Intrinsic blood-brain barrier dysfunction contributes to multiple sclerosis pathogenesis

Abstract

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the CNS are early hallmarks of multiple sclerosis (MS). The mechanisms leading to BBB dysfunction are incompletely understood and generally thought to be a consequence of neuroinflammation. Here, we have challenged this view and asked if intrinsic alterations in the BBB of MS patients contribute to MS pathogenesis. To this end, we made use of human induced pluripotent stem cells derived from healthy controls and MS patients and differentiated them into brain microvascular endothelial cell (BMEC)-like cells as in vitro model of the BBB. MS-derived BMEC-like cells showed impaired junctional integrity, barrier properties and efflux pump activity when compared to healthy controls. Also, MS-derived BMEC-like cells displayed an inflammatory phenotype with increased adhesion molecule expression and immune cell interactions. Activation of Wnt/β-catenin signalling in MS-derived endothelial progenitor cells enhanced barrier characteristics and reduced the inflammatory phenotype. Our study provides evidence for an intrinsic impairment of BBB function in MS patients that can be modelled in vitro. Human iPSC-derived BMEC-like cells are thus suitable to explore the molecular underpinnings of BBB dysfunction in MS and will assist in the identification of potential novel therapeutic targets for BBB stabilization.

Keywords: blood–brain barrier; human induced pluripotent stem cells; immune cell migration; multiple sclerosis; permeability.

Figure 1

Morphological and functional differences in MS-patient versus HC-derived DMM-BMEC-like cells. (A) Representative stainings for ZO-1, claudin-5, or occludin (red), and nuclei (DAPI, blue) from Control HC1, Control HC3, Patient MS1 and Patient MS4 are shown. Each staining is representative of at least three independent experiments using three individual differentiations. Scale bar = 50 μm. (B) Transendothelial electrical resistance (TEER) measured with a Volt-Ohm metre. The black line represents mean ± standard deviation (SD) of six clones from three HC and red line represents mean ± SD of seven clones from four MS patients each performed in triplicates and repeated at least twice using two individual differentiations for each donor. (C) Permeability of sodium fluorescein (NaFl) across DMM-BMEC-like cell monolayers was measured. (D) P-gp efflux pump activity was assessed by intracellular accumulation of Rhodamine 123 in the presence or absence of the P-gp inhibitor cyclosporine A (CsA). (E) Cell surface staining for the adhesion molecule ICAM-1 analysed by flow cytometry is shown. Isotype control, non-stimulated (NS) and 16 h pro-inflammatory cytokine-stimulated conditions are shown with the grey, blue and red lines, respectively. Representative data from Controls HC1 and HC3 and Patients MS1 and MS4 are shown from at least two independent differentiations. (F) The change in geometric mean (ΔMFI = MFI staining–MFI isotype) of cell surface ICAM-1 as analysed by flow cytometry. (C, D and F) Bars show the mean of six clones from three HC or seven clones from four MS patients. Each symbol (HC: black, MS: red) represents the mean of at least two independent experiments using two individual differentiations each performed in at least triplicates. Statistical analysis was performed as outlined in the ‘Materials and methods’ section. P-values are indicated in the respective figures.

Figure 2

Impaired barrier characteristics in MS- versus HC-derived EECM-BMEC-like cells. (A) Immunofluorescence stainings of EECM-BMEC-like cells in co-culture with SMLC for 6 days are shown. Junctions were stained for VE-cadherin, claudin-5, or occludin (red), and nuclei were stained with DAPI (blue). Representative images of three HC and four MS from at least three independent experiments using three individual differentiations performed on three distinct filters are shown. Yellow arrows indicate visible disruptions of junctional stainings for claudin-5 and occludin. Scale bar = 50 μm. (B) TEER of EECM-BMEC-like cell monolayers derived from HC versus MS patients is shown. Black line represents mean ± SD of six clones from three HC and red line represents mean ± SD of seven clones from four MS-patients each performed in triplicates and repeated at least twice using two individual differentiations for each donor. (C) TEER at Day 6 after seeding onto filters of EECM-BMEC-like cell monolayers derived from HC versus MS is shown. (D and E) Permeability of NaFl across EECM-BMEC-like cell monolayers: EECM-BMEC-like cells derived from HC (black) or MS patients (red) were cultured to confluency on 0.4 μm pore size Transwell® filters in monoculture (D) or co-culture with SMLC from the same donor (E) for 6 days and permeability of NaFl was measured at Day 6 after seeding onto the filter. (C–E) Bars show the mean of six clones from three HC and seven clones from four MS patients. Each symbol (HC: black, MS: red) shows the mean of at least two independent experiments using two individual differentiations each performed in at least triplicates. Statistical analysis was performed as outlined in the ‘Materials and methods’ section. P-values are indicated in the respective figures.

Figure 3

MS-derived EECM-BMEC-like cells show enhanced cell surface expression of ICAM-1 and VCAM-1. (A) Immunofluorescence stainings of EECM-BMEC-like cell monolayers for ICAM-1, VCAM-1 (red) are shown. Nuclei were stained with DAPI (blue). Each staining is representative of at least three independent experiments using three individual differentiations performed on three distinct filters. NS and 1 ng/ml TNF-α + 20 IU/ml IFN-γ stimulated conditions are shown. Scale bars = 50 μm. (B) Cell surface staining of EECM-BMEC-like cells for the adhesion molecules ICAM-1 and VCAM-1 was analysed by flow cytometry. Isotype control, non-stimulated (NS), and 16 h pro-inflammatory cytokine-stimulated condition are shown in grey, blue, and red lines respectively. Representative data from Controls HC1 and HC2, and Patients MS1 and MS3 are shown. (C) The change in geometric mean (ΔMFI = MFI staining–MFI isotype) of cell surface ICAM-1 and VCAM-1 of EECM-BMEC-like cells was analysed by flow cytometry. Each symbol (HC: black, MS: red) represents the mean of at least two experiments using two independent differentiations. Bars show the mean of six clones from three HC and seven clones from four MS. Statistical analysis was performed as outlined in the ‘Materials and methods’ section. P-values are indicated in the respective figures.

Figure 4

MS-derived EECM-BMEC-like cells support increased immune cells interaction. (A) Schematic representation of the adhesion assay. (B) The number of allogeneic Th1* cells adherent to NS and pro-inflammatory cytokine-stimulated EECM-BMEC-like cell monolayers derived from HC versus MS patients was counted after 30 min under static conditions. In each assay, Th1* cell adhesion to EECM-BMEC-like cells from HC2 clone1 under NS conditions was included and the number of adherent Th1* cells/FOV was normalized to this condition (Control HC2 clone 1 NS condition = 1). (C) Schematic for in vitro live cell imaging of immune cell/BMEC interaction under physiological flow. (D–G) Analysis of the arrest and post-arrest behaviour of allogeneic Th1* cell or autologous PBMC interactions with pro-inflammatory cytokine-stimulated EECM-BMEC-like cells in the field of view of the videos (example in Supplementary Videos 1 and 2 for allogeneic Th1* and Supplementary Videos 3 and 4 for autologous PBMCs) under physiological flow. (D and F) The number of allogeneic Th1* cells (D) or autologous PBMCs (F) remaining arrested on the EECM-BMEC-like cell monolayer were quantified at the end of the accumulation phase at 4 min 30 s. The number of Th1* cells or PBMCs that had migrated across EECM-BMEC-like cells were counted at 20 min. (E and G) Post-arrest allogeneic Th1* cell (E) or autologous PBMCs (G) behaviour on the EECM-BMEC-like cell monolayers under flow was analysed. (F and G) Autologous PBMCs from Control HC2 or Patient MS3 were used in this assay. (B, D–G) Each symbol (HC: black, MS: red) represents the mean of at least three experiments using two independent differentiations. Bars show the mean of six clones from three HC and seven clones from four MS. Statistical analysis was performed as outlined in the ‘Materials and methods’ section. P-values are indicated in the respective figures.

Figure 5

Pre-activation of Wnt/β-catenin signalling in MS-derived EECM-BMEC-like cells restores barrier characteristics. (A) Schematic representation of the activation of the Wnt/β-catenin signalling pathway in EPCs. (B) Immunofluorescence staining for claudin-5 (red) and nuclei (DAPI, blue) of passage 3 EECM-BMEC-like cells is shown. Representative images for clones derived from four MS patients of at least three independent experiments using three individual differentiations performed on three distinct filters are shown. Yellow arrows indicate disruptions in junctional claudin-5 staining. Scale bar = 50 μm. (C) Permeability of NaFl across passage 3 EECM-BMEC-like cell monolayers in the presence of absence of pre-treatment with 4 μM CHIR99021 are shown. (D) Cell surface staining of EECM-BMEC-like cells for the adhesion molecules ICAM-1 and VCAM-1 in the presence of absence of pre-treatment with 4 μM CHIR99021 under NS and pro-inflammatory cytokines-stimulated condition was analysed by flow cytometry. Isotype control, DMSO control and CHIR99021 treatment condition are represented in grey, red, and green lines, respectively. Representative data from Patients MS1–MS4 are shown. (E) The change in geometric mean (ΔMFI = MFI staining–MFI isotype) of cell surface VCAM-1 of EECM-BMEC-like cells were analysed by flow cytometry. (C and E) Bars show the mean of seven clones from four MS patients. Each symbol represents the mean of at least two independent experiments using two individual differentiations each performed in at least triplicates. Statistical analysis was performed as outlined in the ‘Materials and methods’ section. P-values are indicated in the respective figures.