Astrid Haegens

Myeloperoxidase Deficiency Attenuates Lipopolysaccharide-Induced Acute Lung Inflammation and Subsequent Cytokine and Chemokine Production

Lung neutrophilia is common to a variety of lung diseases. The production of reactive oxygen and nitrogen species during neutrophil oxidative burst has been associated with protein and DNA damage. Myeloperoxidase (MPO) is an enzyme stored in the azurophilic granula of neutrophils. It is important in host defense because it generates the reactive oxidant hypochlorous acid and has been described to play a role in the activation of neutrophils during extravasation. We hypothesized that MPO contributes directly to the development of acute lung neutrophilia via stimulation of neutrophil extravasation and indirectly to the subsequent production of cytokines and chemokines in the lung. To test this hypothesis, wild-type (WT) and Mpo−/− mice were given a single LPS instillation, after which the development of neutrophil-dominated lung inflammation, oxidative stress, and cytokine and chemokine levels were examined. Mpo−/− mice demonstrated a decreased lung neutrophilia that peaked earlier than neutrophilia in WT mice, which can be explained by decreased neutrophil chemoattractant levels in LPS-exposed Mpo−/− compared with WT mice. However, oxidative stress levels were not different in LPS-exposed WT and Mpo−/− mice. Furthermore, in vivo findings were confirmed by in vitro studies, using isolated neutrophils. These results indicate that MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production by other cell types in the lung.

Inhibition of LPS-induced pulmonary inflammation by specific flavonoids.

In the present study, the anti-inflammatory effects of the flavonoids flavone, fisetin and tricetin were evaluated in a mouse model of LPS-induced acute pulmonary inflammation. The flavonoid fisetin significantly reduced lung myeloperoxidase-levels and gene-expression of inflammatory mediators such as IL-6, TNF-alpha, IL-1beta, MIP-1alpha and MIP-2. The LPS-induced gene transcription of HO-1 and SOD2 was also significantly reduced by fisetin. Overall, the anti-inflammatory effects of fisetin in this in vivo model were much more pronounced as compared to the observed effects of flavone or tricetin and the anti-inflammatory glucocorticoid dexamethasone. The results of this study indicate that flavonoids such as fisetin might be potential candidates as pharmaceuticals or nutraceuticals in the treatment of pulmonary inflammatory diseases.

NF-κB Activation Is Required for the Transition of Pulmonary Inflammation to Muscle Atrophy

Disease exacerbations and muscle wasting comprise negative prognostic factors of chronic obstructive pulmonary disease (COPD). Transient systemic inflammation and malnutrition have been implicated in skeletal muscle wasting after acute exacerbations of COPD. However, the interactions between systemic inflammation and malnutrition in their contributions to muscle atrophy, as well as the molecular basis underlying the transition of systemic inflammation to muscle atrophy, remain unresolved. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS to model acute disease exacerbation. Systemic inflammation, nutritional intake, and body and muscle weights were determined. Muscle inflammatory signaling and atrophy signaling were examined, and the effect of the muscle-specific inactivation of NF-κB on muscle atrophy was assessed in genetically modified mice. The intratracheal LPS instillation was followed by markedly elevated circulating cytokine concentrations and NF-κB activation in extrapulmonary tissues, including skeletal muscle. The administration of intratracheal LPS increased the expression of muscle E3 ubiquitin ligases, which govern muscle proteolysis, in particular MuRF1, and caused a rapid loss of muscle mass. Reduced food intake only partly accounted for the observed muscle atrophy, and did not activate NF-κB in muscle. Rather, plasma transfer experiments revealed the presence of NF-κB-signaling and atrophy-signaling properties in the circulation of intratracheal LPS-treated mice. The genetic inhibition of muscle NF-κB activity suppressed intratracheal LPS-induced MuRF1 expression and resulted in a significant sparing of muscle tissue. Systemic inflammation and malnutrition contribute to the muscle wasting induced by acute pulmonary inflammation via distinct mechanisms, and muscle NF-κB activation is required for the transition from inflammatory to muscle atrophy signaling.

Airway Epithelial NF-κB Activation Modulates Asbestos-Induced Inflammation and Mucin Production In Vivo

To investigate the role of bronchiolar epithelial NF-κB activity in the development of inflammation and fibrogenesis in a murine model of asbestos inhalation, we used transgenic (Tg) mice expressing an IκBα mutant (IκBαsr) resistant to phosphorylation-induced degradation and targeted to bronchial epithelium using the CC10 promoter. Sham and chrysotile asbestos-exposed CC10-IκBαsr Tg+ and Tg− mice were examined for altered epithelial cell proliferation and differentiation, cytokine profiles, lung inflammation, and fibrogenesis at 3, 9, and 40 days. KC, IL-6 and IL-1β were increased (p ≤ 0.05) in bronchoalveolar lavage fluid (BALF) from asbestos-exposed mice, but to a lesser extent (p ≤ 0.05) in Tg+ vs Tg− mice. Asbestos also caused increases in IL-4, MIP-1β, and MCP-1 in BALF that were more elevated (p ≤ 0.05) in Tg+ mice at 9 days. Differential cell counts revealed eosinophils in BALF that increased (p ≤ 0.05) in Tg+ mice at 9 days, a time point corresponding with significantly increased numbers of bronchiolar epithelial cells staining positively for mucus production. At all time points, asbestos caused increased numbers of distal bronchiolar epithelial cells and peribronchiolar cells incorporating the proliferation marker, Ki-67. However, bronchiolar epithelial cell and interstitial cell labeling was diminished at 40 days (p ≤ 0.05) in Tg+ vs Tg− mice. Our findings demonstrate that airway epithelial NF-κB activity plays a role in orchestrating the inflammatory response as well as cell proliferation in response to asbestos.

Myeloperoxidase modulates lung epithelial responses to pro-inflammatory agents

During extensive inflammation, neutrophils undergo secondary necrosis causing myeloperoxidase (MPO) release that may damage resident lung cells. Recent observations suggest that MPO has pro-inflammatory properties, independent of its enzymatic activity. The aims of the present study were to characterise MPO internalisation by lung epithelial cells and to investigate the effect of MPO on oxidative stress, DNA damage and cytokine production by lung epithelial cells. Human alveolar and bronchial epithelial cells were stimulated with MPO, with or without priming the cells with pro-inflammatory stimuli. MPO protein was detected in the cell cytoplasm. Expression of haemoxygenase (HO)-1 and DNA strand breakage were determined. The production of interleukin (IL)-8 and -6 were measured. Analyses of MPO-stimulated cells demonstrated MPO presence in the cells. HO-1 expression was increased after MPO stimulation and increased further when cells were primed before MPO stimulation. MPO exposure also induced DNA strand breakage. Interestingly, MPO inhibited IL-8 production in bronchial, but not alveolar epithelium. In conclusion, alveolar and bronchial epithelial cells can internalise myeloperoxidase. Stimulation with myeloperoxidase increases haemoxygenase-1 expression and DNA strand breakage, suggesting cell damaging capacity of myeloperoxidase. In addition, myeloperoxidase inhibited interleukin-8 production by bronchial epithelial cells, indicating a negative feedback loop for neutrophil recruitment.

Lung inflammation is associated with reduced pulmonary nucleotide excision repair in vivo

Chronic pulmonary inflammation is associated with increased lung cancer risk, but the underlying process remains unknown. Recently, we showed that activated neutrophils inhibit nucleotide excision repair (NER) in pulmonary epithelial cells in vitro via the release of myeloperoxidase (MPO). To evaluate the effect of neutrophils on NER in vivo, mice were intratracheally instilled with lipopolysaccharide (LPS) (20 microg), causing acute lung inflammation and associated neutrophil influx into the airways. Three days post-exposure, phenotypical NER capacity was assessed in lung tissue homogenate. LPS exposure inhibited pulmonary NER by approximately 50%. This finding was corroborated by down-regulation of the NER-associated genes Xpa and Xpf. To further elicit the role of neutrophils and MPO in this process, we utilized MPO-deficient mice as well as mice in which circulating neutrophils were depleted by antibody treatment. LPS-induced inhibition of pulmonary NER was not affected by either Mpo(-/-) or by depletion of circulating neutrophils. This contrasts with our previous in vitro observations, suggesting that inhibition of pulmonary NER following acute dosing with LPS is not fully mediated by neutrophils and/or MPO. In conclusion, these data show that LPS-induced pulmonary inflammation is associated with a reduction of NER function in the mouse lung.

Rapid pulmonary expression of acute-phase reactants after local lipopolysaccharide exposure in mice is followed by an interleukin-6 mediated systemic acute-phase response

This study investigated local and systemic innate immune responses in lipopolysaccharide (LPS)-induced lung inflammation in mice. Intratracheal LPS exposure resulted in increased pulmonary mRNA expression for acute-phase reactants (APRs) α1-antitrypsin (α1-AT), α1-acid glycoprotein (AGP), and LPS-binding protein (LBP) from 4 hours post exposure. Although pulmonary serum amyloid P component (SAP) mRNA was not increased, systemic levels of SAP, AGP, and LBP were elevated from 24 hours post exposure. Systemic APRs increase was associated with hepatic mRNA expression. As in vivo neutralization of interleukin (IL)-6, but not tumor necrosis factor (TNF)-α, fully ablated hepatic APR mRNA expression, IL-6 may act as signaling molecule between lung and liver. In conclusion, pulmonary LPS exposure induced rapid APR expression in lung, which precedes IL-6–mediated systemic elevation of APRs associated with hepatic APRs expression.

Inhibition of acute pulmonary and systemic inflammation by 1,7-dimethylxanthine.

The nuclear enzyme poly(ADP-ribose) polymerse-1 (PARP-1) has previously been reported to play an important role in lipopolysaccharide (LPS)-induced pulmonary inflammation and is highly activated in COPD patients. In the present study, the anti-inflammatory efficacy of a previously identified poly(ADP-ribose) polymerase-1 (PARP-1) inhibiting caffeine metabolite, 1,7-dimethylxanthine, was both in vivo as well as ex vivo evaluated. Orally administered 1,7-dimethylxanthine significantly attenuated lung myeloperoxidase-levels, transcription of IL-6, TNF-alpha, MIP1alpha and MIP2 genes as well as PAR-polymer formation in a mouse model of intratracheally LPS-induced acute pulmonary inflammation. Serum amyloid P component and plasma IL-6 were also lowered in 1,7-dimethylxanthine treated mice, indicating a reduced systemic inflammatory response. In addition, at 24h after LPS administration anti-inflammatory effects of 1,7-dimethylxanthine appeared more pronounced than those of the orally administered PARP-1 inhibitor 3-aminobenzamide. In the second model, in blood of COPD-patients and healthy controls ex vivo pre-incubated with a physiological concentration of 1,7-dimethylxanthine (10microM), LPS-induced production of the cytokines IL-6 and TNF-alpha was significantly suppressed. 1,7-Dimethylxanthine exerts anti-inflammatory effects, both in vivo mouse as well as ex vivo human. These results suggest that the PARP-1 inhibiting caffeine metabolite 1,7-dimethylxanthine may have therapeutic potential in pulmonary inflammatory diseases such as COPD.