Figure 1.
JEV interacts with CLEC5A and induces cytokine secretion via CLEC5A.
(A) Interaction of JEV with human and murine CLEC5A.Fc fusion proteins was determined by ELISA. Anti-E Ab is a positive control to confirm the capture of JEV particles; HuIgG1: human IgG1. Data are expressed as means ± s.e.m. for three independent experiments; two-tailed Student's t-tests were performed. (B) The kinetics of DAP12 phosphorylation induced by JEV (m.o.i. = 2) and ultraviolet-inactivated JEV (UV-JEV) in human macrophages were determined by western blotting (h.p.i., hours post infection). The intensities of pDAP12 bands were quantified with MetaMorph software (Molecular Devices) and its relative intensity to that of the band of respective corresponding DAP12 and are shown under each picture. (C) Different amounts of antibody 0.1 µg (2 µg/ml; 0.013 µM), 1 µg (20 µg/ml; 0.13 µM) and 10 µg (200 µg/ml; 1.3 µM) were incubated with MoM before addition of JEV. Dose-dependent inhibition of cytokine release from JEV-infected MoM by anti-CLEC5A mAbs (clones: 2B8H11, 6E11A8, 7F3G2) were determined using ELISAs at 48 h post infection. mIgG1 was used as an isotype control. (D) Bone marrow-derived macrophages (5×105 cells/well) from Clec5A−/− Stat1−/− and Clec5A+/+ Stat1−/− mice were infected with JEV (m.o.i. = 2) and supernatants were harvested at 48 hr after JEV infection for cytokine measurement. Data were collected and expressed as mean ± s.e.m. from at least three independent experiments. Two-tailed Student's t-tests were performed.
Figure 2.
Blockade of CLEC5A inhibits neuronal death induced by supernatant from JEV-infected Stat1−/− murine mixed glial cell culture.
(A) Tissue sections of human perilesional brain cortex from glioblastoma multiforme were stained with CLEC5A mAb (20 µg/ml) and isotype control. CLEC5A staining was observed under a light microscope (Nikon) and photographed. (B) Detection of CLEC5A+ cells in F4/80+, CD11b+ and CD45+ mononuclear cells isolated from naïve murine brain. (C) Viral antigen NS3 (red; cells counterstained with Hoechst (blue)) was detected in JEV-infected microglia or mixed glia (m.o.i. = 5) in the presence of anti-CLEC5A mAb (clone: 3E3G4; 200 µg/ml, 1.3 µM) at 48 h post infection. Scale bars, 50 µm. (D) The kinetics of virus replication were unaffected by anti-CLEC5A mAb (3E3G4). Viral titers in culture supernatants were determined by plaque assay. Data were collected from three independent experiments. (E) anti-CLEC5A mAb (clone: 3E3G4; 200 µg/ml, 1.3 µM) were incubated with mixed glia or microglia before addition of JEV. Cytokine secretion from JEV-infected mixed glia and microglia was harvested and analyzed by ELISA at 48 h p.i.. (F) An immunocytochemical analysis (anti-tubulin β III isoform mAb) shows the morphology of JEV-infected neurons (m.o.i. = 5) in the presence of anti-CLEC5A mAb or isotype matched control at 24 h post JEV infection. (G) An immunocytochemical analysis (anti-tubulin β III isoform mAb) shows the morphology of neurons after incubation with UV-inactivated conditioned medium (CM) from JEV-infected mixed glia (UV-JEVCM) (m.o.i. = 5), in the presence of isotype matched control (UV-isotype_JEVCM) or anti-CLEC5A mAb (UV-CLEC5A mAb_JEVCM) for 24 h. The morphology of neurons was observed by immunocytochemical staining with anti-Tubulin β III isoform Ab (TU20), and visualized under a light microscope (Nikon). Five fields of views were randomly photographed, and the numbers of live neurons were counted and represented as mean ± s.e.m. (under each picture) for three independent experiments. Significance compared to mock treatment was tested using a two-tailed Student's t-test (Figure 2F). In Figure 2G, the P<0.0001 indicates a significant difference in UV-MOCK CM vs. UV-JEVCM; P = 0.0007 indicates a significant difference in UV-Isotype JEVCM vs. UV-CLEC5A mAb JEVCM.
Figure 3.
Effect of anti-CLEC5A mAb on dynamic changes in the BBB during JEV infection.
(A) Stat1−/− mice were injected intravenously with 99mTc-DTPA to enable brain SPECT/CT imaging, before (D0) or after JEV challenge (100 pfu/mouse), in the presence of anti-CLECA5 mAb (3E3G4) or an isotype matched control. Antibody (150 µg/mouse) was administrated intraperitoneally on days 0, 2, 4, 6 and 8 to determine any protection effects in reduction to BBB permeability. Image datasets were reconstructed using the ordered-subset expectation maximization algorithm with standard-mode parameters. (B) The extent of BBB breakdown was calculated as the ratio of the mean counts/pixel in the region of the brain with the greatest accumulation of radiotracer divided by the mean counts/pixel in the neck muscle (b/m ratio). (C) Changes in BBB permeability at day 7 post JEV infection (100 pfu/mouse) were determined by Evans Blue assay. (D) Brains of JEV-infected mice (n = 5) with isotype control or anti-CLEC5A Ab treatment were harvested at day 5 and day 9 post infection to analyze the expression of transcripts encoding tight junction proteins and adhesion molecules by quantitative real-time PCR. For tight junction proteins, y-axis units represent the expression level of each target gene relative to mock control after internal control normalization; the expression level of adhesion molecules is displayed as fold increase relative to mock control. Two-tailed Student's t-tests were performed. (E) H&E staining of murine cerebral cortex at day 5 after JEV infection revealed the inhibitory effect of anti-CLEC5A mAb on perivascular cuffing. Scale bars, 200 µm. Five random fields of views in medium power field (original magnification (OM)×200) were photographed, and the numbers of foci and vessel cross sections in each sample were counted, summed up and represented as mean ± s.e.m. (under each picture) of four independent experiments.
Figure 4.
Effect of anti-CLEC5A mAb on recruitment of immune cells into the CNS during JEV-infection.
(A) Flow cytometry analysis of mononuclear cells (MNCs) isolated from JEV-infected murine brain tissue with isotype control or anti-CLEC5A mAb treatment at day 5 post infection; cell populations were determined by staining specific cell surface markers. (B) MNCs were further characterized by determining expression levels of CD45 in the CD11b+CD11c− population to distinguish inflammatory myeloid cells from peripheral (R1, CD45hi) and resident microglia (R2, CD45low), respectively (upper panel). Recruitment of CD11b+CD45+ cells into brain of JEV-infected mice was tracked by in vivo labeling of blood cells with fluorescent dye CFSE. (C) Analysis of the CD45+CD11b+ population for Ly6C and Ly6G expression revealed the major Ly6C+ and minor Ly6G+ populations in CD11b+CD45hi peripheral inflammatory myeloid cells. The percentages indicate the specific region in CD11b+-gated cells (the collected CD11b+ cells in the isotype group and CLEC5A mAb treatment group are approximately 25,000 cells and 1,000 cells, respectively.) Three independent experiments with three mice per group were performed and representative FACS plots are shown. (D) CLEC5A expression in peripheral inflammatory myeloid cells (R1-gated; CD11c−CD11b+CD45hi) and residential microglia (R2-gated; CD11c−CD11b+CD45low) isolated from JEV-infected murine brain.
Figure 5.
Anti-CLEC5A mAb reduces viral load in the CNS and attenuates CNS inflammation.
(A) Viral titer was determined by plaque assay for sera and tissue homogenates from JEV infected mice with or without anti-CLEC5A mAb treatment (two-tailed Student's t-tests). (B) The extent of NS3 expression in the cortex was diminished after peripheral administration of anti-CLEC5A mAb at day 5 post JEV infection. Scale bars, 50 µm. Five random fields of views in medium power field (original magnification (OM)×200) were photographed, and the numbers of NS3+ cells in each sample were counted, summed up and represented as mean ± s.e.m. (under each picture) of four independent experiments. (C) Numbers of NS3+ leukocytes (CD45hiCD11b+ and CD45midCD11b+) in JEV-infected brain were significantly reduced by administration of anti-CLEC5A mAb at day 5 post JEV infection. (D) Quantification of viral copies in CD45hiCD11b+ (R1) and CD45lowCD11b+ (R2) cells sorted from the brain of JEV-infected mice. The average of total cell numbers in R1 and R2 were approximately 5×104 and 2×104, respectively, in the isotype treated group. Q-PCR assay was performed on total RNA from each population and normalized with its respective internal control. Data were collected from four independent experiments. Two-tailed Student's t-tests were performed. Cytokines levels in the sera (E) and CSF (F) were measured by ELISA at day 5–7 post JEV infection. The effects of CLEC5A mAb (3E3G4) and isotype control were compared using two-tailed Student's t-tests. (G) Intracellular TNF-α and IL-6 were detected in CD11b+ gated F4/80+ and CD45+ cells isolated from JEV-infected brain by flow cytometry.
Figure 6.
Anti-CLEC5A mAb rescues JEV-induced neuronal damage.
(A): JEV-induced pathological changes were examined by H&E staining. Arrowheads indicate dysmrophic neurons in the cerebral cortex (upper panel) and damaged Purkinje cells in the cerebellum (lower panel), scale bars, 200 µm. (B) Neuronal damage induced-astrogliosis was characterized by GFAP staining, scale bars, 50 µm. For (A) and (B), tissues were harvested at day 5 post JEV infection. Five random fields of views in medium power field (original magnification (OM)×200) were photographed, and the numbers of dysmorphic neurons or GFAP+ cells in cerebral cortex and cerebellum in each sample were counted, summed up and represented as mean ± s.e.m. (under each picture) of four independent experiments.
Figure 7.
Anti-CLEC5A mAb protects mice from JEV-induced lethality.
(A) Survival of Stat1−/− mice (8–10 weeks) was monitored for 16 days after intraperitoneal inoculation of JEV (100 pfu/mice); data were collected from four independent experiments and are shown as Kaplan–Meier survival curves with log rank test; n = 20 for each group. (B) Titers of anti-JEV specific IgM and IgG antibodies in murine sera were determined by ELISA (C) Pooled sera from surviving mice were serially diluted and analyzed using a for the plaque reduction neutralization test (PRNT); data were collected from four independents experiments with n = 5 for each group. (D) INF-γ secretion from total splenocytes of surviving mice (recovery, day 21 after JEV infection) after incubation with JEV and UV-JEV for 72 h, but not from mock infected mice. (E) The ability of IFN-γ-secreting T-cell was verified by immobilized anti-CD3 mAb to activate T cells in both mock and JEV-infected (recovery) mice. (F) Viral titers in spleen and brain isolated from surviving mice after JEV challenge were determined by plaque assay.