Animals, Parasite, and Ethics Statement
Female C57BL/6 outbred mice aged ~6 weeks and weighted ~20g were obtained from the Medical Laboratory Animal Center of Guangdong, and housed in pathogen-free environment in the Laboratory Animal Center of Guangdong Pharmaceutical University, PR China. PbANKA strain was routinely maintained in liquid nitrogen, and intraperitoneally (i.p.) injected into healthy female C57BL/6 mice that served as parasite donor for the infection of experimental groups. The experimental protocols were performed with the approval of the Animal Ethics Committee of Guangdong Pharmaceutical University (No. gdpulac2020023), and confirmed to the National Guidelines for the Care and Use of Laboratory Animals.
Preparation of PbANKA blood-stage soluble antigen (PbAg)
Blood were harvested from PbANKA infected-C57BL/6 mice (~40% parasitemia). After the leucocytes were removed from blood using Leukocyte Removal Filter (Beijing ZKSK Technology Co., Ltd, China), iRBCs were isolated using density gradient centrifugation with 60% Perco ll solution. The iRBCs were extracted in 0.01% saponin lysis buffer for 30 min at 4°C, followed by the centrifugation at 10,000 rpm for 1 min. The blood-stage parasites were harvested by centrifugation at 10,000 × g for 1 min, followed by 4X washes with PBS to get rid of soluble red blood cells proteins. The whole parasites were suspended in PBS and disrupted with a sonicator probe at 80W on ice. After filtrating through 0.2um Whatman filter devices, PbAg was quantitated using BCA kit assay and stored at −80 °C until use.
Mouse MCs line culture and challenge, Exosome Purification
The mouse mast cells line (P815 cells) were kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China), and cultured in RPMI-1640 medium (GIBCO, No. 31800022#) containing FBS (10%), NaHCO3 (1.5g/L), glucose (2.5g/L), sodium pyruvate (0.11g/L), penicillin (100 U/mL), and streptomycin (100 μg/mL) in 75-cm2 tissue culture flasks at 37 °C in 5% CO2 and 95% air atmosphere. When the cells attained a density of 106 cells/mL, the P815 cells were washed with PBS twice and immediately exposed to PbAg (20 μg/mL) in the serum-free basal medium for 6 h. The culture supernatants were harvested by centrifugation at 2,000 r/min and 4°C for 10 min. Exosomes from the supernatant of the P815 cells were purified using an exosome isolation kit (Invitrogen, USA), according to the manufacturer's instructions.
MCs-Exo analyzed by TEM, Western Blotting, and NanoSight assays
The MCs-Exo were suspended in 2% glutaraldehyde, and then adsorbed at copper grids for 5 min at room temperature. Samples were negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min and observed using a FEI Tecnai G2 Sprit Twin transmission electron microscope (TEM; FEI, USA). The size and the concentration of MCs-Exo suspended in PBS were analyzed using NanoSight NS300 (Malvern Instruments, UK). In addition, the exosomal markers such as CD9, CD81, and CD63 of MCs-Exo were detected by Western Blotting assay. In brief, after the protein levels of MCs-Exo were determined by using a bicinchoninic acid protein assay kit (Beyotime, China), MCs-Exo were lysed in a western blotting lysis buffer, and separated using 10% SDS-PAGE, electrophoretically transferred onto a polyvinylidene fluoride blotting membrane (GE Healthcare Life Sciences, UK), and then blocked using 5% skimmed milk. The membranes were incubated with anti-mouse CD63, CD9 and CD81 antibody (BD Biosciences, USA) overnight at 4°C. Anti-mouse antibodies conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch Labs Inc., USA) were used as secondary antibodies. Membranes were visualized via an enhanced chemiluminescence (ECL) chemiluminescent detection system (Amersham, USA).
In vivo and ex vivo tracking of MCs-Exo
For labeling MCs-Exo, 5 μL of DiR dye (2mg/mL in DMSO; Life Technologies, USA) was completely mixed with 200 µg MCs-Exo in 200 µL of PBS for 30 min in the dark. The mixture was centrifuged at 120,000 × g for 90 min to remove the unincorporated DiR dye, and the final pellet was resuspended in 200 μL of PBS. After being anaesthetized by intraperitoneal (i.p.) injection with chloral hydrate, the naïve C58BL/6 mice intravenously (i.v.) injected with 5 μL of the DiR-labeled MCs-Exo in PBS to verify the distribution of MCs-Exo in mice. After 6 h injection, living animals were imaged by using the Maestro2 In-Vivo Imaging System (Maestro, USA). Upon completion of live imaging, the animals were sacrificed by CO2 asphyxiation, and major organs were harvested for fluorescence imaging.
MCs-Exo treatment in ECM model
To determine whether MCs-Exo mediate pathogenesis of ECM in mice, the experimental procedures were carried out in the following. Female C57BL/6 outbred mice were divided into 4 different groups: the uninfected mice that did not receive MCs-Exo treatment were used as negative controls (naïve group); the uninfected mice received daily i.v. injection of MCs-Exo (50 μg/mouse) as MCs-Exo group; the mice were only injected i.p. with 106PbANKA-iRBCs as Pb group; whereas mice subjected to PbANKA infection received daily i.v. injection of MCs-Exo (50 μg/mouse) as Pb+MCs-Exo group. Parasitemia in different groups was monitored daily by Giemsa-stained thin blood smears of tail blood. The mice were evaluated daily regarding survival time and neurological signs of ECM in mice. Once the PbANKA-infected mice exhibited neurological manifestations (e.g., coma, loss of reflex, prostration, paralysis, and convulsions), and usually moribund within 6-9 days postinfection (p.i.), these mice were classified as ECM mice.
Toluidine blue staining for MCs
Mice in different groups were sacrificed by CO2 asphyxiation, and major organs (cervical lymph node, skin, and brain) were harvested, immediately fixed in 4% neutral buffered formalin for 48 h, embedded in paraffin, and then cut into 5-μm-thick sections using a Leica microtome (Leica, Germany). After being deparaffinized and rehydrated, these sections were stained with 0.5% toluidine blue (Sigma-Aldrich, USA) for 12 h. The positive MCs that show deep blue-purple staining were visualized and analyzed under a light DM 2500B microscope (Leica, Germany). The number of MCs and level of degranulated forms in tissues was calculated as described protocols in our previous report .
Mice in different groups were euthanatized by CO2 asphyxiation, and their liver and brain tissues were removed, immersed in 4% neutral buffered formalin for 48 h, and then embedded in paraffin. Five-millimeter paraffin sections were stained with haematoxylin and eosin (H&E) dye, and analyzed for histopathological changes under a light microscope at a magnification of ×200 (Leica DM 2500B, Germany). To assess liver tissue damage, the number of inflammatory foci per field in liver tissues were calculated at a magnification of ×200 under a light microscopy.
ELISA assay for determining Th1 cytokines and AST/ALT in sera
Blood were harvested from mice in different groups, level of Th1 cytokines (IFN-γ, TNF-α, IL-6, and IL-1α), and liver damage markers (AST and ALT) in sera were measured by Enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s protocols (Gusabio, China). The concentration of cytokines or AST/ALT was calculated by measuring the absorbance at a wavelength of 450 nm using a Model Microplate Reader (iMark, USA) and comparing to standard curves generated from recombinant standard proteins.
Immunohistochemistry for ICAM-1 and VCAM-1 in brain tissue
After being deparaffinized and rehydrated in distilled water, five-millimeter paraffin brain slices were placed in odium citrate buffer for 10 minutes at 95 °C to achieve antigen retrieval. The slices were incubated with 3% H2O2 in dH2O to inhabit the endogenous peroxidase activity, and then place in 10% normal goat serum at 37 °C for 10 min to block non-specific binding. Slices were incubated with polyclonal mouse anti-ICAM-1 antibody (1:200 dilution; Servicebio, China) or polyclonal mouse anti-VCAM-1 antibody (1:200 dilution; Servicebio, China) for 1 h at 37°C. After being rinsed three times with PBS, slices were then incubated with biotinylated goat anti-mouse IgG (5 mg/mL, 1:200 dilution; Zhongshan, Beijing, China) for 1 h in darkness at room temperature. The slides were washed three times with PBS, exposed to avidin–biotin–peroxidase complex (Zhongshan, Beijing, China) for 20 min at 37°C, and then counterstained with hematoxylin. The positive cells on the brain were identified by dark-brown staining under light microscopy.
Assessment of BBB permeability by Evans blue dye staining
BBB integrity damage in mice was checked by Evans blue dye staining as previously described with minor modification . In brief, mice in different groups received a tail vein injection of Evans blue dye (2% in PBS, 4 ml/kg body weight) for 30 min. After being euthanized by i.p. injection of pentobarbital sodium, the left ventricle was perfused with 20 mL of heparin saline to remove intravascular-localized Evans blue dye. The brain tissue was excised, photographed, weighed, and placed in 20 ml of formamide at 60°C for 24 h to extract the Evans blue dye. Formamide supernatant was harvested by centrifugation at 1,000 rpm for 5 min and read at 620 nm using a Model Microplate Reader (iMark, Bio-Rad). The amount of extracted Evans Blue dye was determined in comparison to standard curves and expressed as ng/mg of tissue weight.
Western blotting for ZO-1 and Claudin-5 protein in brain tissue
The protein levels of ZO-1 and Claudin-5 in brain tissues were analyzed in different groups using western blotting assay. In brief, proteins in brain tissue were extracted in RIPA lysis buffer (Beyotime, China) and quantitated using BCA kit assay (Beyotime, China). The protein supernatants from each sample were separated using a 10% SDS-PAGE gels and subsequently electroblotted onto a polyvinylidene diﬂuoride (PVDF) membrane. The membranes were blocked with 5% non-fat milk at 37°C for 2 h and then subsequently exposed to the specific primary antibodies (anti-ZO-1 or anti-Claudin-5; BD Biosciences, USA) overnight at 4°C, followed by incubation with horseradish peroxide enzyme-conjugated goat anti-rabbit immunoglobulin G (1/2000; ab6721) at 37 °C for 1 h. The target bands were visualized and analyzed using an ECL chemiluminescent detection system (Amersham, USA). β-actin protein was used as the internal control.
bEnd.3 cell viability assay
The effect of MCs-Exo on bEnd.3 cells viability were performed using CCK-8 assay. The bEnd.3 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China), and cultured in DMEM medium (GIBCO, No.12800017) containing NaHCO3 (1.5g/L), FBS (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL) in 75-cm2 tissue culture flasks at 37 °C in 5% CO2 and 95% air atmosphere. When the cells attained a density of 106 cells/mL, the bEnd.3 cells were plated in 96-well plates (104 cells/well). The cells were immediately incubated with PbAg (20 µg/mL), MCs-Exo (50 µg/mL), and PbAg (20 µg/mL) plus MCs-Exo (50 µg/mL), respectively. As the MCs-Exo and/or PbAg were suspended in complete medium, and alternatively flasks were added into only complete medium as blank control. At 24 h, 48 h, and 72 h after incubation, the bEnd.3 cells from different groups were then incubated with 10 μL CCK-8 (Beyotime Biotechnology, China) for 1 h at 37 °C, and the absorbance of each well was measured at 450 nm using a Model Microplate Reader (iMark, Bio-Rad).
Quantitative qPCR assay
To assess the effect of MCs-Exo on the expression of ZO-1, Claudin-5, Ang-1, and Ang-2, as well as the release of chemokines (CCL2, CXCL1, and CXCL9) from bEnd.3 cells stimulated with PbAg, qPCR assay was performed. When the cells attained a density of 106 cells/mL in 75-m2 plate, the bEnd.3 cells were plated in 6-well plates (104 cells/well). PbAg (20 µg/mL), MCs-Exo (50 µg/mL), and PbAg (20 µg/mL) plus MCs-Exo (50 µg/mL) were immediately put into bEnd.3 cells, respectively. After 48 h, total RNA of bEnd.3 cells in different groups were harvested using with Trizol reagent (TaKaRa, Japan), quantitated by using a 1.0% agarose gel, and subsequently quantified by determining the ration of absorbance at 260 nm to 280 nm with a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, USA). cDNA was synthesized from 1.0 μg of total RNA by using PrimeScript 1 st Strand cDNA Synthesis Kit (TaKaRa, Japan), and stored at −80 °C until use. To measure the mRNA expression of CCL2, CXCL1, CXCL9, Ang-1, Ang-2, ZO-1 and Claudin-5 in bEnd.3 cells, the SYBR Green qPCR Master Mix (TaKaRa, Japan) was used to perform qPCR reaction with a Lightcycler®480 instrument (Roche Diagnostics, Switzerland). The qPCR reactions were constructed in a total volume of 10 μL mixture comprising 5.0 μL of SYBR® Premix Ex Taq TM (2×), 0.5 μL of forward- or reverse-primer (10 pM), 1.0 μL of cDNA template (100 ng/μL), and 3.0 μL of ddH2O according to the manufacturer’s instructions. The qPCR reaction contained 95°C for 30 s, 43 cycles of 95°C for 5 s and 60°C for 20 s. The Primer sequences used in this study were listed as follows: ZO-1, 5’-GAACGCTCTCATAAGCTTCGTAA-3’ (forward) and 5’- ACCGTACCAACCATCATTCATTG-3’ (reverse); Claudin-5, 5’-TCTGCTGGTTCGCCAACAT-3’ (forward), and 5’-CGGCACCGTCGGATCA-3’
(reverse); Ang-1, 5’-TGCAGCAACCAGCGCCGAAA-3’ (forward) and 5’-CAG GGCAGTTCCCGTCGTGT-3’ (reverse); Ang-2, 5’-GCTTCGGGAGCCCTCTGGGA-3’ (forward) and 5’-CAGCGAATGCGCCTCGTTGC3’ (reverse); CCL2, 5’-GCAGCAGGTG
TCCCAAAGAA-3’ (forward) and 5’-GGTCAGCACAGACCTCTCTCTTG-3’ (reverse);
CXCL1, 5’-CCACACTCAAGAATGGTCGC-3’ (forward) and 5’-TCTCCGTTACTTGG
GGACAC-3’ (reverse); CXCL9, 5’-GAACTCAGCTCTGCCATGAA-3’(forward) and 5’-GCATCGTGCATTCCTTATCA-3’ (reverse);β-actin, 5’-GTGCTATGTTGCTCTAGAC
TTCG-3 (forward) and 5’-ATGCCACAGGATTCCATACC-3’ (reverse). The mRNA levels of target genes were normalized to those of β-actin gene, and the results were indicated as the fold amplification in comparison to those of naive controls by using 2-ΔΔCT method.
All data are reported as the mean ± SEM. Statistical analysis of data was carried out using GraphPad Prism 5 software. Statistical significance between two experimental groups was assessed using the Independent Sample t-test. Log-Rank test, a time-series analysis test, or one-way ANOVA was used to evaluate the significance difference among multiple groups. P<0.05 was considered statistically significant.