1. Reagents and inhibitors
Evans blue (EB), L-(-)-Fucose, withaferin A (WFA), ginsenoside Rg3, caffeic acid phenethyl ester (CAPE), trifluoperazine (TFP) and nystatin (Nys) were purchased from Sigma-Aldrich (St. Louis, MO). Dynabeads M-450 Tosylactivated was purchased from Invitrogen (Carlsbad, CA). Ulex europaeus I (UEA I) lectin and mounting medium with DAPI were purchased from Vector (Buringame, CA). The full-length HIV-1 gp120 protein was obtained from Protein Sciences (Meriden, CT). All primary antibodies (Ab) were purchased from the commercial sources: rabbit anti-MSFD2 Ab (sc-135305) was purchased from Santa Cruz Biotechnology; rabbit anti-CD44 Ab, rat anti-Ly6C Ab, rabbit anti-GFAP Ab and rabbit anti-S100B Ab were obtained from Abcam, USA; rabbit anti-NeuN, rabbit anti-β-actin Ab and rabbit anti-GAPDH Ab were purchased from Proteintech Group, Chicago, USA. PE-conjugated anti-CD146 Ab (12-1469-41) from eBiosciences (San Diego, CA, USA). The rest chemicals were obtained from Sai Guo Biotech Company, Guangzhou, China.
2. Fungi strain
Cryptococcus neoformans (CN) wild strain B-4500FO2 was cultured with a medium containing 1% yeast extract, 2% peptone and 2% dextrose (YPD broth). Cells were grown aerobically at 30°C and harvested at early log phase. The number of the yeast cell was determined by using a hemocytometer.
3. Cell lines and cultures
HBMECs were cultured as described as previously [8]. Cells were detached by trypsin-EDTA and subcultured on collagen-coated Transwell (3 µm pore size, 6.5-mm diameter) (BD Biosciences, San Jose, CA, USA) from T-25 flasks when ~ 70–80% confluent. HBMEC monolayers on Transwell filters were monitored by measuring trans-endothelial electrical resistance (TEER) changes across the endothelial cell monolayer using an End Ohm epithelial voltohmeter (World Precision Instruments, Sarasota, FL, USA). HL60 cells and vimentin-knockdown (Vim-KD) HBMECs were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin G (50 µg/ml) and streptomycin (100 µg/ml) at 37°C in a 5% CO2 incubator. Astrocytes were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) containing 10% heat-inactivated fetal bovine serum, penicillin G (50 µg/ml) and streptomycin (100 µg/ml) at 37°C in 5% CO2.
4. Generation of Vim −/−/gp120 Tg mice
The transgenic mice expressing soluble gp120 using the modified GFAP promoter were described previously [33]. Vim gene knockout (KO) mice, previously obtained from Dr. Albee Messing at the University of Wisconsin, were originally provided by Professor Charles Babinet of the Institute Pasteur. Gp120 transgenic (Tg) mice were kindly provided by Professor Eliezer Masliah (Division of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, USA). All mice were specific pathogen free and kept in the animal facility under a strict 12 h light/dark cycle.
For the purpose of generating Vim−/−/gp120 transgenic mice, Vim +/+/gp120 Tg mice were crossed with homozygous VIM−/− mice resulting in the F1 gp120 Tg containing one copy of Vim (+/−) [Vim +/−/gp120 Tg mice], and heterozygous Vim knock out mice. The VIM+/−/gp120 Tg mice were in turn crossed with Vim +/− mice to obtain F2 gp120 Tg that were homozygous for Vim −/− [Vim −/−/gp120 Tg mice]. The colonies of Vim−/−/gp120 Tg mice were utilized for subsequent experiments.
Vim gene and gp120 are located on different chromosomes. For the generation of Vim −/−/gp120 Tg mice, we employed the following PCR primers for screening:
Gp120 Tg mouse genotyping:
1. Gp120-F: 5-GCGGGAGAATGATAATGGAG-3
2. Gp120-R: 5-TATGGGAATTGGCTCAAAGG-3
VIM mouse genotyping:
Primer1: 5-TGTCCTCGTCCTCCTACCGC-3
Primer2: 5-AGCTGCTCGAGCTCAGCCAGC-3
Primer3: 5-CTGTTCGCCAGGCTCAAGGC-3
PCR amplification was conducted in biometra PCR amplification system (UNOII-Themoblockt TM, Germany) using the following protocol for gp120 Tg mouse genotyping: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 95°C for 30s; annealing at 53°C for 30s, extension at 72°C for 30 s. And the following protocol for VIM mouse genotyping: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 95°C for 30 s; annealing at 68°C for 30s, extension at 72°C for 60 s. PCR products were resolved by electrophoresis in 1.5%(w/v) agarose gels using TAE buffer (40mM Tris, 5mM sodium acetate, 1mM EDTA, pH 8.0) and visualized under UV light after ethidium bromide staining.
5. Cryptococcal meningitis model
Six month-old Vim +/+, Vim −/−, Vim +/+/gp120 Tg and Vim −/−/gp120 Tg mice mice were intravenously injected with Cn cells via the tail vein. Mice were anaesthetized with ketamine and lidocaine after 24 h injection. Mouse brain microvascular endothelial cells were isolated and purified from the blood samples that obtained via heart puncture. After perfusion with 20 ml PBS via heart puncture, the skull was opened and the brain tissues and cranial cavity were washed with 100 µl of PBS respectively for the collection of CSF. CSF samples were discarded as contaminated samples that containing more than 10 erythrocytes per µl. To count the monocytes, the samples were stained with a PE-conjugated rat anti-mouse Ly6C Ab (eBiosciences, CA, USA) and counted under the fluorescence microscope.
6. Western blotting analysis
Cell lysates were separated by SDS-PAGE electrophoresis and then transferred onto polyvinylidene difluoride membranes (pore size, 0.45 µm, Millipore, USA). The membranes were blocked with TBST buffer [50 mM Tris/HCl, pH 7.4–7.6, 150 mM NaCl and 0.1% (vol/vol) Tween-20] containing 5% nonfat milk for 1 h, and then incubated with diluted primary antibody for 12 h at 4°C. After that, the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody was used to incubate with the membranes for 1 h at room temperature (RT). An enhanced chemiluminescence reagent kit (Bio-Rad Laboratories, USA) was to visualize the protein on the membrane.
7. Immunohistochemical staining
Firstly, paraffin sections were deparaffinized and rehydrated, and then boiled with 0.01 M citrate buffer (pH 6.0) for 30 min. Secondly, after being washed and immersed in 3% hydrogen peroxide/methanol at room temperature for 30 min, the sections were blocked with 1% BSA and incubated with the primary antibodies including rabbit anti-NeuN (1:400), rabbit anti-Ly6C (1:400), and rabbit anti-GFAP (1:400). Thirdly, the sections were washed and incubated with the appropriate secondary antibodies, and then visualized with hematoxylin counterstain using DAB (3,3'-diaminobenzidine). The number of neurons with NeuN immunoreactivity and the optical density of the immunostained areas were observed under a microscope, and quantified using ImageJ software (US National Institutes of Health).
8. Isolation and purification of mouse brain microvascular endothelial cells
Dynabeads M-450 were prepared according to the manufacturer’s instructions (Invitrogen Corp., Carlsbad, CA, USA) and adjusted to a concentration of 4×l08 beads/ml with Hanks' balanced salt solution (HBSS) plus 5% fetal calf serum (HBSS + 5%FCS). Ulex-coated beads were used to isolate the endothelial cells from blood samples, and fucose was used to detach the cells that absorbing on the Ulex-coated beads. Then the cells were further selected using MFSD2a-coated beads. Cells adhering to MFSD2a-coated beads were labeled with PE-conjugated CD146 antibody and transferred to glass splices by cytospin for cBMECs counting under a fluorescence microscope. These isolated endothelial cells were positive for MFSD2a and CD146. Total cBMECs were identified based on their CD146 (endothelial cell marker)+/DAPI (nuclei)+phenotypes.
9. Monocytes transmigration assay
HBMECs were cultured in trans-well filters (3 µm pore size, 6 mm diameter, Millipore), and then the confluent monolayers were examined with integrity by TEER and microscopy. For HBMEC stimulation, 50 ng/ml HIV-1 gp120 was added for 24 h. Then, the different doses of CAPE (0–25 µM), TFP (0–25 µM) or Nys (0-100 µM) were added to the upper chambers with 0.8 ml EM (EM; containing 49% M199, 49% Ham’s F12, 1 mM sodium pyruvate and 2 mM L-glutamine) for 1 h. After stimulation, freshly isolated monocyte-like vitamin D3-differentiated HL60 cells were added to the upper chamber and allowed to migrate over for 4 h. The migrated monocytes were collected from the lower chamber and counted using a hemacytometer under the microscope. Monocytes transmigration were expressed as the percentage of monocytes across the HBMEC monolayers.
10. Immunofluorescence analysis
HBMECs were fixed using 4% paraformaldehyde and then blocked with 5% normal goat serum in PBS at room temperature for 1 h. The treated cell monolayers were incubated with primary antibody of rabbit anti-GFAP or S100B antibody at 4℃ overnight, and then incubated with appropriate coupled secondary antibody in dark for 1 h at room temperature. Slides were mounted and observed under a fluorescence microscopy (Nikon Eclipse: TE 2000-E, Japan).
11. Statistical Analysis
Data are shown in mean ± standard deviation and analyzed by one-way analysis of variance (ANOVA) tests. All statistical analyses were carried out using SPSS 13.0 (SPSS Inc., Chicago, IL, USA) and P < 0.05 was considered to be statistically significant.