Fig 1.
RSV infection induces host MMP9 expression and activity in mouse lungs.
FVB/NJ mice were infected with 1x106 pfu of RSV and a group of animals were euthanized at 0, 1, 3, 5 and 7 days post infection (dpi). (A) Mmp9 lung gene expression was analyzed by qPCR. Graph are represented as relative quantification (RQ) of the mean ± S.E.M. (B) BALF MMP9 levels were determined by multiplex analysis on mock treated and RSV infected animals. (C) Gelatinase activity was analyzed in BALF using gelatin zymography. Bands corresponding to MMP-2 and MMP9 are highlighted and densitometry was performed for MMP9 bands from data pooled from 3 separate gels. Graphs are represented as mean ± S.E.M., where each measurement was performed 3 times on 10 animals/group. (A-B) p values shown, comparing both treatments connected by a line. (C) *Represents a p value less than 0.05 compared to mock treated mice on same day. All comparisons were determined by student t-tests.
Fig 2.
MMP9 prevents RSV infectivity of human airway epithelial cells and mouse lungs.
(A) SAE cells were treated with various concentrations of active and inactive human MMP9 and TCID50 assays were performed 48 hours later. (B) RSV was treated with various concentrations of active and inactive MMP9 prior to infecting SAE cells. TCID50 assays were performed to determine the quantity of virus following MMP9 treatment. * Represents a p value less than 0.05 comparing inactive to active MMP9 for each concentration. All comparisons were determined by student t-tests. (C) Mmp9-/- mice and their FVB/NJ WT littermates were infected with 1x106 pfu of RSV and animals were euthanized 1, 3, 5 and 7 dpi. Plaque assays and RSV N copy number, by qPCR (on 7 dpi), confirmed viral titers in lung tissue from all RSV-infected animals. Graphs are represented as mean ± S.E.M, with each measurement performed 3 times on 10 animals/group. Two-way ANOVA was used to compare the time-course curves and multiple comparisons were determined by the Bonferroni method (left panel). p value shown for qPCR (right panel) comparing both treatments connected by a line, determined by student t-tests.
Fig 3.
Loss of Mmp9 expression enhances RSV infection-induced weight loss, AHR and reduces apoptosis in mice.
Mmp9-/- mice and their FVB/NJ WT littermates were infected with 1x106 pfu of RSV and animals were euthanized 7 dpi. (A) Animal body weight and (B) AHR to increasing doses of methacholine were assessed in each animal group 7 dpi. (C) BALF cells undergoing apoptosis were determined 7 dpi. Graphs are represented as mean ± S.E.M, with each measurement performed 3 times on 10 animals/group. Two-way ANOVA was used to compare the time-course curves and multiple comparisons were determined by the Bonferroni method (A and B). p values shown, comparing both treatments connected by a line by student t-tests (C).
Fig 4.
MMP9 expression regulates neutrophil recruitment to the lung during RSV infection.
Mmp9-/- mice and their FVB/NJ WT littermates were infected with 1x106 pfu of RSV and animals were euthanized 0, 1, 3, 5 and 7 dpi. (A) BALF total immune cellularity and alveolar macrophages (7 dpi) were determined. Comparative histology images of infected animals from each background are presented here (scale bar = 40 μm). (B) A typical representation of neutrophils from WT and Mmp9-/- mice 1 dpi of RSV, gated as SSChighCD11b+Gr-1+. Mean number of neutrophils from whole lung of WT and Mmp9-/- mice on days 0, 1, 3, 5, and 7 dpi. (C) Lung protein extracts were assayed for myeloperoxidase (MPO) activity from RSV-infected animals. A typical representation of BALF cells from WT and Mmp9-/- mice 1 dpi following cytospin and Diff-Quik staining. Graphs are represented as mean ± S.E.M, where each measurement was performed 3 times on 10 animals/group. Two-way ANOVA was used to compare the time-course curves and multiple comparisons were determined by the Bonferroni method. * Represents a p value less than 0.05 compared to WT mice on same day.
Fig 5.
MMP9 regulates cytokine/chemokine expression during RSV infection.
Mmp9-/- mice and their FVB/NJ WT littermates were infected with 1x106 pfu of RSV and animals were euthanized 7 dpi. (A) Cytokine gene expression in lung tissue and BALF levels of RANTES, IL-1β, SCF and G-CSF were determined in both mouse genotypes, 7 dpi. (B) BALF levels of CXCL5, IL-13, CXCL2, TNF-α, MCP-1, IL-17, IL-10 and CXCL1 were determined in both mouse genotypes, 7 dpi. Graphs are represented as mean ± S.E.M, where each measurement performed 3 times on 10 animals/group. p values shown, comparing both treatments connected by a line. All comparisons were determined by student t-tests.
Fig 6.
Loss of MMP9 expression blunts p38 responses to RSV infection.
p38 activation was determined in (A) whole lung tissue and (B) bone marrow derived macrophages (BMDM) from WT and Mmp9-/- mice following RSV infection, using multiplex antibodies for the phosphorylated and total forms of each protein. (C) Immunoblots (p-p38, p38 and actin) were performed on SAE cells following MMP9 silencing and RSV infection for 24 hours. Densitometry analysis was determined. Densitometry units (DU) represent pixel intensity of phosphorylated p38 as a ratio of total p38. (A) Two-way ANOVA was used to compare the time-course curves and multiple comparisons were determined by the Bonferroni method. (B-C) p values shown comparing both treatments connected by a line, determined by student t-tests.
Fig 7.
RANTES, G-CSF and IL-8 expressions are p38-dependent during RSV infection.
SAE cells were transfected with p38 siRNA and infected with RSV for 24 hours. Immunoblots (p38 and actin) were performed on SAE cells to confirm reduced p38 protein translation. Cytokine gene expression and release from cells were analyzed for RANTES, IL-1β, G-CSF and SCF. IL-8 release was determined in cell media. Results are represented as relative quantification (RQ) and mean cytokine concentration ± S.E.M. Each measurement was performed 3 times on 6 replicates/group. p values shown, comparing both treatments connected by a line, determined by student t-tests.
Fig 8.
Extracellular active MMP9 induces IL-8 secretion from airway cells.
SAE cells were treated with various concentrations of active and inactive human MMP9 for 24 hours. Cytokine gene expression and release from cells were analyzed for RANTES, IL-1β, G-CSF and SCF. IL-8 levels were determined in cell media. Results are represented as relative quantification (RQ) and mean cytokine concentration ± S.E.M. Each measurement was performed 3 times on 6 replicates/group. p value shown, comparing both treatments connected by a line, determined by student t-tests.
Fig 9.
Proposed pathway for MMP9 signaling during RSV infection.