C. Magnus Sköld

Lung Fibroblast Repair Functions in Patients with Chronic Obstructive Pulmonary Disease Are Altered by Multiple Mechanisms

RATIONALE Fibroblasts are believed to be the major cells responsible for the production and maintenance of extracellular matrix. Alterations in fibroblast functional capacity, therefore, could play a role in the pathogenesis of pulmonary emphysema, which is characterized by inadequate maintenance of tissue structure. OBJECTIVES To evaluate the hypothesis that deficient fibroblast repair characterizes cells obtained from individuals with chronic obstructive pulmonary disease (COPD) compared with control subjects. METHODS Fibroblasts were cultured from lung tissue obtained from individuals undergoing thoracotomy and were characterized in vitro. MEASUREMENTS AND MAIN RESULTS Fibroblasts from individuals with COPD, defined by reduced FEV(1), manifested reduced chemotaxis toward fibronectin and reduced contraction of three-dimensional collagen gels, two bioassays associated with fibroblast repair function. At least two mechanisms appear to account for these differences. Prostaglandin E (PGE), a known inhibitor of fibroblast repair functions, was produced in increased amount by fibroblasts from subjects with COPD, which also expressed increased amounts of the receptors EP2 and EP4, both of which signal through cyclic AMP. Incubation of fibroblasts with indomethacin or with the PKA inhibitor KT-5720 partially restored COPD subject fibroblast function. In addition, fibroblasts from subjects with COPD produced more transforming growth factor (TGF)-beta1, but manifested reduced response to TGF-beta1. The functional alterations in fibroblasts correlated with both lung function assessed by FEV(1) and, for the data available, with severity of emphysema assessed by Dl(CO). CONCLUSIONS Fibroblasts from individuals with COPD have reduced capability to sustain tissue repair, which suggests that this may be one mechanism that contributes to the development of emphysema.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation

Objectives Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA. Patients and methods Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA. Results Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA. Conclusions Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.

Assessing Recent Smoking Status by Measuring Exhaled Carbon Monoxide Levels

Background Cigarette smoke causes both acute and chronic changes of the immune system. Excluding recent smoking is therefore important in clinical studies with chronic inflammation as primary focus. In this context, it is common to ask the study subjects to refrain from smoking within a certain time frame prior to sampling. The duration of the smoking cessation is typically from midnight the evening before, i.e. 8 hours from sampling. As it has been shown that a proportion of current smokers underestimates or denies smoking, objective assessment of recent smoking status is of great importance. Our aim was to extend the use of exhaled carbon monoxide (CObreath), a well-established method for separating smokers from non-smokers, to assessment of recent smoking status. Methods and Findings The time course of CObreath decline was investigated by hourly measurements during one day on non-symptomatic smokers and non-smokers (6+7), as well as by measurements on three separate occasions on non-smokers (n = 29), smokers with normal lung function (n = 38) and smokers with chronic obstructive pulmonary disease (n = 19) participating in a clinical study. We used regression analysis to model the decay, and receiver operator characteristics analysis for evaluation of model performance. The decline was described as a mono-exponential decay (r2 = 0.7) with a half-life of 4.5 hours. CO decline rate depends on initial CO levels, and by necessity a generic cut-off is therefore crude as initial CObreath varies a lot between individuals. However, a cut-off level of 12 ppm could classify recent smokers from smokers having refrained from smoking during the past 8 hours with a specificity of 94% and a sensitivity of 90%. Conclusions We hereby describe a method for classifying recent smokers from smokers having refrained from smoking for >8 hours that is easy to implement in a clinical setting.

Safety and tolerability of acetylcysteine and pirfenidone combination therapy in idiopathic pulmonary fibrosis: a randomised, double-blind, placebo-controlled, phase 2 trial

BACKGROUND Oral acetylcysteine (also known as N-acetylcysteine) is used with pirfenidone to treat idiopathic pulmonary fibrosis (IPF) in Europe. However, no randomised studies have investigated the safety and tolerability of this combination. The PANORAMA study assessed the safety and tolerability of acetylcysteine combined with pirfenidone in patients with IPF. Exploratory efficacy endpoints were also assessed. METHODS We did a double-blind randomised trial at 48 sites in eight countries. Patients with IPF aged 40-80 years and established on pirfenidone (at least 1602 mg/day for 8 weeks or longer) were randomly assigned in a 1:1 ratio by interactive voice response system to receive concomitant oral acetylcysteine (600 mg, three times daily) or placebo for 24 weeks. A stratified blocked randomisation scheme was used with a block size of 4. Randomisation was stratified by dose of pirfenidone (2403 mg/day [the maximum dose] or <2403 mg/day). Patients, physicians, study staff and the sponsor were masked to treatment group allocation. The primary endpoint was assessment of adverse events, which were collected at each visit and for 28 days after the last dose of study drug. Exploratory efficacy measurements included forced vital capacity (FVC), carbon monoxide diffusing capacity, and 6 min walk distance. Analyses were done in the modified intention-to-treat population, which included all patients who were randomised and received at least one dose of study medication. This study is registered with the European Clinical Trials Database (EudraCT number 2012-000564-14) and has been completed. FINDINGS 123 patients participated in the study between June 28, 2013, and Feb 24, 2015. 61 were assigned to the acetylcysteine group (60 received study medication and included in analysis) and 62 were assigned to the placebo group (all included in analysis). The occurrence of at least one adverse event (46 [77%] patients receiving acetylcysteine vs 50 [81%] receiving placebo), adverse events related to study treatment (17 [28%] vs 16 [26%]), and the number of patients experiencing severe adverse events (three [5%] vs two [3%]), life-threatening adverse events (one [2%] vs one [2%]), or death (one [2%] vs three [5%]) was similar between treatment groups. One case of diarrhoea in the acetylcysteine group was considered severe and related to study treatment. Nine serious adverse events were reported by seven patients: dyspnoea, headache, hypertension, intervertebral disc protrusion, and malignant lung neoplasm in the acetylcysteine group, and aortic aneurysm, contusion, forearm fracture, and worsening IPF in the placebo group. The most common adverse events were cough, nasopharyngitis, and diarrhoea. Photosensitivity occurred more frequently with acetylcysteine (eight [13%] patients) than placebo (one [2%] patient; difference 11·7%; 95% CI 2·6-20·9; p=0·016]), and was not attributable to differences in location, season, or concomitant medication. Four (7%) patients receiving acetylcysteine and three (5%) receiving placebo discontinued study treatment due to adverse events. In the exploratory analysis, change in FVC indicated that clinical benefit from addition of acetylcysteine to pirfenidone is unlikely, with the possibility of a harmful effect in patients with IPF (adjusted rate of decline 125·6 mL/6 months for acetylcysteine vs 34·3 mL/6 months for placebo; difference -91·3 mL; 95% CI -174·4 to -8·3; p=0·031). INTERPRETATION Findings from the PANORAMA study suggest that addition of acetylcysteine to pirfenidone does not substantially alter the tolerability profile of pirfenidone, and is unlikely to be beneficial in IPF. FUNDING InterMune International AG (Roche).

PDE4 inhibitors roflumilast and rolipram augment PGE2 inhibition of TGF-β1-stimulated fibroblasts

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-beta1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE(2) metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-beta1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was approximately 10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-beta1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE(2) and TGF-beta1-induced PGE(2) production. PDE4 inhibitors together with TGF-beta1 resulted in augmented PGE(2) production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-beta1-induced fibroblast stimulation.

Glucocorticoids Modulate TGF-β Production by Human Fetal Lung Fibroblasts

TGF-β is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF-β isoforms affect TGF-β production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-β1, -β2, or -β3. Post-culture media were collected for ELISA assays of TGF-β1, -β2, and -β3 . TGF-β mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-β2 and -β3 stimulated TGF-β1 production significantly (p < 0.01 relative to control). TGF-β1 stimulated TGF-β2 production (p < 0.01 relative to control). TGF-β3 was undetectable. Glucocorticoids significantly inhibited TGF-β1 and TGF-β2 production and reduced expression of the up-regulated TGF-β1 and TGF-β2 mRNA induced by exogenous TGF-β1, -β2, or -β3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-β-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-β isoforms may stimulate production of various TGF-β isoforms in the lung. Glucocorticoids then may block TGF-β production by modulating mRNA levels and c-Jun.

Cell Recovery in Bronchoalveolar Lavage Fluid in Smokers Is Dependent on Cumulative Smoking History

Background Smoking is a risk factor for various lung diseases in which BAL may be used as a part of a clinical investigation. Interpretation of BAL fluid cellularity is however difficult due to high variability, in particular among smokers. In this study we aimed to evaluate the effect of smoking on BAL cellular components in asymptomatic smokers. The effects of smoking cessation, age and gender were also investigated in groups of smokers and exsmokers. Methods We performed a retrospective review of BAL findings, to our knowledge the largest single center investigation, in our department from 1999 to 2009. One hundred thirty two current smokers (48 males and 84 females) and 44 ex-smokers (16 males and 28 females) were included. A group of 295 (132 males and 163 females) never-smokers served as reference. Result The median [5–95 pctl] total number of cells and cell concentration in current smokers were 63.4 [28.6–132.1]×106 and 382.1 [189.7–864.3]×106/L respectively and correlated positively to the cumulative smoking history. Macrophages were the predominant cell type (96.7% [90.4–99.0]) followed by lymphocytes (2% [0.8–7.7]) and neutrophils (0.6% [0–2.9]). The concentration of all inflammatory cells was increased in smokers compared to never smokers and ex-smokers. BAL fluid recovery was negatively correlated with age (p<0.001). Smoking men had a lower BAL fluid recovery than smoking women. Conclusion Smoking has a profound effect on BAL fluid cellularity, which is dependent on smoking history. Our results performed on a large group of current smokers and ex-smokers in a well standardized way, can contribute to better interpretation of BAL fluid cellularity in clinical context.

Remodeling in asthma and COPD – differences and similarities

Background:  Asthma and chronic obstructive pulmonary disease (COPD) are both inflammatory disorders. Diagnosis of these diseases is based upon limitation of expiratory airflow. The pathophysiological correlates to this impaired lung function are complex but they are associated with the development of structural changes in the airways and lung parenchyma. These remodeling processes differ between the two diseases. In asthma, airways obstruction is predominately located in the large airways, although recent studies indicate that inflammation and structural changes also is present in other compartments of the lungs. In COPD, remodeling of the small airways and lung parenchyma are the main correlates to the limitation of expiratory airflow. However, both asthma and COPD are heterogeneous disorders including various phenotypes and there is a considerable overlap between the two diseases.

Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease

BACKGROUND Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide and is increasing, primarily among women. Underdiagnosis is common, and because of the heterogeneous disease characteristics, molecular markers of specific disease phenotypes and more efficacious treatment regimens are urgently needed. OBJECTIVE In this study the soluble proteome of bronchoalveolar lavage cells, primarily consisting of macrophages, was investigated with the aim of identifying phenotypic differences in early disease development. METHODS Two-dimensional difference gel electrophoresis was used for relative quantification of protein levels, and multivariate modeling was applied to identify proteins of interest that were subsequently identified by means of liquid chromatography-mass spectrometry. RESULTS Significant gender differences were unveiled, with numerous alterations in the bronchoalveolar lavage cell proteome occurring in female but not male patients with COPD. Specifically, a subset of 19 proteins provided classification of female healthy smokers from female patients with COPD with 78% predictive power. Subsequent pathway analyses linked the observed alterations to downregulation of the lysosomal pathway and upregulation of the oxidative phosphorylation pathway, possibly linking dysregulation of macroautophagy to a female-dominated COPD disease phenotype. CONCLUSION This investigation makes an important contribution to the elucidation of putative molecular mechanisms underlying gender-based differences in the pathophysiology of COPD, linking alterations of specific molecular pathways to previously observed gender differences in clinical COPD phenotypes. Furthermore, these results stress the importance of the gender-specific search for biomarkers, diagnosis, and treatment in COPD.

Glucocorticoids Modulate TGF-β Production

TGF-β is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB-β isoforms affect TGF-β production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-β1, -β2, and -β3. TGF-β mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-β2 and -β3 stimulated TGF-β1 production significantly (p < 0.01 relative to control). TGF-β1 stimulated TGF-β2 production (p < 0.01 relative to control). TGF-β3 was undetectable. Glucocorticoids significantly inhibited TGF-β1 and -β2 production and reduced expression of the upregulated TGF-β1 and -β2 mRNA induced by exogenous TGF-β1, -β2 or -β3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF-β-stimulated cells, it was reduced by glucocorticoids. Thus, TGF-β isoforms may stimulate production of various TGF-β isoforms in the lung. Glucocorticoids then may block TGF-β production by modulating mRNA levels and c-Jun.