Intermittent Cough and Hemoptysis With Tree-in-Bud Opacities on Imaging.

CASE PRESENTATION: A 44-year-old man with hyperthyroidism and no smoking history presented to his internist with 5 months of intermittent cough and hemoptysis. The patient's family history was remarkable only for non-Hodgkin's lymphoma in his father. He had a history of a 25-day exposure to a home renovation at work 2 years prior to presentation. He was treated with oral clarithromycin with no improvement in his symptoms. A chest radiograph showed bilateral nodular opacities with a left lower lobar consolidative opacity (Fig 1A, 1B); the patient underwent CT scanning of the chest, which showed areas of nodular infiltration in the lower lobes with tree-in-bud-like opacities. He was referred to a pulmonologist.

Persistence of circulating endothelial microparticles in COPD despite smoking cessation.

INTRODUCTION: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. METHODS: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry. RESULTS: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. CONCLUSIONS: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. TRIAL REGISTRATION NUMBER: NCT00974064; NCT01776398.

Persistence of smoking-induced dysregulation of miRNA expression in the small airway epithelium despite smoking cessation.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/ß-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

Cilia dysfunction in lung disease.

A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes composed of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, to repeated chest infections, and to the progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease.

Smoking accelerates aging of the small airway epithelium.

BACKGROUND: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking. METHODS: Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function. RESULTS: Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers. CONCLUSION: These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

FOXJ1 prevents cilia growth inhibition by cigarette smoke in human airway epithelium in vitro.

Airway epithelium ciliated cells play a central role in clearing the lung of inhaled pathogens and xenobiotics, and cilia length and coordinated beating are important for airway clearance. Based on in vivo studies showing that the airway epithelium of healthy smokers has shorter cilia than that of healthy nonsmokers, we investigated the mechanisms involved in cigarette smoke-mediated inhibition of ciliogenesis by assessing normal human airway basal cell differentiation in air-liquid interface (ALI) cultures in the presence of nontoxic concentrations of cigarette smoke extract (CSE). Measurements of cilia length from Day 28 ALI cultures demonstrated that CSE exposure was associated with shorter cilia (P < 0.05), reproducing the effect of cigarette smoking on cilia length observed in vivo. This phenotype correlated with a broad CSE-mediated suppression of genes involved in cilia-related transcriptional regulation, intraflagellar transport, cilia motility, structural integrity, and basal body development but not of control genes or epithelial barrier integrity. The CSE-mediated inhibition of cilia growth could be prevented by lentivirus-mediated overexpression of FOXJ1, the major cilia-related transcription factor, which led to partial reversal of expression of cilia-related genes suppressed by CSE. Together, the data suggest that components of cigarette smoke are responsible for a broad suppression of genes involved in cilia growth, but, by stimulating ciliogenesis with the transcription factor FOXJ1, it may be possible to maintain close to normal cilia length despite the stress of cigarette smoking.

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.

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

BACKGROUND: Mucus 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. METHODS: Flexible 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. RESULTS: MUC5AC 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. CONCLUSION: The 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 SUMMARY: Mucus 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.

Biologic phenotyping of the human small airway epithelial response to cigarette smoking.

BACKGROUND: The first changes associated with smoking are in the small airway epithelium (SAE). Given that smoking alters SAE gene expression, but only a fraction of smokers develop chronic obstructive pulmonary disease (COPD), we hypothesized that assessment of SAE genome-wide gene expression would permit biologic phenotyping of the smoking response, and that a subset of healthy smokers would have a "COPD-like" SAE transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: SAE (10th-12th generation) was obtained via bronchoscopy of healthy nonsmokers, healthy smokers and COPD smokers and microarray analysis was used to identify differentially expressed genes. Individual responsiveness to smoking was quantified with an index representing the % of smoking-responsive genes abnormally expressed (I(SAE)), with healthy smokers grouped into "high" and "low" responders based on the proportion of smoking-responsive genes up- or down-regulated in each smoker. Smokers demonstrated significant variability in SAE transcriptome with I(SAE) ranging from 2.9 to 51.5%. While the SAE transcriptome of "low" responder healthy smokers differed from both "high" responders and smokers with COPD, the transcriptome of the "high" responder healthy smokers was indistinguishable from COPD smokers. CONCLUSION/SIGNIFICANCE: The SAE transcriptome can be used to classify clinically healthy smokers into subgroups with lesser and greater responses to cigarette smoking, even though these subgroups are indistinguishable by clinical criteria. This identifies a group of smokers with a "COPD-like" SAE transcriptome.

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

Do airway epithelium air-liquid cultures represent the in vivo airway epithelium transcriptome?

Human airway epithelial cells cultured in vitro at the air-liquid interface (ALI) form a pseudostratified epithelium that forms tight junctions and cilia, and produces mucin. These cells are widely used in models of differentiation, injury, and repair. To assess how closely the transcriptome of ALI epithelium matches that of in vivo airway epithelial cells, we used microarrays to compare the transcriptome of human large airway epithelial cells cultured at the ALI with the transcriptome of large airway epithelium obtained via bronchoscopy and brushing. Gene expression profiling showed that global gene expression correlated well between ALI cells and brushed cells, but with some differences. Gene expression patterns mirrored differences in proportions of cell types (ALIs have higher percentages of basal cells, whereas brushed cells have higher percentages of ciliated cells), that is, ALI cells expressed higher levels of basal cell-related genes, and brushed cells expressed higher levels of cilia-related genes. Pathway analysis showed that ALI cells had increased expression of cell cycle and proliferation genes, whereas brushed cells had increased expression of cytoskeletal organization and humoral immune response genes. Overall, ALI cells provide a good representation of the in vivo airway epithelial transcriptome, but for some biologic questions, the differences between in vitro and in vivo environments need to be considered.

Threshold of biologic responses of the small airway epithelium to low levels of tobacco smoke.

RATIONALE: Epidemiologic data demonstrate that individuals exposed to low levels of tobacco smoke have decrements in lung function and higher risk for lung disease compared with unexposed individuals. Although this risk is small, low-level tobacco smoke exposure is so widespread, it is a significant public health concern. OBJECTIVES: To identify biologic correlates of this risk we hypothesized that, compared with unexposed individuals, individuals exposed to low levels of tobacco smoke have biologic changes in the small airway epithelium, the site of the first abnormalities associated with smoking. METHODS: Small airway epithelium was obtained by bronchoscopy from 121 individuals; microarrays were used to assess genome-wide gene expression; urine nicotine and cotinine were used to categorize subjects as "nonsmokers," "active smokers," and "low exposure." Gene expression data were used to determine the threshold and induction half maximal level (ID50) of urine nicotine and cotinine at which the small airway epithelium showed abnormal responses. MEASUREMENTS AND MAIN RESULTS: There was no threshold of urine nicotine without a small airway epithelial response, and only slightly above detectable urine cotinine threshold with a small airway epithelium response. The ID50 for nicotine was 25 ng/ml and for cotinine it was 104 ng/ml. CONCLUSIONS: The small airway epithelium detects and responds to low levels of tobacco smoke with transcriptome modifications. This provides biologic correlates of epidemiologic studies linking low-level tobacco smoke exposure to lung health risk, identifies the genes most sensitive to tobacco smoke, and defines thresholds at which the lung epithelium responds to low levels of tobacco smoke.

Smoking is associated with shortened airway cilia.

BACKGROUND: Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6-7 microm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers. METHODOLOGY/PRINCIPAL FINDINGS: Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers. CONCLUSIONS/SIGNIFICANCE: Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.

Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.

RATIONALE: The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. OBJECTIVES: We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). METHODS: We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. CONCLUSIONS: These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

Trachea epithelium as a "canary" for cigarette smoking-induced biologic phenotype of the small airway epithelium.

The initial site of smoking-induced lung disease is the small airway epithelium, which is difficult and time consuming to sample by fiberoptic bronchoscopy. We developed a rapid, office-based procedure to obtain trachea epithelium without conscious sedation from healthy nonsmokers (n= 26) and healthy smokers (n= 19, 27 +/- 15 pack-year). Gene expression differences (fold change >1.5, p < 0.01, Benjamini-Hochberg correction) were assessed with Affymetrix microarrays. A total of 1,057 probe sets were differentially expressed in healthy smokers versus nonsmokers, representing >500 genes. Trachea gene expression was compared to an independent group of small airway epithelial samples (n= 23 healthy nonsmokers, n= 19 healthy smokers, 25 +/- 12 pack-year). The trachea epithelium is more sensitive to smoking, responding with threefold more differentially expressed genes than small airway epithelium. The trachea transcriptome paralleled the small airway epithelium, with 156 of 167 (93%) genes that are significantly up- and downregulated by smoking in the small airway epithelium showing similar direction and magnitude of response to smoking in the trachea. Trachea epithelium can be obtained without conscious sedation, representing a less invasive surrogate "canary" for smoking-induced changes in the small airway epithelium. This should prove useful in epidemiologic studies correlating gene expression with clinical outcome in assessing smoking-induced lung disease.