Although implicated in a variety of pulmonary diseases, most notably asthma and tuberculosis, a detailed understanding of the involvement of gelatinases in lung disease pathology is lacking. In this study, we addressed the contribution of MMP9 during influenza virus pathogenesis, which had been implicated recently by virtue of increased concentrations in the serum of patients with influenza and elevated enzymatic activity in mouse lung homogenates 
. However, the cellular origin and direct role of MMPs in the pathogenesis of influenza virus have not been previously examined. Thus, we focused on elucidating the effects of MMP9 in a pathological mouse model and identified neutrophils as the predominant pulmonary source of MMP9. We demonstrated that MMP9 mediated neutrophil migration into the infected respiratory tract and that it was required for viral clearance. Although MMP9 release was TLR signaling-dependent, MyD88-mediated signals in non-hematopoietic cells, rather than neutrophil TLRs themselves, were important for neutrophil recruitment. This is most likely due to MyD88-controlled induction of the known neutrophil chemoattractant, CCL3, as well as TNFα, which we found induced MMP9 secretion in neutrophils. Our data demonstrate a previously unknown role of MMP9 to influenza virus pathogenesis by contribution to excessive neutrophil migration into the respiratory tract.
Using a model wherein a lethal dose of virus mimics many of the characteristics observed after pandemic infections, with greater cytokine/chemokine levels, lung pathology, and inflammatory cells 
, we showed that MMP9 activity was upregulated in infected lungs, confirming data published recently 
. We extended these studies by demonstrating that MMP9 was exclusively produced by hematopoietic cells. It is surprising that non-hematopoietic cells did not contribute to MMP9 secretion, as it can be induced under inflammatory conditions in lung epithelial and endothelial cells 
. Nevertheless, our data further indicated that MMP9 was predominantly secreted in the lung by neutrophils. Neutrophil depletion caused a significant reduction in the number of cells being able to produce MMP9, but did not abrogate all MMP9 production, suggesting that other immune cells can also contribute. In a recent study, neutrophil depletion did not affect MMP9 levels in the BAL 5 days after sublethal influenza virus infection, which is at odds with our results 
. However, contributing factors to this discrepancy most likely involve the timing, which was later, and the infective dose, which was lower. Nevertheless, excessive neutrophil infiltration into the lung correlated with increased MMP9 production and viral replication when macrophages were depleted 
, which supports our findings. However, the source of MMP9 was not identified. We are currently investigating the potential role of other cell populations that are capable of producing MMP9 under inflammatory conditions, such as lymphocytes 
. However, unlike these cells, neutrophils do not require de novo
generation of MMP9 and instead are preloaded in the bone marrow and exocytose their granules following stimulation. In addition, they can synthesize and secrete cytokines and chemokines 
, although we were unable to demonstrate a significant contribution of this mechanism to airway inflammation, as depletion of neutrophils did not cause a significant difference in an extensive panel of potential candidates.
While neutrophils are a major cell population recruited to the lung early in the immune response to pandemic influenza viruses 
, their role during pathogenesis remains unclear. Depletion studies demonstrated enhanced susceptibility of mice to infection, exacerbated inflammation, and increased viral titers and mortality rates 
, which suggested a critical contribution of neutrophils to innate immunity. In contrast, other studies parallel ours and show that neutrophils contribute to disease during severe influenza virus infection 
. The disparate results may be a consequence of differential infective doses and genetic background. Nevertheless, we show for the first time that MMP9 may contribute to pathogenesis by mediating excessive neutrophil migration into the respiratory tract in response to viral infection. While depletion of neutrophils reduced morbidity, MMP9 was still required for viral clearance. It is likely that the same mechanism(s) that contribute to immunity cause pathology in a more virulent setting 
. Neutrophils are able to inhibit virus replication in vitro
and the release of anti-viral molecules by neutrophils could contribute to viral clearance from the lung 
. However, the mechanisms that allow for neutrophil influx in response to uncontrolled or high viral load, such as MMP9 proteolytic activity, may exacerbate pathology due to collateral destruction of the lung ECM. This could explain the apparent contradicting reports on the role of neutrophils in viral infections and suggests that, while they contribute to viral clearance, excessive numbers contribute to pathology.
We showed that MMP9 functions by regulating neutrophil localization to the respiratory tract. Cellular influx into the airways is a complex process whereby cells cross the endothelial cell layer, the basement membrane, and the epithelial cell layer. Although rolling and tethering on the endothelium has been described in detail, the means by which cells further progress through the basement membrane to reach the airways remain unclear 
. Whereas gelatinase activity has been implicated in neutrophil migration through artificial basement membranes in vitro
, its relevance in vivo
remains controversial 
. Chemokine-induced migration of neutrophils in an induced lung inflammation model was demonstrated to be independent of MMP9 
, whereas that in a dermal model did require MMP9 
. In part, this discrepancy may be due to the artificial nature of the experiments: while chemokines may be able to mimic inflammation and attract cells, crucial factors necessary for the release of MMP9 may not be available as they are not induced by the chemokine itself (see below). The presence of these required accessory molecules is assured in the influenza model, but at the same time can be difficult to define. Nevertheless, we were able to identify at least one, TNFα, which was able to induce MMP9. It is still of great interest to identify other factors during influenza virus infection as they have obvious clinical relevance. Thus, our data not only demonstrate a role for MMP9 in influenza pathogenesis, but also provide further in vivo
evidence for MMP9 activity as a mechanism by which neutrophils digest the extracellular matrix to traverse the basement membrane in order to gain access to the infected lung epithelium. The colocalization of MMP9 and viral antigen and its expression between the endothelium and areas of viral replication support this hypothesis. In addition to its role in mediating proteolytic cleavage of the basement membrane to allow for motility within the lung, MMP9 may also modulate chemokine expression itself by digesting CXCL-1 
. Our finding that CXCL-1, a potent neutrophil chemoattractant, is significantly upregulated in the Mmp9−/−
mice following infection hints at a negative feedback loop in which MMP9 release by neutrophils disrupts the CXCL-1 gradient and dampens further neutrophil recruitment in the absence of influenza virus.
We hypothesized that TLRs are important to induce MMP9 secretion from neutrophils. These innate pattern recognition receptors are expressed by neutrophils 
. Furthermore, viral RNA has been detected in neutrophils 
, which could facilitate access to TLRs inside endosomes, such as TLR3 and TLR7, which have both been shown to be able to recognize influenza virus. Indeed, TLR7 signaling was demonstrated to be able to activate neutrophils in response to influenza virus, although this was dependent on GM-CSF 
. Therefore, we examined the role of TLR signaling in neutrophil activation in Tlr3−/−
mice. MyD88 is the adapter molecule for all known TLRs with the exception of TLR3, and the inclusion of both strains therefore encompassed all potential TLR-mediated effects. Although both neutrophil accumulation and MMP9 secretion were reduced following high dose influenza virus infection, our bone marrow chimera approach demonstrated that this was not a direct effect as Myd88−/−
neutrophils still migrated into the respiratory tract. Furthermore, in vitro
stimulation with influenza virus of neutrophils enriched from Myd88−/−
mice did not affect MMP9 secretion (data not shown). Rather, Myd88−/−
signaling in non-hematopoietic cells was important for neutrophil accumulation. When we examined chemokine levels in the airways of Myd88−/−
mice, CCL3, a known neutrophil chemoattractant, was downregulated. Thus, it appears that rather than a direct effect on MMP9 activation, ablation of MyD88 signaling may have affected the chemokine gradient that would normally attract the neutrophils to the sites of viral replication. In addition, TNFα induced the release of MMP9 from neutrophils and antibody blocking of TNFα prevented their recruitment into the lungs in response to influenza virus infection in vivo
. TNFα has been demonstrated previously to induce MMP9 exocytosis in a protein kinase C-dependent fashion 
and we hypothesize that the reduced levels in the Myd88−/−
airways prevented MMP9 release and resulted in reduced neutrophil numbers.
The role of neutrophils in viral infections has often been overlooked, on the pretext that they are mostly involved in the clearance of bacterial and fungal pathogens. Their contribution to influenza virus-induced pathology is unclear, but we showed in our pathological model that they contributed to morbidity. Furthermore, we demonstrated that the mechanism for neutrophil-mediated migration to the respiratory tract in response to influenza required MMP9 and depended on extrinsic TLR-signaling. Collectively, our results suggest that innate sensing of viral infection results in a MyD88-dependent induction of chemotactic factors that induce the recruitment of neutrophils. Following diapedesis into the lung parenchyma, MyD88-induced TNFα in non-hematopoietic cells induces MMP9 release in neutrophils and MMP9 enzymatic activity is utilized by these cells to degrade the basement membrane and facilitate their motility within the lung. These novel findings are probably not restricted to influenza pathogenesis, but may also have implications for other viral infections. The dichotomy of the necessity for MMP9-mediated immune cell migration and its role in immunopathology provides real challenges for targeting MMP9 for therapeutic purposes after influenza virus infection. Nevertheless, finding the balance to modulate neutrophils allows for innate immunity to pandemic strains to be boosted whilst preventing potential pathology.