Véronique Leçon-Malas

Altered Nrf2/Keap1-Bach1 equilibrium in pulmonary emphysema

Background: Oxidative stress, resulting from the increased oxidative burden and decreased level of antioxidant proteins, plays a role in the pathophysiology of smoking-related pulmonary emphysema. Expression of several antioxidant proteins, such as heme oxygenase-1 (HO-1), glutathione peroxidase 2 (GPX2) and NAD(P)H:quinone oxidoreductase 1 (NQO1), results from an equilibrium created by positive or negative regulation by the transcription factors Nrf2, Keap1 and Bach1, respectively. However, whether the expression of these transcription factors is altered in emphysema and could account for decreased expression of antioxidant proteins is not known. A study was undertaken to investigate the expression and subcellular localisation of Nrf2, Keap1 and Bach1 as potential regulators of HO-1, GPX2 and NQO1 in alveolar macrophages, a key cell in oxidative stress, in lung surgical specimens from non-smokers without emphysema and smokers with and without emphysema. Methods and results: Western blot, immunohistochemical and laser scanning confocal analysis revealed that the Nrf2 protein level decreased significantly in whole lung tissue and alveolar macrophages (cytosol and nucleus) in patients with emphysema compared with those without emphysema. Conversely, Bach1 and Keap1 levels were increased in patients with emphysema. These modifications were associated with a parallel decrease in the expression of HO-1, GPX2 and NQO1 at the cellular level, which was inversely correlated with airway obstruction and distension indexes, and increased macrophage expression of the lipid peroxidation product 4-hydroxy-2-nonenal. Silencing RNA experiments in vitro in THP-1 cells were performed to confirm the cause-effect relation between the loss of Nrf2 and the decrease in HO-1, NQO1 and GPX2 expression. Nrf2/Keap1-Bach1 equilibrium was altered in alveolar macrophages in pulmonary emphysema, which points to a decreased stress response phenotype. Conclusions: This finding opens a new view of the pathophysiology of emphysema and could provide the basis for new therapeutic approaches based on preservation and/or restoration of such equilibrium.

Halogenated Anesthetics Reduce Interleukin-1β-induced Cytokine Secretion by Rat Alveolar Type II Cells in Primary Culture

Background Alveolar epithelial type II (ATII) cells participate in the intraalveolar cytokine network by secreting cytokines and are widely exposed to volatile anesthetics during general anesthesia. The aim of the current study was to evaluate the effects of halothane, enflurane, and isoflurane on rat ATII cell cytokine secretions in ATII primary cell cultures. Methods Alveolar epithelial type II primary cell cultures were obtained from adult rat lungs. ATII cells were stimulated by recombinant murine interleukin-1&bgr; (rmIL-1&bgr;) to mimic an inflammatory response, and immediately exposed for various duration to different concentration of halothane, enflurane, or isoflurane. Interleukin-6, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1) protein concentrations were then measured in cell culture supernatants. Recombinant mIL-1&bgr;-stimulated ATII cells exposed to air served as control. Results Halothane, isoflurane, and enflurane (1 minimum alveolar concentration [MAC], 4 h) decreased rmIL-1&bgr;-stimulated ATII cell secretions of interleukin-6, MIP-2, and MCP-1, but did not modify total protein secretion. Halothane exposure decreased rmIL-1&bgr;-stimulated ATII cell secretions of interleukin-6, MIP-2, and MCP-1 in a dose- and time-dependent manner. Total protein concentrations remained unchanged except at 1.5 MAC of halothane, and no cytotoxic effect could be evidenced by lactate dehydrogenase release. These effects were transient as rmIL-1&bgr;-stimulated ATII cell secretions of interleukin-6 and MIP-2 progressively reached control values between 4 and 24 h after the end of halothane exposure. However, MCP-1 inhibition persisted until 24 h. rmIL-1&bgr;-induced MIP-2 and tumor necrosis factor-&agr; mRNA expression were decreased by 36 and 24%, respectively, after halothane exposure. Conclusions The current study shows that exposure of rmIL-1&bgr;-stimulated ATII cells to volatile anesthetics reversibly alters their cytokine secretion. Therefore, volatile anesthesia, by modulating pulmonary epithelial cell secretion of inflammatory cytokines, might affect the lung inflammatory response.

Cardiopulmonary bypass decreases cytokine production in lipopolysaccharide-stimulated whole blood cells: roles of interleukin-10 and the extracorporeal circuit.

Objective To determine whether cardiopulmonary bypass (CPB) alters the ex vivo cytokine production of whole blood cells stimulated by lipopolysaccharide (LPS) and to assess the roles of interleukin (IL)-10 and an extracorporeal circuit (ECC) in the alteration. Design Prospective, controlled study. Setting Biochemistry laboratory and surgical intensive care unit in a university hospital. Patients Seventeen consecutive adult patients undergoing coronary artery bypass grafting or valve surgery with normothermic CPB and eight healthy volunteers. Interventions Blood samples for cytokine measurement were drawn from patients before and during (at 60, 90, 120, 180 and 360 mins) CPB and were cultured with and without LPS and with and without anti-IL-10 antibodies. Blood was also drawn from healthy subjects and sampled for cytokine analysis before and during circulation in an isolated ECC. Measurements and Main Results The concentrations of ex vivo tumor necrosis factor (TNF)-&agr;, IL-6, IL-8, and IL-10, measured by enzyme-linked immunosorbent assay, were reduced in both experimental settings. In patients on CPB, LPS hyporesponsiveness was detected at 60 mins after the onset of CPB and was maximal at 120 mins (78% to 86% decreases from pre-CPB levels) but was transient, except for TNF-&agr;. The plasma concentration of IL-10 peaked at 90 mins after the start of CPB, but the role of IL-10 in LPS hyporesponsiveness appears limited because anti-IL-10 antibodies significantly increased ex vivo production of IL-6 but not TNF-&agr; or IL-8. In the isolated ECC study, no IL-10 was detected in plasma, yet the ex vivo production of the cytokines (except IL-8) was decreased (by 66% to 95%). Conclusion Our results demonstrate the following: a) CPB induces an early and transient LPS hyporesponsiveness of whole blood as measured by cytokine production; b) IL-10 seems only partly involved in this process, and its role is restricted to an in vivo situation; and c) contact of blood with an ECC is sufficient to induce LPS hyporesponsiveness.

Decreased expression of interleukin 13 in human lung emphysema

Background: The overexpression of interferon (IFN)γ or interleukin (IL)-13 in the adult murine lung induces the development of changes that mirror human lung emphysema. Methods: IL-13 and IFNγ expression was determined in lung samples from five groups of patients: severe emphysema without α1-antitrypsin deficiency (SE+, nu200a=u200a10); severe emphysema with α1-antitrypsin deficiency (SE−, nu200a=u200a5); mild localised emphysema (ME, nu200a=u200a8); non-emphysema smokers (NE-S, nu200a=u200a9), and non-emphysema non-smokers (NE-NS, nu200a=u200a11). Lung IL-13 and IFNγ mRNA were analysed by RT-PCR. Lung concentrations of IL-13 protein were assessed by ELISA. Results: The expression of IFNγ mRNA was similar in patients with or without emphysema. IL-13 mRNA was markedly decreased in the SE+ group compared with the SE− (pu200a=u200a0.04), ME (pu200a=u200a0.02), and non-emphysema groups (pu200a=u200a0.01). IL-13 mRNA correlated with forced expiratory volume in 1 second (ru200a=u200a0.5, pu200a=u200a0.04) and arterial oxygen tension (ru200a=u200a0.45, pu200a=u200a0.03) in emphysema patients. In contrast to the non-emphysematous lung, IL-13 protein was below the detection limit of the assay in most emphysematous lung homogenates. Conclusion: The lung IL-13 content is reduced in patients with severe emphysema without α1-antitrypsin deficiency.

Induction of Heme Oxygenase-1, Biliverdin Reductase and H-Ferritin in Lung Macrophage in Smokers with Primary Spontaneous Pneumothorax: Role of HIF-1α

Background Few data concern the pathophysiology of primary spontaneous pneumothorax (PSP), which is associated with alveolar hypoxia/reoxygenation. This study tested the hypothesis that PSP is associated with oxidative stress in lung macrophages. We analysed expression of the oxidative stress marker 4-HNE; the antioxidant and anti-inflammatory proteins heme oxygenase-1 (HO-1), biliverdin reductase (BVR) and heavy chain of ferritin (H-ferritin); and the transcription factors controlling their expression Nrf2 and HIF-1α, in lung samples from smoker and nonsmoker patients with PSP (PSP-S and PSP-NS), cigarette smoke being a risk factor of recurrence of the disease. Methodology/Principal Findings mRNA was assessed by RT-PCR and proteins by western blot, immunohistochemistry and confocal laser analysis. 4-HNE, HO-1, BVR and H-ferritin were increased in macrophages from PSP-S as compared to PSP-NS and controls (C). HO-1 increase was associated with increased expression of HIF-1α mRNA and protein in alveolar macrophages in PSP-S patients, whereas Nrf2 was not modified. To understand the regulation of HO-1, BVR and H-ferritin, THP-1 macrophages were exposed to conditions mimicking conditions in C, PSP-S and PSP-NS patients: cigarette smoke condensate (CS) or air exposure followed or not by hypoxia/reoxygenation. Silencing RNA experiments confirmed that HIF-1α nuclear translocation was responsible for HO-1, BVR and H-ferritin induction mediated by CS and hypoxia/reoxygenation. Conclusions/Significance PSP in smokers is associated with lung macrophage oxidative stress. The response to this condition involves HIF-1α-mediated induction of HO-1, BVR and H-ferritin.

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

BackgroundHepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema.MethodsHepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction.ResultsHepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients groups.ConclusionThe expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

Régulation de l’hème oxygénase-1 dans les macrophages humains exposés à la fumée de cigarette

Introductionxa0: L’intoxication tabagique est un facteur de risque important de developpement de l’emphyseme pulmonaire, probleme majeur de sante publique. Les macrophages alveolaires contribuent a l’inflammation pulmonaire dans l’emphyseme en produisant de nombreuses metalloproteases matricielles et des derives reactifs de l’oxygene impliques dans le desequilibre des balances protease/antiprotease et oxydant/anti-oxydant. Les facteurs de transcription Nrf2/ Bach1 (inducteur/inhibiteur) et la proteine Keap1, impliquee dans la degradation de Nrf2 par le proteasome, jouent un role primordial dans la regulation de l’expression d’enzymes anti-oxydantes comme l’heme oxygenase-1 (HO-1). Nous avons montre qu’une anomalie de la voie Nrf2-Keap1/Bach1 etait associee a une diminution de l’expression d’HO-1, enzyme cle du systeme anti-oxydant pulmonaire, dans les macrophages alveolaires de patients fumeurs presentant un emphyseme severe (Goven Thorax 2008). Le but de ce travail est d’etudier in vitro l’effet de l’exposition prolongee des macrophages a la fumee de cigarette (FC) sur l’expression d’HO-1 et sa regulation via les mitogen-activated protein kinase (MAPK). Methodesxa0: Les macrophages humains derives des monocytes de la lignee THP1 ont ete exposes au condensat de FC (0,01 a 20xa0µg/ml) en presence ou non des inhibiteurs specifiques des kinases. Les ARNm d’HO-1 et Keap1 ont ete analyses par PCR quantitative en temps reel. Les proteines HO-1, Nrf2, Bach1, Keap1 et les proteines kinases (p38, Erk1/2, JNK) ont ete etudiees par western-blot. Resultatsxa0: La FC conduit a une induction precoce d’HO-1 (6xa0h- 24xa0h) associee a une translocation nucleaire de Nrf2. Apres 72xa0heures d’exposition au condensat de FC, l’expression d’HO-1 diminue simultanement a une augmentation de la translocation nucleaire de Bach1 et une diminution de celle de Nrf2, associee a une accumulation cytoplasmique de Keap1. Cette diminution d’HO-1 n’est pas observee avec d’autres inducteursxa0: H 2 O 2 , IL1-β, TNF-α et elastase neutrophile. La diminution d’HO-1 induite par l’exposition prolongee au condensat de FC est associee a une augmentation de l’expression de l’ARNm de Keap1 et de la phosphorylation des proteines kinases (p38, Erk et JNK) qui controlent la degradation de Nrf2 par le proteasome. L’effet de la FC sur l’expression d’HO-1 est aboli uniquement en presence des inhibiteurs specifiques des kinases Erk et JNK, ce qui suggere une implication de ces kinases dans la modulation de l’expression d’HO-1 par le tabac. Conclusionsxa0: Ces resultats indiquent que l’exposition prolongee des macrophages humains a la FC conduit a une diminution de l’expression d’HO-1 telle qu’on l’observe dans l’emphyseme pulmonaire severe et qui semble liee a une alteration de l’equilibre Nrf2-Keap1/Bach1 modulee par Erk et JNK.