C57BL/6 (JAX stock #000664) and IFNAR−/− (MMRRC stock #32045) mice were purchased from Jackson Laboratory. IL-22−/− mice were kindly provided by Genentech Inc. Mice were housed under 12-hour day/night cycle in the specific pathogen-free, AAALAC-accredited animal facility of UTMB.
ZIKV (Asian lineage FSS13025) was obtained from the World Reference Center for Emerging Viruses and Arboviruses (Galveston, TX). Virus was amplified in Vero cells and viral titer was calculated as fluorescent focus units (FFU) per ml. All newborn mice were born from pathogen-free parents and inoculated one day after birth with 4 × 103 FFU ZIKV in 2 µL by Hamilton microliter syringes through subcutaneous (s.c.) inoculation at the lateral side of body part. For in vivo treatment, neonatal WT mice were s.c. injected with recombinant IL-22 treatment (1 µg in 5 µL, s.c.) every other day. In some experiments, mice that were 3 weeks old were intraperitoneally infected with 1 × 105 FFU ZIKV. Animals were monitored daily for bodyweight changes and clinical signs of pathology. Moribund animals were euthanized in accordance with the UTMB IACUC guidelines.
Clinical Symptom Evaluation
The clinical evaluation of infected neonatal mice was modified according to another report . Briefly, Mice were weighed and examined for signs of infection daily. Examination criteria included appearance, stance, and motility. The description of clinical presentations included stagger step (increased spread of hind legs and unusual pauses during movement), paralysis (loss of muscle function of one or two hind legs), and seizure (sudden stiffening of muscles in the back and legs).
Cell Lines And Primary Mouse Glial Cells
Vero and U-87 MG cells were cultured in Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum. All cells were cultured in the presence of 100 U/mL penicillin and 100 µg/mL streptomycin in 37 °C incubator with 5% CO2 and 95% humidity control.
For primary mouse glial cell preparation, B6 mouse pups (1–4 days old) were used for mixed cortical cell isolation . Briefly, mouse brains were taken out and placed into a dish containing cold HBSS. The meninges from the cortex hemispheres were pulled with the fine forceps under a stereomicroscope to avoid contamination with meningeal cells and fibroblasts. The cortex hemispheres were cut into small pieces, followed by 2.5% trypsin digestion for 30 min at 37 °C. Cortex tissue pieces were harvested and dissociated into a single-cell suspension. Mixed cortical cells were counted and cultured with DMEM/F12 (1:1) medium plus 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin in a T75 culture flask. Medium was replaced every 5 days. For microglia isolation, the mild trypsinization method was used. Briefly, mixed cortical cells were cultured for 12–15 days and incubated with a trypsin solution (0.25% trypsin, 1 mM EDTA in HBSS) diluted 1:4 in DMEM/F12 medium. The upper layer of cells was detached in one piece and removed from the flask. Microglia remained attached to the bottom and were harvested by further trypsinization for 15–20 min. The purity of microglia was 98.5% as confirmed by CD11b, CX3CR1 and CD45 flow cytometric analysis (Fig. S1A). For astrocyte isolation , after 7–8 days culture, the flask was shaken at 180 rpm for 30 min to remove microglia. New medium was added into the flask followed by shaking at 240 rpm for 6 hours (hrs) to remove oligodendrocyte precursor cells. Astrocytes were detached by trypsin-EDTA, washed with PBS, and plated into two T75 culture flasks. Medium was changed every three days and astrocytes were harvested at 12–14 days after the first split. Astrocytes were then identified using IF staining of GFAP (Fig. S1B). For in vitro infections, a single dose of ZIKV (MOI of 1) was used.
RNA Isolation And Quantitative Real-time PCR (qRT-PCR)
RNA was isolated using Rneasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad). The abundance of target genes was measured by qRT-PCR using a Bio-Rad CFX96 real-time PCR apparatus. SYBR Green Master Mix was from Bio-Rad, and TaqMan Universal Master Mix, including gene-specific probes and primers were from Integrated DNA Technologies (IDT). The relative level of gene expression was calculated using the 2 −ΔΔct method. The sequences of primers and probes are listed in Supplementary table 1.
Measurement Of Viral Burden
For measuring viral burden in vivo, mouse tissues were weighed and homogenized using a Tissue-Tearor (BioSpec). ZIKV RNA levels were determined by TaqMan quantitative reverse transcriptase PCR on the real-time PCR detection system (Bio-Rad). Virus burden was determined by interpolation onto an internal standard curve composed of serial 10-fold dilutions of a synthetic ZIKV RNA fragment. A previously published primer set was used to detect ZIKV RNA . For measuring viral burden in infected cells in vitro, the relative level of gene expression was calculated based on Ct values using GAPDH as a housekeeping gene.
Lymphocytes Isolation And Purification
Lymphocytes were isolated according to our previously reported method . Briefly, brains were cut into pieces and digested with 0.05% collagenase IV (Roche, Indianapolis, IN) at 37 °C for 30 min. Cell suspensions were passed through a 70-µm nylon cell strainer to yield single-cell suspensions. Lymphocytes were enriched by centrifugation (400 g) at room temperature for 30 min over a 30/70% discontinuous Percoll gradient (Sigma). Spleens were collected from mice and gently mashed in the RPMI-1640 medium through a cell strainer. Red blood cells were removed by using Red Cell Lysis Buffer (Sigma, St. Louis, MO). Cells were harvested by centrifugation (300 g, 10 min, 4 °C) and resuspended in RPMI-1640 medium plus 10% fetal bovine serum.
Intracellular staining was performed with flow cytometry as in our previous report . Briefly, for IL-22 and IL-17A detection, lymphocytes were cultured with rIL-23 (20 ng/ml) for 12 hrs. Brefeldin A solution (eBioscience) was added for the last 4 hrs of culture. For detecting IFN-γ and TNF-α in ZIKV-specific CD8 T cells, lymphocytes were incubated with ZIKV peptide E294 − 302 (1 mg/mL, GenScript) in the presence of Brefeldin A solution for 5 hrs. Cells were then stained for anti-CD16/32 (Clone 2.4G2) and surface markers, fixed by using an IC fixation buffer, and followed by staining for intracellular cytokines (Thermo Fisher Scientific, Waltham, MA). Fixable viability dye, efluor 506 (Thermo Fisher Scientific), was also used to exclude dead cells. All samples were processed on an LSRII FACS Fortessa (Becton Dickinson, San Jose, CA) and analyzed using FlowJo software (TreeStar, Ashland, OR). The flow cytometry antibodies PE-Cy7-conjugated anti-CD3 (17A2), efluor450-conjugated anti-CD4 (GK1.5), APC-eFlour780-conjugated anti-CD8 (53 − 6.7), FITC- conjugated anti-NK1.1 (OK136), FITC- conjugated anti-TCR gamma/delta (GL3), PerCp-eFlour710-conjugated anti-TNF-α (MP6-XT22), APC-conjugated anti-IFN-γ (XMG1.2), APC-conjugated anti-CD45 (30-F11), Pacific Blue-conjugated anti-CD11b (M1/70), APC-conjugated anti-IL-17 (eBio17B7) and PE-conjugated anti-IL-22 (1H8PWSR) were purchased from Thermo Fisher Scientific. Purified anti-CD16/32 (2.4G2) and PE-conjugated anti-CX3CR1 (SA011F11) were purchased from Biolegend (San Diego, CA). CFSE dye was used for the cell proliferation assay.
Tissue proteins were extracted using RIPA buffer (Cell Signaling Technology, Danvers, MA) and quantified using a BCA kit (Thermo Fisher Scientific). Mouse IL-22 ELISA kit was purchased from Thermo Fisher Scientific.
Immunofluorescence Staining And Confocal Microscopy
Mice were euthanized with CO2 and perfused transcardially with cold PBS. Frontal cortices were collected and were immediately placed in 4% PFA in PBS at 4 °C overnight and then cryoprotected in a 30% sucrose solution in PBS for at least 24 hrs at 4 °C. Tissues were embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). Transverse sections (35 µm) were prepared on a cryostat (Leica CM 1900). The sections were kept in Hito floating section storage solution (Hitobiotec Corp) at -20 °C until they were stained for immunocytochemistry. For immunostaining, tissue sections were rinsed with PBS twice to remove the storage solution and blocked with 5% BSA and 0.3% Triton X-100 in PBS for 2 hrs at room temperature, followed by 48 hrs incubation with primary antibodies. After five washes with PBS, the sections were incubated with fluorophore-conjugated secondary antibodies at 4 °C overnight prior to section mounting. Confocal Z-stacks images were captured within the layer I-II of the cortex using a confocal microscope (Nikon A1). For each mouse, at least 3 fixed-frozen sections were included for each experiment, and at least 3 z-stacks images at 20 x, 40 x or 60 x magnification were taken. Thirty to fifty consecutive optical sections with 1 µm interval thickness at 40 x and 60 x magnification were captured for each Z-stacks image. To process images, “Subtract Background” (50 pixels) was applied to remove the background and a threshold (50–225) was set to remove outliers. The analysis of positive areas and cell sizes were performed using Image J software. The rabbit-anti-Iba1 (Abcam, ab178846) and goat-anti-GFAP (Abcam, ab53554) antibodies were purchased from Abcam. The secondary antibodies, including goat anti-rabbit IgG (H + L) Alexa Fluor 488 and donkey anti-goat IgG (H + L) Alexa Fluor 555, were purchased from Thermo Fisher Scientific.
Western Blot Analysis
Total brain protein was homogenized in RIPA buffer including a 1% protease inhibitor cocktail (Sigma-Aldrich), and protein concentrations of the lysates were quantified using a BCA kit (Thermo Fisher Scientific). Five to twenty µg of proteins per sample were loaded onto a 12% Novex Tris-Glycine Gel and subsequently transferred to a PVDF membrane. The membrane was blotted with primary antibodies at 4 °C overnight. Antibody detection was accomplished using horseradish peroxidase conjugated secondary antibodies and visualized with ECL. Markers were used to identify the target protein band. As loading control, the expression of GAPDH was also measured. The signal intensity was quantified with Image Studio Lite. The primary antibodies anti-VE-cadherin, anti-ZO1, anti-Occluding, anti-Claudin 1 and anti-Claudin 3 were from Abcam. Anti-GAPDH was purchased from Cell Signaling Technology.
Data were shown as mean ± SEM and analyzed using the two-tailed Student’s t-test when compared between two groups. One-way ANOVA was used for statistical analysis of more than two groups. A log-rank (Mantel-Cox) test was used for survival curve analysis. *, ** or *** means P-value < 0.05, < 0.01 or < 0.001, respectively. Statistical analyses were operated by GraphPad Prism software 7.0 (GraphPad Software Inc., San Diego, CA).