Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells as a Cellular Model to Study Neisseria meningitidis Infection

Abstract

Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.

Keywords: Neisseria meningitidis; bacteria; blood-brain barrier; blood-cerebrospinal fluid barrier; brain endothelial cells; meningococcus; stem cells.

FIGURE 1

Characterization of Nm interaction with iPSC-BECs. (A) Schematic cartoon of Nm strains used. MC58 is a serogroup B strain, MC58ΔsiaD is an isogenic non-capsulated mutant, 8013/12 is a serogroup C strain and 8013/12ΔpilT is a highly piliated mutant. (B) Gentamicin protection assay of Nm on iPSC-BECs showing invasion of MC58ΔsiaD and 8013/12 relative to MC58 into iPSC-BECs at the indicated time points and Multiplicity of Infection (MOI) of 10. (C) Confocal microscopy images of iPSC-BECs infected with the Nm strains mentioned in (B), at 4 h p.i. and MOI 100. Image is a maximum image projection. Scale bar = 20 μm. (D) Gentamicin protection assay showing invasion of ΔpilT mutant relative to WT 8013/12 Nm strains into iPSC-BECs at 4 h p.i. and MOI 10. For (B,D) data is presented as mean ± S.E.M of three independent experiments done in technical duplicate and triplicate, respectively. Student’s t-test was used to determine significance. ^∗p < 0.05; ^∗∗p < 0.01. (E) Immunofluorescence staining showing areas of recruitment of receptor CD147 (red) around MC58 and MC58ΔsiaD colonies (green) highlighted with white arrow heads in iPSC-BECs at 4 h p.i. and MOI of 100. Scale bar = 5 μm.

FIGURE 2

Neisseria meningitidis infection impacts barrier properties of iPSC-BECs. (A,B) iPSC-BEC monolayers seeded onto 0.4 μm pore size transwells with or without Nm challenge at MOI 10 were used for (A) monitoring of TEER values (Ω × cm²) over a time course of 32 h and (B) determination of NaF permeability coefficient (cm/min) at 24 and 32 h p.i. The data are represented as mean ± S.E.M. of three independent experiments each done in technical triplicate. Student’s t-test was used to determine significance. ^∗p < 0.05; ^∗∗∗p < 0.001.

FIGURE 3

Neisseria meningitidis induces tight junction disruption and bacterial transmigration in iPSC-BECs. (A) Confocal microscopy images showing tight junction staining for Occludin, ZO-1 and Claudin-5 in iPSC-BECs seeded on ibidi microscopy slides with and without Nm challenge at MOI 10 at 24 h of infection. Areas outlined with dashed lines indicate gaps between cells. Image is a maximum image projection. Yellow = tight junctions, Blue = DAPI. Scale bar = 50 μm. (B,C) qPCR showing relative expression of (B) tight junction-coding genes OCLN, TJP1, and CLDN5 at 8 and 24 h p.i. and (C) tight junction repressor gene SNAI1 over a time course of 24 h, performed on iPSC-BECs with (dark gray bars) or without (light gray bars) Nm challenge at MOI 10. (D) Transmigration of Nm across monolayers of polarized iPSC-BECs seeded onto 3 μm pore size transwells at MOI 10. The number of CFUs traversing the layer was determined by assessing bacteria in the basolateral chamber at 2, 4, 6, 8, and 24 h. For B, C and D the data are presented as mean ± S.E.M. of three independent experiments done in triplicate. Student’s t-test was used to determine significance. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗∗p < 0.0001.

FIGURE 4

Neisseria meningitidis infection leads to activation of iPSC-BECs. (A) Detection of RANTES, IFN-γ and IL-8 on supernatants of mock- (light gray bars) and bacteria-infected (dark gray bars) iPSC-BECs by Luminex bead-based multiplex assays during a time course of 2, 4, 6, 8 and 24 h of infection. (B) Quantitative PCR showing relative expression of CXCL8, CXCL1, CXCL2, CCL20, and IL6 transcripts during a time course of 2, 4, 6, 8, and 24 h of infection for mock- (light gray bars) and Nm-infected (dark gray bars) monolayers. Data are presented as mean ± S.E.M of three independent experiments done in duplicate (A) or triplicate (B). Student’s t-test was used to determine significance. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001.

FIGURE 5

RNA-Seq of sort-enriched, infected iPSC-BECs. (A) Experimental workflow. iPSC-BECs were infected with Nm-GFP at MOI 10, detached through Accutase treatment, and fixed in RNAlater©. An outline of the gating strategy is provided in Supplementary Figure 5A. Mock controls without Nm challenge were sorted and collected in parallel (i.e., GFP-negative gated population P3 of mock, Supplementary Figure 5A). Total RNA was extracted from each sample and polyadenylated transcripts were converted into cDNA libraries and sequenced to ∼25–30 million reads/library. (B) Heat map showing differentially expressed mRNAs between GFP-positive, infected cells and mock controls (without Nm challenge) at 24 h of infection. Plotted are all genes that were significantly differentially expressed (adjusted P-value < 0.1; DESeq2). Sequencing data was derived from two biological replicates. (C) Gene Ontology analysis of differentially expressed mRNAs shown in (B), acquired with DAVID bioinformatics resources. Enriched GO terms with P-value < 0.05 are shown as well as the differentially expressed mRNAs corresponding to the respective pathway. (D) Validation of RNA-Seq data by qPCR. VEGFA and TNFAIP2 mRNA levels were increased at 24 h after MC58 infection (MOI 10) as compared to mock-treated cultures. RNA was isolated from monolayers of iPSC-BECs without sorting. The data are presented as mean ± S.E.M of three independent experiments done in triplicate. Student’s t-test was used to determine significance. ^∗p < 0.05; ^∗∗∗p < 0.001.