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La pollution de l’air ‒ ou pollution atmospherique ‒ est une modification de la composition de l’air par des polluants nuisibles a la sante et a l’environnement ; elle constitue la plus grande cause environnementale de maladies et de deces prematures dans le monde actuel. Negligee initialement car consideree comme un effet adverse de la pollution atmospherique, la relation entre la pollution et la survenue et/ou la severite d’infections respiratoires est devenue ces dernieres annees une preoccupation croissante en sante publique. En ce qui concerne les infections virales, qui constituent une majorite des infections respiratoires, differentes etudes montrent que leur incidence et/ou leur severite peuvent etre correlees aux concentrations des polluants atmospheriques, tels que le dioxyde d’azote (NO2), l’ozone (O3) et les particules. Les mecanismes sous-jacents ne sont pas completement elucides a l’heure actuelle, ils font probablement intervenir une facilitation de la transmission des virus et/ou une susceptibilite accrue aux effets des virus. Bien que d’autres etudes soient necessaires pour mieux comprendre ces phenomenes, les donnees disponibles doivent inciter la prise de mesures pour diminuer la concentration des polluants dans l’air afin de contrer la surmortalite liee a la pollution, y compris les infections virales.

International audience; Obstructive sleep apnea is a common chronic disorder that leads to chronic intermittent hypoxia described as an important factor contributing to the pathogenesis of OSA-related comorbidities. Besides, recent data suggest that intermittent hypoxia can induce adaptative cardiovascular pathways inducing a relative resistance to ischemic insults. Adaptative pathways induced by hypoxia could implicate autophagic processes and Beclin-1, one of the first mammalian autophagy effectors. Thus, activation of autophagy could protect against cardiovascular events in patients with OSA and could be considered as biomarker of a better prognosis.

International audience; Background: Mineralogical analyses of bronchoalveolar lavage (BAL) may help in assessing past exposure to mineral particles. However, their interpretation relies on their quality, meaning their representativeness of the alveolar compartment. The aim of this study was to find predictive factors of BAL samples quality allowing a reliable mineralogical analysis. Methods: All BAL samples analyzed between 2018 and 2020 in the Asbestos Fibers and Particles Laboratory from Paris City were included. They were read by an experienced cyto-pathologist and validated according to their representativeness of the alveolar region compartment. Univariate and stratified analyses were conducted to identify factors associated with the samples’ cytological quality. Results: On the 780 samples included, 64.4% were deemed of good cytological quality and 17.9% were not interpretable. Injected volume and BAL yield (recovery volume on injected volume ratio) were associated with cytological quality. Injecting at least 100mL with a ≥60% yield or injecting at least 150mL with a ≥30% yield allowed having a good proportion of BAL with sufficient cytological quality. Conclusions: Injected volume greater than 100mL with sufficient BAL yield are essential factors to ensure a reliable mineralogical analysis of BAL samples.

La pollution atmospherique constitue un risque environnemental majeur pour la sante. Une exposition a des niveaux eleves de pollution atmospherique entraine des effets respiratoires, mais aussi au niveau cardiovasculaire ou du systeme nerveux, notamment. Un important constat est que ces effets dependent non seulement de la qualite de l’air, mais aussi des sujets concernes et de leur vulnerabilite individuelle (enfants, personnes âgees, patients avec pathologies prealables...). Du point de vue de la sante, les constituants de la pollution atmospherique le plus souvent associes a des effets deleteres sont les polluants gazeux (ozone, dioxyde de soufre, oxydes d’azote...) et les particules. Cependant, l’une des principales difficultes tient a l’extreme complexite du melange atmospherique, le developpement de dispositifs experimentaux innovants est donc necessaire pour mieux apprehender ces effets. Les efforts doivent etre poursuivis pour proteger les populations des effets deleteres de la pollution de l’air sur leur sante.

Introduction: Alveolar type 2 cells (AT2) play a key role in alveolar regeneration after alveolar damage. Mechanisms involved in this process are not well known. Aims and Objectives: Targeting p16INK4a, a cell cycle inhibitor, in AT2 could be a way to promote alveolar regeneration. Methods: In vitro alveolar organoids were obtained by co-culturing EpCAM+ cells (sorted with magnetic beads from lungs of p16INK4a-/- or wild type (WT) mice) with WT lung primary fibroblasts. Colony forming units (CFU), organoid size, organoid types (alveolar, bronchoalveolar by pro-SpC and ACT staining) were quantified at D14 and D21. Organoids from emphysematous mice (elastase model) were compared to organoids from PBS-injected mice as a control with the same genetic background (p16INK4a-/- or WT). In vivo, morphological analysis (mean linear intercept) and AT2 number were quantified in elastase or PBS injected mice at D21 and D90 to assess alveolar destruction and regeneration according to WT or p16INK4a-/- genotype. Results: Organoids made with p16INK4a-/- EpCAM+ cells were as numerous but had an increased size compared to those made from WT EpCAM+ cells at D14 and D21. CFU and organoid size from emphysematous mice were significantly decreased compared to control mice. Number and size of organoids made with p16INK4a-/- EpCAM+cells from emphysematous mice were higher than organoids made with WT EpCAM + cells from emphysematous mice. In vivo, p16INK4a deletion was associated with an increase of AT2 at D21 without a protective effect on alveolar architecture. At D90, p16INK4a-/- mice had less emphysema than WT mice. Conclusions: p16INK4a deletion increases endogenous alveolar regeneration by AT2 cells. Mechanisms involved remain to be determined.

Gold (Au) can be deposited as nanoparticles (NPs) smaller than 10 nm in diameter on a variety of metal oxide (MOx) NPs. Au/MOx NPs have high catalytic performance and selective oxidation capacity which could have implications in terms of biological activity, and more specifically in modulation of the inflammatory reaction. Therefore, the aim of this study was to examine the effect of Au/TiO2, Au/ZrO2 and Au/Ce/O2 on viability, phagocytic capacity and inflammatory profile (TNF-α and IL-1β secretion) of murine macrophages. The most important result of this study is an anti-inflammatory effect of Au/MOx NPs depending on the MOx nature with particle internalization and no alteration of cell viability and phagocytosis. The effect was dependent on the MOx NPs chemical nature (Au/TiO2> Au/ZrO2> Au/CeO2 if we consider the number of cytokines whose concentration was reduced by the NPs), and on the inflammatory mediator considered. The effect of Au/TiO2 NPs was not related to Au NPs size (at least in the case of Au/TiO2 NPs in the range of 3-8 nm). To the best of our knowledge, this is the first demonstration of an anti-inflammatory effect of Au/MOx NPs.

BackgroundCerium (Ce) is a rare earth element, rapidly oxidizing to form CeO2, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are yet not completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO2 NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO2 NP by single oropharyngeal administration and sacrificed up to one month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFβ1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype). Results: Such pulmonary exposure to CeO2 NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockade of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype. Conclusion: In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as potential new therapeutic targets for future anti-fibrotic therapies.

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (β-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.

SummaryUsing CESAM, an atmospheric simulation chamber (cesam.cnrs.fr), we have developed a totally innovative platform for exposing mice to realistic atmospheric conditions. Here we present the first toxicological analyses of the organs of these mice after 48 hours to several days of exposure, carried out as part of feasibility experiments aimed at testing this experimental concept. This platform has received funding from the European Union’s Horizon 2020 research and innovation programme through the EUROCHAMP-2020 Infrastructure Activity under grant agreement N° 730997, and is now supporting the new REMEDIA H-2020 project (call H2020 “Exposome”) IntroductionThe World Health Organization (WHO) estimated that there were 3.7 million premature deaths due to air pollution in 2014, and confirmed that air pollution is the greatest environmental risk to health (responsible for a loss of more than 3% of productivity).The studies conducted so far show that the effects of air pollution on health depend not only on the quality of the surrounding air, but also on the subjects exposed and their individual vulnerability (asthma, obesity, period of life, etc.). Despite the evidence on the adverse health effects of exposure to air micro-pollutants, there are still uncertainties about the nature of these effects, and progress need to be made on their quantification. This limitation of knowledge is mainly attributed to the complexity of the polluted atmospheres, and to the great difficulty to model the impact of realistic situations of exposure. MethodologyThe innovative approach we set up is to realistically simulate, at the laboratory, the atmospheric mixture in all its complexity, thus keeping the ability to control, reproduce and carefully characterize the experimental conditions. We used the CESAM chamber (4.2 m3 stainless steel atmospheric simulation, evacuable down to a few 10-7 atm, temperature controlled between +15°C and +60°C) in order to study the myriad of products arising from the atmospheric oxidation of primary organic compounds.The experimental protocol consists in the continuous injection of relevant mixtures of primary pollutants (mainly nitrogen oxides, organic compounds from a representative mix of anthropogenic emissions, sulphur dioxide, soot, inorganic salts and potentially mineral dust particles if needed - e.g. to simulate Beijing’s atmosphere) at low concentrations (ppb levels) in air in the CESAM simulation chamber operated as a slow flow reactor. The residence time of simulated air parcels in the experimental volume is fixed to 4 hours, in order to represent air masses of regional scale. During this time the synthetic mixture is exposed to an artificial solar irradiation, allowing secondary pollutants such as ozone, nitric acid, formaldehyde, peroxyacetyl nitrate as well as complex polyfunctional organics including SOA to be produced and to reach their chemical steady state. Mice are exposed to constant flows of such a mixture during time scales of week to address their effects on health. ConclusionsHere we present the first toxicological analyses related to organs/tissue of these mice after exposure of 48h to several day, carried out with a representative atmosphere of Beijing or a representative atmosphere of Paris. ReferencesColl P. et al., 2018, WIT Transactions on Ecology and the Environment, 230.

Whether the systemic manifestations observed in Chronic Obstructive Pulmonary Disease (COPD) are ascribable to lung dysfunction or direct effects of smoking is in debate. Structural Equations Modeling (SEM), a causal-oriented statistical approach, could help unraveling the pathways involved, by enabling estimation of direct and indirect associations between variables. The objectives of the study was to investigate the relative impact of smoking and COPD on systemic manifestations, inflammation and telomere length. In 292 individuals (103 women; 97 smokers with COPD, 96 smokers without COPD, 99 non-smokers), we used SEM to explore the pathways between smoking (pack-years), lung disease (FEV1, KCO), and the following parameters: arterial stiffness (aortic pulse wave velocity, PWV), bone mineral density (BMD), appendicular skeletal muscle mass (ASMM), grip strength, insulin resistance (HOMA-IR), creatinine clearance (eGFR), blood leukocyte telomere length and inflammatory markers (Luminex assay). All models were adjusted on age and gender. Latent variables were created for systemic inflammation (inflammatory markers) and musculoskeletal parameters (ASMM, grip strength, BMD). SEM showed that most effects of smoking were indirectly mediated by lung dysfunction: e.g. via FEV1 on musculoskeletal factor, eGFR, HOMA-IR, PWV, telomere length, CRP, white blood cells count (WBC) and inflammation factor, and via KCO on musculoskeletal factor, eGFR and PWV. Direct effects of smoking were limited to CRP and WBC. Models had excellent fit. In conclusion, SEM highlighted the major role of COPD in the occurrence of systemic manifestations while smoking effects were mostly mediated by lung function.

Introduction Les pneumocytes de type 2 (p2) jouent un role cle dans les processus de regeneration apres des dommages alveolaires. Les mecanismes sous-jacents sont peu connus. L’objectif de ce travail est d’examiner si l’inhibiteur du cycle cellulaire p16INK4a module des proprietes des p2 et augmentant leurs capacites a induire une regeneration alveolaire. Methodes Des organoides pulmonaires ont ete realises par la mise en co-culture de cellules EpCAM+ obtenues par tri cellulaire magnetique (CD31-, CD45-, CD16/32-, CD90-, CD271-, Ter119-, EpCAM+) a partir de poumon de souris p16INK4a-/− ou WT avec des fibroblastes primaires pulmonaires WT. Une analyse de la taille et du nombre de colonies d’organoides formees (colony forming unit : CFU) et du type d’organoides (alveolaires ou bronchiques avec les marquages proSpC et acetylated tubulin-ACT) a ete realisee a 14 et 21 jours de culture. Dans un deuxieme temps, ces organoides ont ete obtenus a partir de poumon de souris emphysemateuses (J21 apres instillation d’elastase 2UI) compare a des souris controles (serum physiologique), p16INK4a-/− ou WT. Des analyses morphometriques (mean linear intercept) et le nombre de p2 ont ete realises 21 et 90 jours apres elastase. Resultats Les organoides formes de cellule EpCAM- p16INK4a-/− avaient une taille plus importante que ceux forme a partir d’EpCAM-WT a J14 (p = 0,046) et J21 (p = 0,0001). La majorite des organoides etaient alveolaires. La taille des organoides issus de souris emphysemateuses etait plus basse par rapport aux controles (p = 0,019), avec une tendance a la diminution du nombre de CFU. Les organoides issus de souris emphysemateuses p16INK4a-/− etaient de taille plus importante et plus nombreux que ceux issus de souris emphysemateuses WT. In vivo, la deletion de p16 etait associee 21 jours apres instillation d’elastase a un nombre plus important de p2 dans le poumon sans impact sur l’emphyseme. 90 jours apres elastase, les souris p16INK4a-/− avaient moins d’emphyseme (mean linear intercept) que les souris WT. Conclusions Inhiber p16INK4a augmente les capacites de regeneration endogene alveolaire des p2. Les mecanismes impliques restent a determiner.

Background: Fetal determinants of airway function, such as in utero exposure to maternal cigarette smoke (CS), may create a predisposition to adult airflow obstruction and chronic obstructive pulmonary disease (COPD) in adulthood. It has been suggested that active smoking in adolescence and preexisting airflow obstruction have synergistic deleterious effects. Objective: We used a mouse model to investigate whether there is a synergistic effect of exposure to CS in utero and during adolescence on lung function. Methods: Female C57Bl/6J mice were exposed to CS or to filtered room air during pregnancy. Exposure to CS began 2 weeks before mating and continued until delivery. After birth, the pups were not exposed to CS until day 21 (D21). Between D21 and D49, corresponding to “adolescence,” litters were randomized for an additional 4 weeks of exposure to CS. Lung morphometry, lung mechanics, and the expression of genes involved in senescence were evaluated in different subsets of mice on D21 and D49. Results: In utero exposure to CS induced significant lung function impairment by D21. CS exposure between D21 and D49 induced significant functional impairment only in mice exposed to CS prenatally. On D49, no difference was observed between subgroups in terms of lung p53, p16, p21, and Bax mRNA levels. Conclusions: Our findings suggest that prenatal and adolescent CS exposure have a synergistic effect on lung function in mice. The combined effect did not appear to be a consequence of early pulmonary senescence. Citation: Drummond D, Baravalle-Einaudi M, Lezmi G, Vibhushan S, Franco-Montoya ML, Hadchouel A, Boczkowski J, Delacourt C. 2017. Combined effects of in utero and adolescent tobacco smoke exposure on lung function in C57Bl/6J mice. Environ Health Perspect 125:392–399; http://dx.doi.org/10.1289/EHP54

International audience; Rationale: Promoting endogenous pulmonary regeneration is crucial after damage to restore normal lungs and prevent the onset of chronic adult lung diseases.Objectives: To investigate whether the cell-cycle inhibitor p16INK4a limits lung regeneration after newborn bronchopulmonary dysplasia (BPD), a condition characterized by the arrest of alveolar development, leading to adult sequelae.Methods: We exposed p16INK4a-/- and p16INK4a ATTAC (apoptosis through targeted activation of caspase 8) transgenic mice to postnatal hyperoxia, followed by pneumonectomy of the p16INK4a-/- mice. We measured p16INK4a in blood mononuclear cells of preterm newborns, 7- to 15-year-old survivors of BPD, and the lungs of patients with BPD.Measurements and Main Results: p16INK4a concentrations increased in lung fibroblasts after hyperoxia-induced BPD in mice and persisted into adulthood. p16INK4a deficiency did not protect against hyperoxic lesions in newborn pups but promoted restoration of the lung architecture by adulthood. Curative clearance of p16INK4a-positive cells once hyperoxic lung lesions were established restored normal lungs by adulthood. p16INK4a deficiency increased neutral lipid synthesis and promoted lipofibroblast and alveolar type 2 (AT2) cell development within the stem-cell niche. Besides, lipofibroblasts support self-renewal of AT2 cells into alveolospheres. Induction with a PPARγ (peroxisome proliferator-activated receptor γ) agonist after hyperoxia also increased lipofibroblast and AT2 cell numbers and restored alveolar architecture in hyperoxia-exposed mice. After pneumonectomy, p16INK4a deficiency again led to an increase in lipofibroblast and AT2 cell numbers in the contralateral lung. Finally, we observed p16INK4a mRNA overexpression in the blood and lungs of preterm newborns, which persisted in the blood of older survivors of BPD.Conclusions: These data demonstrate the potential of targeting p16INK4a and promoting lipofibroblast development to stimulate alveolar regeneration from childhood to adulthood.

Chronic exposure to environmental pollutants is often associated with systemic inflammation. As such, cigarette smoking contributes to inflammation and lung diseases by inducing senescence of pulmonary cells such as pneumocytes, fibroblasts, and endothelial cells. Yet, how smoking worsens evolution of chronic inflammatory disorders associated with Th17 lymphocytes, such as rheumatoid arthritis, psoriasis, Crohn’s disease, and multiple sclerosis, is largely unknown. Results from human studies show an increase in inflammatory CD4+ Th17 lymphocytes at blood- and pulmonary level in smokers. The aim of the study was to evaluate the sensitivity of CD4+ Th17 lymphocytes to cigarette smoke-induced senescence. Mucosa-homing CCR6+ Th17- were compared to CCR6neg -and regulatory T peripheral lymphocytes after exposure to cigarette smoke extract (CSE). Senescence sensitivity of CSE-exposed cells was assessed by determination of various senescence biomarkers (β-galactosidase activity, p16Ink4a- and p21 expression) and cytokines production. CCR6+ Th17 cells showed a higher sensitivity to CSE-induced senescence compared to controls, which is associated to oxidative stress and higher VEGFα secretion. Pharmacological targeting of ROS- and ERK1/2 signalling pathways prevented CSE-induced senescence of CCR6+Th17 lymphocytes as well as VEGFα secretion. Altogether, these results identify mechanisms by which pro-oxidant environmental pollutants contribute to pro-angiogenic and pathogenic CCR6+Th17 cells, therefore potential targets for therapeutic purposes.

Introduction: Lung regeneration is essential for damage recovering after lung injury at different age of life. Whether lipofibroblasts (LIF) are able to enhance endogenous alveolar regeneration once alveoli are damaged remains to be determined. Aim: To determine in a mouse model of bronchopulmonary dysplasia if a lipid switch of fibroblasts to LIF induce a curative lung regeneration. Methods: Wt C57BL/6J mice were exposed to hyperoxia (85% O2) or room air from days 3 to 14 of post-natal life. Rosiglitazone (RGZ), a PPARγ agonist which activates fibroblasts differentiation into LIF, was administered intraperitoneally at a dose of 5µg/g of mice daily, 5 days a week, starting after hyperoxia exposure, until adulthood (D60). Tissues were immunostained with anti-ADRP and anti-proSpC antibodies for LIF and AT2 detection respectively. Number of alveoli were estimated by the mean linear intercept and secondary septation by septal crest number (Weigert). Results: Compared to mice exposed to room air, mice exposed to hyperoxia presented a significant decreased number of LIF in lung associated with an arrested alveolar development revealed by a reduced number of alveoli and a concomitant disruption of secondary septation. Compared to hyperoxic, non-treated mice, RGZ administration increased the number of LIF and AT2 cells in the lung, associated with an increase in the number of alveoli and septal crests at adulthood. Conclusion: Switching fibroblasts towards LIF through PPARγ agonist administration induces lung regeneration after hypoalveolization due to post-natal hyperoxia. Administration of PPARγ agonists can be a new pharmacologic strategy to promote lung recovery after injury.

Chronic obstructive pulmonary disease, a disease of increasing incidence, is related mainly to smoking. Although symptoms only appear at adulthood, the disease can develop from early life events. For example, bronchopulmonary dysplasia, which occurs in preterm infants, is characterized by airspace enlargement and could lead to late lung consequences. Once the lesions are established, no curative treatment is available. Stimulating lung regeneration from endogenous stem cells is therefore an exciting research domain, particularly through the activation of the mesenchymal contingent located in the lung stem cell niche., La bronchopneumopathie chronique obstructive, affection d’incidence croissante, est liée principalement au tabagisme. Bien que les symptômes n’apparaissent qu’à l’âge adulte, la maladie peut se développer à partir d’événements précoces de la vie. La dysplasie bronchopulmonaire, par exemple, survenant chez le nouveau-né prématuré, est caractérisée par un élargissement des espaces aériens et peut avoir des conséquences pulmonaires tardives. Une fois les lésions établies, aucun traitement curatif n’est disponible. Stimuler la régénération pulmonaire à partir de cellules souches endogènes est donc une perspective de recherche séduisante, notamment par le biais de l’activation du contingent mésenchymateux présent dans la niche des cellules souches pulmonaires.

Baarsma et al. report increased expression and posttranslational modification of the noncanonical ligand WNT-5A in COPD. Fibroblast-derived WNT-5A inhibits canonical WNT–β-catenin–driven alveolar epithelial cell–mediated wound healing and transdifferentiation, and thus contributes to impaired lung regeneration and COPD pathogenesis., Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. One main pathological feature of COPD is the loss of functional alveolar tissue without adequate repair (emphysema), yet the underlying mechanisms are poorly defined. Reduced WNT–β-catenin signaling is linked to impaired lung repair in COPD; however, the factors responsible for attenuating this pathway remain to be elucidated. Here, we identify a canonical to noncanonical WNT signaling shift contributing to COPD pathogenesis. We demonstrate enhanced expression of noncanonical WNT-5A in two experimental models of COPD and increased posttranslationally modified WNT-5A in human COPD tissue specimens. WNT-5A was increased in primary lung fibroblasts from COPD patients and induced by COPD-related stimuli, such as TGF-β, cigarette smoke (CS), and cellular senescence. Functionally, mature WNT-5A attenuated canonical WNT-driven alveolar epithelial cell wound healing and transdifferentiation in vitro. Lung-specific WNT-5A overexpression exacerbated airspace enlargement in elastase-induced emphysema in vivo. Accordingly, inhibition of WNT-5A in vivo attenuated lung tissue destruction, improved lung function, and restored expression of β-catenin–driven target genes and alveolar epithelial cell markers in the elastase, as well as in CS-induced models of COPD. We thus identify a novel essential mechanism involved in impaired mesenchymal–epithelial cross talk in COPD pathogenesis, which is amenable to therapy.

The transcription factor p53 is overexpressed in the lung of patients with emphysema, but it remains unclear if it has a deleterious or protective effect in disease progression. We investigated the role of p53 in the elastase-induced emphysema model and the molecular underlining mechanisms. Wild-type (WT) and p53(-/-) mice were instilled with pancreatic porcine elastase. We quantified emphysema (morphometric analysis), chemokine (C-C motif) ligand 2 (CCL2), and TNF-α in bronchoalveolar lavage (BAL) (ELISA), oxidative stress markers [heme oxygenase 1 (HO1), NAD(P)H dehydrogenase quinone 1 (NQO1), and quantitative RT-PCR], matrix metalloproteinase 12 (MMP12) expression, and macrophage apoptosis (cleaved caspase-3, immunofluorescence). p53 gene expression was up-regulated in the lung of elastase-instilled mice. p53 deletion aggravated elastase-induced emphysema severity, pulmonary inflammation (macrophage and neutrophil numbers and CCL2 and TNF-α levels in BAL), and lung oxidative stress. These findings, except for the increase in CCL2, were reproduced in WT mice transplanted with p53(-/-) bone marrow cells. The increased number of macrophages in p53(-/-) mice was not a consequence of reduced apoptosis or an excess of chemotaxis toward CCL2. Macrophage expression of MMP12 was higher in p53(-/-) mice compared with WT mice after elastase instillation. These findings provide evidence that p53(-/-) mice and WT mice grafted with p53(-/-) bone marrow cells are more prone to developing elastase-induced emphysema, supporting a protective role of p53, and more precisely p53 expressed in macrophages, against emphysema development. The pivotal role played by macrophages in this phenomenon may involve the MMP12-TNF-α pathway.

Introduction: Pulmonary emphysema is an aging-related disease. However, the mechanisms underlying this phenomenon still remain poorly unknown. P16Ink4 is a key factor implicated in aging-related diseases. Whether p16 is involved in elastase-induced emphysema in mice remains to be determined. Aim: To determine if p16 deletion protects against elastase-induced emphysema in mice. Methods: Wild-type (WT) and p16-/- mice were instilled with pancreatic porcine elastase and sacrificed three weeks or three months later to address the degree of emphysema (morphometric analysis), alveolar septal thickness, collagen deposition (sirius red) and myofibroblasts content (α smooth muscle actin immunostaining). Results: Three weeks after instillation, elastase induced airspace enlargement in the same way in WT and p16-/- mice. However, three months later, elastase-instilled WT mice showed airspace enlargement and, without modification of collagen deposition while elastase-instilled p16-/- mice have reduced airspace enlargement, and increased septal thickness with enhanced collagen deposition, and more myofibroblasts. Conclusions: p16-/- mice are not immediately but later protected against elastase-induced emphysema, and develop a concomitant alveolar fibrosis suggesting a process of repair partially defective.

Introduction: Air pollution (AP) represents the largest environmental risk for health. Such considerations however rely on the static quantification of only a few individual components of AP, without considering their interactions, chemical reactivity and synergistic toxicity. We therefore developed an innovative approach to analyze the health effects of “real life” model atmospheres at the preclinical level. Here, we report the first results of the feasibility study. Methods: A realistic atmosphere, representative of a 2017 pollution event in Paris, was generated (CESAM atmospheric simulation chamber - cesam.cnrs.fr). The chamber was connected to stalling cabinets where mice were exposed during 6 or 48 hours. Lungs, spleen, adipose tissue, heart, mesenteric ganglions were then harvested to address the expression of detoxification and antioxidant genes, as well as total cell count (TCC) and inflammatory cytokines expression in BAL fluid Results: The atmosphere contained 57.3 µg/m3 particulate matter, 114±11 ppb NO2 and 242±101 ppb O3. No mortality and no weight loss was observed. Increased expression of Ahr, Cyp1a1, Hmox1 and NQO1 was detected in the lungs, and increased TCC and KC concentrations in the BAL. Interestingly, we found extra pulmonary modifications; increased expression of Ahr, Cyp1a1, Hmox1 and NQO1 in the spleen, Hmox1, Cyp1a1 and Cyp1b1 in ganglions, and Hmox1 in the adipose tissue and heart. Limited modifications were detected after 6h. Conclusion: These preliminary results demonstrate the feasibility of our innovative experimental approach, which represents a versatile tool to get a better understanding of AP impacts on health.

International audience; Nanoparticles (NPs) can be toxic, depending on their physico-chemical characteristics. Macroautophagy/autophagy could represent a potential underlying mechanism of this toxicity. We therefore set up a study aimed to characterize in depth the effects, on autophagy, of macrophage exposure to NPs, with a particular attention paid to the role of NP physico-chemical characteristics (specifically chemical composition, shape, size, length, crystal phase, and/or surface properties). We demonstrate that exposure to carbon nanotubes (CNT) but not to spherical NPs leads to the blockage of the autophagic flux. We further identified lysosomal dysfunction, in association with the downregulation of SNAPIN expression, as the underlying mechanism responsible for the CNT-induced autophagy blockade. These results identify for the first time the shape as a major determinant of the interaction of NPs with the autophagy pathway. Moreover, identifying the lysosomes and SNAPIN as primary targets of MWCNT toxicity opens new directions in the interpretation and understanding of nanomaterial toxicity.

International audience

Obese patients could be more susceptible to mechanical ventilation (MV)-induced lung injury than non-obese patients due to weight-dependent changes in lung properties. The aim of this study was therefore to evaluate the pulmonary effects of 2 hours low VT MV in a diet-induced obese mice model, with VT calculated on either the actual body weight (VTaw) or the ideal body weight (VTiw) . First, we hypothesized that a MV with VTaw would be associated with altered lung mechanics and an increased lung inflammation. Second, we hypothesised that a MV with a VTiw would preserve lung mechanics and limit lung inflammation. We analyzed lung mechanics and inflammation using bronchoalveolar lavage (BAL) cell counts, flow cytometry tissue analysis and histology. Lung mechanics and inflammation were comparable in control and obese mice receiving VTiw. By contrast, obese mice receiving VTaw had significantly more alterations in lung mechanics, BAL cellularity and lung influx of monocytes as compared to control mice. Their monocyte expression of Gr1 and CD62L was also increased. Alveolar neutrophil infiltration was significantly increased in all obese mice as compared to controls. In conclusion, our findings suggest that protective MV with a VTaw is deleterious, with a marked alteration in lung mechanics and associated lung inflammation as compared to lean mice. With VTiw, lung mechanics and inflammation were close to that of control mice, except for an increased alveolar infiltrate of polymorphonuclear neutrophils. This inflammation might be attenuated by a blunted recruitment of inflammatory cells associated with obesity.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung cell senescence may be a major pathogenic factor. Here, we investigated the potential role for mTOR signaling in lung cell senescence and alterations in COPD using lung tissue and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the proinflammatory senescence-associated secretory phenotype. To explore whether mTOR activation was a causal pathogenic factor, we developed transgenic mice exhibiting mTOR overactivity in lung vascular cells or alveolar epithelial cells. In this model, mTOR activation was sufficient to induce lung cell senescence and to mimic COPD lung alterations, with the rapid development of lung emphysema, pulmonary hypertension, and inflammation. These findings support a causal relationship between mTOR activation, lung cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a potentially new therapeutic target in COPD.

International audience; No abstract available

Anthropic nanoparticles (NP) are increasingly produced and emitted, with accompanying concerns for human health. Currently there is no global understanding as to the exact mechanistics of NP toxicity, as the traditional nanotoxicological approaches only provide a restricted overview. To address this issue, we performed an in-depth transcriptomic analysis of human macrophages exposed to a panel of welding-related metal oxide NP that we previously identified in welders lungs (Fe2O3, Fe3O4, MnFe2O4 and CrOOH NP). Utilizing the specified analysis criteria (|fold change| ≥1.5, p ≤ 0.001), a total of 2164 genes were identified to be differentially expressed after THP-1 macrophage exposure to the different NP. Performing Gene Ontology enrichment analysis, for cellular content, biological processes and Swiss-Prot/Protein Information Resource keywords the data show for the first time a profound modification of gene differential expression in response to the different NP, among which MnFe2O4 NP were the most potent to induce THP-1 macrophage activation. The transcriptomic analysis utilized in the study, provides novel insights into mechanisms that could contribute to NP-induced adverse effects and support the need for widened approaches to supplement existing knowledge of the processes underlying NP toxicity which would have not been possible using traditional nanotoxicological studies.

BackgroundAn excessive pulmonary inflammatory response could explain the poor prognosis of chronic obstructive pulmonary disease (COPD) patients submitted to invasive mechanical ventilation. The aim of this study was to evaluate the response to normal tidal volume (Vt) mechanical ventilation in a murine model of pulmonary emphysema, which represents the alveolar component of COPD. In this model, two time points associated with different levels of lung inflammation but similar lung destruction, were analyzed.MethodsC57BL/6 mice received a tracheal instillation of 5 IU of porcine pancreatic elastase (Elastase mice) or the same volume of saline (Saline mice). Fourteen (D14) and 21 (D21) days after instillation, mice were anesthetized, intubated, and either mechanically ventilated (MV) with a normal Vt (8 mL/kg) or maintained on spontaneous ventilation (SV) during two hours. We analyzed respiratory mechanics, emphysema degree (mean chord length by lung histological analysis), and lung inflammation (bronchoalveolar lavage (BAL) cellularity, proportion and activation of total lung inflammatory cells by flow cytometry).ResultsAs compared with Saline mice, Elastase mice showed a similarly increased mean chord length and pulmonary compliance at D14 and D21, while BAL cellularity was comparable between groups. Lung mechanics was similarly altered during mechanical ventilation in Elastase and Saline mice. Activated alveolar macrophages CD11bmid were present in lung parenchyma in both Elastase SV mice and Elastase MV mice at D14 but were absent at D21 and in Saline mice, indicating an inflammatory state with elastase at D14 only. At D14, Elastase MV mice showed a significant increase in percentage of neutrophils concomitant with a decrease in percentage of alveolar macrophages in total lung, as compared with Elastase SV mice. Furthermore, alveolar macrophages of Elastase MV mice at D14 overexpressed Gr1, and monocytes showed a trend to overexpression of CD62L, compared with Elastase SV mice.ConclusionsIn an elastase-induced model of pulmonary emphysema, normal Vt mechanical ventilation produced an increase in the proportion of pulmonary neutrophils, and an activation of alveolar macrophages and pulmonary monocytes. This response was observed only when the emphysema model showed an underlying inflammation (D14), reflected by the presence of activated alveolar macrophages CD11bmid.

We hypothesized that O2 tension influences the redox state and the immunomodulatory responses of inflammatory cells to dimethyl fumarate (DMF), an activator of the nuclear factor Nrf2 that controls antioxidant genes expression. This concept was investigated in macrophages permanently cultured at either physiological (5% O2) or atmospheric (20% O2) oxygen levels and then treated with DMF or challenged with lipopolysaccharide (LPS) to induce inflammation. RAW 264.7 macrophages cultured at 20% O2 exhibited a pro-oxidant phenotype, reflected by a lower content of reduced glutathione, higher oxidized glutathione and increased production of reactive oxygen species when compared to macrophages continuously grown at 5% O2. At 20% O2, DMF induced a stronger antioxidant response compared to 5% O2 as evidenced by a higher expression of heme oxygenase-1, NAD(P)H:quinone oxydoreductase-1 and superoxide dismutase-2. After challenge of macrophages with LPS, several pro-inflammatory (iNOS, TNF-α, MMP-2, MMP-9), anti-inflammatory (arginase-1, IL-10) and pro-angiogenic (VEGF-A) mediators were evaluated in the presence or absence of DMF. All markers, with few interesting exceptions, were significantly reduced at 5% O2. This study brings new insights on the effects of O2 in the cellular adaptation to oxidative and inflammatory stimuli and highlights the importance of characterizing the effects of chemicals and drugs at physiologically relevant O2 tension. Our results demonstrate that the common practice of culturing cells at atmospheric O2 drives the endogenous cellular environment towards an oxidative stress phenotype, affecting inflammation and the expression of antioxidant pathways by exogenous modulators. J. Cell. Physiol. 230: 1128–1138, 2015. © 2014 Wiley Periodicals, Inc., A Wiley Company

Chronic obstructive pulmonary disease (COPD) is a highly prevalent and devastating condition for which no curative treatment is available. Exaggerated lung-cell senescence may contribute substantially to the pathogenesis of COPD. Here, we explored the potential role for the mTOR signaling pathway in cell senescence and lung alterations in COPD, using lung specimens and derived cultured cells from patients with COPD and from age- and sex-matched control smokers. Cell senescence in COPD was linked to mTOR activation, and mTOR inhibition by low-dose rapamycin prevented cell senescence and inhibited the pro-inflammatory senescence-associated secretory phenotype. To assess whether mTOR activation was causal in lung pathology, we generated mice with constitutive or conditional deletion of the tuberous sclerosis complex heterodimer TSC1 (a negative regulator of mTOR complex 1), in smooth muscle cells (SMCs), endothelial cells (ECs), or alveolar epithelial cells (AECs). In this model, mTOR activation was sufficient to induce early-onset replicative cell senescence in cultured pulmonary artery SMCs from mice with TSC1 deletion. Mice with conditional TSC1 deletion in ECs or AECs developed lung emphysema, pulmonary hypertension, and inflammation in the absence of any other stimuli and concomitantly with the accumulation of senescent lung cells. Rapamycin prevented all these effects. These results support causal links between mTOR activation, cell senescence, and lung alterations in COPD, thereby identifying the mTOR pathway as a new therapeutic target for COPD. Keywords: mTOR, senescence, COPD

Introduction: Bronchopulmonary dysplasia (BPD), characterized by airspace enlargement, develops in premature infants. BPD leads to persistent lung consequences in adulthood, being considered as an early determinant of adult lung diseases. P16Ink4 is a key factor implicated in aging-related lung diseases and its levels are increased in cord blood cells of preterm infants. Whether p16 is involved in BPD and its persistent consequences at the adult age remains to be determined. Aims: To determine in a mouse model of BPD: 1) if p16 deletion protects against lung alterations in new born and adult mice, and 2) if these effects are mediated by cellular senescence. Methods: Mouse pups were exposed to hyperoxia (85% O2) or room air from day 3 to 14 of life. Morphometry (mean linear intercept), elastin content (Weigert), collagen deposition (sirius red), cell senescence (βgalactosidase activity and p21) and cell proliferation (Ki67) were evaluated. Results: At day 14, hyperoxia induced airspace enlargement and cellular senescence, without modification of collagen deposition, nor cell proliferation. Hyperoxic p16-/- mice were not protected against alveolar disruption, but presented an increased alveolar wall thickness and cell proliferation. At day 60, hyperoxic mice showed persistence of airspace enlargement and senescence, without modification of collagen deposition, nor cellular proliferation. Hyperoxic p16-/- mice have reduced airspace enlargement, but increased collagen deposition, without modulation of senescence markers. Conclusions: P16 did not modulated hyperoxia-induced alveolar disruption at young age but could play a crucial role in BPD consequences in adults. This effect may be independent of cellular senescence.

Telomere shortening is associated with COPD and impaired lung function in cross-sectional studies, but there is no longitudinal study. We used data from 448 participants recruited as part of the French follow-up of the European Community Respiratory Health Survey. We found no relationship between telomere length at baseline and FEV1 decline after 11 years of follow-up. However, heavy smoking was associated with an accelerated FEV1 decline in individuals with short telomeres, but not in subjects with longer telomeres (p for interaction p=0.08). Our findings suggest that short telomere length in peripheral leucocytes might be a marker for increased susceptibility to the effect of smoking.

Introduction La bronchopneumopathie chronique obstructive represente la 4e cause de mortalite dans le monde et ne dispose a ce jour d’aucun traitement curatif. L’emphyseme, qui en est une des composantes, resulte de la destruction progressive des septums inter-alveolaires, conduisant a une insuffisance respiratoire. Parmi les marqueurs de senescence, des etudes montrent une surexpression du suppresseur de tumeurs p16 Ink4a dans les poumons de patients ayant un emphyseme compare aux fumeurs ou non-fumeurs sains. D’autres etudes ont montre l’influence de p16 Ink4a sur l’adipogenese. L’objectif est de determiner si la deletion de p16 Ink4a previent le developpement d’un emphyseme pulmonaire induit par une instillation d’elastase a un temps precoce ou tardif ainsi que les mecanismes impliques, en particulier l’induction de la lipogenese et l’activation des lipofibroblastes. Methodes Des souris adultes p16 Ink4a ko et sauvages (WT) ont recu une instillation intratracheale de 2UI d’elastase porcine pancreatique. Les souris ont ete sacrifiees 21 jours ou 90 jours apres l’instillation pour quantification morphometrique de l’emphyseme, evaluation de la septation secondaire et analyse de l’expression des genes de l’adipogenese et des marqueurs de pneumocytes. Evaluation dans un second temps des remaniements de la matrice extracellulaire. Resultats A 21 jours, la deletion de p16 Ink4a n’influence pas le degre d’emphyseme mais entraine une proliferation des pneumocytes de types 2, atteste par qPCR ou immunohistochimie ainsi qu’une activation de la lipogenese par rapport aux souris WT. A 90 jours, les souris p16 Ink4a presentaient un emphyseme significativement moins severe, des marqueurs de proliferation de pneumocytes 2 mais pas d’activation de l’adipogenese. A ce temps tardif, apparaissaient chez les souris p16 Ink4a des remaniements fibrotiques. Conclusion La deletion de p16 Ink4a ne protege pas de l’emphyseme induit par l’elastase dans un premier temps mais est associee a une amelioration des lesions a un temps tardif. Cet effet sous-tend un processus de regeneration alveolaire avec un remaniement secondaire la matrice extracellulaire.

The biological mechanisms of aging, and more specifically cellular senescence, are increasingly a subject of research. Cellular senescence may be a common determinant of many age-related diseases, including some chronic lung diseases such as chronic obstructive pulmonary disease (COPD) or idiopathic pulmonary fibrosis. Many arguments suggest that these diseases are associated with premature senescence of lung cells, which may be involved in the pathophysiology of respiratory alterations. Furthermore, these diseases are associated with systemic manifestations, such as bone loss, muscle wasting and atherosclerosis, which impact on symptoms and prognosis. Whether these alterations are related to a common pathogenic mechanism or develop independently in patients with COPD remains an open question. In this review, we will focus on cellular senescence and COPD. Two concepts will be discussed: (1) the role of cell senescence in the pathophysiology of lung destruction, vascular remodeling and inflammation in COPD, (2) the possible link between the pulmonary and systemic manifestations of COPD which could reflect a general process of accelerated aging.