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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.

The COVID-19 pandemic is associated with severe pneumonia and acute respiratory distress syndrome leading to death in susceptible individuals. For those who recover, post-COVID-19 complications may include development of pulmonary fibrosis. Factors contributing to disease severity or development of complications are not known. Using computational analysis with experimental data, we report that idiopathic pulmonary fibrosis (IPF)- and chronic obstructive pulmonary disease (COPD)-derived lung fibroblasts express higher levels of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 entry and part of the renin-angiotensin system that is antifibrotic and anti-inflammatory. In preclinical models, we found that chronic exposure to cigarette smoke, a risk factor for both COPD and IPF and potentially for SARS-CoV-2 infection, significantly increased pulmonary ACE2 protein expression. Further studies are needed to understand the functional implications of ACE2 on lung fibroblasts, a cell type that thus far has received relatively little attention in the context of COVID-19.

PURPOSE: To determine if the mean curvature of isophotes (MCI), a standard computer vision technique, can be used to improve detection of chronic obstructive pulmonary disease (COPD) at chest CT. MATERIALS AND METHODS: In this retrospective study, chest CT scans were obtained in 243 patients with COPD and 31 controls (among all 274: 151 women [mean age, 70 years; range, 44–90 years] and 123 men [mean age, 71 years; range, 29–90 years]) from two community practices between 2006 and 2019. A convolutional neural network (CNN) architecture was trained on either CT images or CT images transformed through the MCI algorithm. Separately, a linear classification based on a single feature derived from the MCI computation (called hMCI1) was also evaluated. All three models were evaluated with cross-validation, using precision-macro and recall-macro metrics, that is, the mean of per-class precision and recall values, respectively (the latter being equivalent to balanced accuracy). RESULTS: Linear classification based on hMCI1 resulted in a higher recall-macro relative to the CNN trained and applied on CT images (0.85 [95% CI: 0.84, 0.86] vs 0.77 [95% CI: 0.75, 0.79]) but with a similar reduction in precision-macro (0.66 [95% CI: 0.65, 0.67] vs 0.77 [95% CI: 0.75, 0.79]). The CNN model trained and applied on MCI-transformed images had a higher recall-macro (0.85 [95% CI: 0.83, 0.87] vs 0.77 [95% CI: 0.75, 0.79]) and precision-macro (0.85 [95% CI: 0.83, 0.87] vs 0.77 [95% CI: 0.75, 0.79]) relative to the CNN trained and applied on CT images. CONCLUSION: The MCI algorithm may be valuable toward the automated detection and diagnosis of COPD on chest CT scans as part of a CNN-based pipeline or with stand-alone features. Keywords: Chronic Obstructive Pulmonary Disease, Quantification, Lung, CT Supplemental material is available for this article. See also the invited commentary by Vannier in this issue. © RSNA, 2021

Pulmonary fibrosis is a chronic, fibrotic lung disease affecting 3 million people worldwide. The ACE2/Ang-(1–7)/MasR axis is of interest in pulmonary fibrosis due to evidence of its anti-fibrotic action. Current scientific evidence supports that inhibition of ACE2 causes enhanced fibrosis. ACE2 is also the primary receptor that facilitates the entry of SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. COVID-19 is associated with a myriad of symptoms ranging from asymptomatic to severe pneumonia and acute respiratory distress syndrome (ARDS) leading to respiratory failure, mechanical ventilation, and often death. One of the potential complications in people who recover from COVID-19 is pulmonary fibrosis. Cigarette smoking is a risk factor for fibrotic lung diseases, including the idiopathic form of this disease (idiopathic pulmonary fibrosis), which has a prevalence of 41% to 83%. Cigarette smoke increases the expression of pulmonary ACE2 and is thought to alter susceptibility to COVID-19. Cannabis is another popular combustible product that shares some similarities with cigarette smoke, however, cannabis contains cannabinoids that may reduce inflammation and/or ACE2 levels. The role of cannabis smoke in the pathogenesis of pulmonary fibrosis remains unknown. This review aimed to characterize the ACE2-Ang-(1–7)-MasR Axis in the context of pulmonary fibrosis with an emphasis on risk factors, including the SARS-CoV-2 virus and exposure to environmental toxicants. In the context of the pandemic, there is a dire need for an understanding of pulmonary fibrotic events. More research is needed to understand the interplay between ACE2, pulmonary fibrosis, and susceptibility to coronavirus infection.

COVID-19 has rapidly spread through the world, and is caused by SARS-CoV-2. Patients with chronic obstructive pulmonary disease (COPD) face an increased risk for severe illness from COVID-19 because of angiotensin-converting enzyme 2 (ACE2) upregulation, an entry receptor for SARS-CoV-2. Our group recently revealed that COPD-derived lung fibroblasts express higher level of ACE2, and provide direct evidence that chronic cigarette smoke (CS) exposure significantly increases pulmonary ACE2 protein. CS is the primary risk factor for COPD. ACE2 expression may be regulated at the transcriptional, post-transcriptional and translational levels. Changes in protein expression can be controlled by RNA-binding proteins (RBPs). One of the best studied RBPs is human antigen R (HuR), a ubiquitously expressed RBP, that regulates the stability, localization and/or translation of target mRNAs. To stabilize target mRNA, HuR translocates from the nucleus to the cytoplasm. CS may increases HuR translocation to the cytoplasm, thereby indirectly augmenting cellular protein levels. Therefore, we hypothesized that HuR regulates ACE2 expression in response to CS, thereby accounting for higher ACE2 in COPD-derived cells. Aims: 1)- Evaluate HuR expression and localization in Normal, Smoker and COPD lung tissues;2) Establish that HuR induces ACE2 expression in primary human lung fibroblasts (HLF);3)- Determine if HuR controls ACE2 expression by regulating mRNA stability. Methods: 1)- Multiplex Immunohistochemistry was performed on lung tissue from Normal, Smoker and COPD subjects. The localization of HuR was assessed in HLF exposed to 2% cigarette smoke extract (CSE) for 4h by subcellular fractionation-western blot. 2)- Normal, Smoker and COPD HLF were transfected with either small interfering RNA (siRNA) against HuR or Control siRNA. ACE2 protein was evaluated by western blot. 3)- RNA-binding protein immunoprecipitation-qPCR was used to assess the association of HuR to ACE2 mRNA in Normal, Smoker and COPD HLF. The effect of HuR on the stability of ACE2 mRNA was assessed by using actinomycin D pulse-chase followed by qPCR. Results: 1)- HuR cytoplasmic localization is higher in Smoker and COPD lung tissues. There was an increase in cytoplasmic HuR in HLF exposed to 2%CSE for 4h. 2)- Knockdown of HuR slightly increases ACE2 protein. 3)- HuR associates with ACE2 mRNA in Normal and Smoker HLF. Significance: Our work is the first to highlight the association between ACE2 and HuR in COPD. The regulation of ACE2 by HuR could provide the basis to understand the upregulation of ACE2 expression in COPD HLF and the potential consequence towards COVID-19.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate toxic responses to dioxin. However, the role of the AhR in the regulation of cellular physiology has only recently been appreciated, including its ability to control cell cycle progression and apoptosis by unknown mechanisms. We hypothesized that the AhR enhances the activation of the AKT serine/threonine kinase (Akt) pathway to promote cell survival. Utilizing AhR knock-out (Ahr−/−) and wild-type (Ahr+/+) mouse lung fibroblasts (MLFs), we found that Ahr−/− MLFs have significantly higher basal Akt phosphorylation but that AhR did not affect Akt phosphorylation in MLFs exposed to growth factors or AhR ligands. Basal Akt phosphorylation was dependent on PI3K but was unaffected by changes in intracellular glutathione (GSH) or p85α. There was no significant decrease in cell viability in Ahr−/− MLFs treated with LY294002—a PI3K inhibitor—although LY294002 did attenuate MTT reduction, indicating an affect on mitochondrial function. Using a mass spectrometry (MS)-based approach, we identified several proteins that were differentially phosphorylated in the Ahr−/− MLFs compared to control cells, including proteins involved in the regulation of extracellular matrix (ECM), focal adhesion, cytoskeleton remodeling and mitochondrial function. In conclusion, Ahr ablation increased basal Akt phosphorylation in MLFs. Our results indicate that AhR may modulate the phosphorylation of a variety of novel proteins not previously identified as AhR targets, findings that help advance our understanding of the endogenous functions of AhR.

Idiopathic pulmonary fibrosis (IPF) is a disease of progressive scarring caused by excessive extracellular matrix (ECM) deposition and activation of α-SMA-expressing myofibroblasts. Human antigen R (HuR) is an RNA binding protein that promotes protein translation. Upon translocation from the nucleus to the cytoplasm, HuR functions to stabilize messenger RNA (mRNA) to increase protein levels. However, the role of HuR in promoting ECM production, myofibroblast differentiation, and lung fibrosis is unknown. Human lung fibroblasts (HLFs) treated with transforming growth factor β1 (TGF-β1) showed a significant increase in translocation of HuR from the nucleus to the cytoplasm. TGF-β-treated HLFs that were transfected with HuR small interfering RNA had a significant reduction in α-SMA protein as well as the ECM proteins COL1A1, COL3A, and FN1. HuR was also bound to mRNA for ACTA2, COL1A1, COL3A1, and FN. HuR knockdown affected the mRNA stability of ACTA2 but not that of the ECM genes COL1A1, COL3A1, or FN. In mouse models of pulmonary fibrosis, there was higher cytoplasmic HuR in lung structural cells compared to control mice. In human IPF lungs, there was also more cytoplasmic HuR. This study is the first to show that HuR in lung fibroblasts controls their differentiation to myofibroblasts and consequent ECM production. Further research on HuR could assist in establishing the basis for the development of new target therapy for fibrotic diseases, such as IPF.

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.

E-cigarettes have a liquid that may contain flavors, solvents, and nicotine. Heating this liquid generates an aerosol that is inhaled into the lungs in a process commonly referred to as vaping. E-cigarette devices can also contain cannabis-based products including tetrahydrocannabinol (THC), the psychoactive component of cannabis (marijuana). E-cigarette use has rapidly increased among current and former smokers as well as youth who have never smoked. The long-term health effects are unknown, and emerging preclinical and clinical studies suggest that e-cigarettes may not be harmless and can cause cellular alterations analogous to traditional tobacco smoke. Here, we review the historical context and the components of e-cigarettes and discuss toxicological similarities and differences between cigarette smoke and e-cigarette aerosol, with specific reference to adverse respiratory outcomes. Finally, we outline possible clinical disorders associated with vaping on pulmonary health and the recent escalation of acute lung injuries, which led to the declaration of the vaping product use-associated lung injury (EVALI) outbreak. It is clear there is much about vaping that is not understood. Consequently, until more is known about the health effects of vaping, individual factors that need to be taken into consideration include age, current and prior use of combustible tobacco products, and whether the user has preexisting lung conditions such as asthma and chronic obstructive pulmonary disease (COPD).

Background Asthma is a heterogenous disease characterized by chronic inflammation and airway remodeling. An increase in the severity of airway remodeling is associated with a more severe form of asthma. There is increasing interest in the epithelial to mesenchymal transition process and mechanisms involved in the differentiation and repair of the airway epithelium, especially as they apply to severe asthma. Growing evidence suggests that Epithelial-Mesenchymal transition (EMT) could contribute to airway remodeling and fibrosis in asthma. Severe asthmatic patients with remodeled airways have a neutrophil driven inflammation. Neutrophils are an important source of TGF-β1, which plays a role in recruitment and activation of inflammatory cells, extracellular matrix (ECM) production and fibrosis development, and is a potent inducer of EMT. Objective As there is little data examining the contribution of neutrophils and/or their mediators to the induction of EMT in airway epithelial cells, the objective of this study was to better understand the potential role of neutrophils in severe asthma in regards to EMT. Methods We used an in vitro system to investigate the neutrophil-epithelial cell interaction. We obtained peripheral blood neutrophils from severe asthmatic patients and control subjects and examined for their ability to induce EMT in primary airway epithelial cells. Results Our data indicate that neutrophils from severe asthmatic patients induce changes in morphology and EMT marker expression in bronchial epithelial cells consistent with the EMT process when co-cultured. TGF-β1 levels in the culture medium of severe asthmatic patients were increased compared to that from co-cultures of non-asthmatic neutrophils and epithelial cells. Conclusions and clinical relevance As an inducer of EMT and an important source of TGF-β1, neutrophils may play a significant role in the development of airway remodeling and fibrosis in severe asthmatic airways.

Inflammation is a response to injury and infection. Although protective under physiological conditions, excessive and persistent inflammation is linked to numerous diseases. As the lungs are continuously exposed to the external environment, the respiratory system is particularly liable to damage from inflammation. RelB is a member of the non-canonical NF-κB pathway that may control lung inflammation caused by cigarette smoke (CS), a leading cause of morbidity and mortality worldwide. Our lab has previously shown that RelB protects against CS-induced inflammation in vitro, leading us to hypothesize that RelB would protect against acute CS-induced pulmonary inflammation in vivo. We exposed wild-type (Relb+/+) and RelB-deficient mice (Relb-/-) mice to room air or to CS and found that CS exposure caused a sustained decrease in pulmonary granulocytes in Relb-/- mice that was predominated by a decrease in neutrophils. Pulmonary inflammation caused by other irritants, including chlorine, ovalbumin (OVA; to mimic features of asthma) and lipopolysaccharide (LPS) was not controlled by RelB. Differential cytokine analysis suggests that alterations in chemotactic cytokines do not fully account for the CS-specific decrease in neutrophils in Relb-/- mice. Flow cytometric analysis of the bronchoalveolar lavage and bone marrow cells also reveal that it is unlikely that the sustained decrease is caused by excessive cell death or decreased hematopoiesis from the bone marrow. Overall, our results indicate that RelB regulates acute CS-induced pulmonary inflammation. Understanding how RelB regulates CS-induced inflammation may potentiate the discovery of new therapeutic strategies for many of the inflammatory diseases caused by CS.

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.

We’ve shown FOT staged COPD correlates better with patient reported outcomes (PROs) than spirometry (SP) staged COPD. We hypothesized non-obstructed subjects (non-OB, FEV 1 /FVC≥0.7) with either MMEF 130% predicted would be more similar to obstructed subjects (OB, FEV 1 /FVC X >9 cmH2O/L/s) by FOT. 300 COPD subjects (>10 pack-year smoking and either FEV 1 /FVC 130%predicted) had PROs (mMRC, COPD Assessment Tests, chronic bronchitis questionnaires and AERs), PFTs (Jaeger or Vmax22, Carefusion, Germany), FOT (tremoFlo C-100, Thorasys, Canada) and in a subset, quantitative CT scan lung density (LD) abstracted from charts. Subjects were assigned to 1 of 4 groups based on FEV 1 /FVC≥/ X >/≤9. Differences between continuous variables were determined with ANOVA and dichotomous with χ2. Significance was set at p While concordant phenotypes of non-VI/non-OB and VI/OB had a clear separation of disease burden in terms of PROs and emphysema, the discordant phenotypes of VI/non-OB and non-VI/OB resembled more their VI status than their OB status. Our data suggests that up to 19% of COPD subjects may be misclassified using SP.

Background Bronchial vascular remodelling may contribute to the severity of airway narrowing through mucosal congestion. Interleukin (IL)-17A is associated with the most severe asthmatic phenotype but whether it might contribute to vascular remodelling is uncertain. Objective To assess vascular remodelling in severe asthma and whether IL-17A directly or indirectly may cause endothelial cell activation and angiogenesis. Methods Bronchial vascularization was quantified in asthmatic subjects, COPD and healthy subjects together with the number of IL-17A+ cells as well as the concentration of angiogenic factors in the sputum. The effect of IL-17A on in vitro angiogenesis, cell migration and endothelial permeability was assessed directly on primary human lung microvascular endothelial cells (HMVEC-L) or indirectly with conditioned medium derived from normal bronchial epithelial cells (NHBEC), fibroblasts (NHBF) and airway smooth muscle cells (ASMC) after IL-17A stimulation. Results Severe asthmatics have increased vascularity compared to the other groups, which correlates positively with the concentrations of angiogenic factors in sputum. Interestingly, we demonstrated that increased bronchial vascularity correlates positively with the number of subepithelial IL-17A+ cells. However IL-17A had no direct effect on HMVEC-L function but it enhanced endothelial tube formation and cell migration through the production of angiogenic factors by NHBE and ASMC. Conclusions & clinical relevance Our results shed light on the role of IL-17A in vascular remodelling, most likely through stimulating the synthesis of other angiogenic factors. Knowledge of these pathways may aid in the identification of new therapeutic targets.

Heightened inflammation, including expression of COX-2, is associated with chronic obstructive 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 for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived 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 fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α–deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC’s ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK–signaling pathway.

Almost 200 years ago, a general hospital and a medical school were established with the goal of addressing the needs of the growing community of immigrants who came to Montreal in search of prosperity in furs and timber. Within a few years, the school became the Faculty of Medicine of the newly created McGill University, named for the fur trader who gave the land and the initial endowment to establish it.

Structural cell migration plays a central role in the pathophysiology of several diseases, including asthma. Previously, we established that IL-17–induced (CXCL1, CXCL2, and CXCL3) production promoted airway smooth muscle cell (ASMC) migration, and consequently we sought to investigate the molecular mechanism of CXC-induced ASMC migration. Recombinant human CXCL1, CXCL2, and CXCL3 were used to assess migration of human primary ASMCs from normal and asthmatic subjects using a modified Boyden chamber. Neutralizing Abs or small interfering RNA (siRNA) knockdown and pharmacological inhibitors of PI3K, ERK1/2, and p38 MAPK pathways were used to investigate the receptors and the signaling pathways involved in CXC-induced ASMC migration, respectively. We established the ability of CXCL2 and CXCL3, but not CXCL1, to induce ASMC migration at the tested concentrations using normal ASMCs. We found CXCL2-induced ASMC migration to be dependent on p38 MAPK and CXCR2, whereas CXCL3-induced migration was dependent on p38 and ERK1/2 MAPK pathways via CXCR1 and CXCR2. While investigating the effect of CXCL2 and CXCL3 on asthmatic ASMC migration, we found that they induced greater migration of asthmatic ASMCs compared with normal ones. Interestingly, unlike normal ASMCs, CXCL2- and CXCL3-induced asthmatic ASMC migration was mainly mediated by the PI3K pathway through CXCR1. In conclusion, our results establish a new role of CXCR1 in ASMC migration and demonstrate the diverse mechanisms by which CXCL2 and CXCL3 mediate normal and asthmatic ASMC migration, suggesting that they may play a role in the pathogenesis of airway remodeling in asthma.

Nitric oxide (NO) plays an important role in innate host defense and inflammation. In response to infection, NO is generated by inducible nitric oxide synthase (iNOS), a gene product whose expression is highly modulated by different stimuli, including lipopolysaccharide (LPS) from gram-negative bacteria. We reported recently that LPS from Pseudomonas aeruginosa altered Na+transport in alveolar epithelial cells via a suramin-dependent process, indicating that LPS activated a purinergic response in these cells. To further study this question, in the present work, we tested whether iNOS mRNA and protein expression were modulated in response to LPS in alveolar epithelial cells. We found that LPS induced a 12-fold increase in iNOS mRNA expression via a transcription-dependent process in these cells. iNOS protein, NO, and nitrotyrosine were also significantly elevated in LPS-treated cells. Ca2+chelation and protein kinase C (PKCα-β1) inhibition suppressed iNOS mRNA induction by LPS, implicating Ca2+-dependent PKC signaling in this process. LPS evoked a significant increase of extracellular ATP. Because PKC activation is one of the signaling pathways known to mediate purinergic signaling, we evaluated the hypothesis that iNOS induction was ATP dependent. Although high suramin concentration inhibited iNOS mRNA induction, the process was not ATP dependent, since specific purinergic receptor antagonists could not inhibit the process. Altogether, these findings demonstrate that iNOS expression is highly modulated in alveolar epithelial cells by LPS via a Ca2+/PKCα-β1 pathway independent of ATP signaling.

Chronic obstructive pulmonary disease (COPD) is a chronic and prevalent respiratory disease caused primarily by long term inhalation of cigarette smoke. A major hallmark of COPD is elevated apoptosis of structural lung cells including fibroblasts. The NF-κB member RelB may suppress apoptosis in response to cigarette smoke, but its role in lung cell survival is not known. RelB may act as a pro-survival factor by controlling the expression of superoxide dismutase 2 (SOD2). SOD2 is also regulated by the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that suppresses cigarette smoke-induced apoptosis. As the AhR is also a binding partner for RelB, we speculate that RelB suppresses cigarette smoke-induced apoptosis by regulating the AhR. Using an in vitro model of cigarette smoke exposure (cigarette smoke extract [CSE]), we found that CSE down-regulated RelB expression in mouse lung fibroblasts, which was associated with elevated levels of cleaved PARP. Genetic ablation of RelB elevated CSE-induced apoptosis, including chromatin condensation, and reduced mitochondrial function. There was also more reactive oxygen species production in RelB-/- cells exposed to CSE. While there was no alteration in Nrf2 expression or localization between RelB-/- and wild type cells in response to CSE, RelB-/- cells displayed significantly decreased AhR mRNA and protein expression, concomitant with loss of AhR target gene expression (Cyp1a1, Cyp1b1, Nqo1). Finally, we found that RelB binds to the Ahr gene at 3 sites to potentially increase its expression via transcriptional induction. These data support that RelB suppresses cigarette smoke-induced apoptosis, potentially by increasing the AhR. Together, these two proteins may comprise an important cell survival signaling pathway that reduces apoptosis upon cigarette smoke exposure.

Quantitative CT is used to estimate LD but is not always available. We hypothesized that non-radiological parameters gathered in clinical practice may predict LD. 136 COPD subjects from a respirology practice completed PRO (mMRC and COPD Assessment Tests), PFT (Jaeger or Vmax22, Carefusion, Germany), OS (tremoFlo C-100, Thorasys, Canada) and a non-research CT scan within 1 year. LD (at 15th percentile) was determined from CT scans using AirwayInspector (airwayinspector.acil-bwh.org). Multiple linear regressions were performed (Stata, USA) using LD vs. BM, PRO, ln transformed OS parameters and PFT from a derivation group (DG) of 68 subjects. The resulting model was applied to data from a validation group (VG) of the remaining 68 subjects. With significance set at p LDmodel=0.37(CAT)-3.91(mMRC)+0.27(BMI)+0.19(FEV1/FVC)+0.72(lnAX)-0.32(TLC %predicted)+ 0.03(FRC %predicted)+0.32(Kco %predicted)+50.17 resulted in a strong correlation (r=0.75, p

Increased airway smooth muscle (ASM) mass is a hallmark of airway remodeling in severe asthma. Th17-associated cytokines, particularly IL-17A, IL-17F, and IL-22, have been postulated to play a role in the pathogenesis of asthma. To investigate the in vitro effect of Th17 cytokines on the proliferation and survival of airway smooth muscle cells (ASMCs), human ASMCs from asthmatic and nonasthmatic subjects were incubated with IL-17A, IL-17F, or IL-22. The aforementioned cytokines demonstrated an ability to promote proliferation and survival of ASMCs from asthmatic and nonasthmatic subjects, which were mediated by selective activation of their corresponding receptors on ASMCs, including IL-17RA, IL-17RC, or IL-22R1, respectively. IL-17A and IL-17F-induced proliferation of ASMCs was dependent on ERK1/2 MAPK pathway, while IL-22-induced proliferation involved both ERK1/2 MAPK and NF-κB pathways. The involvement of signaling pathways was further confirmed by the inhibition of proliferation by knockdown of ERK1/2 MAPK or NF-κB p65 expression with pathway-specific siRNA. Together, our results show that Th17-associated cytokines promote proliferation and reduce the apoptotic rate of human ASMCs, raising the possibility that Th17 cytokines may contribute to increasing airway smooth muscle mass and airway remodeling in asthma.

Patients with severe asthma have asthma symptoms which are difficult to control, require high dosages of medication, and continue to experience persistent symptoms, asthma exacerbations or airflow obstruction. Epidemiological and clinical evidences point to the fact that severe asthma is not a single phenotype. Cluster analyses have identified subclasses of severe asthma using parameters such as patient characteristics, and cytokine profiles have also been useful in classifying moderate and severe asthma. The IL-4/IL-13 signalling pathway accounts for the symptoms experienced by a subset of severe asthmatics with allergen-associated symptoms and high serum immunoglobulin E (IgE) levels, and these patients are generally responsive to anti-IgE treatment. The IL-5/IL-33 signalling pathway is likely to play a key role in the disease pathogenesis of those who are resistant to high doses of inhaled corticosteroid but responsive to systemic corticosteroids and anti-IL5 therapy. The IL-17 signalling pathway is thought to contribute to 'neutrophilic asthma'. Although traditionally viewed as players in the defence mechanism against viral and intracellular bacterial infection, mounting evidence supports a role for Th1 cytokines such as IL-18 and IFN-γ in severe asthma pathogenesis. Furthermore, these cytokine signalling pathways interact to contribute to the spectrum of clinical pathological outcomes in severe asthma. To date, glucocorticoids are the most effective anti-asthma drugs available, yet severe asthma patients are typically resistant to the effects of glucocorticoids. Glucocorticoid receptor dysfunction and histone deacetylase activity reduction are likely to contribute to glucocorticoid resistance in severe asthma patients. This review discusses recent development in different cytokine signalling pathways, their interactions and steroid resistance, in the context of severe asthma pathogenesis.

Background: Recent evidence suggests that cells other than Th-17 lymphocytes express interleukin (IL)-17A and IL-17F and contribute to the production of these cytokines in immunologically mediated diseases. B lymphocytes are known to be an important source of cytokines in chronic inflammatory diseases. We therefore investigated the potential of human B lymphocytes to produce IL-17A and IL-17F. Methods: Highly purified B cells were obtained using a multiple-step separation procedure which included rosette depletion, adherence depletion, CD3+ cell magnetic activated depletion and CD19+ magnetic activated positive cell selection. In these CD19+ B cell fractions, CD3+/CD4+ and CD14+ cells were negligible (Results: Evidence of expression of IL-17A and IL-17F in purified B cells was obtained using RT-PCR, flow cytometry, immunofluorescence microscopy, Western immunoblotting and ELISA. Stimulation of B cells with IL-6, IL-23 or TGF-β upregulated the expression of both IL-17A and F cytokines. Conclusions: These novel findings provide evidence that cytokine-stimulated B lymphocytes could be a significant source of IL-17A and IL-17F and support the notion that these cells actively participate in immune responses via alternative mechanisms in addition to the classic release of antibodies.

Summary Background Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-β (GR-β) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-β in bronchial epithelial cells. Objectives To confirm the expression of the GR-β and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-β in bronchial epithelial cells obtained from normal subjects and asthmatic patients. Methods The expression of IL-17A and F, GR-α and GR-β was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-β was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. Results We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-α expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-β in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-β in response to IL-17A and F in the cells from asthmatics (P0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P0.05). Conclusion Evidence of an increased GR-β expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects. Cite this as: A. Vazquez-Tello, A. Semlali, J. Chakir, J. G. Martin, D. Y. Leung, D. H. Eidelman and Q. Hamid, Clinical & Experimental Allergy, 2010 (40) 1312–1322.

To investigate peroxidase induced 3'-nitrotyrosine (3NT) formation, neutrophil derived myeloperoxidase (MPO) (0.025 microM) was directly administered to A549 epithelial cells with or without H(2)O(2) (150 microM). Little evidence of 3NT was found. In contrast, there was a dose dependent increase in intracellular NO (p0.001, n=8) following MPO (0.025 microM) treatment, which was further enhanced (p0.0003, n=8) by addition of H(2)O(2). Extracellular NO also increased after MPO (p0.002, n=5) and with MPO and H(2)O(2) (p0.004, n=5). Substantial 3NT formation was only detected following addition of nitrite (NO(2)(-),or =100 microM), which induced a dose dependent increase in epithelial 3NT. In contrast, protein carbonyl formation and increased GSSG/GSH ratios were associated with MPO treatment even in the absence of NO(2)(-). Co-culture of A549 epithelial cells with polymorphonuclear leukocytes (PMN) (10(6)/ml) led to immunocytochemical detection of epithelial 3NT and induction of nitric oxide synthase (NOS2). However, in a Transwell system direct contact between PMN and A549 cells was necessary for immunodetection of 3NT but not of NOS2 consistent with a role for high local nitrite concentrations. These findings demonstrate dissociation between epithelial endogenous NO production and 3NT formation. Although MPO can influence cellular oxidative stress, particularly in the presence of H(2)O(2), 3NT formation requires the presence of high concentrations of NO(2)(-) in the milieu.

The effect of muscle activation on muscle nitric oxide (NO) production remains controversial. Whereas NO release increases in in vitro activated muscles and in vivo limb muscles, diaphragmatic NO synthase (NOS) activity declines after 3 h of inspiratory resistive loading (IRL). We tested in this study the hypotheses that acute IRL decreases diaphragmatic NO derivatives levels and reduces protein expression of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) NO synthases, as well as 3-nitrotyrosine formation. Anesthetized, tracheostomized, spontaneously breathing adult rats were subjected to IRL (50% of the maximum inspiratory pressure) for 1, 3, or 6 h. Quietly breathing rats served as controls. After 3 h of IRL, muscle eNOS and nNOS protein levels rose by 80 and 60% of control values, respectively. Whereas eNOS expression did not change any further, nNOS expression reached 550% of control values after 6 h of IRL. Strong iNOS protein expression was detected in the diaphragms after 6 h of IRL. Total NO derivatives levels in the diaphragm declined during IRL as a result of reduction in nitrate, nitrite, and nitrosothiols. Diaphragmatic protein tyrosine nitration decreased in response to IRL, and this reduction was mainly due to reduced tyrosine nitration of enolase and aldolase. We conclude that diaphragmatic NO derivatives levels decline in response to IRL and that the rise in diaphragmatic NOS protein expression may be a compensatory response designed to counterbalance the decline in NOS activity.

SummaryBackground Chronic inflammation, typified by increased expression of IL-17A, together with airway and parenchymal remodelling are features of chronic lung diseases. Emerging evidence suggests that phenotypic heterogeneity of repair and inflammatory capacities of fibroblasts may contribute to the differential structural changes observed in different regions of the lung. Objective To investigate phenotypic differences in parenchymal and bronchial fibroblasts, either in terms of inflammation and remodelling or the ability of these fibroblasts to respond to IL-17A. Methods Four groups of primary fibroblasts were used: normal human bronchial fibroblast (NHBF), normal human parenchymal fibroblast (NHPF), COPD human bronchial fibroblast (CHBF) and COPD human parenchymal fibroblast (CHPF). Cytokine and extracellular matrix (ECM) expression were measured at baseline and after stimulation with IL-17A. Actinomycin D was used to measure cytokine mRNA stability. Results At baseline, we observed higher protein production of IL-6 in NHPF than NHBF, but higher levels of IL-8 and GRO-α in NHBF. IL-17A induced a higher expression of GRO-α (CXCL1) and IL-6 in NHPF than in NHBF, and a higher level of IL-8 expression in NHBF. IL-17A treatment decreased the mRNA stability of IL-6 in NHBF when compared with NHPF. CHPF expressed higher protein levels of fibronectin, collagen-I and collagen-III than CHBF, NHBF and NHPF. IL-17A increased fibronectin and collagen-III protein only in NHPF and collagen-III protein production in CHBF and CHPF. Conclusions and Clinical Relevance These findings provide insight into the inflammatory and remodelling processes that may be related to the phenotypic heterogeneity of fibroblasts from airway and parenchymal regions and in their response to IL-17A.

Oscillometry (OS) is typically measured seated. Body position (BoP) influence on PFTs is well known but not on OS. We wished to determine BoP effect on OS and if PFTs or lung density (LD) would predict any changes. COPD and heathy subjects (HS) performed OS (tremoFlo C-100, 5-37Hz, Thorasys) sitting, supine and sitting again, and OS was determined; resistance at 5 Hz (R 5 ), frequency dependence of resistance (R 5-19 ), reactance at 5 Hz (X 5 ), resonance frequency (F res ) and reactance area (A X ). Absolute change (Δ) and %change (Δ%) sitting to supine OS was calculated. PFTs and CT scan LD (15 th percentile density+1000HU, AirwayInspector.acil-bwh.org) were measured. Sitting vs. supine OS was compared with Holm9s corrected Student9s t-tests. Δ and Δ% were correlated with PFTs and LD by linear regression. R 5 Δ correlated with TLC (r=0.55, p=0.03, n=15), R 5-19 Δ with TLC (r=0.85, p 5-19 Δ% with RV/TLC (r=0.64, p=0.01, n=15) and A X Δ% with LD (r=0.62, p=0.05, n=10). OS changes with BoP in both COPD and HS. BoP changes in R 5 , R 5-19 , and A X correlate moderately to very strongly with TLC, RV/TLC or LD. These findings impact on the use of OS in supine patients and supine CT data to model airways for comparison with sitting OS, and suggest airway-parenchymal interdependence influences airway caliber. Independent confirmation is necessary before using R 5-19 Δ as a predictor of TLC or LD.