Article Figures & Data
Figures
- FIG. 1.
MMP2 and -9 expression in macrophages. (A) Peritoneal macrophages from mice were infected with WNV (MOI = 1) for 6 and 24 h. Total RNA was purified and converted into cDNA, and selected transcripts were assessed by quantitative RT-PCR. Bars represent the changes in the levels in infected samples compared to the levels in uninfected controls. Data shown represent the results of one of three experiments with similar results. *, P < 0.01. (B) Expression of MMP2 and -9 in human macrophages. Amounts of 1 × 106 PBMCs were isolated from fresh blood and grown in vitro for 7 days. Cells were infected with WNV (MOI = 1) for the indicated time intervals. Data represent the means ± standard errors of the pooled results (n = 5). *, P values were between <0.05 and 0.0001.
- FIG. 2.
MMP9 in the blood of WNV-infected mice. Wild-type mice (n = 8) were infected with 500 PFU of WNV, and whole-blood samples were drawn at the indicated time points. Levels of WNV E (A) and MMP9 (B) gene expression in the whole blood were assessed by quantitative RT-PCR. Bars represent the means ± standard errors of the results. MMP9 transcripts were significant higher at days 1 and 2 than at day 0 (P < 0.001). (C) MMP9 protein levels in the sera were quantified by ELISA. MMP9 levels were significantly elevated at days 2 and 5 compared to the levels on day 0 (P < 0.01). Horizontal lines, means of results.
- FIG. 3.
MMP9 expression in the brains of WNV-infected mice and the CSF of WNV patients. Wild-type mice were infected with 500 PFU of WNV and perfused with PBS at days 3, 5, and 7 postinfection. WNV E (A) and MMP9 (B) transcripts were examined by quantitative RT-PCR. Bars represent the means ± standard errors of the results (n = 5 for each time point). (C) Zymography results. The transparent bands indicate the presence of gelatinase, MMP9, or MMP2. WT, wild type. (D) Localization of MMP9. Infected mice were sacrificed and perfused at days 0, 3, 5, and 7. Brain sections (cerebral cortex) were stained for MMP9. Images were acquired by using a Zeiss AxioCam fluorescence microscope. Original magnification is ×100. Arrows indicate MMP9 staining on blood vessels, and arrowheads point to MMP9 staining in brain parenchyma. (E) Total MMP9 levels in the WNV-negative and -positive CSF samples were determined by ELISA (n = 7/group). Bars represent the means ± standard errors of the results. *, P < 0.04.
- FIG. 4.
Phenotypic analyses of MMP9−/− mice upon WNV infection. (A) Wild-type and MMP9−/− mice were infected with 500 PFU of WNV and monitored for mortality daily. Data are pooled from the results of two experiments (n = 27/group). P < 0.04. (B) Viremia in the whole blood at selected time points (left panel) was examined by quantitative RT-PCR using the WNV E gene and normalized with murine actin (n = 10/group). Virus titers in sera were determined by plaque assay and are expressed as PFU per ml of sera (right panel). Bars indicate the means ± standard errors of the results from two experiments (n = 10/group). Blood IL-6 (C) and TNF-α (D) levels were examined by using quantitative RT-PCR. Bars represent the means ± standard errors of the results from two experiments (n = 10/group). WT, wild type; KO, knockout.
- FIG. 5.
Brain viral loads and histopathology of MMP9−/− mice infected with WNV. Wild-type and MMP9−/− mice were infected with 500 PFU of WNV for 5 and 7 days, euthanized, and perfused. (A) RNA was extracted from brains, and quantitative RT-PCR was performed to measure the transcripts of WNV E at days 5 and 7. *, P value of <0.04 by Mann-Whitney test. Horizontal lines, means of results. (C to E) The transcripts of TNF-α (C), alpha interferon (D), and IL-6 (E) in the brain at day 7 were measured by quantitative RT-PCR. Bars indicate the means ± standard errors of pooled results from two experiments (n = 10/group). *, P value of <0.04 by Mann-Whitney test. (B, F, and G) Results of immunofluorescence staining of WNV E (B), staining of CD45 by IHC (F), and H&E staining of cerebral cortex sections (G). Open triangle, neurons with condensed and aggregated nuclear chromatin; arrow, healthy neuron; closed triangle, infiltrating leukocytes; star, microglial nodules formed by clusters of microglial/infiltrating cells around necrotic brain tissues. All images were acquired by using a Zeiss AxioCam fluorescence microscope at a magnification of ×100. Shown are representative cerebral cortex sections from 10 mice/group. WT, wild type.
- FIG. 6.
BBB permeability in MMP9−/− mice infected with WNV. (A) Wild-type and MMP9−/− mice were infected with 500 PFU of WNV for 7 days, euthanized, and perfused. Brain samples (nondenatured) were resolved by SDS-PAGE, and Western blotting was performed to detect IgGs (∼150 kDa), type IV collagen alpha chain (∼160 kDa), and β-actin by using specific antibodies. (B) The intensities of type IV collagen bands were quantified and normalized with actin. Representative data from 3 out of 10 mice are shown here. (C) Wild-type and MMP9−/− mice were infected with 500 PFU of WNV and at day 4 injected with Evans blue and perfused 1 h later. Data are representative of the results of two experiments (n = 10/group). (D) Quantification of Evans blue in the mouse brain. Evans blue was extracted from whole brains, and absorbance at a wavelength of 611 nm (A611 nm) was measured, using uninfected-mouse brain extract as a blank. Bars represent the means ± standard errors of the results (n = 10/group). *, P < 0.04. WT, wild type.
Tables
- TABLE 1.
Primers and probesa
Gene product Sequence M. musculus Mmp9 F: 5-TGGACGCGACCGTAGTTG-3 R: 5-GCTTGCCCAGGAAGACGAA-3 P: 5-6FAM-CTGTGCGTCTTCCCC-MGB-3 M. musculus Mmp2 F: 5-GAGACCGCTATGTCCACTGT-3 R: 5-CTTGTTGCCCAGGAAAGTGAAG-3 P: 5-6FAM-CACCTTCTGAATTTCC-MGB-3 M. musculus β-actin F: 5-AGAGGGAAATCGTGCGTGAC-3 R: 5-CAATAGTGATGATGACCTGGCCGT-3 P: 5-6FAM-CACTGCCGCATCCTCTTCCTCCC-TAMRA-3 M. musculus TNF-α F: 5-CTCCAGGCGGTGCCTATGT-3 R: 5-GAAGAGCGTGGTGGCCC-3 P: 5-6FAM-CAGCCTCTTCTCATTCCTGCTTGTGGC-TAMRA-3 M. musculus IL-6 F: 5-CCAGAAACCGCTATGAAGTTCC-3 R: 5-TCACCAGCATCAGTCCCAAG-3 P: 5-6FAM-TCTGCAAGAGACTTCCATCCAGTTGCCT-TAMRA-3 M. musculus IFN-α F: 5-CTTCCACAGGATCACTGTGTACCT-3 R: 5-TTCTGCTCTGACCACCTCCC-3 P: 5-6FAM-AGAGAGAAGAAACACAGCCCCTGTGCC-TAMRA-3 WNV E F: 5-TTCTCGAAGGCGACAGCTG-3 R: 5-CCGCCTCCATATTCATCATC-3 P: 5-6FAM-ATGTCTAAGGACAAGCCTACCATC-TAMRA-3 ↵ a Primers and probes for Homo sapiens MMP2, MMP9, and TIMP-2 and M. musculus MMP3, MMP13, TIMP-1, and TIMP-2 are inventoried products of Applied Biosystems. F, forward; R, reverse; P, probe; 6FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine; MGB, minor groove binding.
