Cell cultures
Human brain microvascular endothelial cells (HBMEC).
HBMEC used in the present study represent a stable, well characterized, and differentiated human brain endothelial cell line (32). Briefly, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40T antigen. Among several stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, the hCMEC/D3 cell line (referred here as HBMEC) was selected as expressing normal endothelial markers and demonstrating blood–brain barrier characteristics. HBMEC for the present study were supplied by Dr. Couraud (Institut Cochin, Paris, France). HBMEC were cultured on collagen type I (BD Biosciences Pharmingen, San Jose, CA) coated dishes in EBM-2 medium (Clonetics, East Rutherford, NJ) supplemented with VEGF, IGF-1, EGF, basic FGF, hydrocortisone, ascorbate, gentamycin and 0.5% exosome depleted fetal bovine serum (Exo-FBS; System Biosciences, Mountain View, CA).
Human neural progenitor cells (NPCs)
An immortalized NPC line ReNcell VM, derived from 10-week human ventral mesencephalon, was obtained from Millipore and cultured according to the manufacturer’s protocols. The cells were validated for high expression of Sox2 and nestin as well as for their self-renewal and differentiation capacity. Cells were grown on laminin coated tissue culture dishes in a maintenance medium (Millipore), containing 20 ng/ml FGF-2 and 20 ng/ml of rhEGF. Cells were used for experiments at < 80% confluence, three days after plating.
HIV infection and treatment factors
HIV stock was generated using human embryonic kidney (HEK) 293T cells (ATCC) transfected with pYK-JRCSF plasmid containing full-length proviral DNA. Throughout the study, HBMEC were exposed to HIV particles at the p24 level of 30 ng/ml as previously reported (33). Treatment was terminated by removing cell culture media containing HIV, followed by washing the cells in PBS.
Aβ (1–40) and Aβ (1–40) HiLyte were purchased from Anaspec (San Jose, CA) and Aβ (1–40) was dissolved in PBS. Freshly solubilized Aβ solutions without pre-aggregation were used for experiments as such a form of Aβ was demonstrated to induce proinflammatory reactions in isolated rat brain microvessels (34). Aβ (1–40) HiLyte was dissolved first in a basic buffer (0.1 M NH4OH) and then diluted further in PBS as suggested by the manufacturer. Cells were treated with Aβ (1–40) or Aβ (1–40) HiLyte at the concentration of 100 nM in complete medium.
FPS-ZM1 (FPS, Cayman Chemicals, Ann Arbor, MI, USA) is a high-affinity RAGE-specific inhibitor (35). 1 mM stock solution was prepared in DMSO. Cells were pretreated with 500 nM FPS for 2 h followed by coexposure with HIV and/or 100 nM Aβ (1-40).
GW 4869 (Millipore Sigma, Burlington, MA, USA) is a cell-permeable, non-competitive inhibitor of neutral sphingomyelinases. 5 mM stock solution of GW4869 was prepared in DMSO. Because of low solubility, the stock was incubated at 37°C for 15 min and supplemented with 1/20 volume of 5% methane-sulfonic acid (MSA) before adding GW4869 to cell culture medium (36). Control cultures were exposed to vehicle, i.e., 0.4% DMSO supplemented with 1/20 volume of 5% MSA. Cells were pretreated with the solubilized GW4869 (20 μM) for 1 h followed by coexposure with HIV and/or 100 nM Aβ (1-40).
Treatment of brain endothelial cells and ECV isolation
Confluent HBMEC were exposed to HIV and/or Aβ (1–40)/Aβ (1–40) HiLyte for 48 h. ECVs were isolated from conditioned medium using ExoQuick-TC exosome precipitation solution (System Biosciences) according to the manufacturer's specifications. Briefly, 10 ml culture medium (from 1.7 x 107 cells at confluency cultured in a P100 dish) was centrifuged at 3000 g for 15 min to remove cells and debris. Then, the samples were mixed thoroughly with 2 ml of Exo-Quick exosome precipitation solution and incubated overnight at 4°C. The next day, the samples were centrifuged at 1500 g for 30 min, the supernatants were removed and centrifuged again at 1500 g for 5 min. The ECV pellets were resuspended in 400 μl PBS and used for further studies. The aliquots of 20 μl of ECV suspension for every 100 μl of cell culture media was used for NPC treatment.
Nanoparticle tracking analysis (NTA)
ECVs were analyzed by NanoSight model NS300 (Malvern Instruments Company, Nanosight, Malvern, United Kingdom) as described earlier (22). Briefly, ECV pellet samples obtained in the process of ECV isolation were resuspended in 100 μl 4% paraformaldehyde-PBS and further diluted 100 fold in PBS for analysis. Three 90 s videos were recorded for each sample. The obtained data were analyzed using Nanosight NTA 2.3 Analytical Software (Malvern Instruments Company) with the detection threshold optimized for each sample and screen gain at 10 to track the maximal number of particles with minimal background. Most of isolated ECVs carry fluorescent Aβ (1-40) cargo. In addition, Aβ is associated with ECVs of different size (see supplemental Figure S1).
Protein isolation and western blot
Proteins from NPCs were extracted with Radio Immuno Precipitation Assay (RIPA) buffer (Pierce, IL, USA) with freshly added protease inhibitors and 1% Triton-X 100 to inactivate HIV. ECV protein concentration was measured by BCA protein assay kit (Pierce). Equal amount of proteins (8-16 μg/lane) was loaded on sodium dodecyl sulfate polyacrylamide 4-20% ready gels (BioRad, Hercules, CA) and electrotransferred to a nitrocellulose membrane using a transfer pack system (BioRad). The blots were probed at 4°C with rabbit anti-ASC antibody (Catalog # AL177, Adipogen Life Sciences, San Diego, CA), rabbit anti-NLRP3 antibody (Catalog # LSB4321, LSBio, Seattle, WA) or mouse anti-caspase-1 antibody (Catalog # sc-514, Santa Cruz Biotechnology Inc) (1:400) in 5% milk-TBS-T. After washing 3 times with TBS-T, the samples were incubated with secondary antibodies diluted at 1:20,000 (anti-mouse 800CW or anti-rabbit 680RD). Anti-GAPDH antibody conjugated with Dylight 680 (1:20,000; Novus Biologicals, Littleton, CO, USA) was employed as an internal control. Signal detection was performed using Image Studio 4.0 software (LI-COR). In our cells, the 16.5 kDa ASC band corresponded to a splice variant according to the manufacturer. For NLRP3, additional bands were found above and below the manufacturer-predicted 101, 114 kDa bands. For caspase-1 a very weak band of ~40 kDa was found (see supplemental Figure S2).
Caspase-1 activity assay
Caspase-1 activity was measured after a 5 h and 24 h exposure to ECVs with the Caspase-Glo 1 Inflammasome assay (Catalog # G9951, Promega, Madison, WI) following the manufacturer's instructions.
Enzyme Linked Immunosorbent Assay (ELISA)
ELISA was used to determine levels of total human Aβ (1–40) (R&D Systems, Minneapolis, MN, USA) in the isolated brain endothelial ECVs. IL-1β, VEGF-A and BDNF levels were also measured by ELISA from the cell culture medium after 3 days of NPC differentiation (R & D Systems).
Fluorescence microscopy
NPCs (28,000 cells/well) were cultured and treated on laminin coated 8-chambered glass slides (ibidi USA, Madison, WI). Then, the cells were washed with PBS and fixed with absolute ethanol for 30 min at 4°C. After washing with PBS, nuclei were stained with DRAQ5 for 5 min followed by PBS wash. Slides were mounted using ProLong Gold antifade reagent (Life Technologies). Green fluorescence (originating from Aβ HiLyte Alexa Fluor488) and red fluorescence (from DRAQ5) were acquired directly using a Nikon Eclipse Ti-U fluorescence microscope.
Confocal microscopy
ECV-treated NPCs cultured on laminin coated chambered glass slides (Becton Dickinson Biosciences, San Jose, CA) were fixed with ethanol for 30 min at 4°C. After washing with PBS and blocking with 3% bovine serum albumin in PBS for 30 min, samples were incubated overnight at 4°C with the primary antibody: rabbit anti-ASC polyclonal antibody (Catalog # AL177, 1:400), rabbit anti-NLRP3 polyclonal antibody (Catalog # LSB4321, 1:400), mouse anti-Hu C/D monoclonal antibody (Catalog # A-21271, 1:300) (from Invitrogen, Carlsbad, CA), rabbit anti-NeuN monoclonal antibody (Catalog # 24307, 1:200, Cell Signaling Technology, Danvers, MA) or mouse anti-doublecortin (DCX) monoclonal antibody (Catalog # GTX60612, 1:200, GeneTex, Irvine, CA). Then, the excess of primary antibody was removed, slides were washed with PBS, and incubated with Alexa Fluor488/594-conjugated secondary antibody (1:200, Invitrogen) for 2 h at room temperature. In some experiments, nuclei were stained with DRAQ5 followed by PBS wash. Slides were mounted using ProLong Gold Antifade reagent with or without 4',6-diamidino-2-phenylindole (DAPI, Invitrogen) to visualize the nuclei. Specimens were covered with coverslips and the immunofluorescent images were evaluated and captured under confocal microscopy. Red fluorescence originating from DRAQ5, blue fluorescence from DAPI, and green fluorescence from ECV-Aβ HiLyte-Alexa Fluor488 was acquired directly using confocal microscopy (Olympus, Fluoview 1200, dry lens UPLFLN 40x NA: 0.75 or 60x oil immersion lens, room temperature) and did not require the use of primary or secondary antibody.
For Aβ measurements, fields were chosen at random and acquisition and quantification were performed using the FV10-ASW3.1 software. For the nuclear Aβ measurements, mean fluorescence intensity was measured in the nuclear areas outlined by the DRAQ5 or DAPI staining. For ASC and NLRP3 measurements, the total area fluorescence intensity on the acquired images was normalized to the number of nuclei. For the nuclear ASC, NLRP3 measurements, mean fluorescence intensity was measured in random nuclear areas outlined by the DAPI staining.
NPC differentiation
NPCs were seeded on laminin coated 8-well chambered glass slides (28,000 cells/well) and incubated overnight at 37 °C in maintenance culture medium (Millipore), containing 20 ng/ml FGF-2 and 20 ng/ml of rhEGF. The following day, the medium was changed to maintenance medium without growth factors to induce differentiation. Cells were allowed to differentiate for 3 days in the presence or absence of the employed ECVs. To block RAGE signaling, selected NPC cultures were pretreated with 500 nM FPS for 2 h, followed by coexposure with ECVs for 3 days. After 3 days, cells were washed with PBS, fixed with ethanol for 30 min at 4°C, and subjected to immunostaining for Hu C/D, NeuN, and DCX markers to assess neurons at different stages of development. Fluorescence images were acquired randomly through a confocal laser-scanning microscope (Olympus Fluoview 1200) and analyzed using FV10-ASW3.1 software. Hu C/D, NeuN positive and NeuN/DCX double positive cells were counted and expressed as percentage of the total cell number.
Luminex MagPix Assay
Cell culture media samples collected after 3 days of differentiation were analyzed for cytokine/chemokine panel (G-CSF, IL-4, CCL2, Fractalkine, PDGF-AA, PDGF-AB/BB, VEGF-A, BDNF, NGF-β; Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel, Millipore Corp., Billerica, MA) following the kit-specific protocols provided by Millipore. Analytes were quantified using a Magpix analytical test instrument, which utilizes xMAP technology (Luminex Corp., Austin, TX), and xPONENT 4.1 software (Luminex). xMAP technology uses fluorescent-coded magnetic microspheres coated with analyte-specific capture antibodies to simultaneously measure multiple analytes in a specimen. After micro-spheres have captured the analytes, a biotinylated detection antibody binds to that complex. Streptavidin PE then attaches as a reporter molecule. Inside the instrument, magnetic beads are held in a monolayer by a magnet, where two LEDs are used to excite the internal micro-sphere dye and the dye of the reporter molecule, respectively. A CCD camera captures these images, which are then analyzed by Milliplex Analyst software (Millipore).
Concentrations of cytokines (pg/ml) were determined on the basis of the fit of a standard curve for mean fluorescence intensity versus pg/ml. Two quality controls were run with each assay (control 1, low level; control 2, high level.) All cytokines were found to fall within the quality control ranges except for VEGF-A. Therefore VEGF-A levels were separately determined with ELISA.
Statistical Analysis
Data were analyzed using GraphPad Prism 6.0 (Graphpad Software, San Diego, CA). ANOVA was used to compare responses among treatments. Treatment means were compared using All Pairwise Multiple Comparison Procedures and p<0.05 was considered significant.