Matrix Metalloproteinase 9 Exerts Antiviral Activity against Respiratory Syncytial Virus

<div><p>Increased lung levels of matrix metalloproteinase 9 (MMP9) are frequently observed during respiratory syncytial virus (RSV) infection and elevated MMP9 concentrations are associated with severe disease. However little is known of the functional role of MMP9 during lung infection with RSV. To determine whether MMP9 exerted direct antiviral potential, active MMP9 was incubated with RSV, which showed that MMP9 directly prevented RSV infectivity to airway epithelial cells. Using knockout mice the effect of the loss of <i>Mmp9</i> expression was examined during RSV infection to demonstrate MMP9’s role in viral clearance and disease progression. Seven days following RSV infection, <i>Mmp9</i>^-/- mice displayed substantial weight loss, increased RSV-induced airway hyperresponsiveness (AHR) and reduced clearance of RSV from the lungs compared to wild type mice. Although total bronchoalveolar lavage fluid (BALF) cell counts were similar in both groups, neutrophil recruitment to the lungs during RSV infection was significantly reduced in <i>Mmp9</i>^-/- mice. Reduced neutrophil recruitment coincided with diminished RANTES, IL-1β, SCF, G-CSF expression and p38 phosphorylation. Induction of p38 signaling was required for RANTES and G-CSF expression during RSV infection in airway epithelial cells. Therefore, MMP9 in RSV lung infection significantly enhances neutrophil recruitment, cytokine production and viral clearance while reducing AHR.</p></div

MMP9 regulates cytokine/chemokine expression during RSV infection.

<p><i>Mmp9</i>^-/- 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.</p

RSV infection induces host MMP9 expression and activity in mouse lungs.

<p>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) <i>Mmp9</i> 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.</p

MMP9 expression regulates neutrophil recruitment to the lung during RSV infection.

<p><i>Mmp9</i>^-/- 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 <i>Mmp9</i>^-/- mice 1 dpi of RSV, gated as SSC^highCD11b⁺Gr-1⁺. Mean number of neutrophils from whole lung of WT and <i>Mmp9</i>^-/- 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 <i>Mmp9</i>^-/- 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.</p

Loss of MMP9 expression blunts p38 responses to RSV infection.

<p>p38 activation was determined in (A) whole lung tissue and (B) bone marrow derived macrophages (BMDM) from WT and <i>Mmp9</i>^-/- 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.</p

RANTES, G-CSF and IL-8 expressions are p38-dependent during RSV infection.

<p>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.</p

MMP9 prevents RSV infectivity of human airway epithelial cells and mouse lungs.

<p>(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) <i>Mmp9</i>^-/- 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.</p

Proposed pathway for MMP9 signaling during RSV infection.

<p>Proposed pathway for MMP9 signaling during RSV infection.</p