Small Airway Epithelium Research Articles

Intraflagellar Transport Gene Expression Associated with Short Cilia in Smoking and COPD

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

High Correlation of the Response of Upper and Lower Lobe Small Airway Epithelium to Smoking

The distribution of lung disease induced by inhaled cigarette smoke is complex, depending on many factors. With the knowledge that the small airway epithelium (SAE) is the earliest site of smoking-induced lung disease, and that the SAE gene expression is likely sensitive to inhaled cigarette smoke, we compared upper vs. lower lobe gene expression in the SAE within the same cigarette smokers to determine if the gene expression patterns were similar or different. Active smokers (n = 11) with early evidence of smoking-induced lung disease (normal spirometry but low diffusing capacity) underwent bronchoscopy and brushing of the upper and lower lobe SAE in order to compare upper vs lower lobe genome-wide and smoking-responsive gene expression by microarray. Cluster and principal component analysis demonstrated that, for each individual, the expression of the known SAE smoking-responsive genes were highly correlated in upper and lower lobe pairs, although, as expected, there were differences in the smoking-induced changes in gene expression from individual to individual. These observations support the concept that the heterogeneity observed among smokers in the anatomic distribution of smoking-induced disease are not secondary to the topographic differences in the effects of cigarette smoke on the airway epithelium.

ICOS-Expressing Lymphocytes Promote Resolution of CD8-Mediated Lung Injury in a Mouse Model of Lung Rejection

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8+ T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8+ T cell–mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG-/- mice, we found that CD4+ T cells and ICOS+/+ T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4+Foxp3 + ICOS+ T cells were enriched in the lungs of animals surviving lung injury and ICOS+/+ Tregs promoted survival in animals that received ICOS-/- T cells. Direct comparison of ICOS-/- Tregs to ICOS+/+ Tregs found defects in vitro but no differences in the ability of ICOS-/- Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

Cigarette smoking induces small airway epithelial epigenetic changes with corresponding modulation of gene expression

The small airway epithelium (SAE), the first site of smoking-induced lung pathology, exhibits genome-wide changes in gene expression in response to cigarette smoking. Based on the increasing evidence that the epigenome can respond to external stimuli in a rapid manner, we assessed the SAE of smokers for genome-wide DNA methylation changes compared with nonsmokers, and whether changes in SAE DNA methylation were linked to the transcriptional output of these cells. Using genome-wide methylation analysis of SAE DNA of nonsmokers and smokers, the data identified 204 unique genes differentially methylated in SAE DNA of smokers compared with nonsmokers, with 67% of the regions with differential methylation occurring within 2 kb of the transcriptional start site. Among the genes with differential methylation were those related to metabolism, transcription, signal transduction and transport. For the differentially methylated genes, 35 exhibited a correlation with gene expression, 54% with an inverse correlation of DNA methylation with gene expression and 46% a direct correlation. These observations provide evidence that cigarette smoking alters the DNA methylation patterning of the SAE and that, for some genes, these changes are associated with the smoking-related changes in gene expression.

Outdoor Smoking Areas: Does the Science Support a Ban?

Inhaling secondhand cigarette smoke (SHS), also known as passive smoking, can cause cancer and respiratory and cardiovascular disease.1 Indoor smoking is banned at many public places and worksites; at others, smoking areas have been moved outdoors. But is keeping cigarette smoke outdoors enough to dissipate the health risks associated with SHS exposure? A review in this issue of EHP explores the current state of knowledge about outdoor SHS exposure.2 “Outdoor SHS is an emerging topic in the tobacco control community,” says review coauthor Esteve Fernandez, an epidemiologist at the Institut Catala d’Oncologia–ICO in Barcelona, Spain. Surveys indicate that public support for banning outdoor smoking has increased in recent years,3 although some opponents argue that such bans are unsustainable, unduly restrictive, and unsupported by the evidence to date.4,5 To impose outdoor smoking laws, tobacco control advocates need “evidence-based results from valid and representative epidemiological studies about levels of SHS in different outdoor areas,” says Fernandez. For the current review Fernandez and colleagues analyzed data from 18 scientific papers published between 2005 and 2012, which measured SHS exposure at outdoor settings in Europe, the United States, Canada, Australia, and New Zealand. Sites included hospitality venues (e.g., restaurants and bars), airports, parks, streets, entrances to buildings, and college campuses.2 In most of the studies reviewed, the main marker for SHS was fine particulate matter (PM2.5). Measured average levels of PM2.5 ranged from 8.32 μg/m3 to 124 μg/m3 at outdoor hospitality venues where smokers were present, and from 4.6 μg/m3 to 17.8 μg/m3 at other outdoor settings. Individual point measurements exceeded 1,000 μg/m3 in some cases. Densely packed smokers, partially enclosed outdoor areas, low wind speeds, and closeness to people smoking all contributed to high levels of outdoor SHS. Smoke-free indoor settings near outdoor smoking areas also had elevated PM2.5 levels, with mean concentrations ranging from 4 μg/m3 to 120.51 μg/m3. Measured levels exceeded the median level for irritation from secondhand smoke PM2.5 reported for brief exposures.6 Most studies detected outdoor concentrations of PM2.5 exceeding 10 μg/m3, the annual outdoor average that the World Health Organization sets as the lowest cutoff at which lung cancer and cardiopulmonary deaths are likely to increase7—a particular concern for chronically exposed hospitality workers. “Although outdoor SHS levels are more transient than indoor levels, and can quickly drop to background levels in the absence of active smoking,” the authors wrote, “potential health effects of these exposures merit consideration and need to be further studied.” Although more transient than indoor levels, outdoor levels of secondhand smoke can be quite high, depending on the setting. The review shows that “depending on the microenvironment, you can get very high levels of secondhand smoke outdoors. A cigarette is a point source of outdoor pollution,” says Stanton Glantz, director of the Center for Tobacco Control Research and Education at the University of California, San Francisco. Fernandez’s compilation of data scattered across different journals “will be useful to policy makers. The evidence points to banning outdoor SHS where smokers congregate,” Glantz says. The compiled data also highlight the need for better standardized methods in future studies. PM2.5, although cheap and easy to measure, is a common traffic pollutant and not specific to SHS. More precise and sensitive markers such as salivary cotinine (a metabolic by-product of nicotine) better reflect personal exposure to SHS.2 An ideal study would combine both environmental markers such as airborne nicotine and biological markers such as cotinine in saliva. “Such studies, although more complicated to implement, would be of extreme relevance,” says Fernandez. SHS contains more than 7,000 chemicals, including about 70 known and probable carcinogens, as well as toxicants and irritants.1 In the United States, an estimated 46,000 premature deaths from heart disease and 3,400 lung cancer deaths in nonsmokers are caused by SHS exposure yearly.1 Gene microarray scans of cells lining the small airways suggest there are no safe levels of SHS exposure. One study showed that even very low exposure was associated with changes in gene expression that may reflect early smoking-induced damage, potentially setting the stage for lung disease and cancer.8

The role of uPAR in epithelial-mesenchymal transition in small airway epithelium of patients with chronic obstructive pulmonary disease

BackgroundEpithelial-mesenchymal transition (EMT) plays a crucial role in small airway fibrosis of patients with chronic obstructive pulmonary disease (COPD). Increasing evidence suggests that the urokinase plasminogen activator receptor (uPAR) is involved in the pathogenesis of COPD. Increased uPAR expression has been implicated in the promotion of EMT in numerous cancers; however the role of uPAR in EMT in small airway epithelial cells of patients with COPD remains unclear. In this study, we investigated the degree of EMT and uPAR expression in lung epithelium of COPD patients, and verified the effect of uPAR on cigarette smoke extract (CSE)-induced EMT in vitro.MethodsThe expression of EMT biomarkers and uPAR was assessed in lung epithelium specimens from non-smokers (n = 25), smokers (n = 25) and non-smokers with COPD (n = 10) and smokers with COPD (n = 18). The role of uPAR on CSE-induced EMT in human small airway epithelial cells (HSAEpiCs) was assessed by silencing uPAR expression in vitro.ResultsMarkers of active EMT and uPAR expression were significantly increased in the small airway epithelium of patients with COPD compared with controls. We also observed a significant correlation between uPAR and vimentin expression in the small airway epithelium. In vitro, CSE-induced EMT in HSAEpiCs was associated with high expression of uPAR, and targeted silencing of uPAR using shRNA inhibited CSE-induced EMT. Finally, we demonstrate that the PI3K/Akt signaling pathway is required for uPAR-mediated EMT in HSAEpiCs.ConclusionsA uPAR-dependent signaling pathway is required for CSE-induced EMT, which contributes to small airway fibrosis in COPD. We propose that increased uPAR expression in the small airway epithelium of patients with COPD participates in an active EMT process.

A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment

Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function. We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy. Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays. We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts. Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.

Transbronchial Brush (TBBr) Detection of Activated T Cells Identifies Patients at Risk of Progressive Bronchiolitis Obliterans Syndrome (BOS)

Purpose Lymphocytic bronchiolitis (LB) is one of the strongest risk factors for subsequent BOS, but the operating characteristics of transbronchial biopsy (TBBx) for LB diagnosis are poor. We hypothesised that combining transbronchial brush (TBBr) with flow cytometric phenotyping of epithelial lymphocyte populations would better assess allograft health. Methods and Materials TBBr and bronchial brushings were performed to obtain small and large airway epithelium respectively at surveillance and diagnostic bronchoscopy. Cells were stained with cytokeratin, CD3, CD8, CD103 (intraepithelial T cells) and granzyme B (GrB) before flow cytometry was performed. Data are presented as median (interquartile range) percentage of total cells. Results 67 patients (23 CF, 21 COPD, 13 IPF, 91% bilateral, median age 49.7 yrs, 51% male, median 14.3 (3.9-49.5) months post-transplant) were assessed. Bronchial CD3+(p=0.006), CD8+ (p=0.044), CD3+CD103+ (p=0.022) and CD3+GrB+ (p=0.009) cells were increased in those undergoing diagnostic bronchoscopy (n=23) and those with A grade rejection (n=5 A1, n=3 A2, n=1 A3) compared to those having a surveillance bronchoscopy (n=35). In contrast, only the CD3+GrB+ subset was increased in the bronchiolar region (1.44 (0.9-3.0) vs 0.73 (0.4-1.5), p=0.005). There was no correlation between any subset and immunosuppressant dose or level, however treatment of A grade rejection with augmented immunosuppression led to a reduction in the bronchial CD3+ (p=0.043) and bronchiolar CD3+GrB+ (p=0.012) subsets. Only the bronchiolar CD3+GrB+ fraction was increased (1.92 (1.0-4.1) vs 1.0 (0.4-1.7), p=0.028) in the 18% of the cohort who experienced progressive BOS in the following 13.3 (5.8-18.3) months. TBBx did not predict outcome. Conclusions Infiltration of the bronchiolar region by activated CD3+GrB+ T cells is associated with subsequent allograft dysfunction. As this subset is responsive to augmented immunosuppression, vigilant immune monitoring using TBBr could prevent BOS.

Distinct Lymphocyte Subsets Infiltrate the Small and Large Airway Epithelium after Lung Transplantation

Purpose Alloreactive epitheial injury is thought to be the main driver of lung allograft loss due to obliterative bronchiolitis, a process largely confined to the bronchiolar region. We hypothesised that the bronchiolar region would be more prone to lymphocytic infiltration after lung transplantation. Methods and Materials Small and large airway bronchial brushings were obtained from 74 patients (28 CF, 22 COPD, 14 IPF, 92% bilateral, median age 48.6 yrs, 46% male) undergoing post-transplant surveillance and diagnostic bronchoscopy at a median 14.3 (3.9-49.5) months. Cells were stained with cytokeratin, CD3, CD8, CD103 (intraepithelial T cell marker) and granzyme B (GrB) before flow cytometric analysis was performed. Data are presented as median (interquartile range) percentage of total cells. Results While there was no difference in total CD3+ cells, CD8+ and resident (CD103+) T cells were enriched in the bronchi compared to the bronchioles ( table , p 0.6, p Conclusions The CD103+ T cell fraction is reduced in the bronchiolar region of the lung allograft. Since this fraction has been associated with a regulatory phenotype and front-line antiviral defence, this deficiency may explain why the small airway region is particularly prone to alloreactive and other injury. Bronchial region Bronchiolar region P value CD3+ 3.36 (2.0-6.9) 3.64 (1.8-5.9) 0.933 CD8+ 2.38 (1.3-4.8) 1.74 (0.9-3.0) 0.010 CD3+CD103+ 1.95 (1.0-4.0) 1.17 (0.5-2.2) CD3+CD103- 1.34 (0.8-2.4) 1.81 (1.1-3.8) 0.012 CD3+CD103+GrB+ 0.91 (0.4-1.6) 0.36 (0.2-1.0)

Year in review 2012: Asthma and chronic obstructive pulmonary disease

Year in review 2012: Asthma and chronic obstructive pulmonary disease

Immune Modulatory Effects of IL-22 On Allergen-Induced Pulmonary Inflammation

RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system.MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined.ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE.ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. RationaleAllergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. Allergen-induced pulmonary inflammation in asthma is mainly TH2 dominated. A recently discovered TH17/TH22 cell-derived cytokine, IL-22, has been found to be increased in asthma. However, several studies showed controversial findings in the immune modulatory effects of IL-22 in animal models of allergic asthma. Our objective was to determine the regulatory role of IL-22 in OVA-induced allergic inflammation using a novel lung-specific IL-22 transgenic overexpression system. MethodsInducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. Inducible IL-22 transgenic (CC10-rtTA- and SPC-rtTA-TRE-tight-IL-22) mice on C57BL/6 genetic background were generated. IHC of lung tissue and ELISA of bronchoalveolar lavage (BAL) fluid for IL-22 were performed to confirm the location and quantity of IL-22 expression. OVA-induced pulmonary inflammatory responses were compared between IL-22 transgenic and wild type control mice. BAL total and differential cell counts, lung histology, mucous metaplasia, IHC of major basic protein for eosinophils, and serum OVA-specific IgE were determined. ResultsIL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. IL-22 transgene was successfully activated in the large (CC10 promoter) and small (SPC promoter) airway epithelium cells by doxycycline in the drinking water. OVA sensitization and challenge did not induce endogenous IL-22 expression. Compared to wild type mice, IL-22 transgenic mice showed decreased percentage of eosinophils in the BAL and slight reduction in eosinophilic inflammation and mucus metaplasia in the airways. Interestingly, inflammatory cell infiltration in lung parenchyma appeared slightly accentuated. In addition, there was no statistic difference in serum OVA-specific IgE. ConclusionsThese findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma. These findings indicate that IL-22 may have immune modulatory effects on pulmonary inflammatory responses in the development of allergen-induced asthma.

The role of the liver X receptor in chronic obstructive pulmonary disease.

BackgroundThere is a need for novel anti-inflammatory therapies to treat COPD. The liver X receptor (LXR) is a nuclear hormone receptor with anti-inflammatory properties.MethodsWe investigated LXR gene and protein expression levels in alveolar macrophages and whole lung tissue from COPD patients and controls, the effect of LXR activation on the suppression of inflammatory mediators from LPS stimulated COPD alveolar macrophages, and the effect of LXR activation on the induction of genes associated with alternative macrophage polarisation.ResultsThe levels of LXR mRNA were significantly increased in whole lung tissue extracts in COPD patients and smokers compared to non-smokers. The expression of LXR protein was significantly increased in small airway epithelium and alveolar epithelium in COPD patients compared to controls. No differences in LXR mRNA and protein levels were observed in alveolar macrophages between patient groups. The LXR agonist GW3965 significantly induced the expression of the LXR dependent genes ABCA1 and ABCG1 in alveolar macrophage cultures. In LPS stimulated alveolar macrophages, GW3965 suppressed the production of CXCL10 and CCL5, whilst stimulating IL-10 production.ConclusionsGW3965 did not significantly suppress the production of TNFα, IL-1β, or CXCL8. Our major finding is that LXR activation has anti-inflammatory effects on CXC10, CCL5 and IL-10 production from alveolar macrophages.

Role of epithelial mesenchymal transition (EMT) in chronic obstructive pulmonary disease (COPD)

Small airway fibrosis is the main contributor to physiological airway dysfunction in COPD. One potential mechanism contributing to small airway fibrosis is epithelial mesenchymal transition (EMT). When associated with angiogenesis (so called EMT-Type-3) it may well also be the link with the development of cancer, which is closely associated with COPD and predominantly in large airways. In a recent study published in Respiratory Research, Qin Wang and colleagues investigated the role of urokinase plasminogen activator receptor (uPAR) in EMT in small airway epithelium of COPD patients. However, there are some issues with the paper which we wish to comment on.

Airway Epithelial Expression of TLR5 Is Downregulated in Healthy Smokers and Smokers with Chronic Obstructive Pulmonary Disease

The TLRs are important components of the respiratory epithelium host innate defense, enabling the airway surface to recognize and respond to a variety of insults in inhaled air. On the basis of the knowledge that smokers are more susceptible to pulmonary infection and that the airway epithelium of smokers with chronic obstructive pulmonary disease (COPD) is characterized by bacterial colonization and acute exacerbation of airway infections, we assessed whether smoking alters expression of TLRs in human small airway epithelium, the primary site of smoking-induced disease. Microarrays were used to survey the TLR family gene expression in small airway (10th to 12th order) epithelium from healthy nonsmokers (n = 60), healthy smokers (n = 73), and smokers with COPD (n = 36). Using the criteria of detection call of present (P call) ≥ 50%, 6 of 10 TLRs (TLRs 1-5 and 8) were expressed. Compared with nonsmokers, the most striking change was for TLR5, which was downregulated in healthy smokers (1.4-fold, p < 10⁻¹⁰) and smokers with COPD (1.6-fold, p < 10⁻¹¹). TaqMan RT-PCR confirmed these observations. Bronchial biopsy immunofluorescence studies showed that TLR5 was expressed mainly on the apical side of the epithelium and was decreased in healthy smokers and smokers with COPD. In vitro, the level of TLR5 downstream genes, IL-6 and IL-8, was highly induced by flagellin in TLR5 high-expressing cells compared with TLR5 low-expressing cells. In the context that TLR5 functions to recognize pathogens and activate innate immune responses, the smoking-induced downregulation of TLR5 may contribute to smoking-related susceptibility to airway infection, at least for flagellated bacteria.

Expression of acute-phase cytokines, surfactant proteins, and epithelial apoptosis in small airways of human acute respiratory distress syndrome

PurposeRecent studies suggest a role for distal airway injury in acute respiratory distress syndrome (ARDS). The epithelium lining the small airways secretes a large number of molecules such as surfactant components and inflammatory mediators. There is little information on how these small airway secretory functions are altered in ARDS. Materials and MethodsWe studied the lungs of 31 patients with ARDS (Pao2/fraction of inspired oxygen ≤200, 45 ± 14 years, 16 men) and 11 controls (52 ± 16 years, 7 men) submitted to autopsy and quantified the expression of interleukin (IL) 6, IL-8, surfactant proteins (SP) A and SP-B in the epithelium of small airways using immunohistochemistry and image analysis. In addition, an index of airway epithelial apoptosis was determined by the terminal deoxynucleotidyl transferase-mediated deoxyuridine-triphosphatase nick-end labeling assay, caspase 3, and Fas/Fas ligand expression. The density of inflammatory cells expressing IL-6 and IL-8 within the small airway walls was also quantified. ResultsAcute respiratory distress syndrome airways showed an increase in the epithelial expression of IL-8 (P = .006) and an increased density of inflammatory cells expressing IL-6 (P = .004) and IL-8 (P < .001) compared with controls. There were no differences in SP-A and SP-B epithelium expression or in epithelial apoptosis index between ARDS and controls. ConclusionDistal airways are involved in ARDS lung inflammation and show a high expression of proinflammatory interleukins in both airway epithelial and inflammatory cells. Apoptosis may not be a major mechanism of airway epithelial cell death in ARDS.

Genes associated with MUC5AC expression in small airway epithelium of human smokers and non-smokers

BackgroundMucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Despite progress in animal studies, the genes and their expression pattern involved in mucus production and secretion in human airway epithelium are not well understood. We hypothesized that comparison of the transcriptomes of the small airway epithelium of individuals that express high vs low levels of MUC5AC, the major macromolecular component of airway mucus, could be used as a probe to identify the genes related to human small airway mucus production/secretion.MethodsFlexible bronchoscopy and brushing were used to obtain small airway epithelium (10th to 12th order bronchi) from healthy nonsmokers (n=60) and healthy smokers (n=72). Affymetrix HG-U133 plus 2.0 microarrays were used to assess gene expression. Massive parallel sequencing (RNA-Seq) was used to verify gene expression of small airway epithelium from 5 nonsmokers and 6 smokers.ResultsMUC5AC expression varied 31-fold among the healthy nonsmokers. Genome-wide comparison between healthy nonsmokers (n = 60) grouped as “high MUC5AC expressors” vs “low MUC5AC expressors” identified 528 genes significantly up-regulated and 15 genes significantly down-regulated in the high vs low expressors. This strategy identified both mucus production and secretion related genes under control of a network composed of multiple transcription factors. Based on the literature, genes in the up-regulated list were used to identify a 73 “MUC5AC-associated core gene” list with 9 categories: mucus component; mucus-producing cell differentiation-related transcription factor; mucus-producing cell differentiation-related pathway or mediator; post-translational modification of mucin; vesicle transport; endoplasmic reticulum stress-related; secretory granule-associated; mucus secretion-related regulator and mucus hypersecretory-related ion channel. As a validation cohort, we assessed the MUC5AC-associated core gene list in the small airway epithelium of an independent set of healthy smokers (n = 72). There was up-regulation of MUC5AC in the small airway epithelium of smokers (2.3-fold, p < 10-8) associated with a coordinated up-regulation of MUC5AC-associated core gene expression pattern in the small airway epithelium of smokers (p < 0.01). Deep sequencing confirmed these observations.ConclusionThe identification of the genes associated with increased airway mucin production in humans should be useful in understanding the pathogenesis of airway mucus hypersecretion and identifying therapeutic targets.Author summaryMucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Little is known about the gene networks associated with the synthesis and secretion of mucins in the human small airway epithelium. Taking advantage of the knowledge that MUC5AC is a major mucin secreted by the small airway epithelium, the expression of MUC5AC in small airway epithelium is highly regulated at the transcriptional level and our observation that healthy nonsmokers have variable numbers of MUC5AC+ secretory cells in the human small airway epithelium, we compared genome-wide gene expression of the small airway epithelium of high vs low MUC5AC expressors from 60 nonsmokers to identify the genes associated with MUC5AC expression. This novel strategy enabled identification of a 73 “MUC5AC-associated core gene” list with 9 categories, which control a series of processes from mucin biosynthesis to mucus secretion. The coordinated gene expression pattern of MUC5AC-associated core genes were corroborated in an independent cohort of 72 healthy smokers. Deep sequencing of small airway epithelium RNA confirmed these observations. This finding will be useful in identifying therapeutic targets to treat small airway mucus hypersecretion.

Naphthalene as a model to study repair processes of the murine tracheal epithelium in vivo

The naphthalene model of damage and repair of the small intrapulmonary airway epithelium is well established. We aimed to determine if naphthalene can also be used to study repair processes in murine tracheal epithelium. Explanted mouse tracheae were kept in Hepes-Ringer solution at 37°C and were incubated with naphthalene (200µg/ml) for 2h. In mice, a dose of 300mg/kg naphthalene or vehicle was injected intraperitoneally. Tracheae were explanted and fixed at 12h, 24h, 48h, 60h, 72h, 4d, 5d, 6d and 15d and examined using semi-thin sections, scanning electron microscopy, immunohistochemistry, real-time RT-PCR and high speed video microscopy. Lungs were obtained as positive control. Ex vivo application of naphthalene resulted in detachment of secretory cells from the basement membrane leaving islets of ciliated cells. In vivo after 12h, the detachment of few secretory as well as ciliated cells was observed. After 24h, the epithelium was destroyed with the majority of secretory and ciliated cells being lost. After 48h, no ciliated and secretory cells were found. However, basal cells still covered the basement membrane. Cell proliferation in the remaining cells increased from less than 1% to >20% and remained high until 4d. After 60h a continuous epithelium with short cilia was observed. Ciliary beat frequency was nearly normal at 4d but cilia-driven transport was impaired. Transcripts for CK5 (expressed in basal cells) remained constant throughout. In contrast, transcripts for CC10 (expressed in secretory cells) were significantly increased at 60h, 72h, 4d and 6d and returned to baseline at 15d. However, CC10-immunoreactivity was detectable only from day 5 on. After 15d, repair of the epithelium was completed and cilia reached their initial length.

MiR‐146a regulates mechanotransduction and pressure‐induced inflammation in small airway epithelium

Mechanical ventilation generates biophysical forces, including high transmural pressures, which exacerbate lung inflammation. This study sought to determine whether microRNAs (miRNAs) respond to this mechanical force and play a role in regulating mechanically induced inflammation. Primary human small airway epithelial cells (HSAEpCs) were exposed to 12 h of oscillatory pressure and/or the proinflammatory cytokine TNF-α. Experiments were also conducted after manipulating miRNA expression and silencing the transcription factor NF-κB or toll-like receptor proteins IRAK1 and TRAF6. NF-κB activation, IL-6/IL-8/IL-1β cytokine secretion, miRNA expression, and IRAK1/TRAF6 protein levels were monitored. A total of 12 h of oscillatory pressure and TNF-α resulted in a 5- to 7-fold increase in IL-6/IL-8 cytokine secretion, and oscillatory pressure also resulted in a time-dependent increase in IL-6/IL-8/IL-1β cytokine secretion. Pressure and TNF-α also resulted in distinct patterns of miRNA expression, with miR-146a being the most deregulated miRNA. Manipulating miR-146a expression altered pressure-induced cytokine secretion. Silencing of IRAK1 or TRAF6, confirmed targets of miR-146a, resulted in a 3-fold decrease in pressure-induced cytokine secretion. Cotransfection experiments demonstrate that miR-146a's regulation of pressure-induced cytokine secretion depends on its targeting of both IRAK1 and TRAF6. MiR-146a is a mechanosensitive miRNA that is rapidly up-regulated by oscillatory pressure and plays an important role in regulating mechanically induced inflammation in lung epithelia.

CFTR Dependent Bicarbonate Secretion in Native Small Airway Epithelia

Loss of bicarbonate (HCO3−) transport may play a major role in pathological changes in airways, especially in Cystic Fibrosis (CF). In the small intestine normal mucus release requires CFTR dependent HCO3− secretion. HCO3− may also be crucial for effective bactericidal activities of antibiotics as well as innate antimicrobials. Evidence indicates that HCO3− secretion is present in normal, but defective in CF cultured airway cells. However, whether HCO3− is secreted in native small airways is not known. We have developed a mini‐Ussing system and assayed HCO3− transport in native small airways of pigs and humans indirectly as the changes of transepithelial potential and equivalent short circuit current. With bilateral 25 mM NaHCO3− plus 125 mM NaGlu Ringers’ with amiloride in the lumen, we stimulated with different cAMP and Ca2+ mediated agonists and inhibited with known blockers of HCO3− transport. Results showed that the native small airway epithelia are capable of secreting CFTR dependent HCO3−. HCO3− secretion is physiologically regulated, and probably critical to normal innate defense mechanism of the airways. Failure to secrete HCO3− may cause poor mucociliary and pathogen clearance and may be a target for therapy.Supported by the Nancy Olmsted Trust, NIH (R01‐HL084042) and Cystic Fibrosis Foundation.

Abstract B33: Identification of the Early Molecular Cancerization Features in the Airway Epithelium of Smokers without Cancer

Abstract Purpose: The field cancerization concept proposes that molecular alterations caused by oncogenic stressors are present throughout the airway epithelium. Cigarette smoking, the major risk factor for the development of lung cancer, induces molecular changes in the airway epithelium prior to the development of disease; these molecular changes may indicate the risk for lung cancer development. Given that gene expression changes reflect genomic and epigenomic alterations preceding the development of pathologic lesions, we hypothesized that the earliest features of molecular cancerization can be detected in the airway epithelium of a subset of healthy smokers based on the expression of genes associated with the progression toward lung cancer. We also asked whether chronic obstructive pulmonary disease (COPD), a smoking-induced lung disease representing an independent risk factor for lung cancer, is associated with further acquisition of the molecular cancerization features in the airway epithelium. Methods: Large airway epithelium (LAE; 3rd-4th order bronchi) of healthy nonsmokers (LAE-NS, n=21) and healthy smokers (LAE-S, n=31) and small airway epithelium (SAE; 10th-12th order bronchi) of healthy nonsmokers (SAE-NS, n=63), healthy smokers (SAE-S, n=73) and smokers with COPD (SAE-COPD, n=37) were obtained by bronchoscopic brushings and analyzed using Affymetrix HG-U133 Plus 2.0 arrays. LAE/SAE common airway epithelium-expressed genes (“AE genes”) were defined as a set of genes expressed in at least 20% of any of the LAE/SAE groups. Lung cancer samples (adenocarcinoma, adenoCa, n=45; squamous cell carcinoma, SqCa, n=27; large cell carcinoma, LCC, n=19) and adjacent nonmalignant lung tissue (n=65) were from the published data set of Hou et al (PLoS ONE 2010; 5:e10312). “Cancerization” genes were identified by comparison of expression of the AE genes in lung cancer samples vs nonmalignant adjacent tissue (fold-increase &amp;gt;2, p&amp;lt;0.01 with Benjamini-Hochberg correction). A “cancerization index” (CI) was calculated for each LAE/SAE sample as a number of cancerization genes having expression level &amp;gt; mean ± 2 standard deviations of healthy nonsmokers. Results: Gene expression analysis identified 641 AE genes up-regulated in lung cancer vs nonmalignant adjacent tissue. This “cancerization” gene set was enriched in biological processes related to cell cycle (M phase, p&amp;lt;10−30) and p53 pathway (p&amp;lt;10−5). Progressive up-regulation of these genes in the tumor tissue was confirmed using 50 lung cancer samples of the original cohort having matched adjacent nonmalignant tissue samples (p&amp;lt;0.001). The top 10 cancerization genes were MMP12, SPP1, GREM1, KRT6A, TOP2A, ANLN, MMP1, AKR1B10, DLGAP5, and GPX2 (p values 10−12–10−34). Notably, 20% of the identified cancerization genes overlapped with our previously described signature of human airway basal progenitor cells (Hackett et al, PLoS ONE 2011;6:e18378). In the LAE-S, there was significantly higher overall expression of the cancerization genes vs LAE-NS (p&amp;lt;0.001), whereas in the SAE, only SAE-COPD, but not SAE-S, demonstrated significantly higher overall expression of these genes (p&amp;lt;0.003 vs SAE-NS, p&amp;lt;0.03 vs SAE-S). Among 23 healthy smokers with paired LAE/SAE samples, 14 had high CI in the LAE only, 1 in the SAE only, and 2 in both regions. Conclusion: Smoking is associated with acquisition of the molecular features of cancerization in the LAE, which likely precedes that in the SAE, where the molecular cancerization has a stronger association with the development of COPD. These molecular cancerization features in the airway epithelium of subjects without cancer are relevant to understanding the early pathogenesis of lung cancer and for the development of strategies to prevent lung cancer in susceptible individuals.