Lorenza Franciosi

Cigarette smoke induces endoplasmic reticulum stress response and proteasomal dysfunction in human alveolar epithelial cells

•  What is the central question of this study? The endoplasmic reticulum stress response caused by cigarette smoke may lead to excessive apoptosis with disruption of the epithelial barrier, thus contributing to chronic obstructive pulmonary disease. One way of promoting cell survival is to facilitate degradation of cigarette smoke‐induced protein damage through the ubiquitin–proteasome pathway. Direct effects of gas‐phase cigarette smoke on proteasomal activities have not been demonstrated previously. •  What is the main finding and what is its importance? We show that cigarette smoke induces protein damage and triggers the endoplasmic reticulum stress response in human alveolar epithelial cells. A significant reduction of all three proteasomal activities was found. Ineffective degradation of damaged proteins could lead to a sustained epithelial stress response and development of chronic obstructive pulmonary disease.

Acute and chronic inflammatory responses induced by smoking in individuals susceptible and non-susceptible to development of COPD: from specific disease phenotyping towards novel therapy. Protocol of a cross-sectional study

Introduction Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with pulmonary and extra-pulmonary manifestations. Although COPD is a complex disease, diagnosis and staging are still based on simple spirometry measurements. Different COPD phenotypes exist based on clinical, physiological, immunological and radiological observations. Cigarette smoking is the most important risk factor for COPD, but only 15–20% of smokers develop the disease, suggesting a genetic predisposition. Unfortunately, little is known about the pathogenesis of COPD, and even less on the very first steps that are associated with an aberrant response to smoke exposure. This study aims to investigate the underlying local and systemic inflammation of different clinical COPD phenotypes, and acute effects of cigarette smoke exposure in individuals susceptible and non-susceptible for the development of COPD. Furthermore, we will investigate mechanisms associated with corticosteroid insensitivity. Our study will provide valuable information regarding the pathogenetic mechanisms underlying the natural course of COPD. Methods and analysis This cross-sectional study will include young and old individuals susceptible or non-susceptible to develop COPD. At a young age (18–40 years) 60 ‘party smokers’ will be included who are called susceptible or non-susceptible based on COPD prevalence in smoking family members. In addition, 30 healthy smokers (age 40–75 years) and 110 COPD patients will be included. Measurements will include questionnaires, pulmonary function, low-dose CT scanning of the lung, body composition, 6 min walking distance and biomarkers in peripheral blood, sputum, urine, exhaled breath condensate, epithelial lining fluid, bronchial brushes and biopsies. Non-biased approaches such as proteomics will be performed in blood and epithelial lining fluid. Ethics and dissemination This multicentre study was approved by the medical ethical committees of UMC Groningen and Utrecht, the Netherlands. The study findings will be presented at conferences and will be reported in peer-reviewed journals. Trial registration ClinicalTrials.gov, NCT00807469 (study 1) and NCT00850863 (study 2).

Susceptibility to COPD: differential proteomic profiling after acute smoking.

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as “susceptible individuals”. Here we perform unbiased analyses of proteomic profiles to assess how “susceptible individuals” differ from age-matched “non-susceptible individuals” in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD.

Proteomic analysis of human epithelial lining fluid by microfluidics-based nanoLC-MS/MS: a feasibility study.

Microfluidics‐based nanoLC‐MS/MS (chipLC‐MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D‐PAGE followed by in‐gel tryptic digestion and chipLC‐MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non‐COPD controls using chemical stable isotope labeling (iTRAQ®‐8Plex) showed that the levels of lactotransferrin, high‐mobility group protein B1 (HMGB 1), alpha 1‐antichymotrypsin and cofilin‐1 differed significantly in ELF from COPD patients and non‐COPD controls (p‐values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non‐COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC‐MS/MS and quantitative proteomics in human ELF.

Proteomics of Epithelial Lining Fluid Obtained by Bronchoscopic Microprobe Sampling

Epithelial lining fluid (ELF) forms a thin fluid layer that covers the mucosa of the alveoli, the small airways, and the large airways. Since it constitutes the first barrier between the lung and the outer world, it is an interesting target for proteomics studies that focus on lung disease. Bronchoscopic microprobe (BMP) sampling of ELF uses small probes with an absorptive tip that are introduced bronchoscopically. In contrast to other methods used so far for the collection of biofluids from the lung (e.g., bronchoalveolar lavage fluid, induced sputum), this technique has the advantage that ELF is not diluted and contains high concentrations of biomolecules. In addition, the investigated location in the tracheobronchial tree is well defined, and there is no contamination with oropharyngeal bacteria or saliva. Despite occasional blood contamination of the probes by scratching the mucosa of the airways, the proteomic analysis of microprobe-sampled ELF opens new possibilities for research in lung diseases. Our work focuses particularly on the induction and progression of cigarette smoke-induced Chronic Obstructive Pulmonary Disease (COPD). In this chapter, we describe the practical aspects of sampling ELF followed by a detailed description of proteomics analysis by LC-MS/MS after protein separation by SDS-PAGE and in-gel digestion. As an example, we apply this proteomic platform to the identification and quantification of proteins in ELF from COPD patients and healthy subjects.

GM-CSF and TNFα modulate protein expression of human neutrophils visualized by fluorescence two-dimensional difference gel electrophoresis

Increased serum levels of TNFα and GM-CSF are found in various chronic inflammatory diseases and these cytokines affect the function of circulating and tissue neutrophils. TNFα- and GM-CSF-induced protein expression profiles could, therefore, serve as biomarker for the action of these cytokines in vivo. We stimulated human peripheral neutrophils with TNFα and GM-CSF in vitro and analyzed changes in their proteome by fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). We report the differential expression of 3 and 18 protein spots following TNFα and GM-CSF stimulation, respectively. Differences in protein expression induced by TNFα were limited and did not show discriminatory power in a principal component analysis, whereas the profile induced by GM-CSF did. TNFα- and GM-CSF-induced both de novo IL-1β and sIL-1Ra protein expression as detected by Western blot analysis, which confirmed proper neutrophil activation by these cytokines in vitro. Mass spectrometry analysis of cytokine-regulated protein spots resulted in the identification of 8 proteins. Among the identified proteins, enolase 1 and annexin A1 might function as markers for peripheral neutrophil activation. In conclusion, a proteomic analysis of neutrophils by 2D-DIGE provides proof-of-principle that cytokine-induced protein profiles can serve as biomarkers for the action of individual cytokines in vivo.

Proteomic analysis of human epithelial lining fluid by microfluidics-based nanoLC-MS/MS: A feasibility study: Microfluidics and Miniaturization

Microfluidics‐based nanoLC‐MS/MS (chipLC‐MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D‐PAGE followed by in‐gel tryptic digestion and chipLC‐MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non‐COPD controls using chemical stable isotope labeling (iTRAQ®‐8Plex) showed that the levels of lactotransferrin, high‐mobility group protein B1 (HMGB 1), alpha 1‐antichymotrypsin and cofilin‐1 differed significantly in ELF from COPD patients and non‐COPD controls (p‐values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non‐COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC‐MS/MS and quantitative proteomics in human ELF.

Proteomic analysis of human epithelial lining fluid by microfluidics-based nanoLC-MS/MS

Microfluidics‐based nanoLC‐MS/MS (chipLC‐MS/MS) was used to identify and quantify proteins in epithelial lining fluid (ELF), collected during bronchoscopy from the main bronchi of chronic obstructive pulmonary disease (COPD) patients and healthy controls using microprobes. ELF is a biofluid that is well suited to study pathophysiological processes in the lung, because it contains high concentrations of biologically active molecules. 1D‐PAGE followed by in‐gel tryptic digestion and chipLC‐MS/MS resulted in identification of approximately 300 proteins. A comparative study of ELF from COPD patients and non‐COPD controls using chemical stable isotope labeling (iTRAQ®‐8Plex) showed that the levels of lactotransferrin, high‐mobility group protein B1 (HMGB 1), alpha 1‐antichymotrypsin and cofilin‐1 differed significantly in ELF from COPD patients and non‐COPD controls (p‐values < 0.05). These results were reproduced in another, independent set of ELF samples from COPD patients and non‐COPD controls and further validated by immunohistochemistry. This study shows the feasibility of performing chipLC‐MS/MS and quantitative proteomics in human ELF.

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.