Oxidized Phosphatidylcholines Trigger TRPA1 and Ryanodine Receptor-dependent Airway Smooth Muscle Contraction.

Asthma pathobiology includes oxidative stress that modifies cell membranes and extracellular phospholipids. Oxidized phosphatidylcholines (OxPCs) in lung lavage from allergen-challenged human participants correlate with airway hyperresponsiveness and induce bronchial narrowing in murine thin-cut lung slices. OxPCs activate many signaling pathways, but mechanisms for these responses are unclear. We hypothesize that OxPCs stimulate intracellular free Ca2+ flux to trigger airway smooth muscle contraction. Intracellular Ca2+ flux was assessed in Fura-2-loaded, cultured human airway smooth muscle cells. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) induced an approximately threefold increase in 20 kD myosin light chain phosphorylation. This correlated with a rapid peak in intracellular cytoplasmic Ca2+ concentration ([Ca2+]i) (143 nM) and a sustained plateau that included slow oscillations in [Ca2+]i. Sustained [Ca2+]i elevation was ablated in Ca2+-free buffer and by TRPA1 inhibition. Conversely, OxPAPC-induced peak [Ca2+]i was unaffected in Ca2+-free buffer, by TRPA1 inhibition, or by inositol 1,4,5-triphosphate receptor inhibition. Peak [Ca2+]i was ablated by pharmacologic inhibition of ryanodine receptor (RyR) Ca2+ release from the sarcoplasmic reticulum. Inhibiting the upstream RyR activator cyclic adenosine diphosphate ribose with 8-bromo-cyclic adenosine diphosphate ribose was sufficient to abolish OxPAPC-induced cytoplasmic Ca2+ flux. OxPAPC induced ∼15% bronchial narrowing in thin-cut lung slices that could be prevented by pharmacologic inhibition of either TRPA1 or RyR, which similarly inhibited OxPC-induced myosin light chain phosphorylation in cultured human airway smooth muscle cells. In summary, OxPC mediates airway narrowing by triggering TRPA1 and RyR-mediated mobilization of intracellular and extracellular Ca2+ in airway smooth muscle. These data suggest that OxPC in the airways of allergen-challenged subjects and subjects with asthma may contribute to airway hyperresponsiveness.

Dysregulation of endocytic machinery and ACE2 in small airways of smokers and COPD patients can augment their susceptibility to SARS-CoV-2 (COVID-19) infections.

Lungs of smokers and chronic obstructive pulmonary disease (COPD) are severely compromised and are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attack. The dangerous combination of enhanced SARS-CoV-2 attachment receptor protein ACE2 along with an increase in endocytic vacuoles will enable viral attachment, entry, and replication. The objective of the study was to identify the presence of SARS-CoV-2 host attachment receptor angiotensin-converting enzyme-2 (ACE2) along with endocytic vacuoles, early endosome antigen-1 (EEA1), late endosome marker RAB7, cathepsin-L, and lysosomal associated membrane protein-1 (LAMP-1) as lysosome markers in the airways of smokers and COPD patients. The study design was cross-sectional and involved lung resections from 39 patients in total, which included 19 patients with Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage I or GOLD stage II COPD, of which 9 were current smokers with COPD (COPD-CS) and 10 were ex-smokers with COPD (COPD-ES), 10 were normal lung function smokers, and 10 were never-smoking normal controls. Immunostaining for ACE2, EEA1, RAB7, and cathepsin-L was done. A comparative description for ACE2, EEA1, RAB7, and cathepsin-L expression pattern is provided for the patient groups. Furthermore, staining intensity for LAMP-1 lysosomes was measured as the ratio of the LAMP-1-stained areas per total area of epithelium or subepithelium, using Image ProPlus v7.0 software. LAMP-1 expression showed a positive correlation to patient smoking history while in COPD LAMP-1 negatively correlated to lung function. The active presence of ACE2 protein along with endocytic vacuoles such as early/late endosomes and lysosomes in the small airways of smokers and COPD patients provides evidence that these patient groups could be more susceptible to COVID-19.

Second harmonic generation imaging of collagen scaffolds within the alveolar ducts of healthy and emphysematous mouse lungs.

The alveolar ducts are connected to peripheral septal fibers which extend from the visceral pleura into interlobular septa, and are anchored to axial fibers in the small airways. Together these axial and septal fibers constitute a fiber continuum that provides tension and integrity throughout the lung. Building on the observations that alveolar ducts associated with sub-pleural alveoli are orientated perpendicular to the visceral pleura, and in parallel to each other, the goal of the present study was to investigate the nature of the collagen fiber organization within sub-pleural alveolar ducts in healthy control and elastase-induced emphysema murine lungs. Employing three-dimensional second harmonic generation imaging, the structural arrangement of fibrilar collagen fibers could be visualized in cleared murine lungs. In healthy control lungs, fibrilar collagen fibers within alveolar mouths formed the coiled collagen structure within the alveolar duct. In the elastase-treated emphysema lungs, there was loss of fibrilar collagen fibers (p < 0.01) and disruption of collagens structural organization as measured by the fibrillar collagen length (p < 0.01) and entropy (p < 0.01). Compared to the alveolar ducts from healthy controls, there was a significant increase in the area of cells (nm2, p < 0.001), and area of cells with cytoplasmic granules (nm2, p < 0.001) compared to emphysematous lungs. These results are consistent with the idea that one of the major contributors to the progressive loss of alveolar surfaces and elastic recoil in the emphysematous lung is loss of the structural integrity of the collagen scaffold that maintains the spatial relationships important for cell survival and lung function.

IL-4Rα blockade reduces influenza-associated morbidity in a murine model of allergic asthma.

BACKGROUND: Asthma was identified as the most common comorbidity in hospitalized patients during the 2009 H1N1 influenza pandemic. We determined using a murine model of allergic asthma whether these mice experienced increased morbidity from pandemic H1N1 (pH1N1) viral infection and whether blockade of interleukin-4 receptor α (IL-4Rα), a critical mediator of Th2 signalling, improved their outcomes. METHODS: Male BALB/c mice were intranasally sensitized with house dust mite antigen (Der p 1) for 2 weeks; the mice were then inoculated intranasally with a single dose of pandemic H1N1 (pH1N1). The mice were administered intraperitoneally anti-IL-4Rα through either a prophylactic or a therapeutic treatment strategy. RESULTS: Infection with pH1N1 of mice sensitized to house dust mite (HDM) led to a 24% loss in weight by day 7 of infection (versus 14% in non-sensitized mice; p < .05). This was accompanied by increased viral load in the airways and a dampened anti-viral host responses to the infection. Treatment of HDM sensitized mice with a monoclonal antibody against IL-4Rα prior to or following pH1N1 infection prevented the excess weight loss, reduced the viral load in the lungs and ameliorated airway eosinophilia and systemic inflammation related to the pH1N1 infection. CONCLUSION: Together, these data implicate allergic asthma as a significant risk factor for H1N1-related morbidity and reveal a potential therapeutic role for IL-4Rα signalling blockade in reducing the severity of influenza infection in those with allergic airway disease.

Noninvasive Imaging Biomarker Identifies Small Airway Damage in Severe Chronic Obstructive Pulmonary Disease.

Rationale: Evidence suggests damage to small airways is a key pathologic lesion in chronic obstructive pulmonary disease (COPD). Computed tomography densitometry has been demonstrated to identify emphysema, but no such studies have been performed linking an imaging metric to small airway abnormality.Objectives: To correlate ex vivo parametric response mapping (PRM) analysis to in vivo lung tissue measurements of patients with severe COPD treated by lung transplantation and control subjects.Methods: Resected lungs were inflated, frozen, and systematically sampled, generating 33 COPD (n = 11 subjects) and 22 control tissue samples (n = 3 subjects) for micro-computed tomography analysis of terminal bronchioles (TBs; last generation of conducting airways) and emphysema.Measurements and Main Results: PRM analysis was conducted to differentiate functional small airways disease (PRMfSAD) from emphysema (PRMEmph). In COPD lungs, TB numbers were reduced (P = 0.01); surviving TBs had increased wall area percentage (P < 0.001), decreased circularity (P < 0.001), reduced cross-sectional luminal area (P < 0.001), and greater airway obstruction (P = 0.008). COPD lungs had increased airspace size (P < 0.001) and decreased alveolar surface area (P < 0.001). Regression analyses demonstrated unique correlations between PRMfSAD and TBs, with decreased circularity (P < 0.001), decreased luminal area (P < 0.001), and complete obstruction (P = 0.008). PRMEmph correlated with increased airspace size (P < 0.001), decreased alveolar surface area (P = 0.003), and fewer alveolar attachments per TB (P = 0.01).Conclusions: PRMfSAD identifies areas of lung tissue with TB loss, luminal narrowing, and obstruction. This is the first confirmation that an imaging biomarker can identify terminal bronchial pathology in established COPD and provides a noninvasive imaging methodology to identify small airway damage in COPD.

Heterogeneity of airway wall dimensions in humans: a critical determinant of lung function in asthmatics and nonasthmatics.

Airway remodeling, a key feature of asthma, alters every layer of the airway wall but most strikingly the airway smooth muscle (ASM) layer. Airway remodeling in asthmatics contributes to fixed airflow obstruction and can amplify airway narrowing caused by ASM activation. Previous modeling studies have shown that the increase in ASM mass has the largest effect on increasing maximal airway narrowing. Simulated heterogeneity in the dimensions and properties of the airway wall can further amplify airway narrowing. Using measurements made on histological sections from donor lungs, we show for the first time that there is profound heterogeneity of ASM area and wall area in both nonasthmatics and asthmatics. Using a mathematical model, we found that this heterogeneity, together with changes in the mean values, contributes to an increased baseline resistance and elastance in asthmatics as well as a leftward shift in the responsiveness of the airways to a simulated agonist in both nonasthmatics and asthmatics. The ability of heterogeneous wall dimensions to shift the dose-response curve is largely due to an increased susceptibility for the small airways to close. This research confirms that heterogeneity of airway wall dimensions can contribute to exaggerated airway narrowing and provides an actual assessment of the magnitude of these effects.

miR-146a-5p plays an essential role in the aberrant epithelial-fibroblast cross-talk in COPD.

We previously reported that epithelial-derived interleukin (IL)-1α drives fibroblast-derived inflammation in the lung epithelial-mesenchymal trophic unit. Since miR-146a-5p has been shown to negatively regulate IL-1 signalling, we investigated the role of miR-146a-5p in the regulation of IL-1α-driven inflammation in chronic obstructive pulmonary disease (COPD).Human bronchial epithelial (16HBE14o-) cells were co-cultured with control and COPD-derived primary human lung fibroblasts (PHLFs), and miR-146a-5p expression was assessed with and without IL-1α neutralising antibody. Genomic DNA was assessed for the presence of the single nucleotide polymorphism (SNP) rs2910164. miR-146a-5p mimics were used for overexpression studies to assess IL-1α-induced signalling and IL-8 production by PHLFs.Co-culture of PHLFs with airway epithelial cells significantly increased the expression of miR-146a-5p and this induction was dependent on epithelial-derived IL-1α. miR-146a-5p overexpression decreased IL-1α-induced IL-8 secretion in PHLFs via downregulation of IL-1 receptor-associated kinase-1. In COPD PHLFs, the induction of miR-146a-5p was significantly less compared with controls and was associated with the SNP rs2910164 (GG allele) in the miR-146a-5p gene.Our results suggest that induction of miR-146a-5p is involved in epithelial-fibroblast communication in the lungs and negatively regulates epithelial-derived IL-1α induction of IL-8 by fibroblasts. The decreased levels of miR-146a-5p in COPD fibroblasts may induce a more pro-inflammatory phenotype, contributing to chronic inflammation in COPD.

Acute cigarette smoke exposure activates apoptotic and inflammatory programs but a second stimulus is required to induce epithelial to mesenchymal transition in COPD epithelium.

BACKGROUND: Smoking and aberrant epithelial responses are risk factors for lung cancer as well as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis. In these conditions, disease progression is associated with epithelial damage and fragility, airway remodelling and sub-epithelial fibrosis. The aim of this study was to assess the acute effects of cigarette smoke on epithelial cell phenotype and pro-fibrotic responses in vitro and in vivo. RESULTS: Apoptosis was significantly greater in unstimulated cells from COPD patients compared to control, but proliferation and CXCL8 release were not different. Cigarette smoke dose-dependently induced apoptosis, proliferation and CXCL8 release with normal epithelial cells being more responsive than COPD patient derived cells. Cigarette smoke did not induce epithelial-mesenchymal transition. In vivo, cigarette smoke exposure promoted epithelial apoptosis and proliferation. Moreover, mimicking a virus-induced exacerbation by exposing to mice to poly I:C, exaggerated the inflammatory responses, whereas expression of remodelling genes was similar in both. CONCLUSIONS: Collectively, these data indicate that cigarette smoke promotes epithelial cell activation and hyperplasia, but a secondary stimulus is required for the remodelling phenotype associated with COPD.

Gene expression analysis in asthma using a targeted multiplex array.

BACKGROUND: Gene expression changes in the structural cells of the airways are thought to play a role in the development of asthma and airway hyperresponsiveness. This includes changes to smooth muscle contractile machinery and epithelial barrier integrity genes. We used a targeted gene expression arrays to identify changes in the expression and co-expression of genes important in asthma pathology. METHODS: RNA was isolated from the airways of donor lungs from 12 patients with asthma (8 fatal) and 12 non-asthmatics controls and analyzed using a multiplexed, hypothesis-directed platform to detect differences in gene expression. Genes were grouped according to their role in airway dysfunction: airway smooth muscle contraction, cytoskeleton structure and regulation, epithelial barrier function, innate and adaptive immunity, fibrosis and remodeling, and epigenetics. RESULTS: Differential gene expression and gene co-expression analyses were used to identify disease associated changes in the airways of asthmatics. There was significantly decreased abundance of integrin beta 6 and Ras-Related C3 Botulinum Toxin Substrate 1 (RAC1) in the airways of asthmatics, genes which are known to play an important role in barrier function. Significantly elevated levels of Collagen Type 1 Alpha 1 (COL1A1) and COL3A1 which have been shown to modulate cell proliferation and inflammation, were found in asthmatic airways. Additionally, we identified patterns of differentially co-expressed genes related to pathways involved in virus recognition and regulation of interferon production. 7 of 8 pairs of differentially co-expressed genes were found to contain CCCTC-binding factor (CTCF) motifs in their upstream promoters. CONCLUSIONS: Changes in the abundance of genes involved in cell-cell and cell-matrix interactions could play an important role in regulating inflammation and remodeling in asthma. Additionally, our results suggest that alterations to the binding site of the transcriptional regulator CTCF could drive changes in gene expression in asthmatic airways. Several asthma susceptibility loci are known to contain CTCF motifs and so understanding the role of this transcription factor may expand our understanding of asthma pathophysiology and therapeutic options.

Interleukin-1α drives the dysfunctional cross-talk of the airway epithelium and lung fibroblasts in COPD.

Chronic obstructive pulmonary disease (COPD) has been associated with aberrant epithelial-mesenchymal interactions resulting in inflammatory and remodelling processes. We developed a co-culture model using COPD and control-derived airway epithelial cells (AECs) and lung fibroblasts to understand the mediators that are involved in remodelling and inflammation in COPD.AECs and fibroblasts obtained from COPD and control lung tissue were grown in co-culture with fetal lung fibroblast or human bronchial epithelial cell lines. mRNA and protein expression of inflammatory mediators, pro-fibrotic molecules and extracellular matrix (ECM) proteins were assessed.Co-culture resulted in the release of pro-inflammatory mediators interleukin (IL)-8/CXCL8 and heat shock protein (Hsp70) from lung fibroblasts, and decreased expression of ECM molecules (e.g. collagen, decorin) that was not different between control and COPD-derived primary cells. This pro-inflammatory effect was mediated by epithelial-derived IL-1α and increased upon epithelial exposure to cigarette smoke extract (CSE). When exposed to CSE, COPD-derived AECs elicited a stronger IL-1α response compared with control-derived airway epithelium and this corresponded with a significantly enhanced IL-8 release from lung fibroblasts.We demonstrate that, through IL-1α production, AECs induce a pro-inflammatory lung fibroblast phenotype that is further enhanced with CSE exposure in COPD, suggesting an aberrant epithelial-fibroblast interaction in COPD.

Morphometric analysis of inflammation in bronchial biopsies following exposure to inhaled diesel exhaust and allergen challenge in atopic subjects.

BACKGROUND: Allergen exposure and air pollution are two risk factors for asthma development and airway inflammation that have been examined extensively in isolation. The impact of combined allergen and diesel exhaust exposure has received considerably less attention. Diesel exhaust (DE) is a major contributor to ambient particulate matter (PM) air pollution, which can act as an adjuvant to immune responses and augment allergic inflammation. We aimed to clarify whether DE increases allergen-induced inflammation and cellular immune response in the airways of atopic human subjects. METHODS: Twelve atopic subjects were exposed to DE 300 µg.m(-3) or filtered air for 2 h in a blinded crossover study design with a four-week washout period between arms. One hour following either filtered air or DE exposure, subjects were exposed to allergen or saline (vehicle control) via segmental challenge. Forty-eight hours post-allergen or control exposure, bronchial biopsies were collected. The study design generated 4 different conditions: filtered air + saline (FAS), DE + saline (DES), filtered air + allergen (FAA) and DE + allergen (DEA). Biopsies sections were immunostained for tryptase, eosinophil cationic protein (ECP), neutrophil elastase (NE), CD138, CD4 and interleukin (IL)-4. The percent positivity of positive cells were quantified in the bronchial submucosa. RESULTS: The percent positivity for tryptase expression and ECP expression remained unchanged in the bronchial submucosa in all conditions. CD4 % positive staining in DEA (0.311 ± 0.060) was elevated relative to FAS (0.087 ± 0.018; p = 0.035). IL-4% positive staining in DEA (0.548 ± 0.143) was elevated relative to FAS (0.127 ± 0.062; p = 0.034). CD138 % positive staining in DEA (0.120 ± 0.031) was elevated relative to FAS (0.017 ± 0.006; p = 0.015), DES (0.044 ± 0.024; p = 0.040), and FAA (0.044 ± 0.008; p = 0.037). CD138% positive staining in FAA (0.044 ± 0.008) was elevated relative to FAS (0.017 ± 0.006; p = 0.049). NE percent positive staining in DEA (0.224 ± 0.047) was elevated relative to FAS (0.045 ± 0.014; p = 0.031). CONCLUSIONS: In vivo allergen and DE co-exposure results in elevated CD4, IL-4, CD138 and NE in the respiratory submucosa of atopic subjects, while eosinophils and mast cells are not changed. TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01792232.

Ultrastructure of human tracheal smooth muscle from subjects with asthma and nonasthmatic subjects. Standardized methods for comparison.

A characteristic feature of asthma is exaggerated airway narrowing, termed airway hyper-responsiveness (AHR) due to contraction of airway smooth muscle (ASM). Although smooth muscle (SM)-specific asthma susceptibility genes have been identified, it is not known whether asthmatic ASM is phenotypically different from nonasthmatic ASM in terms of subcellular structure or mechanical function. The present study is the first to systematically quantify, using electron microscopy, the ultrastructure of tracheal SM from subjects with asthma and nonasthmatic subjects. Methodological details concerning tissue sample preparation, ultrastructural quantification, and normalization of isometric force by appropriate morphometric parameters are described. We reasoned that genetic and/or acquired differences in the ultrastructure of asthmatic ASM could be associated with functional changes. We recently reported that asthmatic ASM is better able to maintain and recover active force generation after length oscillations simulating deep inspirations. The present study was designed to seek structural evidence to account for this observation. Contrary to our hypotheses, no significant qualitative or quantitative differences were found in the subcellular structure of asthmatic versus nonasthmatic tracheal SM. Specifically, there were no differences in average SM cell cross-sectional area; fraction of the cell area occupied by nonfilamentous area; amounts of mitochondria, dense bodies, and dense plaques; myosin and actin filament densities; basal lamina thickness; and the number of microtubules. These results indicate that functional differences in ASM do not necessarily translate into observable structural changes.

Lung eQTLs to help reveal the molecular underpinnings of asthma.

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55 × 10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.

Airway epithelial regulation of pulmonary immune homeostasis and inflammation.

Recent genetic, structural and functional studies have identified the airway and lung epithelium as a key orchestrator of the immune response. Further, there is now strong evidence that epithelium dysfunction is involved in the development of inflammatory disorders of the lung. Here we review the characteristic immune responses that are orchestrated by the epithelium in response to diverse triggers such as pollutants, cigarette smoke, bacterial peptides, and viruses. We focus in part on the role of epithelium-derived interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), as well as CC family chemokines as critical regulators of the immune response. We cite examples of the function of the epithelium in host defense and the role of epithelium dysfunction in the development of inflammatory diseases.

E-cadherin: gatekeeper of airway mucosa and allergic sensitization.

The airway epithelium plays a role in immune regulation during environmental challenge, which is intertwined with its barrier function and capacity to limit submucosal access of environmental factors. In asthma, mucosal barrier function is often compromised, with disrupted expression of the adhesion molecule E-cadherin. Recent progress suggests that E-cadherin contributes to the structural and immunological function of airway epithelium, through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators that can modulate the immune response. Here, we discuss this novel role for E-cadherin in mediating the crucial immunological decision between maintenance of tolerance versus induction of innate and adaptive immunity.

Pathological changes in the COPD lung mesenchyme--novel lessons learned from in vitro and in vivo studies.

Chronic obstructive pulmonary disease (COPD) is currently the fourth leading cause of death worldwide and, in contrast to the trend for cardiovascular diseases, mortality rates still continue to climb. This increase is in part due to an aging population, being expanded by the "Baby boomer" generation who grew up when smoking rates were at their peak and by people in developing countries living longer. Sadly, there has been a disheartening lack of new therapeutic approaches to counteract the progressive decline in lung function associated with the disease that leads to disability and death. COPD is characterized by irreversible chronic airflow limitation that is caused by emphysematous destruction of lung elastic tissue and/or obstruction in the small airways due to occlusion of their lumen by inflammatory mucus exudates, narrowing and obliteration. These lesions are mainly produced by the response of the tissue to the repetitive inhalational injury inflicted by noxious gases, including cigarette smoke, which involves interaction between infiltrating inflammatory immune cells, resident cells (e.g. epithelial cells and fibroblasts) and the extra cellular matrix. This interaction leads to tissue destruction and airway remodeling with changes in elastin and collagen, such that the epithelial-mesenchymal trophic unit is dysregulated in both the disease pathologies. This review focuses on: 1--novel inflammatory and remodeling factors that are altered in COPD; 2--in vitro and in vivo models to understand the mechanism whereby the extra cellular matrix environment in altered in COPD; and 3--COPD in the context of wound-repair tissue responses, with a focus on the regulation of mesenchymal cell fate and phenotype.

Formation of a stable mimic of ambient particulate matter containing viable infectious respiratory syncytial virus and its dry-deposition directly onto cell cultures.

Epidemiological associations of worse respiratory outcomes from combined exposure to ambient particulate matter (PM) and respiratory viral infection suggest possible interactions between PM and viruses. To characterize outcomes of such exposures, we developed an in vitro mimic of the in vivo event of exposure to PM contaminated with respiratory syncytial virus (RSV). Concentration of infectious RSV stocks and a particle levitation apparatus were the foundations of the methodology developed to generate specific numbers of PM mimics (PM(Mimics)) of known composition for dry, direct deposition onto airway epithelial cell cultures. Three types of PM(Mimics) were generated for this study: (i) carbon alone (P(C)), (ii) carbon and infectious RSV (P(C+RSV)), and (iii) aerosols consisting of RSV (A(RSV)). P(C+RSV) were stable in solution and harbored infectious RSV for up to 6 months. Unlike A(RSV) infection, P(C+RSV) infection was found to be dynamin dependent and to cause lysosomal rupture. Cells dosed with PM(Mimics) comprised of RSV (A(RSV)), carbon (P(C)), or RSV and carbon (P(C+RSV)) responded differentially as exemplified by the secretion patterns of IL-6 and IL-8. Upon infection, and prior to lung cell death due to viral infection, regression analysis of these two mediators in response to incubation with A(RSV), P(C), or P(C+RSV) yielded higher concentrations upon infection with the latter and at earlier time points than the other PM(Mimics). In conclusion, this experimental platform provides an approach to study the combined effects of PM-viral interactions and airway epithelial exposures in the pathogenesis of respiratory diseases involving inhalation of environmental agents.

Epithelial-mesenchymal transition changes in nonsmall cell lung cancer patients with early COPD.

Background: Epithelial-mesenchymal transition (EMT) might be central to lung cancer development in smokers and COPD. We illustrate EMT changes in a broader demographic of patient groups who were diagnosed with nonsmall cell lung cancer (adenocarcinoma and squamous cell carcinoma). These included COPD current and ex-smokers, patients with small airway disease and normal lung function smokers compared to normal controls. Methods: We had access to surgically resected small airway tissue from 46 subjects and assessed for airway wall thickness and immunohistochemically for the EMT biomarkers E-cadherin, N-cadherin, S100A4, vimentin and epidermal growth factor receptor (EGFR). All tissue analysis was done with a computer and microscope-assisted Image-Pro Plus 7.0 software. Results: Airway wall thickness significantly increased across all pathological groups (p<0.05) compared to normal controls. Small airway epithelial E-cadherin expression markedly decreased (p<0.01), and increases in N-cadherin, vimentin, S100A4 and EGFR expression were observed in all pathological groups compared to normal controls (p<0.01). Vimentin-positive cells in the reticular basement membrane, lamina propria and adventitia showed a similar trend to epithelium across all pathological groups (p<0.05); however, such changes were only observed in reticular basement membrane for S100A4 (p<0.05). Vimentin was higher in adenocarcinoma versus squamous cell carcinoma; in contrast, S100A4 was higher in the squamous cell carcinoma group. EGFR and N-cadherin expression in both phenotypes was markedly higher than E-cadherin, vimentin and S100A4 (p<0.0001). Conclusion: EMT is an active process in the small airway of smokers and COPD diagnosed with nonsmall cell lung cancer, contributing to small airway remodelling and cancer development as seen in these patients.

Transforming growth factor-ß1 and SMAD signalling pathway in the small airways of smokers and patients with COPD: potential role in driving fibrotic type-2 epithelial mesenchymal transition.

Background: COPD is a common disease characterized by respiratory airflow obstruction. TGF-ß1 and SMAD pathway is believed to play a role in COPD pathogenesis by driving epithelial mesenchymal transition (EMT). Methods: We investigated TGF-ß1 signalling and pSmad2/3 and Smad7 activity in resected small airway tissue from patients with; normal lung function and a smoking history (NLFS), current smokers and ex-smokers with COPD GOLD stage 1 and 2 (COPD-CS and COPD-ES) and compared these with normal non-smoking controls (NC). Using immunohistochemistry, we measured activity for these markers in the epithelium, basal epithelium, and reticular basement membrane (RBM). Tissue was also stained for EMT markers E-cadherin, S100A4 and vimentin. Results: The Staining of pSMAD2/3 was significantly increased in the epithelium, and RBM of all COPD groups compared to NC (p <0.0005). There was a less significant increase in COPD-ES basal cell numbers compared to NC (p= 0.02). SMAD7 staining showed a similar pattern (p <0.0001). All COPD group levels of TGF-ß1 in the epithelium, basal cells, and RBM cells were significantly lower than NC (p <0.0001). Ratio analysis showed a disproportionate increase in SMAD7 levels compared to pSMAD2/3 in NLFS, COPD-CS and COPD-ES. pSMAD negatively correlated with small airway calibre (FEF25-75%; p= 0.03 r= -0.36). EMT markers were active in the small airway epithelium of all the pathological groups compared to patients with COPD. Conclusion: Activation of the SMAD pathway via pSMAD2/3 is triggered by smoking and active in patients with mild to moderate COPD. These changes correlated to decline in lung function. Activation of the SMADs in the small airways is independent of TGF-ß1, suggesting factors other than TGF-ß1 are driving these pathways. These factors may have implications for small airway pathology in smokers and COPD through the process of EMT, however more mechanistic work is needed to prove these correlations.

Mucosal immune alterations at the early onset of tissue destruction in chronic obstructive pulmonary disease.

Rationale: COPD is characterized by chronic airway inflammation, small airways changes, with disappearance and obstruction, and also distal/alveolar destruction (emphysema). The chronology by which these three features evolve with altered mucosal immunity remains elusive. This study assessed the mucosal immune defense in human control and end-stage COPD lungs, by detailed microCT and RNA transcriptomic analysis of diversely affected zones. Methods: In 11 control (non-used donors) and 11 COPD (end-stage) explant frozen lungs, 4 cylinders/cores were processed per lung for microCT and tissue transcriptomics. MicroCT was used to quantify tissue percentage and alveolar surface density to classify the COPD cores in mild, moderate and severe alveolar destruction groups, as well as to quantify terminal bronchioles in each group. Transcriptomics of each core assessed fold changes in innate and adaptive cells and pathway enrichment score between control and COPD cores. Immunostainings of immune cells were performed for validation. Results: In mildly affected zones, decreased defensins and increased mucus production were observed, along CD8+ T cell accumulation and activation of the IgA pathway. In more severely affected zones, CD68+ myeloid antigen-presenting cells, CD4+ T cells and B cells, as well as MHCII and IgA pathway genes were upregulated. In contrast, terminal bronchioles were decreased in all COPD cores. Conclusion: Spatial investigation of end-stage COPD lungs show that mucosal defense dysregulation with decreased defensins and increased mucus and IgA responses, start concomitantly with CD8+ T-cell accumulation in mild emphysema zones, where terminal bronchioles are already decreased. In contrast, adaptive Th and B cell activation is observed in areas with more advanced tissue destruction. This study suggests that in COPD innate immune alterations occur early in the tissue destruction process, which affects both the alveoli and the terminal bronchioles, before the onset of an adaptive immune response.