Biomarker Discovery In Chronic Obstructive Pulmonary Disease (COPD) Using Epithelial Lining Fluid:A Proteomic Approach

RATIONALE Chronic Obstructive Pulmonary Disease (COPD) is the third most frequent disease worldwide with increasing mortality. Cigarette smoking is the principle risk factor and 15-20% of smokers develop COPD. Epithelial Lining Fluid (ELF) covers the internal part of the airways and can be collected during bronchoscopy. ELF appears to be well-suited for proteomic analysis, since it contains a higher concentration of proteins (150-300 μg /mL) than other lung fluids and can be obtained from different locations of the lungs. No comprehensive proteomic analysis of human ELF has been performed to date, which makes ELF a highly interesting fluid for biomarker discovery in COPD. AIM To discover proteins that change in abundance in ELF from COPD patients versus healthy controls using a quantitative proteomics approach. METHODS The ELF proteome from COPD patients and healthy controls was studied by 1D polyacrylamide gel electrophoresis in the presence of SDS followed by in-gel tryptic digestion to establish the methodology and assess the feasibility of such an approach. Approximately 40 gel slices were obtained from each lane of the gel (corresponding to one patient). Digested samples were analyzed by nanoChip-LC-MS/MS using an ion trap. We performed a quantitative pilot study of ELF from 4 COPD patients and 4 healthy controls (table 1) to test for statistically significant differences in protein levels. ELF samples were digested by trypsin, labeled with stable isotope-containing reagents (iTRAQ®, 8-plex) and processed by strong cation-exchange chromatography followed by nanoLC-MS/MS. In order to validate the results, a second quantitative analysis of an independent sample set (4 COPD vs 4 healthy) using the same methodological approach was done. RESULTS The 1D electrophoretic approach resulted in more than 300 identified proteins. Most of the identified proteins were present in both COPD and healthy samples, although some proteins were only identified either in healthy control or in COPD samples. The quantitative studies showed that a number of proteins was significantly different between ELF of COPD patients and controls, including 4 up-regulated proteins in common in both studies. CONCLUSIONS This is the first study in ELF of COPD patients and healthy controls in which such a large number of proteins has been identified. The obtained results show the feasibility of this proteomic approach and the possibility to discover proteins that are differentially expressed in ELF of COPD patients and controls. We are currently validating these proteins further by western blot and immunohistochemistry

Mitochondrial dysfunction increases pro-inflammatory cytokine production and impairs repair and corticosteroid responsiveness in lung epithelium

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD

Two sisters with lung emphysema

BACKGROUND: α1-antitrypsin is an antiprotease that is mainly produced in the liver; it plays a crucial role in the protection of lung parenchyma against the destructive effects of proteases. Mutations in the α1-antitrypsin gene can cause α1-antitrypsin deficiency. Individuals homozygous for the Z-genotype have drastically lowered serum α1-antitrypsine concentrations and often develop lung emphysema at an early age.CASE DESCRIPTION: A 38-year-old woman and her 43-year-old sister both developed lung emphysema at an early age; this could be attributed to severe α1-antitrypsin deficiency. The only treatment for this condition is α1-antitrypsin supplement therapy, but this therapy is not reimbursed by health insurance companies in the Netherlands.CONCLUSION: α1-antitrypsin deficiency is a relatively rare cause of lung emphysema and can be seen as an orphan phenotype of chronic obstructive pulmonary disease (COPD). We appeal for reconsideration of coverage of α1-antitrypsine supplement therapy by basic health insurance in the Netherlands, on the basis of a recent randomised placebo-controlled study in which the protective effect of this therapy on progressive emphysema was demonstrated by CT lung densitometry.</p

Gene expression profiling of bronchial brushes is associated with the level of emphysema measured by computed tomography-based parametric response mapping

Parametric response mapping (PRM) is a computed tomography (CT)-based method to phenotype patients with chronic obstructive pulmonary disease (COPD). It is capable of differentiating emphysema-related air trapping with nonemphysematous air trapping (small airway disease), which helps to identify the extent and localization of the disease. Most studies evaluating the gene expression in smokers and COPD patients related this to spirometric measurements, but none have investigated the relationship with CT-based measurements of lung structure. The current study aimed to examine gene expression profiles of brushed bronchial epithelial cells in association with the PRM-defined CT-based measurements of emphysema (PRM(Emph)) and small airway disease (PRM(fSAD)). Using the Top Institute Pharma (TIP) study cohort (COPD = 12 and asymptomatic smokers = 32), we identified a gene expression signature of bronchial brushings, which was associated with PRM(Emph) in the lungs. One hundred thirty-three genes were identified to be associated with PRM(Emph). Among the most significantly associated genes, CXCL11 is a potent chemokine involved with CD8(+) T cell activation during inflammation in COPD, indicating that it may play an essential role in the development of emphysema. The PRM(Emph) signature was then replicated in two independent data sets. Pathway analysis showed that the PRM(Emph) signature is associated with proinflammatory and notch signaling pathways. Together these findings indicate that airway epithelium may play a role in the development of emphysema and/or may act as a biomarker for the presence of emphysema. In contrast, its role in relation to functional small airways disease is less clear

Wnt5a induces ROR1 to complex with HS1 to enhance migration of chronic lymphocytic leukemia cells.

ROR1 (receptor tyrosine kinase-like orphan receptor 1) is a conserved, oncoembryonic surface antigen expressed in chronic lymphocytic leukemia (CLL). We found that ROR1 associates with hematopoietic-lineage-cell-specific protein 1 (HS1) in freshly isolated CLL cells or in CLL cells cultured with exogenous Wnt5a. Wnt5a also induced HS1 tyrosine phosphorylation, recruitment of ARHGEF1, activation of RhoA and enhanced chemokine-directed migration; such effects could be inhibited by cirmtuzumab, a humanized anti-ROR1 mAb. We generated truncated forms of ROR1 and found its extracellular cysteine-rich domain or kringle domain was necessary for Wnt5a-induced HS1 phosphorylation. Moreover, the cytoplamic, and more specifically the proline-rich domain (PRD), of ROR1 was required for it to associate with HS1 and allow for F-actin polymerization in response to Wnt5a. Accordingly, we introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1 PRD at positions 784, 808, 826, 841 or 850 in potential SH3-binding motifs. In contrast to wild-type ROR1, or other ROR1P→︀A mutants, ROR1P(841)A had impaired capacity to recruit HS1 and ARHGEF1 to ROR1 in response to Wnt5a. Moreover, Wnt5a could not induce cells expressing ROR1P(841)A to phosphorylate HS1 or activate ARHGEF1, and was unable to enhance CLL-cell motility. Collectively, these studies indicate HS1 plays an important role in ROR1-dependent Wnt5a-enhanced chemokine-directed leukemia-cell migration

Neutrophilic Asthma Is Associated With Smoking, High Numbers of IRF5+, and Low Numbers of IL10+ Macrophages

Asthma is a heterogenous disease with different inflammatory subgroups that differ in disease severity. This disease variation is hampering treatment and development of new treatment strategies. Macrophages may contribute to asthma phenotypes by their ability to activate in different ways, i.e., T helper cell 1 (Th1)-associated, Th2-associated, or anti-inflammatory activation. It is currently unknown if these different types of activation correspond with specific inflammatory subgroups of asthma. We hypothesized that eosinophilic asthma would be characterized by having Th2-associated macrophages, whereas neutrophilic asthma would have Th1-associated macrophages and both having few anti-inflammatory macrophages. We quantified macrophage subsets in bronchial biopsies of asthma patients using interferon regulatory factor 5 (IRF5)/CD68 for Th1-associated macrophages, CD206/CD68 for Th2-associated macrophages and interleukin 10 (IL10)/CD68 for anti-inflammatory macrophages. Macrophage subset percentages were investigated in subgroups of asthma as defined by unsupervised clustering using neutrophil/eosinophil counts in sputum and tissue and forced expiratory volume in 1 s (FEV1). Asthma patients clustered into four subgroups: mixed-eosinophilic/neutrophilic, paucigranulocytic, neutrophilic with normal FEV1, and neutrophilic with low FEV1, the latter group consisting mainly of smokers. No differences were found for CD206+ macrophages within asthma subgroups. In contrast, IRF5+ macrophages were significantly higher and IL10+ macrophages lower in neutrophilic asthmatics with low FEV1 as compared to those with neutrophilic asthma and normal FEV1 or mixed-eosinophilic asthma. This study shows that neutrophilic asthma with low FEV1 is associated with high numbers of IRF5+, and low numbers of IL10+ macrophages, which may be the result of combined effects of smoking and having asthma.</p