Teresa Peiró

Alternaria-derived serine protease activity drives IL-33-mediated asthma exacerbations.

Background The fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear. Objective We sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation. Methods IL-33 levels were quantified in wild-type and ST2−/− mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later. Results Alternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model. Conclusion Alternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease.

Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition

Background Idiopathic pulmonary fibrosis (IPF) is characterised by the aberrant epithelial to mesenchymal transition (EMT) and myofibroblast accumulation. Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SPHK1) have been implicated in lung myofibroblast transition, but their role in EMT and their expression in patients with IPF is unknown. Methods and results S1P levels were measured in serum (n=27) and bronchoalveolar lavage (BAL; n=15) from patients with IPF and controls (n=30 for serum and n=15 for BAL studies). SPHK1 expression was measured in lung tissue from patients with IPF (n=12) and controls (n=15). Alveolar type II transformation into mesenchymal cells was studied in response to S1P (10−9–10−5 M). The median (IQR) of S1P serum levels was increased in patients with IPF (1.4 (0.4) μM) versus controls (1 (0.26) μM; p<0.0001). BAL S1P levels were increased in patients with IPF (1.12 (0.53) μM) versus controls (0.2 (0.5); p<0.0001) and correlated with diffusion capacity of the lung for carbon monoxide, forced expiratory volume in 1 s and forced vital capacity (Spearmans r=−0.87, −0.72 and −0.68, respectively) in patients with IPF. SPHK1 was upregulated in lung tissue from patients with IPF and correlated with α-smooth muscle actin, vimentin and collagen type I (Spearmans r=0.82, 0.85 and 0.72, respectively). S1P induced EMT in alveolar type II cells by interacting with S1P2 and S1P3, as well as by the activation of p-Smad3, RhoA-GTP, oxidative stress and transforming growth factor-β1 (TGF-β1) release. Furthermore, TGF-β1-induced EMT was partially conducted by the S1P/SPHK1 activation, suggesting crosstalk between TGF-β1 and the S1P/SPHK1 axis. Conclusions S1P is elevated in patients with IPF, correlates with the lung function and mediates EMT.

Matrikines are key regulators in modulating the amplitude of lung inflammation in acute pulmonary infection

Bioactive matrix fragments (matrikines) have been identified in a myriad of disorders, but their impact on the evolution of airway inflammation has not been demonstrated. We recently described a pathway where the matrikine and neutrophil chemoattractant proline–glycine–proline (PGP) could be degraded by the enzyme leukotriene A4 hydrolase (LTA4H). LTA4H classically functions in the generation of pro-inflammatory leukotriene B4, thus LTA4H exhibits opposing pro- and anti-inflammatory activities. The physiological significance of this secondary anti-inflammatory activity remains unknown. Here we show, using readily resolving pulmonary inflammation models, that loss of this secondary activity leads to more pronounced and sustained inflammation and illness owing to PGP accumulation. PGP elicits an exacerbated neutrophilic inflammation and protease imbalance that further degrades the extracellular matrix, generating fragments that perpetuate inflammation. This highlights a critical role for the secondary anti-inflammatory activity of LTA4H and thus has consequences for the generation of global LTA4H inhibitors currently being developed.

Neutrophil Activation in Severe, Early-Onset COPD Patients versus Healthy Non-Smoker Subjects in vitro: Effects of Antioxidant Therapy

Background: Neutrophils and oxidative stress have been implicated in the pathogenesis of COPD. Severe, early-onset COPD is characterized by a rapid decline in the lung function at an early age; however, nothing is known about neutrophil activation in COPD patients. Objectives: The aim of this study was to evaluate peripheral blood neutrophil activation in severe, early-onset COPD patients versus healthy non-smokers and the effect of N-acetyl-L-cysteine (NAC) on neutrophil activation in vitro. Methods: Neutrophils were isolated from 15 severe, early-onset COPD patients and 15 age-matched healthy subjects and stimulated with N-formyl- Met-Leu-Phe (fMLP) in the presence or absence of NAC (10 µM to 10 mM). Neutrophil chemotaxis, elastase release, reactive oxygen species (ROS), intracellular thiols and apoptosis were measured by Boyden chamber, spectrofluorometry, CMFDA and H2DCF-DA dyes and by annexin V-FITC, respectively. Results: Chemotaxis of peripheral blood neutrophils from COPD patients in response to fMLP was 30% more increased than that observed in healthy subjects. Elastase release in response to fMLP was 2-fold higher in neutrophils from COPD patients versus healthy subjects. Intracellular thiol levels were 30% lower in COPD and ROS was approximately 30% higher in COPD versus healthy neutrophils. Spontaneous apoptosis showed no differences in both groups of patients and fMLP-induced apoptosis was higher in COPD. Pre-treatment with the antioxidant NAC effectively attenuated neutrophil chemotaxis, elastase release and ROS as well as effectively increased thiol levels in COPD. Conclusions: Neutrophils in severe, early-onset COPD patients are highly activated and this is alleviated by NAC in vitro.

Aclidinium inhibits cigarette smoke-induced lung fibroblast-to-myofibroblast transition

Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of this remodelling, peribronchiolar fibrosis is observed in the small airways of COPD patients and contributes to airway obstruction. Fibroblast-to-myofibroblast transition is a key step in peribronchiolar fibrosis formation. This in vitro study examined the effect of cigarette smoke on bronchial fibroblast-to-myofibroblast transition, and whether aclidinium bromide inhibits this process. Human bronchial fibroblasts were incubated with aclidinium bromide (10−9–10−7 M) and exposed to cigarette smoke extract. Collagen type I and &agr;-smooth muscle actin (&agr;-SMA) expression were measured by real-time PCR and Western blotting, as myofibroblast markers. Intracellular reactive oxygen species, cyclic AMP (cAMP), extracellular signal-regulated kinase (ERK)1/2 and choline acetyltransferase were measured as intracellular signalling mediators. Cigarette smoke-induced collagen type I and &agr;-SMA was mediated by the production of reactive oxygen species, the depletion of intracellular cAMP and the increase of ERK1/2 phosphorylation and choline acetyltransferase. These effects could be reversed by treatment with the anticholinergic aclidinium bromide, by silencing the mRNA of muscarinic receptors M1, M2 or M3, or by the depletion of extracellular acetylcholine by treatment with acetylcholinesterase. A non-neuronal cholinergic system is implicated in cigarette smoke-induced bronchial fibroblast-to-myofibroblast transition, which is inhibited by aclidinium bromide.

Neutrophils drive alveolar macrophage IL-1β release during respiratory viral infection

Background Alveolar macrophages are sentinels of the airways that must exhibit immune restraint to innocuous antigens but elicit a robust inflammatory response to pathogenic threats. How distinction between these dichotomous functions is controlled is poorly defined. Neutrophils are the first responders to infection, and we hypothesised that they may free alveolar macrophages from their hyporesponsive state, promoting their activation. Activation of the inflammasome and interleukin (IL)-1β release is a key early inflammatory event that must be tightly regulated. Thus, the role of neutrophils in defining inflammasome activation in the alveolar macrophage was assessed. Methods Mice were infected with the X31 strain of influenza virus and the role of neutrophils in alveolar macrophage activation established through administration of a neutrophil-depleting (1A8) antibody. Results Influenza elicited a robust IL-1β release that correlated (r=0.6849; p<0.001) with neutrophil infiltrate and was ablated by neutrophil depletion. Alveolar macrophages were shown to be the prominent source of IL-1β during influenza infection, and virus triggered the expression of Nod-like receptor protein 3 (NLRP3) inflammasome and pro-IL-1β in these cells. However, subsequent activation of the inflammasome complex and release of mature IL-1β from alveolar macrophages were critically dependent on the provision of a secondary signal, in the form of antimicrobial peptide mCRAMP, from infiltrating neutrophils. Conclusions Neutrophils are critical for the activation of the NLRP3 inflammasome in alveolar macrophages during respiratory viral infection. Accordingly, we rationalise that neutrophils are recruited to the lung to confront a viable pathogenic threat and subsequently commit alveolar macrophages to a pro-inflammatory phenotype to combat infection.

Authors’ response to: Epithelial mesenchymal transition (EMT) in small airways of COPD patient

We understand the reservations highlighted by Sohal and Walters in their letter in response to our recent work published.1 In their letter, Sohal and Walters argue that E-cadherin and ZO-1 are absent in our immunohistochemistry analysis of small bronchi of smokers and COPD patients, and that if disappeared completely epithelium would fall apart.1 In our study, we detected downregulation of E-cadherin and ZO-1 as well as a change of intercellular and apical distribution to diffuse cytoplasmic redistribution (figure 3B; in our work published in Thorax on 7 January 2013),2 but not …

An extracellular matrix fragment drives epithelial remodeling and airway hyperresponsiveness

The matrikine PGP is a mediator of pathological epithelial remodeling in asthma. Extracellular matrix extends its influence in asthma The interplay between immune cells and smooth muscle cells in asthma pathogenesis and response to treatment is under increasing scrutiny. Patel et al. focused on how leukotrienes can modulate immune inflammation and airway hypersensitivity. Mice lacking leukotriene A4 hydrolase had exaggerated epithelial remodeling but dampened immune responses in a house dust mite model of asthma. This was due to release of Pro-Gly-Pro (PGP), a component of the extracellular matrix, which was also elevated in the sputum from severe asthmatics in two clinical cohorts. Their results showcase how the extracellular matrix is a dynamic player in disease and reveal a mediator to be targeted in asthma therapy. It is anticipated that bioactive fragments of the extracellular matrix (matrikines) can influence the development and progression of chronic diseases. The enzyme leukotriene A4 hydrolase (LTA4H) mediates opposing proinflammatory and anti-inflammatory activities, through the generation of leukotriene B4 (LTB4) and degradation of proneutrophilic matrikine Pro-Gly-Pro (PGP), respectively. We show that abrogation of LTB4 signaling ameliorated inflammation and airway hyperresponsiveness (AHR) in a murine asthma model, yet global loss of LTA4H exacerbated AHR, despite the absence of LTB4. This exacerbated AHR was attributable to a neutrophil-independent capacity of PGP to promote pathological airway epithelial remodeling. Thus, we demonstrate a disconnect between airway inflammation and AHR and the ability of a matrikine to promote an epithelial remodeling phenotype that negatively affects lung function. Subsequently, we show that substantial quantities of PGP are detectable in the sputum of moderate-severe asthmatics in two distinct cohorts of patients. These studies have implications for our understanding of remodeling phenotypes in asthma and may rationalize the failure of LTA4H inhibitors in the clinic.