Aryl hydrocarbon receptor deficiency causes the development of chronic obstructive pulmonary disease through the integration of multiple pathogenic mechanisms

Emphysema, a component of chronic obstructive pulmonary disease (COPD), is characterized by irreversible alveolar destruction that results in a progressive decline in lung function. This alveolar destruction is caused by cigarette smoke, the most important risk factor for COPD. Only 15%-20% of smokers develop COPD, suggesting that unknown factors contribute to disease pathogenesis. We postulate that the aryl hydrocarbon receptor (AHR), a receptor/transcription factor highly expressed in the lungs, may be a new susceptibility factor whose expression protects against COPD. Here, we report that Ahr-deficient mice chronically exposed to cigarette smoke develop airspace enlargement concomitant with a decline in lung function. Chronic cigarette smoke exposure also increased cleaved caspase-3, lowered SOD2 expression, and altered MMP9 and TIMP-1 levels in Ahr-deficient mice. We also show that people with COPD have reduced expression of pulmonary and systemic AHR, with systemic AHR mRNA levels positively correlating with lung function. Systemic AHR was also lower in never-smokers with COPD. Thus, AHR expression protects against the development of COPD by controlling interrelated mechanisms involved in the pathogenesis of this disease. This study identifies the AHR as a new, central player in the homeostatic maintenance of lung health, providing a foundation for the AHR as a novel therapeutic target and/or predictive biomarker in chronic lung disease

Reduced aryl hydrocarbon receptor (AhR) expression drives the pathogenesis of cigarette smoke-induced emphysema

Chronic Obstructive Pulmonary Disease (COPD) is a prevalent and complex respiratory disorder primarily caused by inhalational exposure to cigarette smoke (CS). However, only approximately 15% of cigarette smokers develop COPD, suggesting that genetic or epigenetic factors may contribute to disease susceptibility. One feature of COPD is emphysema, which is characterized by permanent alveolar wall destruction leading to airspace enlargement. The alveolar wall destruction in emphysema is a consequence of multiple pathogenic mechanisms including chronic inflammation, a protease: anti-protease imbalance and the upregulation of cell death programs such as apoptosis, autophagy and endoplasmic reticulum (ER) stress. However, molecular mechanism(s) that regulate these pathogenic events, and thus the development of emphysema, are poorly understood. This regulation may involve the aryl hydrocarbon receptor (AhR), which is a ligand-activated transcription factor whose involvement in COPD pathogenesis is unknown. Our lab has previously published that AhR-deficiency exacerbates CS-induced cell death and inflammation in lung structural cells. Thus, we hypothesized that AhR-deficiency would promote the development of CS-induced emphysema and COPD pathogenesis.Using a preclinical model of CS-exposure, we demonstrate that AhR deficiency worsens the development of a CS-induced emphysema-like phenotype in the murine lung. AhR ablation promoted the CS-induced: (1) upregulation of pathogenic mechanisms underlying emphysema development (i.e. inflammation, an antiprotease imbalance, and the activation of cell death machinery), (2) lung parenchymal destruction, and (3) declines in lung function. In COPD subjects, there was less pulmonary and systemic AHR expression. In humans, systemic AHR mRNA levels also positively correlated with lung function. There was no alteration in the frequency of AHR single nucleotide polymorphisms (SNPs) that could explain this decrease in AHR in COPD subjects. However, elevated expression of the AhR Repressor (AHRR), which is a negative regulator of the AHR, in the COPD lung may contribute to reduced AHR expression in these subjects.We also utilized an in vitro model of CS exposure to evaluate if the AhR attenuation of CS-induced cell death in lung structural cells involves its regulation of cell death modalities such as autophagy and/or ER stress. We show that the expression of the autophagy and ER stress protein LC3II was significantly elevated in cigarette smoke extract (CSE)-exposed AhR-deficient lung structural cells; these cells were primary mouse lung fibroblasts (MLFs), mouse lung epithelial cells (MLE12) and alveolar epithelial cells (A549). Heightened LC3II expression could not be explained by the transcriptional upregulation of key autophagy genes (Gabarapl1, Beclin1, Lc3b), upregulation of upstream autophagic machinery (ATG5-12, ATG3) or impaired autophagic flux. This suggested that elevated LC3II in CSE-treated AhR-deficient cells is likely mediated by an autophagy-independent mechanism. This was further supported by the absence of autophagosomes in transmission electron micrographs. However, CSE-treated Ahr-/- MLFs showed significantly reduced viability, widespread ER-dilation, elevated expression of ER stress markers (i.e. CHOP and GADD34) and the accumulation of ubiquitinated proteins. Thus, the AhR attenuates an ER stress response that is autophagy-independent yet associated with elevated LC3 expression and processing. Collectively, our data position the AhR as a central player in the homeostatic maintenance of lung health by demonstrating that loss of the AhR promotes the development of CS-induced emphysema. Given that no effective therapeutic options currently exist to stop or slow COPD progression, these findings could provide the basis for the development of new therapeutic agents or biomarkers for COPD.La Maladie Pulmonaire Obstructive Chronique (MPOC) est une maladie respiratoire complexe à prévalence élevée, dont le principal facteur de risque est l'exposition à la fumée de cigarette. Cependant, seuls 15% des fumeurs développent une MPOC, suggérant l'implication de facteurs génétiques ou épigénétiques dans la susceptibilité à la maladie. Une des composantes de la MPOC est l'emphysème, défini par une destruction irréversible des parois alvéolaires aboutissant à un élargissement permanent des espaces aériens distaux. La destruction alvéolaire est la conséquence de multiples mécanismes physiopathologiques incluant l'inflammation chronique, le déséquilibre de la balance protéases : anti-protéases et l'activation de divers programmes de mort cellulaire tels que l'apoptose, l'autophagie ou le stress du réticulum endoplasmique (ER stress). Toutefois, les mécanismes moléculaires régulant ces événements, et par suite le développement de l'emphysème, sont encore imparfaitement connus. Cette régulation pourra impliquer l'aryl hydrocarbon receptor (AhR), qui est un facteur de transcription activé par un ligand et dont son implication dans le développement de MPOC est inconnue. Notre laboratoire a précédemment montré que le déficit en AhR dans les cellules pulmonaires exacerbe la mort cellulaire et l'inflammation induites par la fumée de cigarettes. C'est pour cela, nous avons émis l'hypothèse que la déficiance en AhR favoriserait le développement de l'emphysème et de la pathogenèse de la MPOC induits par la fumée de cigarettes.Dans notre modèle murin, nous démontrons que le déficit en AhR aggrave le développement d'un phénotype semblable à un emphysème induit par l'exposition à la fumée de cigarettes. L'ablation AhR a promu: 1) des mécanismes pathogéniques aboutissant au développement d'emphysème (ex : inflammation, déséquilibre protéases-antiprotéases au niveau pulmonaire et activation de programmes de mort cellulaire), (2) destruction de parenchyme pulmonaire, et (3) le déclin de la fonction respiratoire. L'expression pulmonaire et systémique de l'AHR était diminuée chez les patients atteints de MPOC. De plus, les niveaux d'ARN messagers systémiques de l'AHR étaient positivement corrélés à la fonction respiratoire. Nous avons observé que l'expression du répresseur d'AHR (AHRR), qui est un régulateur négatif d'AHR, est élevée dans les poumons de MPOC. Ceci pourra expliquer la diminution d'AHR observée chez ces sujets.De plus, nous avons utilisé un modèle in vitro d'exposition à la fumée de cigarette afin d'évaluer si AhR réduit le mort des cellules structurales en régulant les programmes de mort cellulaire, comme autophagie et/ou ER. Nous montrons que l'expression du marqueur d'autophagie et d'ER la protéine LC3II était significativement augmentée dans les cellules pulmonaires de structure déficientes pour l'AhR, exposées à de l'extrait de fumée de cigarette (CSE). L'augmentation de l'expression de LC3II n'était pas relative à une sur expression transcriptionnelle des gènes clés mis en jeu dans l'autophagie, une régulation positive de la machinerie autophagique d'amont, ou une altération du flux autophagique. Cependant, les MLFs Ahr-/- présentaient une viabilité significativement réduite, une dilatation diffuse du réticulum endoplasmique, et une élévation de l'expression du marqueur d'ER-stress. Ainsi, l'AhR semble atténuer une réponse de type ER stress de manière indépendante de l'autophagie.L'ensemble de ces données positionne l'AhR comme un facteur central dans la préservation de l'homéostasie pulmonaire en démontrant que la perte de l'AhR favorise le développement de l'emphysème induit par la fumée de cigarettes. Étant donnée l'absence actuelle de traitements efficaces pour arrêter ou ralentir la progression de la MPOC, ces résultats pourraient constituer une base de travail pour le développement de nouveaux traitements

The Aryl Hydrocarbon Receptor and the Maintenance of Lung Health

Much of what is known about the Aryl Hydrocarbon Receptor (AhR) centers on its ability to mediate the deleterious effects of the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin). However, the AhR is both ubiquitously-expressed and evolutionarily-conserved, suggesting that it evolved for purposes beyond strictly mediating responses to man-made environmental toxicants. There is growing evidence that the AhR is required for the maintenance of health, as it is implicated in physiological processes such as xenobiotic metabolism, organ development and immunity. Dysregulation of AhR expression and activity is also associated with a variety of disease states, particularly those at barrier organs such as the skin, gut and lungs. The lungs are particularly vulnerable to inhaled toxicants such as cigarette smoke. However, the role of the AhR in diseases such as chronic obstructive pulmonary disease (COPD)—a respiratory illness caused predominately by cigarette smoking—and lung cancer remains largely unexplored. This review will discuss the growing body of literature that provides evidence that the AhR protects the lungs against the damaging effects of cigarette smoke

Purely Inorganic Highly Efficient Ice Nucleating Particle

Air pollution of anthropogenic origin is largely from the combustion of biomass (e.g., wood), fossil fuels (e.g., cars and trucks), incinerators, landfills, agricultural activities and tobacco smoke. Air pollution is a complex mixture that varies in space and time, and contains hundreds of compounds including volatile organic compounds (e.g., benzene), metals, sulphur and nitrogen oxides, ozone and particulate matter (PM). PM0.1 (ultrafine particles (UFP)), those particles with a diameter less than 100 nm (includes nanoparticles (NP)) are considered especially dangerous to human health and may contribute significantly to the development of numerous respiratory and cardiovascular diseases such as chronic obstructive pulmonary disease (COPD) and atherosclerosis. [...] This paper highlights emerging molecular concepts associated with inhalational exposure to PM0.1 and their ability to contribute to chronic respiratory and systemic disease

The aryl hydrocarbon receptor reduces LC3II expression and controls endoplasmic reticulum stress

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose physiological function is poorly understood. The AhR is highly expressed in barrier organs such as the skin, intestine, and lung. The lungs are continuously exposed to environmental pollutants such as cigarette smoke (CS) that can induce cell death mechanisms such as apoptosis, autophagy, and endoplasmic reticulum (ER) stress. CS also contains toxicants that are AhR ligands. We have previously shown that the AhR protects against apoptosis, but whether the AhR also protects against autophagy or ER stress is not known. Using cigarette smoke extract (CSE) as our in vitro surrogate of environmental tobacco exposure, we first assessed the conversion of LC3I to LC3II, a classic feature of both autophagic and ER stress-mediated cell death pathways. LC3II was elevated in CSE-exposed lung structural cells [mouse lung fibroblasts (MLFs), MLE12 and A549 cells] when AhR was absent. However, this heightened LC3II expression could not be explained by increased expression of key autophagy genes ( Gabarapl1, Becn1, Map1lc3b), upregulation of upstream autophagic machinery (Atg5–12, Atg3), or impaired autophagic flux, suggesting that LC3II may be autophagy independent. This was further supported by the absence of autophagosomes in Ahr −/− lung cells. However, Ahr −/− lung cells had widespread ER dilation, elevated expression of the ER stress markers CHOP and GADD34, and an accumulation of ubiquitinated proteins. These findings collectively illustrate a novel role for the AhR in attenuating ER stress by a mechanism that may be autophagy independent

Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability

Heightened inflammation, including expression of COX-2, is associated with chronicobstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR)is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts.The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor forCOPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulationof microRNA (miRNA). Whether reduced AhR expression is responsible for heightenedCOX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as theexpression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblastsderived from non-smokers (Normal) and smokers (At Risk) with and without COPD.To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblastsand MLE-12 cells was employed as were A549-AhRko cells. [...

The AhR does not control miR-146a expression in response to CSE or IL-1β.

<p>(A) COPD-derived lung fibroblasts have less miR-146a in response to IL-1β- Human lung fibroblasts from Normal, At Risk or COPD subjects were exposed to IL-1β (10 ng/ml) for 6 hours and miR-146a evaluated by qRT-PCR. IL-1β significantly increased miR-146a expression in Norma (fold-increase 33.4 ± 6.8; ***p < 0.0001) and At Risk (34.1 ± 3.9; ** p < 0.01) lung fibroblasts. There was no significant induction in miR-146a in COPD fibroblasts (fold-increase 9.8 ± 5.9; $p < 0.05,$$ p < 0.01 compared to At Risk or Normal fibroblasts, respectively). Results are expressed as the mean ± SEM, n = 3 independent experiments of samples utilizing lung fibroblasts derived from 3–6 different individuals. (B) There was a significant increase in miR-146a in human lung fibroblasts exposed to IL-1β for 6 hours (fold-increase 29 ± 8.5). There was a slight increase with CH-223191 for 6 hours but no effect when both CH-223191 and IL-1β were combined (fold change 31 ± 9.6). Results are expressed as the mean ± SEM, n = 3 separate experiments. (C) <i>Ahr</i>^<i>+/-</i> and <i>Ahr</i>^<i>-/-</i> cells were exposed to IL-1β and miR-146a levels assessed by qRT-PCR. There was a significant induction of miR-146a upon stimulation with IL-1β (*** p < 0.001); there was no significant difference in the magnitude of induction between <i>Ahr</i>^<i>+/-</i> and <i>Ahr</i>^<i>-/-</i> fibroblasts (ns). Results are expressed as the mean ± SEM, n = 12 separate experiments.</p

The AhR does not control RelB induction by CSE or IL-1β.

<p><i>Ahr</i>^<i>+/-</i> and <i>Ahr</i>^<i>-/-</i> mouse lung fibroblasts were exposed to 1% CSE or rmIL-1β and RelB mRNA (A) and protein (B) evaluated by qRT-PCR or western blot respectively. There was no significant difference in RelB mRNA induction between <i>Ahr</i>^<i>+/-</i> and <i>Ahr</i>^<i>-/-</i> cells. Results are expressed as the mean ± SEM, n = 8–10 separate experiments.</p

RelB expression in increased in lung fibroblasts in response to IL-1β.

<p>Human lung fibroblasts were cultured with 10 ng/ml of rhIL-1β for 6 or 24 hours and RelB mRNA assessed by qRT-PCR and cell lysate for detection of RelB protein by western blot. (A) RelB mRNA- There was a significant increase in RelB mRNA in lung fibroblasts derived from Normal, At Risk and COPD subjects compared to respective unstimulated controls (*** p < 0.001). Results are expressed as the mean ± SEM of three strains from each subject group. (B) RelB protein- There was a noticeable and significant increase in RelB protein expression upon stimulation of lung fibroblasts with IL-1β for 24 hours (*p < 0.05 for each fibroblast group compared to their respective unstimulated controls). There was no difference in RelB induction between the three groups. Results are expressed as the mean ± SEM of three independent experiments.</p