BAL Cell Gene Expression in Severe Asthma Reveals Mechanisms of Severe Disease and Influences of Medications.

Rationale: Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.Objectives: To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression.Methods: BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.Measurements and Main Results: Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an in vitro cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and ß-agonist-naive subjects given a ß-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.Conclusions: Gene expression in BAL cells is influenced by factors seldomly considered. Notably, ß-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.

Loss of Twist1 in the Mesenchymal Compartment Promotes Increased Fibrosis in Experimental Lung Injury by Enhanced Expression of CXCL12.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.

Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease.

RATIONALE: Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. OBJECTIVES: Identify networks of genes reflective of underlying biological processes that define SA. METHODS: Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. MEASUREMENTS AND MAIN RESULTS: Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. CONCLUSIONS: In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.

Expression of RXFP1 Is Decreased in Idiopathic Pulmonary Fibrosis. Implications for Relaxin-based Therapies.

RATIONALE: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. OBJECTIVES: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. METHODS: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. MEASUREMENTS AND MAIN RESULTS: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-ß decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. CONCLUSIONS: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.

Gene expression in relation to exhaled nitric oxide identifies novel asthma phenotypes with unique biomolecular pathways.

RATIONALE: Although asthma is recognized as a heterogeneous disease associated with clinical phenotypes, the molecular basis of these phenotypes remains poorly understood. Although genomic studies have successfully broadened our understanding in diseases such as cancer, they have not been widely used in asthma studies. OBJECTIVES: To link gene expression patterns to clinical asthma phenotypes. METHODS: We used a microarray platform to analyze bronchial airway epithelial cell gene expression in relation to the asthma biomarker fractional exhaled nitric oxide (FeNO) in 155 subjects with asthma and healthy control subjects from the Severe Asthma Research Program (SARP). MEASUREMENTS AND MAIN RESULTS: We first identified a diverse set of 549 genes whose expression correlated with FeNO. We used k-means to cluster the patient samples according to the expression of these genes, identifying five asthma clusters/phenotypes with distinct clinical, physiological, cellular, and gene transcription characteristics-termed "subject clusters" (SCs). To then investigate differences in gene expression between SCs, a total of 1,384 genes were identified that highly differentiated the SCs at an unadjusted P value < 10(-6). Hierarchical clustering of these 1,384 genes identified nine gene clusters or "biclusters," whose coexpression suggested biological characteristics unique to each SC. Although genes related to type 2 inflammation were present, novel pathways, including those related to neuronal function, WNT pathways, and actin cytoskeleton, were noted. CONCLUSIONS: These findings show that bronchial epithelial cell gene expression, as related to the asthma biomarker FeNO, can identify distinct asthma phenotypes, while also suggesting the presence of underlying novel gene pathways relevant to these phenotypes.

C-X-C motif chemokine 13 (CXCL13) is a prognostic biomarker of idiopathic pulmonary fibrosis.

RATIONALE: C-X-C motif chemokine 13 (CXCL13) mediates B-cell trafficking and is increased, proportionately to disease activity, in many antibody-mediated syndromes. Dysregulated B cells have recently been implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis. OBJECTIVES: To determine if CXCL13 is associated with IPF progression. METHODS: CXCL13 was measured in lungs by DNA microarray and immunohistochemistry, and in plasma by ELISA. MEASUREMENTS AND MAIN RESULTS: CXCL13 mRNA was threefold and eightfold greater in IPF lungs (n = 92) compared with chronic obstructive pulmonary disease (COPD) (n = 191) and normal (n = 108) specimens, respectively (P < 0.0001). IPF lungs also showed increased CXCL13 staining. Plasma CXCL13 concentrations (pg/ml) were greater in 95 patients with IPF (94 ± 8) than in 128 subjects with COPD (53 ± 9) and 57 normal subjects (35 ± 3) (P < 0.0001). Circulating CXCL13 levels were highest in patients with IPF with pulmonary artery hypertension (P = 0.01) or acute exacerbations (P = 0.002). Six-month survival of patients with IPF in the highest quartile of plasma CXCL13 was 65 ± 10% versus 93 ± 10% in the others (hazard ratio, 5.5; 95% confidence interval, 1.8-16.9; P = 0.0008). CXCL13 increases by more than 50% in IPF serial assays, irrespective of initial values, also presaged respiratory failure (hazard ratio, 7.2; 95% confidence interval, 1.3-40.0; P = 0.008). In contrast, CXCL13 clinical associations in subjects with COPD were limited to modest correlations with FEV1 (P = 0.05) and progression of radiographic emphysema (P = 0.05). CONCLUSIONS: CXCL13 is increased and is a prognostic biomarker in patients with IPF, and more so than in patients with COPD. This contrast indicates CXCL13 overexpressions are intrinsic to IPF, rather than an epiphenomenon of lung injury. The present data implicate CXCL13 and B cells in IPF pathogenesis, and support considerations for trials of specific B-cell-targeted therapies in patients with this intractable disease.

Relationship of DNA methylation and gene expression in idiopathic pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. OBJECTIVES: To identify methylation marks that modify gene expression in IPF lung. METHODS: We assessed DNA methylation (comprehensive high-throughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. MEASUREMENTS AND MAIN RESULTS: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 × 10(-16)). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 × 10(-16)). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. CONCLUSIONS: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.

Pneumocystis jirovecii colonization is associated with enhanced Th1 inflammatory gene expression in lungs of humans with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a complex disease, the pathogenesis of which remains incompletely understood. Colonization with Pneumocystis jirovecii may play a role in COPD pathogenesis; however, the mechanisms by which such colonization contributes to COPD are unknown. The objective of this study was to determine lung gene expression profiles associated with Pneumocystis colonization in patients with COPD to identify potential key pathways involved in disease pathogenesis. Using COPD lung tissue samples made available through the Lung Tissue Research Consortium (LTRC), Pneumocystis colonization status was determined by nested PCR. Microarray gene expression profiles were performed for each sample and the profiles of colonized and non-colonized samples compared. Overall, 18 participants (8.5%) were Pneumocystis-colonized. Pneumocystis colonization was associated with fold increase in expression of four closely related genes: INF-γ and the three chemokine ligands CXCL9, CXCL10, and CXCL11. These ligands are chemoattractants for the common cognate receptor CXCR3, which is predominantly expressed on activated Th1 T-lymphocytes. Although these ligand-receptor pairs have previously been implicated in COPD pathogenesis, few initiators of ligand expression and subsequent lymphocyte trafficking have been identified: our findings implicate Pneumocystis as a potential trigger. The finding of upregulation of these inflammatory genes in the setting of Pneumocystis colonization sheds light on infectious-immune relationships in COPD.

Assessment of lung volume collapsibility in chronic obstructive lung disease patients using CT.

OBJECTIVE: To investigate the collapsibility of the lung and individual lobes in patients with COPD during inspiration/expiration and assess the association of whole lung and lobar volume changes with pulmonary function tests (PFTs) and disease severity. METHODS: PFT measures used were RV/TLC%, FEV1% predicted, FVC, FEV1/FVC%, DLco% predicted and GOLD category. A total of 360 paired inspiratory and expiratory CT examinations acquired in 180 subjects were analysed. Automated computerised algorithms were used to compute individual lobe and total lung volumes. Lung volume collapsibility was assessed quantitatively using the simple difference between CT computed inspiration (I) and expiration (E) volumes (I-E), and a relative measure of volume changes, (I-E)/I. RESULTS: Mean absolute collapsibility (I-E) decreased in all lung lobes with increasing disease severity defined by GOLD classification. Relative collapsibility (I-E)/I showed a similar trend. Upper lobes had lower volume collapsibility across all GOLD categories and lower lobes collectively had the largest volume collapsibility. Whole lung and left lower lobe collapsibility measures tended to have the highest correlations with PFT measures. Collapsibility of lung lobes and whole lung was also negatively correlated with the degree of air trapping between expiration and inspiration, as measured by mean lung density. All measured associations were statistically significant (P < 0.01). CONCLUSION: Severity of COPD appears associated with increased collapsibility in the upper lobes, but change (decline) in collapsibility is faster in the lower lobes. KEY POINTS: • Inspiratory and expiratory computed tomography allows assessment of lung collapsibility • Lobe volume collapsibility is significantly correlated with measures of lung function. • As COPD severity increases, collapsibility of individual lung lobes decreases. • Upper lobes exhibit more severe disease, while lower lobes decline faster.

Optimal threshold in CT quantification of emphysema.

OBJECTIVES: To determine the optimal threshold by quantitatively assessing the extent of emphysema at the level of the entire lung and at the level of individual lobes using a large, diverse dataset of computed tomography (CT) examinations. METHODS: This study comprises 573 chest CT examinations acquired from subjects with different levels of airway obstruction (222 none, 83 mild, 141 moderate, 63 severe and 64 very severe). The extent of emphysema was quantified using the percentage of the low attenuation area (LAA%) divided by the total lung or lobe volume(s). The correlations between the extent of emphysema, and pulmonary functions and the five-category classification were assessed using Pearson and Spearman's correlation coefficients, respectively. When quantifying emphysema using a density mask, a wide range of thresholds from -850 to -1,000 HU were used. RESULTS: The highest correlations of LAA% with the five-category classification and PFT measures ranged from -925 to -965 HU for each individual lobe and the entire lung. However, the differences between the highest correlations and those obtained at -950 HU are relatively small. CONCLUSION: Although there are variations in the optimal cut-off thresholds for individual lobes, the single threshold of -950 HU is still an acceptable threshold for density-based emphysema quantification.

VCAM-1 is a TGF-ß1 inducible gene upregulated in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease of unknown etiology. We previously reported that high plasma levels of vascular cell adhesion molecule 1 (VCAM-1) predict mortality in IPF subjects. Here we investigated the cellular origin and potential role of VCAM-1 in regulating primary lung fibroblast behavior. VCAM-1 mRNA was significantly increased in lungs of subjects with IPF compared to lungs from control subjects (p=0.001), and it negatively correlated with two markers of lung function, forced vital capacity (FVC) and pulmonary diffusion capacity for carbon monoxide (DLCO). VCAM-1 protein levels were highly expressed in IPF subjects where it was detected in fibrotic foci and blood vessels of IPF lung. Treatment of human lung fibroblasts with TGF-ß1 significantly increased steady-state VCAM1 mRNA and protein levels without affecting VCAM1 mRNA stability. Further, cellular depletion of VCAM-1 inhibited fibroblast cell proliferation and reduced G2/M and S phases of the cell cycle suggestive of cell cycle arrest. These effects on cell cycle progression triggered by VCAM1 depletion were associated with reductions in levels of phosphorylated extracellular regulated kinase 1/2 and cyclin D1. Thus, these observations suggest that VCAM-1 is a TGF-ß1 responsive mediator that partakes in fibroblast proliferation in subjects with IPF.

Suppression of NLRX1 in chronic obstructive pulmonary disease.

Cigarette smoke (CS) and viruses promote the inflammation and remodeling associated with chronic obstructive pulmonary disease (COPD). The MAVS/RIG-I-like helicase (MAVS/RLH) pathway and inflammasome-dependent innate immune pathways are important mediators of these responses. At baseline, the MAVS/RLH pathway is suppressed, and this inhibition must be reversed to engender tissue effects; however, the mechanisms that mediate activation and repression of the pathway have not been defined. In addition, the regulation and contribution of MAVS/RLH signaling in CS-induced inflammation and remodeling responses and in the development of human COPD remain unaddressed. Here, we demonstrate that expression of NLRX1, which inhibits the MAVS/RLH pathway and regulates other innate immune responses, was markedly decreased in 3 independent cohorts of COPD patients. NLRX1 suppression correlated directly with disease severity and inversely with pulmonary function, quality of life, and prognosis. In murine models, CS inhibited NLRX1, and CS-induced inflammation, alveolar destruction, protease induction, structural cell apoptosis, and inflammasome activation were augmented in NLRX1-deficient animals. Conversely, MAVS deficiency abrogated this CS-induced inflammation and remodeling. Restoration of NLRX1 in CS-exposed animals ameliorated alveolar destruction. These data support a model in which CS-dependent NLRX1 inhibition facilitates MAVS/RHL activation and subsequent inflammation, remodeling, protease, cell death, and inflammasome responses.

Failure of trabecular bone with simulated lytic defects can be predicted non-invasively by structural analysis.

Pathologic fracture is a significant risk for patients afflicted with metastatic or benign skeletal tumors. The quandary for physicians who treat these patients is that after making the diagnosis they must try to predict the load bearing capacity of the involved bone and the fracture risk from images seen in radiological examinations. Since bone fails at a relatively constant strain independent of density we demonstrate that using a mechanics of materials approach that the cross-sectional structural properties of the bone most affected by the lytic defect governs the load bearing capacity of the entire bone. Homogeneous cylindrical cores of trabecular bone were harvested from the vertebral bodies of whale spines, and prepared with circular or slotted through-hole defects of varying sizes to simulate lytic skeletal tumors. Each specimen was imaged using quantitative computed tomography (CT), dual energy X-ray absorptiometry (DXA), and magnetic resonance imaging (MRI) to obtain data for calculating cross-sectional structural properties: axial, flexural, and torsional rigidity. The specimens were then divided into groups uniformly distributed with respect to defect sizes and shapes, and subjected to uniaxial tension, four-point bending or torsion until failure. A strong positive relationship was found between measured tensile yield loads, bending, and torsional yield moments vs. axial, flexural and torsional structural rigidities respectively, calculated from QCT, DXA, and MRI data [QCT: tension r2=0.951 , bending r2=0.909, torsion r2=0.914; DXA: tension r2=0.926, bending r2=0.841, torsion r2=0.916 (p<0.001); MRI: tension r2=0.916; bending r2=0.856, torsion r2=0.852]. For cylindrical cores of trabecular bone with simulated lytic defects, the load bearing capacity of the entire core was directly proportional to the axial, bending, or torsional rigidity at the weakest cross-section through the core containing the defect. Therefore structural rigidity analysis of cross-sectional geometric data measured non-invasively by QCT, DXA, and MRI of bones containing lytic defects may be used to predict the load bearing capacity of the involved bone and the relative fracture risk in vivo.

Human adult bone marrow-derived stem cells decrease severity of lipopolysaccharide-induced acute respiratory distress syndrome in sheep.

INTRODUCTION: Acute respiratory distress syndrome (ARDS) is the most common cause of respiratory failure among critically ill subjects, sepsis and severe bacterial pneumonia being its most common causes. The only interventions that have proven beneficial are protective ventilation strategies and fluid conservation approaches. New therapies are needed to address this common clinical problem. Others and we have previously shown the beneficial effect of infusion of exogenous adult stem cells in different pre-clinical models of ARDS. METHODS: In the present study endotoxin was infused intravenously into 14 sheep from which 6 received different doses of adult stem cells by intrabronchial delivery to evaluate the effect of stem cell therapy. RESULTS: After administration of endotoxin, there was a rapid decline in oxygenation to hypoxemic values, indicative of severe-to-moderate ARDS. None of the animals treated with saline solution recovered to normal baseline values during the 6 hours that the animals were followed. In contrast, sheep treated with a dose of 40 million adult stem cells returned their levels of oxygen in their blood to baseline two hours after the cells were infused. Similarly, improvements in carbon dioxide (CO2) clearance, pulmonary vascular pressures and inflammation were observed and confirmed by histology and by the decrease in lung edema. CONCLUSIONS: We concluded that instillation of adult non-hematopoietic stem cells can diminish the impact of endotoxin and accelerate recovery of oxygenation, CO2 removal and inflammation in the ovine model, making the use of adult stem cells a real alternative for future therapies for ARDS.