. suis ± Plg or Control) using the RNeasy Mini Kit (Qiagen). Three separate batches (sampling three organoids per batch) were used for each condition. Organoids were dissociated using Accutase (STEMCELL, 07920) by pipetting up and down 10 times at 5 min intervals. Cells suspension was pelleted at 300 × g for 5 min, resuspended in 350 µL of Buffer RLT, and thoroughly disrupted by vortexing. 350 µL of 70% ethanol was added to tube and up to 700 µL of the sample was transferred to an RNeasy Mini spin column placed in a 2 mL collection tube. The remaining steps were carried out according to the manufacturer’s recommended protocol. Subsequently, genomic DNA was eliminated from RNA samples using DNA-free™ DNA Removal Kit (Invitrogen, AM 1906) following the manufacturer’s recommended protocol.

RNA-seq and data analysis

Messenger RNA (mRNA) was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers, followed by the second-strand cDNA synthesis using dTTP for non-directional library construction. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled in an equimolar ratio for multiplexed sequencing on Illumina platforms (Hi-Seq 4000, Novogene, Hong Kong). Subsequently, raw reads obtained as FASTQ files were filtered by removing adapter sequences, poly-N stretches, and low-quality reads. Index of the reference genome was downloaded from genome website and built using Hisat2 v2.0.5. Paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. The mapped reads of each sample were assembled by StringTie (v1.3.3b) according to a reference-based approach [44]. To estimate gene expression levels, reads numbers that mapped to each gene were counted using FeatureCounts v1.5.0-p3 [45] and then Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads numbers. A cutoff of 1 FPKM was used in this study. Differential expression analysis of S. suis vs. Control and S. suis + Plg vs. S. suis (three batches per condition) was performed using the DESeq2R package (1.20.0) [46]. Genes with unadjusted P values < 0.05 found by DESeq2 were considered differentially expressed genes (DEGs).

A more generic overview of biological processes in ChP organoids infected by S. suis (with or without Plg) was generated by Gene Set Enrichment Analysis (GSEA) using ErmineJ [47] on log2fold change (log2FC) values with the receiver operating characteristic (ROC) method. GSEA ranks genes by pertinent signals (e.g. log2FC or associated P values) for the comparisons of S. suis vs. Control and S. suis + Plg vs. S. suis to subsequently identify biological pathways or processes, such as gene ontology (GO) terms, that are enriched in highly ranked genes. GO terms significantly enriched (P values < 0.05) in comparisons of S. suis vs. Control and S. suis + Plg vs. S. suis are visualized using Cytoscape (v. 3.9.1) [48, 49].

Statistical analysis

Unpaired two-tailed Student’s t-test was implemented to compare the FITC intensity of CSF between Control and S. suis infected ChP organoids in the permeability assay, as well as the S. suis translocation (CFUs) between S. suis treated and S. suis + Plg treated ChP organoids. One-way ANOVA was used to compare means across various conditions within S. suis translocation assay and cytotoxicity assay. All graphs and statistical analyses were performed within the GraphPad Prism software version 9 (GraphPad Software Inc.). A P value of < 0.05 was considered statistically significant.

Results

Generation and characterization of iPSC-derived ChP organoids

We generated ChP organoids using human iPSC as previously described [27]. At day 0, single-cell suspension of iPSC was seeded in the presence of ROCK inhibitor to produce aggregates called embryoid bodies (EB). After addition of Induction Medium for 2 days, the EB formed neural ectoderm and were then expanded in Matrigel droplets for a further 3 days to form the neuroepithelium. From day 10 to 14, dorsalizing factor Bmp4 [50] and the Wnt-activator molecule CHIR [51] were added to pattern the organoid neuroepithelium to develop into ChP epithelium. After day 30, ChP organoids formed cystic structures surrounded by a cuboidal epithelium and filled with CSF-like fluid (Fig. 1A), closely mimicking the BCSFB located at the ChP in vivo (Fig. 1B) [27]. From day 30, organoids expressed the specific ChP markers transthyretin (TTR) and chloride intracellular channel 6 (CLIC6), as well as tight junction protein ZO1 (Fig. 1C). From day 30 to 40, the size of the cystic compartment and expression of ZO1 increased (Fig. 1D).

Fig. 1
figure 1

Generation and characterization of ChP organoids. (A) Timeline of ChP organoids generation from iPSC EDi002-A with representative images. The ChP organoid differentiation started with EB formation, followed by neural induction and neuroepithelial expansion. After 30 days, mature ChP organoids developed fluid-filled cysts (arrowhead) containing CSF-like fluid. (B) Schematic of the BCSFB at the ChP. ChP organoids were generated to mimic the ChP in vivo. (C) Representative images of ChP organoids stained for TTR, ZO1, and CLIC6; nuclei were counterstained by DAPI. Arrows point to the apical side of the ChP epithelium, and asterisks mark the lumen of fluid-filled cystic compartment. (D) Comparison of ChP organoid staining at day 30 and day 40. Epithelial cells surrounding cystic compartments displayed polarized apical staining for ZO1 (left). A big cyst filled with CSF-like fluid appeared by day 40 (middle). The right image depicting part of the cyst is a magnified view of the area indicated by the dotted line in the middle image. Staining revealed increased apical localization of ZO1. (E) Heatmap of genes expressed in our ChP organoids that were annotated as ChP specific genes in the Human Protein Atlas. The expression level of each gene was displayed in FPKM (with a cutoff of > 1 FKPM). Typical ChP marker genes are indicated

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To further characterize the ChP organoid, we carried out sequencing of ChP organoid transcriptomes. From RNA-seq data generated in Table S1, we observed that all 371 ChP specific genes retrieved from the Human Protein Atlas (HPA) (Human Protein Atlas proteinatlas.org; https://www.proteinatlas.org/search/brain_category_rna%3Achoroid+plexus%3BRegion+enriched%2CGroup+enriched%2CRegion+enhanced+AND+sort_by%3Atissue+specific+score) were also expressed in our ChP organoids with FPKM > 1 (Fig. 1E). Genes previously shown to be specifically expressed in ChP organoids such as TTR and CLIC6 [27] were also expressed in our ChP organoids, and proteins TTR, MSX1 and CLIC6 were detected using immunofluorescent staining (Figs. 1 and 2). The expression of claudin 1 (CLDN1) and CLDN3 indicated the potential of ChP organoids to form and maintain tight junctions (Table S1).

Fig. 2
figure 2

ChP organoids challenged with S. suis. (A) Schematic of the experimental design: [1] The growth curve of S. suis in CSF collected from mature ChP organoids; [2] S. suis translocation of ChP organoids into the internal cystic compartment after 6 h; [3] Cytotoxicity assay. After 6-hour S. suis challenge, LDH release in the medium was assayed and compared to control; [4] ChP organoid permeability assay. After 2-hour incubation with S. suis, FITC-dextran was incubated for 2 h, after which the fluorescence intensity of the extracted CSF was measured and compared to control. (B) The growth curve of S. suis in CSF. The growth of S. suis in THY was used as reference. Error bars represent SD; n = 3 independent experiments. (C) S. suis translocation assay. The number of S. suis in external medium surrounding the cyst was counted at t0 and t6 to determine replication of S. suis in culture medium, antibiotics were then added for 1 h to kill extracellular S. suis. At t7, no S. suis was found in the medium but around 2 × 104 CFU/ml of S. suis were present in CSF removed from the cyst. Error bars represent SD. ****P < 0.0001; n = 3 independent experiments. (D) Representative images of ChP organoids challenged with GFP-expressing S. suis and stained for TTR, MSX1, and CLIC6; nuclei were counterstained with DAPI. Arrows point to the apical side of the ChP epithelium, and asterisks mark the lumen of fluid-filled cystic compartment. (E) Cytotoxicity assay of ChP organoids. The positive control represents maximum LDH release for calculating cytotoxicity %. Error bars represent SD. ***P < 0.001; n = 3 independent experiments. (F) Permeability assay of ChP organoids. Error bars represent SD; n = 3 independent experiments

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S. suis translocates across the ChP epithelium and replicates in CSF

We first determined whether S. suis would be able to replicate in CSF after translocation by inoculating S. suis into the CSF recovered from the organoid lumen (Fig. 2A-(1)). S. suis proliferated in CSF at a rate which was not significantly different from that in bacterial THY medium for the first 3-hour incubation (Fig. 2B). However, S. suis did not reach the same final density in CSF as in THY (Fig. 2B). We then investigated whether inoculation of S. suis in the organoid culture medium would result in translocation into the lumen of ChP organoids using the procedure depicted in Fig. 2A-(2)]. Indeed, S. suis translocated across the ChP epithelium into the cystic compartment of the ChP organoid, with approximately 0.04% of the initial S. suis inoculum present in the medium (Fig. 2C). After fixing, embedding, and cryosectioning ChP organoids, we detected GFP-expressing S. suis in contact with ChP epithelium and inside the ChP organoids by immunofluorescent antibody staining and fluorescent microscopy (Fig. 2D).

S. suis translocation does not induce cell death or increase barrier permeability

To investigate the mechanism by which S. suis translocates across the ChP epithelium, we measured cytotoxicity and permeability changes in ChP epithelium as depicted in Fig. 2A(3) and 2A-(4)]. After 6-hour S. suis infection at an MOI of 10, the LDH concentration in the supernatant was not significantly different to the control (Fig. 2E). This suggested that S. suis does not induce significant cell death enabling it to enter the ChP lumen. Furthermore, the permeability of ChP organoids to low molecular weight FITC-dextran (4 kDa) was not significantly different between S. suis challenged ChP organoids and Control ChP organoids (Fig. 2F). These findings indicated that S. suis can translocate across the ChP epithelium without inducing epithelial cell death or increasing epithelial barrier permeability.

Plg binding to S. suis facilitates bacterial translocation across the ChP epithelium without increasing barrier permeability

Our previous studies have demonstrated that Plg binding to the surface of S. suis significantly increases bacterial translocation across in vitro models of the BBB, including hCMEC/D3 cell line [7] and iPSC-derived brain endothelial cells (iBEC) (data are unpublished). To assess the role of Plg binding in S. suis translocation across the ChP organoid epithelium, we added Plg and tPA to the S. suis inoculum. After 6-hour incubation with S. suis + Plg, we collected CSF from ChP organoids and enumerated translocated S. suis as performed for S. suis challenged ChP organoids (Fig. 2A-b). As hypothesized, Plg binding to bacterial surface significantly increased the translocation of S. suis (Fig. 3A), but did not increase the permeability of ChP organoids compared to Control and S. suis challenged ChP organoids (Fig. 3B).

Fig. 3
figure 3

Plg binding to S. suis facilitates translocation across ChP organoids without increasing barrier permeability compared to S. suis infected organoids. (A) S. suis translocation assay. Error bars represent SD. ***P < 0.001. (B) The permeability assay of ChP organoids challenged with S. suis (with or without Plg). Error bars represent SD; n = 3 independent batches (three organoids per batch, days 36, 40, and 41)

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Transcriptional response of S. suis infected ChP organoids

To determine the transcriptional response of ChP organoids, RNA was isolated from 3 independent batches from each treatment condition (Control, S. suis infection, or S. suis + Plg infection at MOI of 10) after 6-hour incubation and used for RNA-seq.

The correlation matrix of the normalized gene expression values (FPKM) from all samples (Table S1) showed high Spearman correlation coefficients between the expression levels of the same gene across different samples (Fig. 4A). Principal component analysis (PCA) was used to analyze gene expression data from 9 RNA samples and visualize their group relationships and variations (Fig. 4B). The first and second principal components accounted for 20.74% and 26.32% of the variance, respectively. Samples from each group clustered together and separately from the other groups when plotted against the principal components. Transcriptomes of ChP organoids challenged with S. suis were similar to those challenged with S. suis + Plg in the first dimension and clearly separated in the second dimension, supporting the hypothesis that the addition of Plg and tPA to S. suis would alter the gene expression response compared to S. suis alone at the same MOI. A heatmap of gene expression with hierarchical clustering was constructed across samples within the same group or between different groups (Fig. 4C). Transcriptomes of ChP organoids challenged with S. suis and S. suis + Plg clustered together at the first main split in the distance tree, away from transcriptomes of Control group, and transcriptomes of triplicates in each group clustered together.

Fig. 4
figure 4

Overview of transcriptome sequencing results. (A) Correlation matrix of all 9 samples displayed high correlation of transcriptomes of ChP organoids in the same treatment group. (B) Principal component analysis (PCA) of transcriptomes of ChP organoids for S. suis challenged, S. suis + Plg challenged, and Control samples without logarithmic transformation of the input data. The centroid (average position) of each cluster is shown as red triangle with the group name. (C) Heatmap of gene expression in each sample combined with hierarchical clustering. The rows represent genes and their expression value is indicated by a heatmap color scale (shown on the right-hand side of panel). The result of the hierarchical clustering is represented as a dendrogram shown at the top of the heatmap. The variation between replicates in the same group is likely due to the variation in the size of ChP organoids (see Materials and Methods)

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Identification of differentially expressed genes (DEGs)

A total of 3070 DEGs were identified in ChP organoids for the comparison of S. suis vs. Control with a P value of < 0.05 unadjusted for multiple testing (Table S2). 1317 DEGs exhibited at least a 2-fold up- (595) or downregulation (722). Expression levels of all DEGs are shown in the heatmap (Fig. 5A) and DEGs found in S. suis challenged ChP organoids compared to Control ChP organoids are depicted in a volcano plot (Fig. 5B) with their unadjusted P values in Table S2. The genes most highly upregulated (i.e. FOS, ZFP36, EGR1, FOSB, ICAM1, JUNB, CEBPD, GDF15, and PCK1) or downregulated (i.e. FAM111B, OLIG3, and MT-TT) in S. suis vs. Control are highlighted in Fig. 5B. These genes include transcription factors known to influence cell stress and genes involved in innate inflammatory responses.

Fig. 5
figure 5

Differentially expressed genes (DEGs) in ChP organoids for the comparisons of S. suis vs. Control and S. suis + Plg vs. S. suis. (A) Hierarchical clustering of the heatmap of DEGs in Control and S. suis challenged ChP organoids. Upregulated DEGs are displayed in red and downregulated DEGs are displayed in blue. (B) Volcano plot showing DEGs for the comparison of S. suis vs. Control. (C) Hierarchical clustering of the heatmap of DEGs in S. suis and S. suis + Plg challenged ChP organoids. (D) Volcano plot of DEGs for the comparison of S. suis + Plg vs. S. suis. DEGs in (B) and (D) are labeled with their corresponding gene symbols

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For the comparison between S. suis + Plg and S. suis challenged ChP organoids, 533 DEGs were identified with an unadjusted P value of < 0.05 (Table S2), of which 280 DEGs were upregulated and 253 DEGs were downregulated. Expression levels of all DEGs are shown in the heatmap (Fig. 5C) and DEGs found in S. suis + Plg challenged ChP organoids compared to S. suis challenged ChP organoids are displayed in a volcano plot (Fig. 5D) with their unadjusted P values are shown in Table S2. The genes most highly upregulated (i.e. CXCL8, CD53, PANK4, RN7SL1, IRX6, RLBP1, COL8A1, SELE, CXCL1, and CXCL2) or downregulated (i.e. LHX5, FOXL2, BRD2, and GPR6) in S. suis + Plg vs. S. suis are involved in innate immune responses and processes to meet the cellular energy and secretory requirements for responses to infection.

Gene set enrichment analysis (GSEA) of gene expression data

GSEA of GO terms from the transcriptome comparisons are depicted in Figs. 6 and 7. P values and genes associated with the GO terms within each cluster are provided in Table S3 and S4.

Fig. 6
figure 6

Significantly enriched GO terms (FDR-corrected P value ≤ 0.05) with 10 or more assigned genes in the comparison of S. suis vs. Control. Nodes represent GO terms and are colored based on the GSEA P value. Edges represent GO terms sharing > 50% genes. Pink dashed circles group similar GO terms into common themes, which are identified by visually inspecting all associated GO terms. Network was visualized with Cytoscape. (A) GO terms enriched in highly expressed genes in S. suis infected ChP organoids. (B) GO terms enriched in highly expressed genes in Control

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Fig. 7
figure 7

Significantly enriched GO terms (FDR-corrected P value ≤ 0.05) with 10 or more assigned genes in the comparison of S. suis + Plg vs. S. suis visualized by Cytoscape. Nodes represent GO terms and are colored based on the GSEA P value. Edges represent GO terms sharing > 50% genes. Pink dashed circles group similar GO terms into common themes, which are identified by visually inspecting all associated GO terms. (A) GO terms related to highly expressed genes in S. suis + Plg infected ChP organoids. (B) GO terms related to highly expressed genes in S. suis infected ChP organoids

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The GO terms for genes which are highly expressed in S. suis infected ChP organoids vs. Control involve a large cluster related to “Regulation of gene expression and protein synthesis” and the cluster “Regulation of growth and metabolism”. These GO functionalities are consistent with increased signaling and regulation of gene expression and metabolism in response to the infection. Other GO terms link to “Immune and stress responses” and the clusters “Cytoskeletal dynamics and vesicle trafficking”, “Altered cell-cell adhesion”, and “Structural integrity and intermediate filaments”, which are directed at maintaining the epithelial barrier through cell movement, wound healing, and maintenance of cell integrity (Fig. 6A).

The GO terms for genes highly expressed in Control vs. S. suis infected ChP organoids include the clusters related to “Ribosome structure and activity”, “Mitochondrial respiration”, “Extracellular matrix assembly”, and “STAT protein signaling”, which are normal cellular metabolic and regulatory processes (Fig. 6B). Higher expression of genes in these GO terms in the Control vs. S. suis infected ChP organoids suggests these normal cellular activities are disrupted by the strong immune response to the infection (signaling and secretion of chemokines and cytokines) and diversion of energy resources to maintain the ChP epithelial barrier integrity (depicted in Fig. 6A).

The GO terms for genes which are higher expressed in ChP organoids infected with S. suis + Plg compared to S. suis are mainly found in the clusters associated with “DNA replication”, “DNA repair”, and “Monooxygenase activity” which are linked to detoxification. This was accompanied by high expression of genes in GO terms for “Chromatin organization” and “RNA processing” (Fig. 7A). We speculated that genes in all these GO terms are more highly expressed likely because of increased contact of S. suis with ChP epithelial cells through the proteolytic activity of Pln on the ECM and cell junctions.

The GO terms for genes higher expressed in S. suis vs. S. suis + Plg infected ChP organoids include the cluster “Immune responses”, which suggests the inflammatory and cell defense against the infection. Other GO terms link to “Cilium motility” and “Cilium assembly”, indicating that genes have higher expression in S. suis infected ChP organoids contribute to recover the epithelial barrier integrity through cell movement and remodeling (Fig. 7B). Additionally, the cluster “Extracellular matrix organization” suggests that the integrity of ECM is maintained in S. suis infected ChP organoids compared to S. suis + Plg infected ChP organoids due to the degradation of ECM by proteolytic Pln.

For the comparison between S. suis + Plg and Control ChP organoids, 3951 DEGs were identified with an unadjusted P value of < 0.05 (Table S2). The GO terms for genes highly expressed in S. suis + Plg infected ChP organoids vs. Control are like to those in the S. suis vs. Control comparison, except for a few unique terms linked to “eye development”, “visual perception”, and “neuron projection”, which do not provide any additional insights to the mechanism. The GO terms for genes highly expressed in Control vs. S. suis + Plg ChP only suggest differences in RNA splicing and G protein coupled-receptor activity (Table S5).

Discussion

Human ChP organoid models have only been reported recently [27] and here we described their first application as a model of the BCSFB to investigate the infection with a meningitis-causing bacterium, S. suis. As previously reported, ChP organoids recapitulate the BCSFB in vivo, forming large cystic structures with the mature polarized epithelium separating the organoid culture medium from the CSF-like fluid secreted into the lumen.

In vivo S. suis entry into the brain via the ChP would have to occur from the basolateral side, where the porous fenestrated blood vessels lie beneath the ChP epithelium. Thus, we inoculated S. suis at an MOI of 10 into the organoid culture medium and measured bacterial translocation inside the lumen of the organoid by removing the CSF-like fluid with a syringe and plating for CFU counting. Translocation of S. suis across the ChP epithelium was observed without increasing permeability of the ChP organoid epithelium to low molecular weight FITC-dextran (4 kDa). Although GFP-S. suis were shown to be adhering to the ChP epithelium in low numbers, we did not observe a change in organoid morphology due to leakage of the CSF into the medium or significant epithelial cell death by measuring the concentration of cellular LDH in the surrounding medium.

In concert with fibroblasts, epithelial cells secrete ECM proteins which form the basement membrane, a dense matrix of extracellular proteins on the basolateral side of the epithelium. The basement membrane plays a critical role in cell adhesion and the mechanical signal transduction from the ECM to the cell [52]. Binding of Plg to S. suis and its proteolytic activation to Pln by tPA facilitated translocation into the ChP lumen. This is most likely due to cleavage of ECM proteins and tight junctions between ChP epithelial cells by Pln, as reported for S. pneumoniae translocation across ECM and endothelial monolayers [38, 53] and S. pyogenes translocation across upper respiratory tract epithelium [54]. Furthermore, a recent study showed that Plg binding by S. suis enhances translocation across endothelial cell monolayers [7].

To investigate the response of ChP organoids to infection with S. suis and additionally the impact of Plg binding to S. suis and its conversion to Pln, we performed RNA-seq on ChP organoids infected and control samples. GSEA of genes highly expressed in S. suis infected vs. Control ChP organoids indicated a large effect on gene regulation and metabolism due to the infection. This is consistent with the enrichment of more highly expressed genes in the clusters of GO terms linked to “Immune and stress responses”. Other clusters of GO terms enriched in genes highly expressed in the S. suis infected organoids vs. Control related to “Cytoskeletal dynamics”, “Altered cell-cell adhesion”, and “Structural integrity and intermediate filaments”. These GO terms are consistent with responses aimed at maintaining the epithelial barrier through cell movement, wound healing, and maintenance of cell integrity.

Some of the most highly upregulated genes in S. suis infected ChP organoids were transcription factors such as FOS, JUNB, and CEBPD, which regulate gene expression in response to various stimuli including stress and infection, as well as RNA binding protein and cytokine regulators of the immune response such as ZFP36 and GDF15. Another highly expressed gene in S. suis infected organoids was ICAM1, an adhesion molecule for leukocytes, which can mediate the adhesion of leukocytes to ChP epithelial cells [55]. In addition, two ChP specific genes AQP1 and TTR were more highly expressed in response to S. suis infection. AQP1 has been shown to transport signaling molecules such as nitric oxide (NO) across cell membranes [56]. NO can directly kill or inhibit the growth of a wide range of pathogens including bacteria and viruses [57]. Additionally, it is a signaling molecule playing an important role in modulating the immune response and increasing blood flow and vascular permeability, thereby facilitating the infiltration of immune cells to the site of infection [58]. TTR facilitates the transport of thyroxine and retinol [59] and may be associated with protecting the brain from oxidative stress.

Most highly downregulated genes in S. suis infected ChP organoids were involved in cell growth and development such as FAM111B which appears to play a role in DNA repair and apoptosis regulation, and MT-TT which is crucial for mitochondrial protein synthesis and energy production. The downregulation of these genes might be a part of a broader response to S. suis infection, which could include apoptosis of infected cells potentially regulating host defense against S. suis, or a redirection of cellular resources towards the innate immune response.

GSEA of genes highly expressed in ChP organoids infected with S. suis + Plg vs. S. suis identified GO terms linked to DNA repair, DNA replication, and monooxygenase activity. This is likely due to higher production of reactive oxygen species in response to bacterial infection [60]. In contrast, the results of GSEA analysis suggested that genes with higher expression in S. suis infected ChP organoids are associated with GO terms related to the immune response. This suggests that Pln might be able to regulate aspects of the immune response, for example, through the known activation of protease-activated receptors PAR-1 and PAR-2.

Some of the genes most highly expressed in ChP organoids infected with S. suis + Plg compared to S. suis were indeed associated with aspects of the immune response. For example, the chemokines CXCL8 and CXCL1 are known for their role in promoting polymorphonuclear leukocyte (PMN) transmigration across ChP epithelial cells [61]. Additionally, SELE, the leukocyte adhesion molecule E-selectin, was more highly expressed in ChP organoids infected with S. suis + Plg compared to S. suis. The role of other most highly expressed genes in the S. suis + Plg group is less clear but includes the metabolic regulator PANK4 and the transcription factor IRX6 which might be directly involved in regulating the energetic demands of the ChP innate immune response.

Our transcriptomics data showed immune response and maintenance of barrier integrity of ChP organoids to S. suis infection, which was in line with previous studies using PCPEC and HIBCPP. For example, in S. suis infected HIBCPP cells, genes ICAM-1, VACM-1, MMP3, CCL2, CXCL10, CXCL2, IL6, LIF, and PTGS2 were found to be significantly upregulated, which were consistent with our results and indicated both pro-inflammatory and anti-inflammatory responses of HIBCPP [19, 62]. Genes encoding tight junction proteins, including CLDN11, CLDN10, CLDN3, and CLDN1, were highly expressed in S. suis infected ChP organoids in our study, whereas CLDN2 was strongly downregulated in S. suis infected primary PCPEC [62]. This result might be due to differences in the transcriptional response in ChP organoids and the primary porcine cell line.

In the future, Enzyme-Linked Immunosorbent Assay (ELISA) can be used to validate the altered expression of specific proteins involved in the pathways identified from our RNA-seq results, providing complementary evidence at the protein level.

The advancement of iPSC technology has revolutionized the development of in vitro models, particularly for studying brain barriers. iPSC-derived brain endothelial cells have been extensively developed to mimic the BBB, enabling investigations of bacterial interactions with the BBB in vitro. These models have provided valuable insights into pathogen-host interactions at the BBB. However, previous studies of S. suis infection at the BCSFB have primarily relied on PCPEC and HIBCPP cell lines. Recently, ChP organoids have emerged as a promising in vitro model for viral infection studies, such as with SARS-CoV-2 [28] and HSV-1 [63]. ChP organoids can be further utilized to study neuroinflammation and immune cell infiltration caused by pathogenic infection, providing a unique tool to explore the immune dynamics at the ChP.

One limitation of the ChP organoid is the lack of immune cells. The in vivo ChP epithelium is encapsulated by the stroma, which is rich with fibroblasts and immune cells such as leukocytes, macrophages, and dendritic cells. The capillaries within the stroma are composed of fenestrated endothelial cells, allowing the free infiltration of immune cells from the blood to the basolateral side of the ChP epithelium. One of the future experiments could be culturing macrophages on the underside of Transwell inserts and mature ChP organoids in the upper chamber of inserts to generate a co-culture model, which more closely recapitulate the epithelial-immune cells interactions within the ChP.

In summary, we utilized ChP organoids as a 3D in vitro model to explore the S. suis infection at the BCSFB. We found that S. suis translocates across the ChP organoid epithelium and replicates in CSF without inducing cell death or disrupting the barrier integrity of ChP organoids. Furthermore, Plg binding to S. suis facilitated translocation without increasing barrier permeability. The transcriptional response of ChP organoids to S. suis infection was largely aimed at recruiting phagocytes to kill S. suis and processes which maintain barrier integrity such as wound healing, cell migration, ECM remodeling, and cell proliferation. The ability of S. suis to bind Plg and Pln altered the gene expression response of ChP organoids to infection. GSEA indicated a higher expression of genes in S. suis + Plg vs. S. suis in GO terms associated with DNA repair and replication as well as monooxygenase activity, suggesting an increased oxidative response. The results also showed that S. suis + Plg reduced expression of genes in GO terms associated with immune response compared to S. suis. The reasons for this are not clear but suggest a potential role of Pln in modulating the immune response. The cellular responses of ChP organoids to S. suis infection revealed that the ChP epithelium has the potential to behave similarly to immune cells by secreting cytokines and chemokines [19, 64]. To our knowledge, this is the first study to utilize ChP organoids to investigate bacterial infection of the ChP. Our findings highlight the potential of ChP organoids as an in vitro model of the BCSFB for studying the mechanisms of bacterial interaction with the human ChP and offering an alternative to animal experimentation. Follow up studies will be focused on further studies to elucidate the mechanism of transmigration by imaging GFP- expressing S. suis and different knockout mutants using confocal microscopy.

Data availability

The dataset supporting the conclusions of this article is available in the ArrayExpress repository, https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-14229?key=a535d232-04e2-40a6-9d33-e05f09b4b80a. The datasets supporting the conclusions of this article are included within the article and its additional files.

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Acknowledgements

We thank Dr. Anda Voulgari-Kokota, for help with qPCR and advice with cell culture. We are also grateful to Prof. Madeleine Lancaster for hosting a visit to her laboratory and for technical advice.

Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by a grant from the China Scholarship Council (NO. 201906350084) to Tiantong Zhao and funding of research materials from the Host-Microbe Interactomics Group, Wageningen University and Research.

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Authors and Affiliations

  1. Host-Microbe Interactomics, Department Animal Science, Wageningen University & Research, De Elst 1, Wageningen, 6708 WD, The Netherlands

    Tiantong Zhao, Bart van der Hee, Jos Boekhorst, Aline Fernandes, Sylvia Brugman, Peter van Baarlen & Jerry M. Wells

  2. Centre for Developmental Neurobiology, King’s College London, Guys Campus, New Hunt’s House, London, UK

    Laura Pellegrini

  3. MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, UK

    Laura Pellegrini

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  1. Tiantong Zhao
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Contributions

JMW and TZ conceptualised the main research questions and experimental approach, with input from PvB, SB. TZ designed the experiments and performed the research with advice and guidance on specific techniques from LP, BvH, JB, SB, PvB and JMW. All authors discussed the results and contributed to the interpretation of the results. TZ wrote the first draft of the manuscript which was revised after suggestions and comments from JMW, PvB and SB. All authors commented and approved the final version of the version of the manuscript.

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Correspondence to Jerry M. Wells.

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Zhao, T., Pellegrini, L., van der Hee, B. et al. Choroid plexus organoids reveal mechanisms of Streptococcus suis translocation at the blood-cerebrospinal fluid barrier. Fluids Barriers CNS 22, 14 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12987-025-00627-y

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Fluids and Barriers of the CNS

ISSN: 2045-8118