<i>Respirology</i> year‐in‐review 2008: Basic science

In this review, we present important findings in basic sciences from articles published in Respirology in 2008. Our aim is to provide a summary and interpretation of new data published in the journal in the basic translational sciences, which will lead to a better understanding of lung diseases, and lead to improved diagnosis, prevention and treatment. COPD is a chronic inflammatory disease of the lungs leading to airflow limitation, gas trapping, breathlessness, respiratory failure, exacerbations and death. Cigarette smoke exposure is the predominant cause of COPD, although there is variable susceptibility to the development of COPD, indicating the effects of gene–environment interaction. While direct cigarette smoke exposure is the predominant causative factor leading to COPD, environmental tobacco smoke exposure may also be important in the development of airway inflammation. The smoking of even a single cigarette releases particulate matter into the adjacent environment. In a granulometry study (the study of the size distribution of ‘grains’ or particles), an enclosed office space was used as the laboratory.1 In each experiment, a single cigarette was smoked, and the resulting suspended particle, dust and microbial load was measured within the room for 3 h afterwards. The concentration of suspended large and medium-sized particles reached a peak soon after smoking, whereas the concentration of small particles (< 0.7 µmol/L) remained elevated after the cigarette was smoked. Bacteria were detected and cultured in the basal conditions prior to the cigarette being smoked. The load of microorganisms sedimented by gravity increased in the first hour after the smoking of the cigarette, followed by a decrease in load. The authors suggested that cigarette smoke may aggregate with bacteria, and (although not specifically tested) that these sedimented aggregates may later be resuspended and inhaled as people move around a room after passive smoking, potentially leading to respiratory infections.1 Genetic risk factors are likely to interact with exposure to cigarette smoke and other environmental irritants, to play a role in the aetiology of COPD. Many studies have examined gene–environment interaction in COPD. The only confirmed genetic risk factor is homozygous ZZ α1-antitrypsin deficiency, although this contributes only a small fraction of COPD and emphysema. Numerous other candidate genes have been postulated;2 however, none has been definitively confirmed in genetic association studies, possibly due to small genetic effects, heterogeneity in study design and phenotyping of COPD, different populations and small sample sizes.3 Genome-wide association studies in COPD are awaited with interest, and will potentially confirm known candidates as well as identifying novel candidate genes. One of the main mechanisms implicated in emphysema is protease–antiprotease imbalance. Cathepsins are cysteine proteases, and cathepsin S is expressed by antigen-presenting cells. The cathepsin S gene (CTSS) was examined in a genetic association study of 204 smokers with COPD and 104 healthy non-smokers.4 Screening of the promoter region of the gene identified a number of polymorphisms, including single nucleotide polymorphisms, variable number of tandem repeats and an insertion/deletion polymorphism. The two main haplotypes (combinations of alleles) differed in promoter function, as determined by luciferase assay in a macrophage-like cell line stimulated by the inflammatory cytokine, interferon-γ. Finally, one of the haplotypes was more frequent in smokers with various severities of airflow limitation, compared with never smokers.4 The precise role of these polymorphisms in the cathepsin S gene in COPD pathogenesis remains to be worked out, particularly the alterations in protease function and immune modulation. Microsomal epoxide hydrolase (mEH) is an enzyme that potentially reduces oxidative stress by detoxifying epoxide compounds such as those found in cigarette smoke. Genotypes at two genetic loci within the mEH gene (EPHX1) appear to influence mEH activity. Of the coding region polymorphisms, the histidine (His) to arginine (Arg) polymorphism at residue 139 exists in exon 4 of EPHX1 (the Arg139 allele has fast enzyme activity), and the tyrosine (Tyr) to histidine (His) polymorphism at residue 113 occurs in exon 3 (the His113 allele has slow enzyme activity). The haplotypes with slow activity (i.e. reduced detoxification) have been associated with increased risk and severity of COPD, but not consistently in all studies. An updated meta-analysis of 16 studies of EPHX1 polymorphisms in COPD pooled results across studies totalling 1847 COPD subjects and 2455 controls.5 The His/His113 genotype was associated with increased risk of COPD (odds ratio 1.59, 95% confidence interval 1.14–2.21), compared with the Tyr/Tyr113 genotype; this association was stronger in Asian populations. Other genotypes altering enzyme activity were also associated with increased or decreased risk of developing COPD. The results of this meta-analysis suggest that polymorphisms in xenobiotic metabolism pathways may contribute to susceptibility to COPD, adding to the support for specific candidate genes such as EPHX1 in COPD. Once COPD has developed, further genetic factors may play a role in the progression of COPD and its phenotypes. The angiotensin-converting enzyme (ACE) gene has functions in the lung related to inflammation and respiratory drive, and has been hypothesized to regulate skeletal muscle function. In a study of 61 COPD patients and 57 controls, ventilator response to exercise was not altered by ACE genotype, whereas the ACE insertion/insertion genotype was associated with maximal work load and aerobic work efficiency.6 The mechanism for this association is not clear, although the authors speculated that changes in muscle fibre type could be an underlying mechanisms resulting from different ACE genotypes. Biomarkers of severity of COPD would be useful for monitoring patients, prognostication and developing therapeutic targets. Induced sputum is a potentially useful, non-invasive tool for studying biomarkers in COPD. In a study of 43 COPD patients, 12 smoking controls and 10 non-smokers, the levels of prostaglandin E2 (PGE2) and matrix metalloproteinase-2 (MMP-2) in induced sputum were higher in COPD patients, and inversely correlated with FEV1 % predicted.7 The cyclo-oxygenase pathway, from which PGE2 is a product, activates MMP-2, lending weight to the notion that these markers are implicated in COPD pathogenesis, through their involvement in inflammation and airway remodelling. Acute exacerbations of COPD contribute substantially to the cost and personal burden of COPD, and are associated with mortality and accelerated lung function decline. Viral and bacterial infections are the predominant cause of exacerbations. Colonization by bacteria, and possibly viruses, may lead to chronic mucus hypersecretion and frequent exacerbations. Innate and acquired immunity are both important in the host defence system of the lungs against microbial infection. There are concerns about impaired host defence with cigarette smoke exposure and COPD, particularly reduced mucociliary clearance. In a mouse model, mice were exposed to cigarettes for 6 months, and the mRNA gene expression of antimicrobial molecules in the lungs was compared with mice without cigarette smoke exposure.8 Cigarette smoke upregulated levels of surfactant protein-A, β-defensins 2 and 3, and secretory leucocyte protease inhibitor, whereas there was no change in lysozyme expression, and a decrease in β-defensin-1 expression. From these data, it can be concluded that, similar to the human airway transcriptome,9 antimicrobial and host defence molecules are in fact activated (rather than impaired) in the airways of mouse models of smoking and COPD. Therefore, other pathways in the immune response should be examined for defects that underpin impaired host defence and frequent exacerbations. Expression of other pattern recognition receptors, such as Toll-like receptors (TLR), has been shown to be altered in COPD. In an elastase-induced mouse model of emphysema, exposure to the bacteria Streptococcus pneumoniae induced expression of TLR2 and TLR4 in alveolar macrophages.10 Subsequently, the numbers of neutrophils increased in the BAL, as did the levels of inflammatory cytokines (tumour necrosis factor (TNF)-α, IL-1-β and IL-6). The reasons for the enhanced immune and inflammatory response in this model should be investigated to further understand differences in the response to bacterial infection in disease models, given that some previous studies have found impaired responses, depending on the model used. The relationship between the presence of bronchodilator reversibility and airway inflammation in COPD is unclear. Traditionally, COPD is characterized as being non-reversible or at least chronic airflow limitation that does not fully reverse with bronchodilator. Sputum eosinophilia is observed in some COPD patients, including in exacerbations, although the underlying mechanisms leading to this are not certain. Thirty-six subjects with mild or moderate COPD were investigated in a randomized controlled trial of inhaled fluticasone of 6 months' duration.11 Airway inflammatory markers, including eosinophilia at baseline did not correlate with the degree of bronchodilator reversibility. Inhaled fluticasone reduced counts of neutrophils and epithelial cells in BAL, whereas airway wall neutrophil counts increased with the inhaled steroid. Bronchodilator reversibility and airway inflammatory markers at baseline did not predict the changes in airway inflammation with fluticasone. Further work is required to predict future treatment response to inhaled steroids and long-acting bronchodilators.12 Pharmacogenomics has emerged as a discipline studying the effects of variation in the human genome on treatment responses, particularly pharmacological therapy. The use of such information may lead to individualized treatment (e.g. choice of bronchodilators) based on the patient's ‘personal genome’. It is recognized that cross-talk may occur between muscarinic acetylcholine receptors (which mediate the bronchodilatation of anticholinergic agents) and β2-adrenergic receptors (which mediate the bronchodilatation of β-agonists). In an 8-week study of 44 COPD patients receiving the long-acting anticholinergic bronchodilator, tiotropium, subjects with the Arg/Arg genotype at amino acid 16 of the β2-adrenergic receptor gene had greater FEV1 and quality of life improvements, compared with the other genotypes carrying the glycine (Gly) allele.13 Whether this effect is due to the desensitization of the Gly form of the β2-adrenergic receptor, and therefore reduced functional capacity for bronchodilatation, is yet to be explored. Given the differential expression of mediators in the airways and lungs in asthma, monitoring of biomarkers of airway inflammation,14 a key feature of asthma, is important. Exhaled breath condensate (EBC) can be collected from cooled exhaled air, in order to measure molecules released into the airways in asthma and other lung diseases. In a study of non-smokers, smokers and asthmatics, levels of CRP, hydrogen peroxide (H2O2) and nitrite/nitrate were increased in EBC of asthmatics.15 The next step now is to find the most useful set of biomarkers in EBC that help to differentiate the specific severities of asthma, and assist with more accurate monitoring of asthma control. CRP may also be a useful biomarker in asthma, when measured in serum. High-sensitivity CRP was measured in 281 asthmatics of varying severities and 177 non-asthmatic controls.16 Increasing levels of CRP were associated with more severe asthma. At present, it is not clear whether CRP is a marker of inflammation in asthma, being an acute phase reactant, or a pathogenic component of the inflammatory process, for example, as in the inflammation of cardiovascular disease. Inhaled steroids are effective as anti-inflammatory agents in asthma, although further understanding is needed of the effects on regulators of airway inflammation and the resulting airway wall remodelling. CD4+CD25+ regulatory T cells (Treg) are T lymphocytes with suppressor function that may modulate allergic inflammation. Cytotoxic T-lymphocyte antigen (CTLA)-4 is a marker of activated T cells. The effect of inhaled steroids on these suppressor cells was tested in a 14-day, placebo controlled trial of 11 mild asthmatics given inhaled fluticasone.17 Fluticasone increased the number of CTLA-4+CD4+CD25+ cells in sputum. While bronchial hyper-responsiveness and sputum eosinophilia also improved with fluticasone, these improvements did not correlate with the increase in CTLA-4+CD4+CD25+ cells. These findings indicate that Treg cells respond by upregulation during treatment with inhaled steroids. It is needed to examine larger cohorts to find associations between Treg cell numbers and other asthma phenotypes. The acute effects of corticosteroids in asthma exacerbations warrant further investigation. While many actions of steroids on gene expression require several hours to take effect, there is increasing recognition of the rapid non-genomic effects of steroids. In an asthmatic guinea pig model, the early effect of steroids on oxidative stress was studied.18 Markers of oxidative stress rapidly increased upon ovalbumin (OVA) challenge. However, inhaled budesonide given 5 min before challenge reduced the degree of oxidant-antioxidant imbalance within 15 min, as measured by trolox-equivalent antioxidant capacity in BAL fluid (BALF) and lung manganese superoxide activity. These novel findings pave the way for more detailed investigation into the rapid non-genomic effects of steroids in asthma, which will provide great insight into the mechanism and time-course of steroid actions, of direct relevance to treatment of exacerbations of asthma. Inhibition of TNF-α is currently being explored as an immunomodulatory treatment for refractory asthma. Clinical trials have provided preliminary evidence of potential benefits of the anti-TNF-α mAb, infliximab, in moderate to severe asthma.19 To further define the mechanisms involved, a mouse model has been used to measure airway effect of infliximab.20 Compared with control, infliximab reduced the number of neutrophils and eosinophils in the BALF and the lung, and also reduced the concentrations of relevant chemokines and cytokines. This mouse model is likely to be useful for characterizing further immunomodulatory effects of infliximab, including defining other pathways altered by infliximab and finding predictive markers of clinical response that would assist with individualizing treatment. Profiling of global gene expression is an approach to understanding the development and severity of asthma. Various methods are available including gene expression profiling using microarrays and serial analysis of gene expression. The latter method has been used to measure mRNA transcripts that are differentially expressed between asthma and non-asthma in a rodent asthma model.21 Serial analysis of gene expression analysis of lung tissue was performed in rats sensitized to and then challenged with OVA. Pathway analysis revealed over-representation of genes involved in signalling, adhesion, immune cell surface receptors and extracellular matrix interaction, among others. This study shows that in an animal model of asthma, there appears to be a specific molecular signature of the early airway response phase of asthma. The cross-talk between bronchial epithelium and the underlying airway submucosa can lead to activation of fibroblasts and the development of airway fibrosis in the face of acute and chronic injury. Bile acid aspiration, a common phenomenon post-lung transplantation, may be a factor in the induction of airway fibrosis. Components of bile acid, when challenged to human bronchial epithelial cells, induced expression of connective tissue growth factor (CTGF or also known as CCN2).22 Autocrine activation via TGF-β1 further contributed to CCN2 production. These results indicate how aspiration of bile acid may lead to airway fibrosis (including bronchiolitis obliterans syndrome and pulmonary fibrosis) and suggest potential therapeutic targets to ameliorate the effects. Mucus hypersecretion occurs commonly in patients with asthma and COPD, particularly in exacerbations but also during stability. Mucus is made up of predominantly mucin, comprised of the proteins MUC5AC and MUC5B. MUC5AC is produced by both goblet cells of the bronchial epithelium, and also by the submucosal glands, as recently observed.23 Various factors induce mucus hypersecretion, including cigarette smoke exposure in COPD and allergic inflammation in asthma. The Th2 cytokine, IL-13, has been shown to induce mucous metaplasia in the airways. A model of murine tracheal epithelial cells, comprising an air-liquid interface, was used to further define mechanisms involved in mucus hypersecretion.24 When IL-13 induced mucous metaplasia in the epithelial cell model, there was early phosphorylation of the transcription factor STAT6, then phosphorylation of p38 mitogen-activated protein kinase (MAPK), finally leading to expression of MUC5AC, one of the major mucin proteins. Hence the p38 MAPK pathway is implicated in mucous hypersecretion, particularly in the presence of allergic inflammation. Macrolide antibiotics have both anti-microbial and also anti-inflammatory effects. Macrolides were tested in several in vitro and in vivo models of respiratory disease. In a rat model, intratracheal LPS was instilled, and measurements were made of mucin and cytokine production.25 LPS upregulated the production of MUC5AC, as well as several inflammatory cytokines (IL-1-β, IL-8 and TNF-α). Furthermore, LPS increased the expression of the pro-inflammatory transcription factor, NFκB, in airway epithelial cells. Roxithromycin, a 14-membered ring macrolide, reduced the degree of upregulation of MUC5AC, cytokines and NFκB induced by LPS, whereas josamycin (a 16-membered ring macrolide) and amoxicillin did not have these effects. These results provide information about a potential mechanism (inhibition of NFκB) through which macrolides may act to reduce the inflammatory effects of exposure to bacteria. In an in vitro model, human tracheal epithelial cells were exposed to rhinovirus, and the mRNA and protein expression of MUC5AC was measured.26 Pre-incubation of cells with erythromycin for 3 h reduced the expression of MUC5AC. This occurred through the p44/42 MAP kinase pathway but only in the presence of rhinovirus. This raised the possibility of the usefulness of erythromycin as prophylaxis against some of the airway effects of viral infection, or possibly early treatment soon after the onset of exacerbation symptoms. The bronchial epithelium acts as a protective barrier in the airways, and is at the first line of defence against infections. The changes in gene expression upon exposure to infectious agents, such as viruses, are still being characterized. In an in vitro study, H292 bronchial epithelial cells were infected with influenza virus and the mRNA expression was profiled using microarrays.27 Compared with uninfected cells, influenza exposed cells had greater expression of apoptosis-related and antiviral genes, in addition to a range of other biological pathways. Inhibitors of p38 MAPK and JNK downregulated some of these responsive genes, indicating a biological role for these pathways in viral-mediated epithelial inflammation. Gene expression profiling is therefore a useful tool for characterizing the response of airway cells to infectious of the pathways involved will be useful in understanding pathogenesis of airway inflammation in response to viral infection. an airway is commonly related to is a that and to weight In a study of patients with patients and controls, levels were elevated in patients compared with treatment reduced the levels of the form of in a potential mechanism for the weight in some patients Acute lung is an important clinical the and was the of a large number of basic published in Respirology in the 2008. The and of corticosteroids for and are well known and matter of The of inflammatory changes and may also after including lung fibrosis. The effect of was examined in a large study on rats with LPS induced in a in The authors were on the between acute inflammation and early up to had a in inflammation and fibrosis compared with LPS, through receptor levels and of the While the effects on TNF-α were clear in this study, it is more to the data activity of rats were examined and the used for and not of (i.e. the study to a higher weight in rats with compared with LPS some of the that may be associated with It is well known that many have anti-inflammatory in addition to their antimicrobial effects. The of mucus genes and inflammatory cytokines by macrolides were also immune and inflammatory responses, and their effect on was investigated in a mouse model of lung The LPS of bacteria to the from the effects of and using that was severe in but not with or This was through of inflammatory cytokines and in improved of the These data at and to and of compounds that more recently molecules of the not is a and has the antioxidant to involved in It was shown that can via of pro-inflammatory was used in in patients with and may also be an approach to the in patients with stress is also an important factor in the development of A study on rats exposed to protective effect of the on oxidative inflammation and airway previous in vitro results from lung cell However, in this model had to be early to be that within min after onset of and before This may the benefits of in clinical trials on and In of patients with it is not only the peak but also the and that in with predominantly lung injury. 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