Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: MMP8 expression is upregulated by lab-attenuated RABV infection in cells and mouse brains. C57BL/6 mice (n = 3) were intracerebrally (i.c.) inoculated with 20 FFU of RABV strain CVS-B2c (CVS), 200 FFU of DRV-Mexico (DRV), or the same volume of DMEM (mock). At 2, 4, and 6 d.p.i., mouse brains were harvested and used for further analysis. (A) At 6 d.p.i., total RNA was isolated from brain tissue and analyzed by qPCR for mRNA levels of MMP2, -3, -7, -8, -9, and -12. (B) At 2, 4, 6, and 8 d.p.i., total RNA from mouse brains was isolated, and the MMP8 mRNA level was analyzed by qPCR. (C) At 6 d.p.i., total RNA from different brain sections was isolated, and the MMP8 mRNA level was analyzed by qPCR. (D) At 6 d.p.i., the protein levels of MMP8 and RABV-N in the mouse cortex, cerebellum, and brain stem were assessed by Western blotting; β-actin was used as the control. (E) BV2, C8, and N2a cells were infected with CVS at an MOI of 1, and MMP8 mRNA levels were measured by qPCR at 48 h postinfection (h.p.i.). (F) MMP8 protein levels in BV2, C8, and N2a cells after CVS infection were measured by Western blotting. (G) BV2, C8, and N2a cells were infected with DRV at an MOI of 1, and MMP8 mRNA levels were measured using qPCR at 48 h.p.i. (H) MMP8 protein levels in BV2, C8, and N2a cells after DRV infection were measured by Western blotting. (I) BV2 cells were infected with CVS at the indicated MOIs, and MMP8 mRNA levels were measured using qPCR. (J) BV2 cells were infected with CVS at indicated MOIs, and MMP8 protein levels were assessed using Western blot. The gray value of MMP8 and β-actin was calculated using ImageJ software. Error bars represent SD (n = 3). Statistical differences between infected group and mock-infected group were determined using the Student t test. Western blot data are representative of those from at least two independent experiments. ns, not significant.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Expressing, Infection, Isolation, Western Blot, Control, Software

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: MMP8 expression is regulated via NF-κB and MAPK pathways. (A) C57BL/6 mice (n = 3) were i.c. inoculated with 20 FFU of CVS or 200 FFU of DRV. Mouse brains from each group at 6 d.p.i. were harvested. The expression and phosphorylation of the key molecules in the NF-κB and MAPK pathways in CVS- or DRV-infected mouse brains were measured by Western blotting. (B) BV2 cells were incubated with LPS (100 ng/mL) for the indicated periods. MMP8 mRNA and protein levels were measured by qPCR and Western blotting. (C) BV2 cells were incubated with IFN-γ at the indicated concentrations. MMP8 mRNA level and protein level were measured by qPCR and Western blotting. (D and E) BV2 cells were infected with RABV at an MOI of 1, and at different time points after RABV infection, the culture medium was replaced with specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM, 24 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 24 h) or dimethyl sulfoxide (DMSO). (D) The mRNA levels of MMP8 were quantified by qPCR. (E) MMP8 protein levels were assessed using Western blotting. The gray value of MMP8 and β-actin was calculated using ImageJ software. Anti-MMP8 antibody, 500 ng/mL; anti-β-actin antibody, 500 ng/mL. Statistical analysis of comparisons between groups was carried out by the Student t test. The bar graph shows the means ± SD (n = 3). The Western blot data are representative of those from at least three independent experiments.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot, Incubation, Software

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: c-Fos and p65 elements regulate MMP8 promoter activity. (A) N2a cells were transfected with MMP8 promoter reporter plasmids (TSS-500, TSS-1000, TSS-1500, TSS-2000, and TSS-2500) for 24 h. Then cells were either infected with CVS at an MOI of 0.1 or left uninfected, and the luciferase activities were measured after a further 24 h of infection. (B) N2a cells were transfected with the MMP8 promoter reporter plasmid: TSS-500. At 24 h posttransfection, cells were infected with CVS at an MOI of 0.1, and at different time points after RABV infection, the culture medium was replaced with different signaling pathway-specific inhibitors (p38 inhibitor, 1 μM, 24 h; NF-κB inhibitor, 5 μM, 24 h; JNK inhibitor, 0.5 μM, 12 h; ERK inhibitor, 5 μM, 24 h; MAPK and NF-κB inhibitor, 20 μM, 24 h) or DMSO. After 24 h infection, MMP8 promoter luciferase activity was measured. (C) Schematic representation of point mutations in the wild-type promoter reporter (TSS-500). N2a cells were transfected with the MMP8 promoter reporters (TSS-500 and its mutants). At 24 h posttransfection, cells were either left uninfected or infected with CVS at an MOI of 0.1, and luciferase activity was measured after 24 h of infection. (D) N2a cells were cotransfected with MMP8 promoter reporter (TSS-500) plasmid or HA-c-Fos- or Flag-p65-expressing plasmid for 24 h. Dual-luciferase assays measured the MMP8 promoter activities. The expression of HA-c-Fos and Flag-p65 was determined by Western blotting. (E and G) N2a cells were mock infected (top) or infected with CVS (bottom) at an MOI of 0.1. (E) At 36 h.p.i., the cells were fixed, and c-Fos proteins (left), RABV-N (middle), and cell nuclei (right) were stained as described in the text. (G) At 36 h.p.i., the cells were fixed, and p65 proteins (left), RABV-N (middle), and cell nuclei (right) were stained as described in the text. (F and H) N2a cells were transfected with plasmids encoding HA-c-Fos or Flag-p65 for 24 h and then infected with CVS at an MOI of 0.1. Fixed chromatin from N2a cells was prepared and immunoprecipitated by anti-HA or anti-Flag antibodies. ChIP primers were designed to amplify the region containing respectively c-FOS and p65 binding site in the MMP8 promoter. PCR products were separated by acrylamide gel electrophoresis. Statistical analysis of comparisons between groups was carried out by the Student t test. The bar graph shows the means ± SD (n = 3). Scale bar = 10 μm. Western blot data are representative of those from at least two independent experiments.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Activity Assay, Transfection, Infection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Staining, Immunoprecipitation, Binding Assay, Acrylamide Gel Assay, Electrophoresis

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: Effect of MMP8 expression on RABV replication and viral pathogenicity. (A) Schematic diagram of the construction of rRABV and rRABV-MMP8. (B) N2a cells were infected with rRABV or rRABV-MMP8 at an MOI of 0.1. At 36 h.p.i., the cells were fixed and stained to detect RABV-N and MMP8. (C) N2a cells were infected with rRABV or rRABV-MMP8 at an MOI of 0.1. At 36 h.p.i., cells were harvested, and the expression levels of RABV-N and MMP8 were detected by Western blotting. (D and E) N2a (D) and BSR cells (E) were infected with rRABV or rRABV-MMP8 at an MOI of 0.01 for the indicated times, and the supernatants were harvested for virus titration. (F) Female C57BL/6 mice (n = 10) were i.c. infected with 20 FFU of rRABV or rRABV-MMP8. The survival ratio was monitored daily (means ± SD; the survival ratio was analyzed by log rank test). (G) The mRNA levels of RABV-N in the whole brain were analyzed using qPCR at 2, 4, and 6 d.p.i. Statistical analysis of comparisons between groups was carried out by the Student t test. The bar graph shows the means ± SD. Scale bar = 200 μm. Western blot data are representative of those from at least two independent experiments. NS, not significant.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Expressing, Infection, Staining, Western Blot, Virus, Titration

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: MMP8 expression enhances BBB permeability during RABV infection. C57BL/6 mice (n = 3) were i.c. inoculated with 20 FFU of rRABV or rRABV-MMP8. (A) At 2, 4, and 6 d.p.i., mice were injected intraperitoneally with 100 μL of NaF (100 mg/mL), and the BBB integrity was calculated based on NaF uptake in the brains. (B) At 4 d.p.i., mouse brains were harvested and used for further analysis. In mouse brains, protein levels of occludin, claudin-5, and ZO-1 were measured by Western blotting, and β-actin was included as a control. (C) At 4 d.p.i., mouse brains were harvested, homogenized, and centrifuged, and then the supernatants were harvested and treated with UV light for 30 min to eliminate live viruses. bEnd.3 cells in a Transwell model were treated with the supernatants, and the transendothelial permeability assay was performed to evaluate the integrity of bEnd.3 cells. (D and E) bEnd.3 cells were treated with supernatants for 48 h and then harvested to measure the occludin, claudin-5, and ZO-1 levels using Western blotting (D) and immunofluorescence (E). The mean fluorescence intensity of occludin, claudin-5, and ZO-1 in the respective groups was calculated using ImageJ software. Statistical analysis of comparisons between groups was carried out by the Student t test. The data are presented as means ± SD (n = 3). Scale bar = 200 μm. Western blot data are representative of those from at least two independent experiments.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Expressing, Permeability, Infection, Injection, Western Blot, Control, Immunofluorescence, Fluorescence, Software

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: MMP8 degrades TJ proteins during RABV infection. C57BL/6 mice (n = 3) were i.c. inoculated with 20 FFU of rRABV or mock infected with DMEM at the same volume. At 6 d.p.i., mouse brains were harvested and used for further analysis. (A and C) bEnd.3 cells were treated with supernatants (prepared as for Fig. 5C) in the presence of MMP8 specific inhibitors (10 μM) or DMSO for 48 h and then harvested to measure the occludin, claudin-5, and ZO-1 levels by Western blotting (A) and immunofluorescence (C). (B) bEnd.3 cells in a Transwell model were treated with supernatants, and a transendothelial permeability assay was performed to evaluate the integrity of bEnd.3 cells. (D) The occludin, claudin-5, and ZO-1 expression levels in bEnd.3 cells were determined by Western blotting after treatment with MMP8 recombinant protein at the indicated concentrations. (E) bEnd.3 cells in a Transwell model were treated with MMP8 recombinant protein at the indicated concentrations, and a transendothelial permeability assay was performed to evaluate the integrity of bEnd.3 cells. (F and G) Two groups of C57BL/6 mice (n = 3) were i.c. inoculated with 20 FFU of CVS. One group of C57BL/6 mice (n = 3) were i.c. inoculated with the same volume of DMEM. MMP8 selective inhibitor was dissolved in 1% DMSO in PBS to a final concentration of 0.01 mg/mL. The CVS-infected mice received a 1-mg/kg (of body weight) dose of inhibitor or the same volume of a vehicle (1% DMSO in PBS) at 2 and 4 d.p.i. At 6 d.p.i., mice were injected intraperitoneally with 100 μL of NaF (100 mg/mL), and the BBB integrity was calculated based on NaF uptake in the brains (F). The infected mouse brains at 6 d.p.i. were collected, and the occludin, claudin-5, and ZO-1 expression levels were determined by Western blotting (G). The mean fluorescence intensity of occludin, claudin-5, and ZO-1 in the respective groups was calculated using ImageJ software. Statistical differences between virus-infected cells and mock-infected cells were determined using the Student t test. The data are presented as means ± SD (n = 3). Scale bar = 200 μm. Western blot data are representative of those from at least two independent experiments.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Infection, Western Blot, Immunofluorescence, Permeability, Expressing, Recombinant, Concentration Assay, Injection, Fluorescence, Software, Virus

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: MMP8 expression increases the cytokine production and infiltration of inflammatory cells into the CNS. C57BL/6 mice (n = 3) were i.c. inoculated with 20 FFU of rRABV or rRABV-MMP8 or the same volume of DMEM (mock). (A to D) At 2, 4, 6 d.p.i., brains were harvested, and the transcription levels of TNF-α (A), IL-6 (B), CCL5 (C), and CXCL10 (D) were analyzed by qPCR. (E) At 6 d.p.i., brains were harvested, and the expression and phosphorylation of the key molecules in the NF-κB and MAPK pathways were measured by Western blotting. (F to J) At 4 d.p.i., mouse brains were homogenized, and leukocytes were isolated for analysis of macrophages (CD45hi CD11b+), B cells (CD11c− CD45+ B220+), neutrophils (CD45hi Ly6G+), CD4+ T cells (CD45+ CD4+), and CD8+ T cells (CD45+ CD8+). A total of 20,000 events were acquired for cytometry data collection. Statistical analysis of comparisons between groups was carried out by the Student t test. The data are presented as means ± SD (n = 3).

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Isolation, Cytometry

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: Schematic representation of mechanisms for MMP8 to regulate BBB permeability change during RABV infection. Lab-attenuated RABV rather than wt RABV can cause the activation of immune cells and lead to the activation of inflammation via the NF-κB and MAPK pathways. Translocated NF-κB (p65) and AP-1 (c-Fos) bind to the MMP8 promoter region and enhance its transcription, resulting in the upregulation of MMP8 production. Secreted MMP8 leads to the degradation of tight junction proteins, including occludin, claudin-5, and ZO-1, between the endothelial cells, resulting in disruption of the BBB.

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Permeability, Infection, Activation Assay, Disruption

Journal: Journal of Virology

Article Title: Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8

doi: 10.1128/jvi.01050-22

Figure Lengend Snippet: Primers used for qPCR

Article Snippet: The JNK inhibitor (no. GC13841), the NF-κB inhibitor (no. GC11751), the p38 inhibitor (no. GC18602), the ERK inhibitor (no. GC43624), the MAPK and NF-κB inhibitor (no. GC35411), and the MMP8 inhibitor (no. GC18616) were all purchased from Glpbio (Montclair, CA, USA).

Techniques: Sequencing