X-Box–Binding Protein 1 and Innate Immune Responses of Human Cystic Fibrosis Alveolar Macrophages

Rationale: Alveolar macrophages (AMs) play a key role in host defense to inhaled bacterial pathogens, in part by secreting inflammatory mediators. Cystic fibrosis (CF) airways exhibit a persistent, robust inflammatory response that may contribute to the pathophysiology of CF. Recent findings have linked endoplasmic reticulum stress responses mediated by inositol-requiring enzyme 1α–dependent messenger RNA splicing (activation) of X-box–binding protein-1 (XBP-1s) to inflammation in peripheral macrophages. However, the role of XBP-1s in CF AM function is not known

Limited effects of Muc1 deficiency on mouse adenovirus type 1 respiratory infection

Muc1 (MUC1 in humans) is a membrane-tethered mucin that exerts anti-inflammatory effects in the lung during bacterial infection. Muc1 and other mucins are also likely to form a protective barrier in the lung. We used mouse adenovirus type 1 (MAV-1, also known as MAdV-1) to determine the role of Muc1 in the pathogenesis of an adenovirus in its natural host. Following intranasal inoculation of wild type mice, we detected increased TNF-α, a cytokine linked to Muc1 production, but no consistent changes in the production of lung Muc1, Muc5ac or overall lung mucus production. Viral loads were modestly higher in the lungs of Muc1−/− mice compared to Muc1+/+ mice at several early time points but decreased to similar levels by 14 days post infection in both groups. However, cellular inflammation and the expression of CXCL1, CCL5, and CCL2 did not significantly differ between Muc1−/− and Muc1+/+ mice. Our data therefore suggest that Muc1 may contribute to a physical barrier that protects against MAV-1 respiratory infection. However, our data do not reveal an anti-inflammatory effect of Muc1 that contributes to MAV-1 pathogenesis.

Gene expression in whole lung and pulmonary macrophages reflects the dynamic pathology associated with airway surface dehydration

Abstract Background Defects in airway mucosal defense, including decreased mucus clearance, contribute to the pathogenesis of human chronic obstructive pulmonary diseases. Scnn1b-Tg mice, which exhibit chronic airway surface dehydration from birth, can be used as a model to study the pathogenesis of muco-obstructive lung disease across developmental stages. To identify molecular signatures associated with obstructive lung disease in this model, gene expression analyses were performed on whole lung and purified lung macrophages collected from Scnn1b-Tg and wild-type (WT) littermates at four pathologically relevant time points. Macrophage gene expression at 6 weeks was evaluated in mice from a germ-free environment to understand the contribution of microbes to disease development. Results Development- and disease-specific shifts in gene expression related to Scnn1b over-expression were revealed in longitudinal analyses. While the total number of transgene-related differentially expressed genes producing robust signals was relatively small in whole lung (n = 84), Gene Set Enrichment Analysis (GSEA) revealed significantly perturbed biological pathways and interactions between normal lung development and disease initiation/progression. Purified lung macrophages from Scnn1b-Tg mice exhibited numerous robust and dynamic gene expression changes. The expression levels of Classically-activated (M1) macrophage signatures were significantly altered at post-natal day (PND) 3 when Scnn1b-Tg mice lung exhibit spontaneous bacterial infections, while alternatively-activated (M2) macrophage signatures were more prominent by PND 42, producing a mixed M1-M2 activation profile. While differentially-regulated, inflammation-related genes were consistently identified in both tissues in Scnn1b-Tg mice, there was little overlap between tissues or across time, highlighting time- and tissue-specific responses. Macrophages purified from adult germ-free Scnn1b-Tg mice exhibited signatures remarkably similar to non-germ-free counterparts, indicating that the late-phase macrophage activation profile was not microbe-dependent. Conclusions Whole lung and pulmonary macrophages respond independently and dynamically to local stresses associated with airway mucus stasis. Disease-specific responses interact with normal developmental processes, influencing the final state of disease in this model. The robust signatures observed in Scnn1b-Tg lung macrophages highlight their critical role in disease pathogenesis. These studies emphasize the importance of region-, cell-type-, and time-dependent analyses to fully dissect the natural history of disease and the consequences of disease on normal lung development

Characterization of Rat Meibomian Gland Ion and Fluid Transport

We establish novel primary rat meibomian gland (MG) cell culture systems and explore the ion transport activities of the rat MG

The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes

Rationale: Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous disease. In COPD, the presence of emphysema is associated with increased mortality and risk of lung cancer. High resolution computed tomography (HRCT) scans are useful in quantifying emphysema but are associated with radiation exposure and high incidence of false positive findings (i.e., nodules). Using a comprehensive biomarker panel, we sought to determine if there was a peripheral blood biomarker signature of emphysema. Methods: 114 plasma biomarkers were measured using a custom assay in 588 individuals enrolled in the COPDGene study. Quantitative emphysema measurements included percent low lung attenuation (%LAA) ≤ −950 HU, ≤ − 910 HU and mean lung attenuation at the 15th percentile on lung attenuation curve (LP15A). Multiple regression analysis was performed to determine plasma biomarkers associated with emphysema independent of covariates age, gender, smoking status, body mass index and FEV1. The findings were subsequently validated using baseline blood samples from a separate cohort of 388 subjects enrolled in the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study. Results: Regression analysis identified multiple biomarkers associated with CT-assessed emphysema in COPDGene, including advanced glycosylation end-products receptor (AGER or RAGE, p < 0.001), intercellular adhesion molecule 1 (ICAM, p < 0.001), and chemokine ligand 20 (CCL20, p < 0.001). Validation in the TESRA cohort revealed significant associations with RAGE, ICAM1, and CCL20 with radiologic emphysema (p < 0.001 after meta-analysis). Other biomarkers that were associated with emphysema include CDH1, CDH 13 and SERPINA7, but were not available for validation in the TESRA study. Receiver operating characteristics analysis demonstrated a benefit of adding a biomarker panel to clinical covariates for detecting emphysema, especially in those without severe airflow limitation (AUC 0.85). Conclusions: Our findings, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema, and when combined with clinical covariates, may be useful clinically in predicting the presence of emphysema compared to just using covariates alone, especially in those with less severe COPD. Ultimately biomarkers may shed light on disease pathogenesis, providing targets for new treatments. Electronic supplementary material The online version of this article (doi:10.1186/s12931-014-0127-9) contains supplementary material, which is available to authorized users

Breaking the In Vitro Alveolar Type II Cell Proliferation Barrier while Retaining Ion Transport Properties

Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative “ATI-like” cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a “conditionally reprogrammed cell” phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and “ATI-like” mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air–liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases

The association of plasma biomarkers with computed tomography-assessed emphysema phenotypes

Abstract Rationale Chronic obstructive pulmonary disease (COPD) is a phenotypically heterogeneous disease. In COPD, the presence of emphysema is associated with increased mortality and risk of lung cancer. High resolution computed tomography (HRCT) scans are useful in quantifying emphysema but are associated with radiation exposure and high incidence of false positive findings (i.e., nodules). Using a comprehensive biomarker panel, we sought to determine if there was a peripheral blood biomarker signature of emphysema. Methods 114 plasma biomarkers were measured using a custom assay in 588 individuals enrolled in the COPDGene study. Quantitative emphysema measurements included percent low lung attenuation (%LAA)≤ - 950 HU, ≤ -910 HU and mean lung attenuation at the 15th percentile on lung attenuation curve (LP15A). Multiple regression analysis was performed to determine plasma biomarkers associated with emphysema independent of covariates age, gender, smoking status, body mass index and FEV1. The findings were subsequently validated using baseline blood samples from a separate cohort of 388 subjects enrolled in the Treatment of Emphysema with a Selective Retinoid Agonist (TESRA) study. Results Regression analysis identified multiple biomarkers associated with CT-assessed emphysema in COPDGene, including advanced glycosylation end-products receptor (AGER or RAGE, p < 0.001), intercellular adhesion molecule 1 (ICAM, p < 0.001), and chemokine ligand 20 (CCL20, p < 0.001). Validation in the TESRA cohort revealed significant associations with RAGE, ICAM1, and CCL20 with radiologic emphysema (p < 0.001 after meta-analysis). Other biomarkers that were associated with emphysema include CDH1, CDH 13 and SERPINA7, but were not available for validation in the TESRA study. Receiver operating characteristics analysis demonstrated a benefit of adding a biomarker panel to clinical covariates for detecting emphysema, especially in those without severe airflow limitation (AUC 0.85). Conclusions Our findings, suggest that a panel of blood biomarkers including sRAGE, ICAM1 and CCL20 may serve as a useful surrogate measure of emphysema, and when combined with clinical covariates, may be useful clinically in predicting the presence of emphysema compared to just using covariates alone, especially in those with less severe COPD. Ultimately biomarkers may shed light on disease pathogenesis, providing targets for new treatments.http://deepblue.lib.umich.edu/bitstream/2027.42/134591/1/12931_2014_Article_127.pd

Lower serum IgA is associated with COPD exacerbation risk in SPIROMICS

Background Decreased but measurable serum IgA levels (≤70 mg/dL) have been associated with risk for infections in some populations, but are unstudied in COPD. This study tested the hypothesis that subnormal serum IgA levels would be associated with exacerbation risk in COPD. Methods Data were analyzed from 1,049 COPD participants from the observational cohort study SPIROMICS (535 (51%) women; mean age 66.1 (SD 7.8), 338 (32%) current smokers) who had baseline serum IgA measured using the Myriad RBM biomarker discovery platform. Exacerbation data was collected prospectively (mean 944.3 (SD 281.3) days), and adjusted linear, logistic and zero-inflated negative binomial regressions were performed. Results Mean IgA was 269.1 mg/dL (SD 150.9). One individual had deficient levels of serum IgA (<7 mg/dL) and 25 (2.4%) had IgA level ≤70 mg/dL. Participants with IgA ≤70 mg/dL were younger (62 vs. 66 years, p = 0.01) but otherwise similar to those with higher IgA. In adjusted models, IgA ≤70 mg/dL was associated with higher exacerbation incidence rates (IRR 1.71, 95% CI 1.01–2.87, p = 0.044) and greater risk for any severe exacerbation (OR 2.99, 95% CI 1.30–6.94, p = 0.010). In adjusted models among those in the lowest decile (<120 mg/dL), each 10 mg/dL decrement in IgA (analyzed continuously) was associated with more exacerbations during follow-up (β 0.24, 95% CI 0.017–0.46, p = 0.035). Conclusions Subnormal serum IgA levels were associated with increased risk for acute exacerbations, supporting mildly impaired IgA levels as a contributing factor in COPD morbidity. Additionally, a dose-response relationship between lower serum IgA and number of exacerbations was found among individuals with serum IgA in the lowest decile, further supporting the link between serum IgA and exacerbation risk. Future COPD studies should more comprehensively characterize immune status to define the clinical relevance of these findings and their potential for therapeutic correction

Design of a multi-center immunophenotyping analysis of peripheral blood, sputum and bronchoalveolar lavage fluid in the Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS)

Background Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument. Methods The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data. Results Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel. Conclusions Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters. Trial registration This study was registered with ClinicalTrials.gov as NCT01969344

Variability in objective and subjective measures affects baseline values in studies of patients with COPD

Rationale: Understanding the reliability and repeatability of clinical measurements used in the diagnosis, treatment and monitoring of disease progression is of critical importance across all disciplines of clinical practice and in clinical trials to assess therapeutic efficacy and safety. Objectives: Our goal is to understand normal variability for assessing true changes in health status and to more accurately utilize this data to differentiate disease characteristics and outcomes. Methods: Our study is the first study designed entirely to establish the repeatability of a large number of instruments utilized for the clinical assessment of COPD in the same subjects over the same period. We utilized SPIROMICS participants (n = 98) that returned to their clinical center within 6 weeks of their baseline visit to repeat complete baseline assessments. Demographics, spirometry, questionnaires, complete blood cell counts (CBC), medical history, and emphysema status by computerized tomography (CT) imaging were obtained. Results: Pulmonary function tests (PFTs) were highly repeatable (ICC’s >0.9) but the 6 minute walk (6MW) was less so (ICC = 0.79). Among questionnaires, the Saint George’s Respiratory Questionnaire (SGRQ) was most repeatable. Self-reported clinical features, such as exacerbation history, and features of chronic bronchitis, often produced kappa values <0.6. Reported age at starting smoking and average number of cigarettes smoked were modestly repeatable (kappa = 0.76 and 0.79). Complete blood counts (CBC) variables produced intraclass correlation coefficients (ICC) values between 0.6 and 0.8. Conclusions: PFTs were highly repeatable, while subjective measures and subject recall were more variable. Analyses using features with poor repeatability could lead to misclassification and outcome errors. Hence, care should be taken when interpreting change in clinical features based on measures with low repeatability. Efforts to improve repeatability of key clinical features such as exacerbation history and chronic bronchitis are warranted