Glucocorticosteroids Differentially Regulate MMP-9 and Neutrophil Elastase in COPD

Background: Chronic Obstructive Pulmonary Disease (COPD) is currently the fifth leading cause of death worldwide. Neutrophilic inflammation is prominent, worsened during infective exacerbations and is refractory to glucocorticosteroids (GCs). Deregulated neutrophilic inflammation can cause excessive matrix degradation through proteinase release. Gelatinase and azurophilic granules within neutrophils are a major source of matrix metalloproteinase (MMP)-9 and neutrophil elastase (NE), respectively, which are elevated in COPD. Methods: Secreted MMP-9 and NE activity in BALF were stratified according to GOLD severity stages. The regulation of secreted NE and MMP-9 in isolated blood neutrophils was investigated using a pharmacological approach. In vivo release of MMP-9 and NE in mice exposed to cigarette smoke (CS) and/or the TLR agonist lipopolysaccharide (LPS) in the presence of dexamethasone (Dex) was investigated. Results: Neutrophil activation as assessed by NE release was increased in severe COPD (36-fold, GOLD II vs. IV). MMP-9 levels (8-fold) and activity (21-fold) were also elevated in severe COPD, and this activity was strongly associated with BALF neutrophils (r = 0.92, p < 0.001), but not macrophages (r = 0.48, p = 0.13). In vitro, release of NE and MMP-9 from fMLP stimulated blood neutrophils was insensitive to Dex and attenuated by the PI3K inhibitor, wortmannin. In vivo, GC resistant neutrophil activation (NE release) was only seen in mice exposed to CS and LPS. In addition, GC refractory MMP-9 expression was only associated with neutrophil activation. Conclusions: As neutrophils become activated with increasing COPD severity, they become an important source of NE and MMP-9 activity, which secrete proteinases independently of TIMPs. Furthermore, as NE and MMP-9 release was resistant to GC, targeting of the PI3K pathway may offer an alternative pathway to combating this proteinase imbalance in severe COPD

Constitutive Activation of the Src Family Kinase Hck Results in Spontaneous Pulmonary Inflammation and an Enhanced Innate Immune Response

To identify the physiological role of Hck, a functionally redundant member of the Src family of tyrosine kinases expressed in myelomonocytic cells, we generated HckF/F “knock-in” mice which carry a targeted tyrosine (Y) to phenylalanine (F) substitution of the COOH-terminal, negative regulatory Y499-residue in the Hck protein. Unlike their Hck−/− “loss-of-function” counterparts, HckF/F “gain-of-function” mice spontaneously acquired a lung pathology characterized by extensive eosinophilic and mononuclear cell infiltration within the lung parenchyma, alveolar airspaces, and around blood vessels, as well as marked epithelial mucus metaplasia in conducting airways. Lungs from HckF/F mice showed areas of mild emphysema and pulmonary fibrosis, which together with inflammation resulted in altered lung function and respiratory distress in aging mice. When challenged transnasally with lipopolysaccharide (LPS), HckF/F mice displayed an exaggerated pulmonary innate immune response, characterized by excessive release of matrix metalloproteinases and tumor necrosis factor (TNF)α. Similarly, HckF/F mice were highly sensitive to endotoxemia after systemic administration of LPS, and macrophages and neutrophils derived from HckF/F mice exhibited enhanced effector functions in vitro (e.g., nitric oxide and TNFα production, chemotaxis, and degranulation). Based on the demonstrated functional association of Hck with leukocyte integrins, we propose that constitutive activation of Hck may mimic adhesion-dependent priming of leukocytes. Thus, our observations collectively suggest an enhanced innate immune response in HckF/F mice thereby skewing innate immunity from a reversible physiological host defense response to one causing irreversible tissue damage

Inhibition of Nox2 Oxidase Activity Ameliorates Influenza A Virus-Induced Lung Inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8+ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2−/y mice. The numbers of influenza-specific CD8+DbNP366+ and DbPA224+ T cells in the BALF and spleen were comparable in WT and Nox2−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner

TLR7 promotes chronic airway disease in RSV-infected mice

Respiratory syncytial virus (RSV) commonly infects the upper respiratory tract (URT) of humans, manifesting with mild cold or flu-like symptoms. However, in infants and the elderly, severe disease of the lower respiratory tract (LRT) often occurs and can develop into chronic airway disease. A better understanding of how an acute RSV infection transitions to a LRT chronic inflammatory disease is critically important to improve patient care and long-term health outcomes. To model acute and chronic phases of the disease, we infected wild-type C57BL/6 and toll-like receptor 7 knockout (TLR7 KO) mice with RSV and temporally assessed nasal, airway and lung inflammation for up to 42 days post-infection. We show that TLR7 reduced viral titers in the URT during acute infection but promoted pronounced pathogenic and chronic airway inflammation and hyperreactivity in the LRT. This study defines a hitherto unappreciated molecular mechanism of lower respiratory pathogenesis to RSV, highlighting the potential of TLR7 modulation to constrain RSV pathology to the URT

Non-essential role for TLR2 and its signaling adaptor Mal/TIRAP in preserving normal lung architecture in mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr42/2 mice by 6 months of age, the lungs of Tlr22/2 mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr42/2 mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal2/2 mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal2/2 mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr42/2 mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema