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ABSTRACT: Rationale: Neutrophilic inflammation is an important pathological feature of COPD and infectious exacerbations of COPD. Serum Amyloid A (SAA) promotes neutrophilic inflammation via its interaction with lung mucosal ALX/FPR2 receptors, however little is known about how this endogenous mediator regulates IL-17A immunity. Objectives: The objectives of this study were to determine whether SAA causes neutrophilic inflammation via IL-17A dependent mechanisms. Methods: The relationship between SAA and neutrophils was investigated in lung sections from COPD patients and a chronic mouse model of SAA exposure. A neutralizing antibody to IL-17A was used to block SAA responses in vivo, and a cell sorting strategy was used to identify cellular sources. Measurements and Main Results: SAA mRNA expression was positively associated with tissue neutrophils in COPD (p < 0.05). SAA predominately promoted expression of the TH17 polarizing cytokine IL-6, which was opposed by 15-epi-lipoxin A4, a counter-regulatory mediator and ALX/FPR2 ligand. SAA-induced inflammation was markedly reduced by a neutralizing antibody to IL-17A in vivo. Cellular sources of IL-17A induced by SAA include CD4(+) T cells, γδ T cells and an Epcam(+)CD45(-) population enriched for epithelial cells. SAA promotes expression of IL-17A in γδ T cells and this innate cell proportionally expressed higher levels of IL-17A transcript than CD4(+) T cells or epithelial cells. Conclusions: The SAA-IL-17A axis represents an important innate defense network that may underlie persistent neutrophilic airway inflammation in COPD and modulating the ALX/FPR2 receptor represents a novel approach to targeting aberrant IL-17A mediated lung immunity.
American Journal of Respiratory and Critical Care Medicine 04/2013; · 11.08 Impact Factor
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ABSTRACT: Influenza A virus infection is an ongoing clinical problem and thus, there is an urgent need to understand the mechanisms that regulate the lung inflammation in order to unravel novel generic pharmacological strategies. Evidence indicates that the Nox2-containing NADPH oxidase enzyme promotes influenza A virus-induced lung oxidative stress, inflammation and dysfunction via ROS generation. In addition, lung epithelial and endothelial cells express the Nox1 isoform of NADPH oxidase, placing this enzyme at key sites to regulate influenza A virus-induced lung inflammation. The aim of this study was to investigate whether Nox1 oxidase regulates the inflammatory response and the oxidative stress to influenza infection in vivo in mice. Male WT and Nox1-deficient (Nox1(-/y)) mice were infected with the moderately pathogenic HkX-31 (H3N2, 1×10(4) PFU) influenza A virus for analysis of bodyweight, airways inflammation, oxidative stress, viral titre, lung histopathology, and cytokine/chemokine expression at 3 and 7 days post infection. HkX-31 virus infection of Nox1(-/y) mice resulted in significantly greater: loss of bodyweight (Day 3); BALF neutrophilia, peri-bronchial, peri-vascular and alveolar inflammation; Nox2-dependent inflammatory cell ROS production and peri-bronchial, epithelial and endothelial oxidative stress. The expression of pro-inflammatory cytokines including CCL2, CCL3, CXCL2, IL-1β, IL-6, GM-CSF and TNF-α was higher in Nox1(-/y) lungs compared to WT mice at Day 3, however, the expression of CCL2, CCL3, CXCL2, IFN-γ and the anti-inflammatory cytokine IL-10 were lower in lungs of Nox1(-/y) mice vs. WT mice at Day 7. Lung viral titre, and airways infiltration of active CD8(+) and CD4(+) T lymphocytes, and of Tregs were similar between WT and Nox1(-/y) mice. In conclusion, Nox1 oxidase suppresses influenza A virus induced lung inflammation and oxidative stress in mice particularly at the early phases of the infection. Nox1 and Nox2 oxidases appear to have opposing roles in the regulation of inflammation caused by influenza A viruses.
PLoS ONE 01/2013; 8(4):e60792. · 4.09 Impact Factor
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ABSTRACT: Reactive oxygen species produced during the innate immune response to LPS are important agents of anti-pathogen defence but may also cause oxidative lung damage. Glutathione peroxidase-1 (gpx-1) is an anti-oxidant enzyme that may protect lungs from such damage. We assessed the in vivo importance of gpx-1 in LPS-induced lung inflammation. Male wild-type (WT) or gpx-1 deficient (gpx-1(-/-)) mice were treated intranasally with PBS or 10 µg LPS and killed 3 and 24 h post LPS. Lungs were lavaged with PBS and then harvested for inflammatory marker expression. LPS caused an intense neutrophilia in WT BALF evident 3 and 24 h post challenge that was reduced in gpx-1(-/-) mice. In addition, LPS-treated gpx-1(-/-) mice had significantly fewer macrophages than LPS-treated WT mice. To understand the basis for this paradoxical reduction we assessed inflammatory cytokines and proteases at protein and transcript levels. MMP-9 expression and net gelatinase activity in BALF of gpx-1(-/-) mice treated with LPS for 3 and 24 h was no different to that found in LPS-treated WT mice. BALF from LPS-treated gpx-1(-/-) mice (3 h) had less TNF-α, MIP-2 and GM-CSF protein than LPS-treated WT mice. In contrast, LPS-induced increases in TNF-α, MIP-2 and GM-CSF mRNA expression in WT mice were similar to those observed in gpx-1(-/-) mice. These attenuated protein levels were unexpectedly not mirrored by reduced mRNA transcripts but were associated with increased 20S proteasome expression. Thus, these data suggest that gpx-1 primes pro-inflammatory cytokine production after LPS challenge in vivo.
PLoS ONE 01/2012; 7(3):e33172. · 4.09 Impact Factor
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ABSTRACT: Oxidants in cigarette smoke inhibit pathogen recognition receptor function and phagocytosis, but the molecular basis of this inhibition remains obscure. We sought to identify the inhibitory mechanisms that impair alveolar macrophage function. Balb/c mice were acutely exposed to four cigarettes for 4 hours before treatment with intranasal LPS (1 μg). The mice exhibited significantly reduced airway neutrophilia and expression of TNF-α. Balb/c-derived MH-S alveolar macrophage cells exposed to cigarette smoke extract (CSE) displayed a similar inhibitory response to stimulation with LPS. The induction of inflammatory genes by recombinant (r) TNF-α (100 ng/ml) was also impaired by CSE. Because both pathways converge on NF-κB, the degradation of IκBα and the phosphorylation of p65 were assessed and shown to be blunted by CSE. CSE also blocked the activity of activator protein-1 (AP-1) by inhibiting p38 mitogen activated protein kinase (MAPK) in a reduced glutathione (GSH)-reversible manner. The induction of specific Toll-like receptor (TLR)-negative regulators (suppressor of cytokine signaling-1 [SOCS-1], interleukin-1 receptor associated kinase-M [IRAK-M], and IL-10) did not account for the impaired responses of TLRs. As free radical species are abundant in CSE and GSH restored function, a panel of oxidative/nitrosative stress markers was screened using immunocytochemistry. The panel identified protein carbonylation as the major CSE-inducible marker. Oxyblot analysis confirmed that CSE potently introduced carbonyl groups to many proteins in a dose-dependent and time-dependent manner that inversely correlated with the expression of TNF-α. The formation of pseudopodia was not prevented, but these membrane extensions were heavily carbonylated, and primary alveolar macrophages were also targeted for carbonylation. Oxidants in cigarette smoke drive a rapid, persistent, and global protein carbonylation that may represent a common pathway to altered immunity in disease.
American Journal of Respiratory Cell and Molecular Biology 10/2010; 45(2):229-36. · 5.13 Impact Factor
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ABSTRACT: Reactive oxygen species (ROS) produced from cigarette smoke cause oxidative lung damage including protein denaturation, lipid peroxidation, and DNA damage. Glutathione peroxidase-1 (gpx-1) is a detoxifying enzyme that may protect lungs from such damage. The aim of this study was to determine whether gpx-1 protects the lung against oxidative stress-induced lung inflammation in vivo. Male wild-type (WT) or gpx-1(-/-) mice were exposed to cigarette smoke generated from nine cigarettes per day for 4 days to induce oxidative stress and lung inflammation. The effect of the gpx mimetic ebselen on cigarette smoke-induced lung inflammation was evaluated when given prophylactically and therapeutically, i.e., during established inflammation. Mice were killed, and the lungs were lavaged with PBS and then harvested for genomic and proteomic analysis. Gpx-1(-/-) mice exposed to cigarette smoke had enhanced BALF neutrophils, macrophages, proteolytic burden, whole lung IL-17A, and MIP1alpha mRNA compared with WT mice. The gpx mimetic ebselen (10 and 100 microM) inhibited cigarette smoke extract-induced oxidation of MH-S cells in vitro and inhibited cigarette smoke-induced increases in BALF macrophages, neutrophils, proteolytic burden, and macrophage and neutrophil chemotactic factor gene expression when administered prophylactically. In addition, ebselen inhibited established BALF inflammation when administered therapeutically. These data show that gpx-1 protects against cigarette smoke-induced lung inflammation, and agents that mimic the actions of gpx-1 may have therapeutic utility in inflammatory lung diseases where cigarette smoke plays a role.
AJP Lung Cellular and Molecular Physiology 09/2010; 299(3):L425-33. · 3.66 Impact Factor
