Protocol for a human in vivo model of acute cigarette smoke inhalation challenge in smokers with COPD: monitoring the nasal and systemic immune response using a network biology approach.

INTRODUCTION: Cigarette smoke contributes to a diverse range of diseases including chronic obstructive pulmonary disease (COPD), cardiovascular disorders and many cancers. There currently is a need for human challenge models, to assess the acute effects of a controlled cigarette smoke stimulus, followed by serial sampling of blood and respiratory tissue for advanced molecular profiling. We employ precision sampling of nasal mucosal lining fluid by absorption to permit soluble mediators measurement in eluates. Serial nasal curettage was used for transcriptomic analysis of mucosal tissue. METHODS AND ANALYSIS: Three groups of strictly defined patients will be studied: 12 smokers with COPD (GOLD Stage 2) with emphysema, 12 matched smokers with normal lung function and no evidence of emphysema, and 12 matched never smokers with normal spirometry. Patients in the smoking groups are current smokers, and will be given full support to stop smoking immediately after this study. In giving a controlled cigarette smoke stimulus, all patients will have abstained from smoking for 12 h, and will smoke two cigarettes with expiration through the nose in a ventilated chamber. Before and after inhalation of cigarette smoke, a series of samples will be taken from the blood, nasal mucosal lining fluid and nasal tissue by curettage. Analysis of plasma nicotine and metabolites in relation to levels of soluble inflammatory mediators in nasal lining fluid and blood, as well as assessing nasal transcriptomics, ex vivo blood platelet aggregation and leucocyte responses to toll-like receptor agonists will be undertaken. IMPLICATIONS: Development of acute cigarette smoke challenge models has promise for the study of molecular effects of smoking in a range of pathological processes. ETHICS AND DISSEMINATION: This study was approved by the West London National Research Ethics Committee (12/LO/1101). The study findings will be presented at conferences and will be reported in peer-reviewed journals.

Cyclooxygenase and cytokine regulation in lung fibroblasts activated with viral versus bacterial pathogen associated molecular patterns.

Cyclooxygenase (COX) is required for prostanoid (e.g. prostaglandin PGE2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1(-/-) and COX-2(-/-) mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1-8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24h) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.

Effect of different interferonα2 preparations on IP10 and ET-1 release from human lung cells.

BACKGROUND: Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity. METHODS: Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL). RESULTS: IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells. CONCLUSIONS: Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.

Endothelin-1 as a mediator and potential biomarker for interferon induced pulmonary toxicity.

Endothelin-1 is a potent vasoconstrictor and a therapeutic target in pulmonary arterial hypertension. Endothelial cells are the physiological source of endothelin-1 but in vitro data from our group shows that interferons (IFNα, IFNß or IFNγ) induce endothelin-1 in pulmonary vascular smooth muscle cells. IFNs are integral to innate immunity and their antiviral and immunomodulatory capability has been harnessed therapeutically; for example, IFNα plays a critical role in the treatment of chronic hepatitis C infection. However, in some patients, IFN causes pneumonitis and possibly irreversible pulmonary arterial hypertension. In this study, we found that of 16 patients undergoing a six-month course of IFNα therapy, two demonstrated considerably increased serum levels of endothelin-1. We propose that IFN therapy results in elevated levels of endothelin-1 in some patients and when clinically significant levels are reached, pulmonary side effects could ensue. This hypothesis can be easily tested in IFN-treated patients by measuring serum endothelin-1 levels and cardiopulmonary physiological parameters.