Matrix Metalloprotease 9 Mediates Neutrophil Migration into the Airways in Response to Influenza Virus-Induced Toll-Like Receptor Signaling

The early inflammatory response to influenza virus infection contributes to severe lung disease and continues to pose a serious threat to human health. The mechanisms by which neutrophils gain entry to the respiratory tract and their role during pathogenesis remain unclear. Here, we report that neutrophils significantly contributed to morbidity in a pathological mouse model of influenza virus infection. Using extensive immunohistochemistry, bone marrow transfers, and depletion studies, we identified neutrophils as the predominant pulmonary cellular source of the gelatinase matrix metalloprotease (MMP) 9, which is capable of digesting the extracellular matrix. Furthermore, infection of MMP9-deficient mice showed that MMP9 was functionally required for neutrophil migration and control of viral replication in the respiratory tract. Although MMP9 release was toll-like receptor (TLR) signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil migration. These results were extended using multiplex analyses of inflammatory mediators to show that neutrophil chemotactic factor, CCL3, and TNFα were reduced in the Myd88−/− airways. Furthermore, TNFα induced MMP9 secretion by neutrophils and blocking TNFα in vivo reduced neutrophil recruitment after infection. Innate recognition of influenza virus therefore provides the mechanisms to induce recruitment of neutrophils through chemokines and to enable their motility within the tissue via MMP9-mediated cleavage of the basement membrane. Our results demonstrate a previously unknown contribution of MMP9 to influenza virus pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral replication that could be exploited for therapeutic purposes

Neutrophils require MMP9 to migrate to the respiratory tract.

<p>(A) Neutrophil numbers in the BAL (<i>left panel</i>) and lung (<i>right panel</i>) were enumerated by flow cytometry 3 days after infection of C57BL/6 mice or <i>Mmp9</i>^−/− mice. Control mice were not infected. Mean recovery numbers per lung ± SEM (n = 3–4, representative of three independent experiments). (B) Viral load was enumerated in left lung lobe by quantitative RT-PCR for the influenza PA gene 3 days after infection. Mean PA copies/lung ± SEM (n = 4). (C) Chemokine levels in airways of <i>Mmp9</i>^−/− mice after infection. BALs were collected 3 and 6 days after infection and supernatants assayed for CXCL1 by bead array. Mean ± SEM (n = 4, representative of two independent experiments). (A–C) Considered significant at *<i>P</i><0.05, **<i>P</i><0.01.</p

MyD88-dependent TNFα expression induces MMP9 in neutrophils.

<p>(A–B) BALs of C57BL/6 (<i>grey bars</i>) and <i>Myd88</i>^−/− (<i>clear bars</i>) mice infected 3 or 6 days earlier were analyzed by bead array. Airway levels of (A) CCL3 and (B) TNFα. Mean ± SEM (n = 3, representative of two independent experiments). (C–D) TNFα function was blocked by injecting C57BL/6 mice with 200 µg anti-TNFα (αTNFα, <i>clear bars</i>) or IgG isotype (IgG, <i>grey bars</i>) daily starting one day before infection. (C) The percentage of neutrophils in lungs of antibody-treated mice was enumerated using flow cytometry 3 days after infection. (D) The number of cells secreting MMP9 in the lungs was assessed by ELISPOT 3 days after infection. Mean ± SEM (n = 5, representative of two independent experiments). (E) Neutrophils were negatively enriched from the bone marrow of C57BL/6 mice and stimulated with different doses of recombinant TNFα for 6 hours. MMP9 expression during the 6 hour incubation was assessed by ELISPOT (n = 3, representative of two independent experiments). (A–C) Considered significant at *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Morbidity, inflammation, and neutrophil number are increased after infection with high dose influenza virus.

<p>(A) Kinetics of weight loss as a percentage of starting weight in C57BL/6 mice infected i.n. with 125 (<i>light-grey squares</i>), 1250 (<i>dark-grey triangles</i>), or 12500 (<i>black circles</i>) EID₅₀ PR8 virus. Mean ± SEM (n = 5, representative of four independent experiments). (B) Cytokine and chemokine levels in the BAL of uninfected mice (‘control’, <i>clear bars</i>) or mice infected 6 days earlier with 125 (<i>light-grey bars</i>), 1250 (<i>grey bars</i>), or 12500 (<i>dark-grey bars</i>) EID₅₀ PR8 virus. Mean ± SEM (n = 3–4, representative of two independent experiments). (C) Histological examination of uninfected lungs (control, <i>left</i>) or lungs 6 days after infection with 125 (<i>middle</i>) or 12500 (<i>right</i>) EID₅₀ PR8 virus. Perfused lungs were fixed in formalin and stained for hematoxylin and eosin (scalebar = 100 µm). Images are representative of multiple mice. b. bronchiole, a. arteriole, av. alveole, and arrowhead. venule. (D) Neutrophil numbers in the BAL and lung of uninfected mice (‘control’, <i>clear bars</i>) or mice infected 6 days earlier with 125 (<i>light-grey bars</i>) or 12500 (<i>dark-grey bars</i>) EID₅₀ PR8 virus. Mean ± SEM (n = 3, representative of two independent experiments). (E) The percentage of blood neutrophils from LysM-GFP mice infected with 125 (<i>light-grey squares</i>) or 12500 (<i>black circles</i>) EID₅₀ PR8 virus compared to control (PBS, <i>open squares</i>) or allantoic fluid at the same dilution as the highest viral dose (<i>open triangles</i>). Mean ± SEM (n = 4–5).</p

MMP9 is produced by neutrophils after infection.

<p>(A) MMP2/9 activity in lungs of uninfected control mice (<i>left panel</i>) or those 6 days after infection (<i>right panel</i>). Gelatinase activity was measured by <i>in situ</i> zymogram and visualized by green fluorescence after enzymatic cleavage to release fluorochrome from a quencher (scalebar = 50 µm). Images are representative of multiple mice. (B) MMP9 secretion by cells from BAL and lung from Ly5.1 recipient mice that received BM from either <i>Mmp9</i>^−/− (<i>clear bars</i>) or C57BL/6 (<i>grey bars</i>) donors was measured by ELISPOT. Mean ± SEM (n = 5–7, representative of two independent experiments). (C) Ly6G+ lung neutrophils (<i>red</i>) and MMP9+ cells (<i>green</i>), and their colocalization (<i>merge, right panel</i>) were visualized by immunofluorescence of infected lung 6 days after infection (scalebar = 50 µm). Images are representative of multiple mice.</p

Depletion of neutrophils abrogates MMP9 secretion after influenza virus infection.

<p>Neutrophils were depleted by injecting C57BL/6 mice with 400 µg anti-Ly6G antibody (αLy6G) or isotype control (IgG) one day before infection and every other day thereafter. (A) Depletion of Ly6G+ cells in αLy6G-treated C57BL/6 mice (<i>bottom panel</i>) compared to IgG (<i>top panel</i>) was verified by flow cytometric analysis 6 days after infection. (B) Kinetics of weight loss as a percentage of starting weight in uninfected (control, <i>open symbols</i>) and infected (<i>closed symbols</i>) mice treated with the αLy6G (<i>squares</i>) or IgG isotype (<i>circles</i>). (C) MMP9 secretion by cells from BAL and lung from infected mice that were treated with αLy6G (<i>clear bars</i>) or IgG isotype (<i>grey bars</i>) was measured by ELISPOT. (D) Inflammatory cytokine release in airways after neutrophil depletion. BALs were collected 6 days after infection and supernatants assayed by bead array. (B–D) Mean ± SEM (n = 3, representative of two independent experiments). (B–C) Considered significant at *<i>P</i><0.05, ***<i>P</i><0.001.</p

Neutrophil recruitment, MMP9 secretion, and viral load are TLR-dependent.

<p>Neutrophil numbers and MMP9 secretion are decreased in TLR-deficient mice. (A–E) C57BL/6 (<i>grey bars</i>), <i>Myd88</i>^−/−, and <i>Tlr3</i>^−/− (<i>both clear bars</i>) mice were infected with 12500 EID₅₀ PR8. (A) The viral load was enumerated in left lung lobes of C57BL/6, <i>Myd88</i>^−/−, and <i>Tlr3</i>^−/− mice by quantitative RT-PCR for the influenza PA gene 3 days after infection. Mean PA copies/lung ± SEM (n = 4, representative of two independent experiments). (B, C) The percentage of neutrophils in lungs of <i>Myd88</i>^−/− and <i>Tlr3</i>^−/− mice, respectively, were enumerated by flow cytometry 6 days after infection. (D, E) The number of cells secreting MMP9 in the airways of mice deficient in TLR signaling. MMP9 ELISPOT analysis of BALs from (D) <i>Myd88</i>^−/− or (E) <i>Tlr3</i>^−/− mice. Mean ± SEM (n = 4–5, representative of two independent experiments). (F) Neutrophil recoveries in lung 3 days after infection of mice receiving bone marrow transfer. The percentage of neutrophils after transfer of C57BL/6 (WT>WT) or <i>Myd88</i>^−/− (KO>WT) bone marrow into Ly5.1 congenic mice (<i>grey bars</i>) or after transfer of Ly5.1 bone marrow cells into Ly5.2+ C57BL/6 (WT>WT) or <i>Myd88</i>^−/− (WT>KO) recipients (<i>clear bars</i>) is shown. (A–E) Mean ± SEM (n = 3, representative of two independent experiments). (A–F) Considered significant at *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p