Patients, IgG-patient purification, and NMO-IgG selection
De-identified serum specimens were obtained from six different relapsing NMOSD patients that were included in the French cohort of NMOSD (NOMADMUS), and stored at NeuroBioTec (Biological Resource Centre of the Hospices Civils de Lyon, France). All patients were tested positive for AQP4-antibodies detected by cell-based assay [13], and all patients fulfilled the 2015 criteria for NMOSD [14]. Serum provided by the French blood service (Etablissement Français du Sang) from healthy blood donors were used as controls. Both NMO-IgG (called NMO-IgG1-6) and healthy donors-IgG (called Control-IgG) were purified from the NMOSD and healthy donor serums, respectively, using chromatography over Protein–A Sepharose as previously described [15]. IgGs were then used at a final concentration of 2 µg/µl.
NMO-IgG from the different patients were first tested on cultured astrocytes in order to select NMO-IgG for the experiments on fresh IBM and for the animal model (NMO-rat). After 24 hours of treatment with NMO-IgG1-6 (number of experiments=39), there was a significant decrease in cultured astrocyte AQP4 expression (mean 114.9±24.3) when compared to treatment with Control-IgG (n=12, mean 70.3±17.9, p<0.001), as measured by western blot (Wb). The effects of NMO-IgG1-6 and Control-IgG were evaluated in duplicate or triplicate. NMO-IgG1,2,6 were the most pathogenic, inducing a higher decrease in AQP4 expression than NMO-IgG3,4,5. Among the most pathogenic NMO-IgG, NMO-IgG1 was randomly selected to carry out experiments.
Intraventricular brain NMO-IgG infusion, and ethical procedures
Animal surgery was performed as previously described [15]. Briefly, rats were anesthetised and mounted in a stereotaxic frame. Two hundred µl of purified IgG (NMO-IgG1) or NaCL (Control-IgG) were infused into the CSF during seven days at a 1 µL/hour using a sterile subcutaneous osmotic pump. After seven days, rats were anesthetised and received an intracardiac perfusion of 100 ml phosphate-buffered saline (PBS; 0.1 M, pH 7.4). Then, rats were sacrificed using pentobarbital, brain was then removed and frozen in isopentane at -30°C, and further stored at -80°C.
Brain tissue was embedded in Tissue-Tek OCT, and subsequently cut on a cryomicrotome [15]. Brain slices were used for immunohistochemical (IHC) study of tight junction proteins.
A total of 7 rats received NMO-IgG1 (NMO-rats) and 9 rats received NaCl (Control-rats) for different experiments. Animal care and procedures were carried out in accordance with the European Directive 2010/63/UE and followed the Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines. The study was approved by the local Lyon 1 University Animal Care Committee (B2012-80 project).
Isolation of brain microvessels
Isolation of brain microvessels was performed from two adult rat brains per experiment according to the procedure described [16]. Adult rats were anesthetised with isoflurane and decapitated. Brains were excised and placed at 4°C in Krebs- Ringer buffer (in Mm: 135NaCl, 4 KCl, 2.2 CaCl2, 1.2 MgCl2,6 NaHCO3, 10 Hepes, 5 Glucose monohydrate, pH 7.4). Meninges and choroid plexuses were dissected. Using oxygen-saturated buffers, cortices were homogenised in a Dounce-type glass-glass homogeniser. The preparation was further homogenised in 5 vol/g tissue of 1% BSA-supplemented Kreps Ringer buffer (AKRB), and then filtered through a 500µm mesh sieve (Netwell, Corning, Corning, NY). The filtrate was diluted with 1% AKRB (1:1) and homogenised again. Then, the homogenate was centrifuged for 10 minutes, the pellet was suspended in Krebs-Ringer buffer containing 17.5% 70 kD-dextran, and further centrifuged for 27 minutes. Myelin was retained at the surface of the gradient and the resulting pellet was suspended in 1% AKRB. Pellet was consecutively filtered through 200 and 74-µm mesh sieves to eliminate larger vessels. The preparation was centrifuged for 15 minutes. Finally, the pellet was suspended and filtered on a 40µm mesh sieve (BD Bioscience, Erembodegem, Belgium). All procedures were performed at 4°C.
The fresh IBM retained were recovered in AKRB 0.1% and 2 µl were used to observe their morphology under the microscope. Immunofluorescence analysis showed the presence of Claudin-5, Occludin, and ZO-1 in fresh IBM preparation (Supplementary Figure 1). Fresh IBM suspended in 0.1% AKRB were centrifuged for 15 min at 800 rpm, and immediately suspended in 2ml of Dulbecco's Modified Eagle's (DMEM) + BSA0.1%. To determine the ex vivo effect of NMO-IgG on tight junction proteins (Claudin-5, Occludin, and ZO-1), fresh IBM together with DMEM/ BSA 0.1% were distributed in 6 different 24-well plates (330 µl in each well) and further treated by adding either 330 µl of DMEM/BSA0.1% (called non-treated IBM), Control-IgG, or NMO-IgG1. Fresh IBM were treated in duplicate and incubated for 20 hours at 37°C. Finally, fresh IBM from each well were suspended in DMEM up to 1ml.
Fresh IBM were prepared for Wb or cytokine/chemokine analysis after 20 hours of incubation.
Brain microvascular endothelial cells
Brain microvascular endothelial cells (BMEC) were isolated as previously described [17]. Briefly, microvessels were isolated from 3 rat brains following mechanical and enzymatic digestion of brain tissue, and plated in a T75 flask coated with collagen and fibronectin (microvessels from 1.5 cortex per T75 flask). BMEC were grown and purified with decreasing concentrations of puromycin for 5 days, followed by 3 days of culture in an insulin, transferrin, and sodium selenite-containing medium. The cells were cultured on plastic for BMEC experiments or harvested for seeding on filters to generate in vitro BBB models, as follows. After dissociation during 30 seconds with trypsin 0.05%-EDTA 0.02% solution at 37°C per T75 flask, the detached BMEC (approximately 3*106cells/T75) were dissociated by pipetting up and down. Re-suspended BMEC were then added to the upper compartment of a pre-coated well (filters filled with endothelial cell media (ECM) at high density (160*103cells/filter of 1.1cm2). Endothelial cells expressed tight junction proteins within 3 days [17]. BMEC were then exposed to DMEM/BSA0.1% (termed non-treated BMEC), Control-IgG or NMO-IgG1-6 for 20 hours at the concentration of 350 µg/ml.
Permeability analyses: Bicameral model
For antibody permeability analyses, BMEC were exposed to three different NMO-IgG in the upper compartment of the culture system. The acceptor compartment was then collected, and frozen for later NMO-IgG analysis by Enzyme-linked immunosorbent assays (ELISA). The apical part of the model simulates blood and the basolateral side simulates the CSF.
To test the integrity of the barrier, [14C] sucrose was added in the upper compartment at the end of the incubation with IgG, and sucrose transfer was measured over a 1-hour period. The radioactivity was measured over time in the lower compartment using a Perkin Elmer TRICARB, 4910TR liquid scintillation analyser, Singapore. Clearance curves were generated and permeability coefficients were calculated as described [18].
Enzyme-linked immunosorbent assays
The level of human IgG were quantified in the compartment acceptor by ELISA set according to the manufacturer's protocols (Human IgG ELISA Quantitation Set, Bethyl Laboratories, Inc, Montgomery, Alabama, USA). Samples were diluted at 1:15. The optical density was measured at 450 nm with λ correction of 570 nm using a Spark spectrophotometric microplate reader (Tecan Trading AG, Switzerland). Based on the optical density value of each sample, the sample concentration was calculated in ng/mL and used to calculate clearance.
Cytokine Array analysis
Evaluation of cytokines and chemokines from fresh IBM supernatants were assayed by using Proteome Profiler Array kit (rat cytokine array panel A, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instruction. Fluorescence signal intensity was measured by ImageJ software.
Astrocyte cultures
As previously described, [15] primary glial cultures were isolated from 1-day-old rat pups (n=24) and dissociated cells were further diluted in DMEM to a density of 2.105 cells. Cells were seeded in 6-well plates and incubated at 37°C in a moist 5% CO2, 95% air atmosphere. In order to obtain pure astrocyte cultures and eliminate microglia and oligodendrocytes, cells were treated with cytosine arabinoside (AraC, 25nM, Sigma-Aldrich).
Immunohistochemistry procedures
Both cytospined IBM and brain slides were fixed in 4% paraformaldehyde for 10 minutes, then washed in PBS, and blocked for 1 hour in blocking solution (10% Normal Goat serum, PBS 1X, BSA 1%, Triton 0.3%). Then, the material was incubated overnight at 4°C with primary antibodies in blocking solution and further washed in PBS. Slides were labelled at room temperature with specific fluorescent secondary antibodies in blocking solution for 30 minutes, washed in PBS and incubated with 4',6-diamidino-2-phenylindole (DAPI). Slides were mounted in tamponed glycerol and stored at 4°C. Axio Imager Z1 Apotome technology (Zeiss, Oberkochen, Germany) was used to image the slides.
Immunoblotting
Fresh IBM, BMEC, or brain tissues were dissociated by ultrasound fragmentation in homogenisation buffer and phosphatases inhibitors, and then protein content was counted [15]. Protein samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gels) and transferred to nitrocellulose membranes. Beta-actin was used as an internal standard. The membranes were treated with blocking buffer for 1 hour and incubated with the specified primary antibodies at 4°C overnight. Membranes were exposed to an anti-IgG antibody for 1-h at room temperature and revealed using chemiluminescence, as described [15]. Quantification of the band intensity was obtained using ImageJ software.
Immunohistochemical reagents
The following IgGs were used for tissue sample immunodetection and lysates. The primary antibodies were; Claudin-5 (mouse monoclonal, 35-2500; Invitrogen), Occludin (rabbit polyclonal, 71-1500; Invitrogen), ZO-1 (rabbit polyclonal, 61-7300; Invitrogen), PDGF Receptor-β (rabbit monoclonal, 28E1; Cell Signalling Technology), AQP4 (rabbit polyclonal AB2218; Merck© KGaA); beta-Actin (mouse monoclonal, A1978; Sigma-Aldrich®). The secondary antibodies were: Peroxidase-AffiniPure F(ab)2 fragment donkey anti-mouse IgG (Heavy,[H]+light [L] chains) (715-036-151, Jackson Immunoresearch), Alexa Fluor®488 goat anti-mouse IgG, A11029; Alexa Fluor®555 goat anti-mouse IgG, A21424; Alexa Fluor®488 goat anti-rabbit IgG, A11034; and Alexa Fluor®455 goat anti-rabbit IgG, A21429 from Molecular Probes Inc.
Statistical analysis
All Wb analyses were performed by investigators blinded for treatment conditions. Data are presented as median (interquartile range [IQR]) and percentages. The non-parametric Kruskal-Wallis test was used to evaluate differences in more than two groups and U-Mann-Whitney test was used to compare two groups. All statistical analyses were performed using Prism 5.0 GraphPad Inc software.