Guy Joos

New insights into the immunology of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a heterogeneous syndrome associated with abnormal inflammatory immune responses of the lung to noxious particles and gases. Cigarette smoke activates innate immune cells such as epithelial cells and macrophages by triggering pattern recognition receptors, either directly or indirectly via the release of damage-associated molecular patterns from stressed or dying cells. Activated dendritic cells induce adaptive immune responses encompassing T helper (Th1 and Th17) CD4+ T cells, CD8+ cytotoxicity, and B-cell responses, which lead to the development of lymphoid follicles on chronic inflammation. Viral and bacterial infections not only cause acute exacerbations of COPD, but also amplify and perpetuate chronic inflammation in stable COPD via pathogen-associated molecular patterns. We discuss the role of autoimmunity (autoantibodies), remodelling, extracellular matrix-derived fragments, impaired innate lung defences, oxidative stress, hypoxia, and dysregulation of microRNAs in the persistence of the pulmonary inflammation despite smoking cessation.

Indirect airway challenges

Indirect challenges act by causing the release of endogenous mediators that cause the airway smooth muscle to contract. This is in contrast to the direct challenges where agonists such as methacholine or histamine cause airflow limitation predominantly via a direct effect on airway smooth muscle. Direct airway challenges have been used widely and are well standardised. They are highly sensitive, but not specific to asthma and can be used to exclude current asthma in a clinic population. Indirect bronchial stimuli, in particular exercise, hyperventilation, hypertonic aerosols, as well as adenosine, may reflect more directly the ongoing airway inflammation and are therefore more specific to identify active asthma. They are increasingly used to evaluate the prevalence of bronchial hyperresponsiveness and to assess specific problems in patients with known asthma, e.g. exercise-induced bronchoconstriction, evaluation before scuba diving. Direct bronchial responsiveness is only slowly and to a modest extent, influenced by repeated administration of inhaled steroids. Indirect challenges may reflect more closely acute changes in airway inflammation and a change in responsiveness to an indirect stimulus may be a clinically relevant marker to assess the clinical course of asthma. Moreover, some of the indirect challenges, e.g. hypertonic saline and mannitol, can be combined with the assessment of inflammatory cells by induction of sputum.

Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema

Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation of the airways and progressive destruction of lung parenchyma, a process that in most cases is initiated by cigarette smoking. Several mechanisms are involved in the development of the disease: influx of inflammatory cells into the lung (leading to chronic inflammation of the airways), imbalance between proteolytic and anti-proteolytic activity (resulting in the destruction of healthy lung tissue) and oxidative stress. Recently, an increasing number of data suggest a fourth important mechanism involved in the development of COPD: apoptosis of structural cells in the lung might possibly be an important upstream event in the pathogenesis of COPD. There is an increase in apoptotic alveolar epithelial and endothelial cells in the lungs of COPD patients. Since this is not counterbalanced by an increase in proliferation of these structural cells, the net result is destruction of lung tissue and the development of emphysema. Data from animal models suggest a role for Vascular Endothelial Growth Factor (VEGF) in the induction of apoptosis of structural cells in the lung. Other mediators of apoptosis, such as caspase-3 and ceramide, could be interesting targets to prevent apoptosis and the development of emphysema.In this review, recent data on the role of apoptosis in COPD from both animal models as well as from studies on human subjects will be discussed. The aim is to provide an up to date summary on the increasing knowledge on the role of apoptosis in COPD and pulmonary emphysema.

Time course of cigarette smoke-induced pulmonary inflammation in mice

Inflammation of the airways and lung parenchyma plays a major role in the pathogenesis of chronic obstructive pulmonary disease. In the present study a murine model of tobacco smoke-induced emphysema was used to investigate the time course of airway and pulmonary inflammatory response, with a special emphasis on pulmonary dendritic cell (DC) populations. Groups of mice were exposed to either cigarette smoke or to control air for up to 24 weeks. In response to cigarette smoke, inflammatory cells (i.e. neutrophils, macrophages and lymphocytes) progressively accumulated both in the airways and lung parenchyma of mice. Furthermore, a clear infiltration of DCs was observed in airways (10-fold increase) and lung parenchyma (1.5-fold increase) of cigarette-exposed mice at 24 weeks. Flow cytometric analysis of bronchoalveolar lavage (BAL) DCs of smoke-exposed mice showed upregulation of major histocompatability complex II molecules and costimulatory molecules CD40 and CD86, compared with BAL DCs of air-exposed mice. Morphometric analysis of lung histology demonstrated a significant increase in mean linear intercept and alveolar wall destruction after 24 weeks of smoke exposure. In conclusion, the time course of the changes in inflammatory and dendritic cells in both bronchoalveolar lavage and the pulmonary compartment of cigarette smoke-exposed mice was carefully characterised.

Azithromycin for prevention of exacerbations in severe asthma (AZISAST): a multicentre randomised double-blind placebo-controlled trial

Background Patients with severe asthma are at increased risk of exacerbations and lower respiratory tract infections (LRTI). Severe asthma is heterogeneous, encompassing eosinophilic and non-eosinophilic (mainly neutrophilic) phenotypes. Patients with neutropilic airway diseases may benefit from macrolides. Methods We performed a randomised double-blind placebo-controlled trial in subjects with exacerbation-prone severe asthma. Subjects received low-dose azithromycin (n=55) or placebo (n=54) as add-on treatment to combination therapy of inhaled corticosteroids and long-acting β2 agonists for 6 months. The primary outcome was the rate of severe exacerbations and LRTI requiring treatment with antibiotics during the 26-week treatment phase. Secondary efficacy outcomes included lung function and scores on the Asthma Control Questionnaire (ACQ) and Asthma Quality of Life Questionnaire (AQLQ). Results The rate of primary endpoints (PEPs) during 6 months was not significantly different between the two treatment groups: 0.75 PEPs (95% CI 0.55 to 1.01) per subject in the azithromycin group versus 0.81 PEPs (95% CI 0.61 to 1.09) in the placebo group (p=0.682). In a predefined subgroup analysis according to the inflammatory phenotype, azithromycin was associated with a significantly lower PEP rate than placebo in subjects with non-eosinophilic severe asthma (blood eosinophilia ≤200/µl): 0.44 PEPs (95% CI 0.25 to 0.78) versus 1.03 PEPs (95% CI 0.72 to 1.48) (p=0.013). Azithromycin significantly improved the AQLQ score but there were no significant between-group differences in the ACQ score or lung function. Azithromycin was well tolerated, but was associated with increased oropharyngeal carriage of macrolide-resistant streptococci. Conclusions Azithromycin did not reduce the rate of severe exacerbations and LRTI in patients with severe asthma. However, the significant reduction in the PEP rate in azithromycin-treated patients with non-eosinophilic severe asthma warrants further study. ClinicalTrials.gov number NCT00760838.

Elevated MMP-12 protein levels in induced sputum from patients with COPD

Background: Several matrix metalloproteinases (MMPs) are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). In mice, MMP-12 plays a crucial role in the development of cigarette smoke induced emphysema. A study was undertaken to investigate the role of MMP-12 in the development of COPD in human smokers. Methods: Induced sputum samples were collected from patients with stable COPD (n = 28), healthy smokers (n = 14), never smokers (n = 20), and former smokers (n = 14). MMP-12 protein levels in induced sputum were determined by ELISA and compared between the four study groups. MMP-12 enzymatic activity in induced sputum was evaluated by casein zymography and by cleaving of a fluorescence quenched substrate. Results: Median (IQR) MMP-12 levels were significantly higher in COPD patients than in healthy smokers, never smokers, and former smokers (17.5 (7.1–42.1) v 6.7 (3.9–10.4) v 4.2 (2.4–11.3) v 6.1 (4.5–7.6) ng/ml, p = 0.0002). MMP-12 enzymatic activity was significantly higher in patients with COPD than in controls (4.11 (1.4–8.0) v 0.14 (0.1–0.2) μg/μl, p = 0.0002). Conclusion: MMP-12 is markedly increased in induced sputum from patients with stable COPD compared with controls, suggesting a role for MMP-12 in the development of COPD in smokers.

Cigarette Smoke-Induced Pulmonary Inflammation and Emphysema Are Attenuated in CCR6-Deficient Mice

Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking, and is characterized by an increase in inflammatory cells in the airways and pulmonary tissue. The chemokine receptor CCR6 and its ligand MIP-3α/CCL20 may be involved in the recruitment of these inflammatory cells. To investigate the role of CCR6 in the pathogenesis of COPD, we analyzed the inflammatory responses of CCR6 knockout (KO) and wild-type mice upon cigarette smoke (CS) exposure. Both subacute and chronic exposure to CS induced an increase in cells of the innate and adaptive immune system in the bronchoalveolar lavage, both in CCR6 KO and wild-type mice. However, the accumulation of dendritic cells, neutrophils, and T lymphocytes, which express CCR6, was significantly attenuated in the CCR6 KO mice, compared with their wild-type littermates. In the lung tissue of CCR6 KO mice, there was an impaired increase in dendritic cells, activated CD8+ T lymphocytes, and granulocytes. Moreover, this attenuated inflammatory response in CCR6 KO mice offered a partial protection against pulmonary emphysema, which correlated with an impaired production of MMP-12. Importantly, protein levels of MIP-3α/CCL20, the only chemokine ligand of the CCR6 receptor, and MCP-1/CCL2 were significantly increased upon CS exposure in wild-type, but not in CCR6 KO mice. In contrast, CCR6 deficiency had no effect on the development of airway wall remodeling upon chronic CS exposure. These results indicate that the interaction of CCR6 with its ligand MIP-3α contributes to the pathogenesis of CS-induced pulmonary inflammation and emphysema in this murine model of COPD.

Role of tachykinins in asthma.

The sensory neuropeptides substance P (SP) and neurokinin A (NKA) are localized to sensory airway nerves, from which they can be released by a variety of stimuli, including allergen, ozone, or inflammatory mediators. Sensory nerves containing these peptides are relatively scarce in human airways, but it is becoming increasingly evident that inflammatory cells such as eosinophils, macrophages, lymphocytes, and dendritic cells can produce the tachykinins SP and NKA. Moreover, immune stimuli can boost the production and secretion of SP and NKA. SP and NKA have potent effects on bronchomotor tone, airway secretions, and bronchial circulation (vasodilation and microvascular leakage) and on inflammatory and immune cells. Following their release, tachykinins are degraded by neutral endopeptidase (NEP) and angiotensin‐converting enzyme. The airway effects of the tachykinins are largely mediated by tachykinin NK1 and NK2 receptors. Tachykinins contract smooth muscle mainly by interaction with NK2 receptors, while the vascular and proinflammatory effects are mediated by the NK1 receptor. In view of their potent effects on the airways, tachykinins have been put forward as possible mediators of asthma, and tachykinin receptor antagonists are a potential new class of antiasthmatic medication.

MicroRNA Expression in Induced Sputum of Smokers and Patients with Chronic Obstructive Pulmonary Disease

RATIONALE Chronic obstructive pulmonary disease (COPD) is characterized by progressive inflammation in the airways and lungs combined with disturbed homeostatic functions of pulmonary cells. MicroRNAs (miRNAs) have the ability to regulate these processes by interfering with gene transcription and translation. OBJECTIVES We aimed to identify miRNA expression in induced sputum and examined whether the expression of miRNAs differed between patients with COPD and subjects without airflow limitation. METHODS Expression of 627 miRNAs was evaluated in induced sputum supernatant of 32 subjects by stem-loop reverse transcription-quantitative polymerase chain reaction. Differentially expressed miRNAs were validated in an independent replication cohort of 41 subjects. Enrichment of miRNA target genes was identified by in silico analysis. Protein expression of target genes was determined by ELISA. MEASUREMENTS AND MAIN RESULTS Thirty-four miRNAs were differentially expressed between never-smokers and current smokers without airflow limitation in the screening cohort. Eight miRNAs were expressed at a significantly lower level in current-smoking patients with COPD compared with never-smokers without airflow limitation. Reduced expression of let-7c and miR-125b in patients with COPD compared with healthy subjects was confirmed in the validation cohort. Target genes of let-7c were significantly enriched in the sputum of patients with severe COPD. The concentration of tumor necrosis factor receptor type II (TNFR-II, implicated in COPD pathogenesis and a predicted target gene of let-7c) was inversely correlated with the sputum levels of let-7c . CONCLUSIONS let-7c is significantly reduced in the sputum of currently smoking patients with COPD and is associated with increased expression of TNFR-II.

Decreased FOXP3 protein expression in patients with asthma.

Background:  T‐regulatory cells (Treg) are important in balancing immune responses and maintaining peripheral tolerance. Current evidence suggests that asthma is characterized by a relative deficiency in Treg, allowing T helper 2 cells to expand. In this study, we aimed to evaluate circulating Treg, defined by the protein FOXP3, in both control subjects and patients with stable asthma.