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1.  A Role for Wnt Signaling Genes in the Pathogenesis of Impaired Lung Function in Asthma 
Rationale: Animal models demonstrate that aberrant gene expression in utero can result in abnormal pulmonary phenotypes.
Objectives: We sought to identify genes that are differentially expressed during in utero airway development and test the hypothesis that variants in these genes influence lung function in patients with asthma.
Methods: Stage 1 (Gene Expression): Differential gene expression analysis across the pseudoglandular (n = 27) and canalicular (n = 9) stages of human lung development was performed using regularized t tests with multiple comparison adjustments. Stage 2 (Genetic Association): Genetic association analyses of lung function (FEV1, FVC, and FEV1/FVC) for variants in five differentially expressed genes were conducted in 403 parent-child trios from the Childhood Asthma Management Program (CAMP). Associations were replicated in 583 parent-child trios from the Genetics of Asthma in Costa Rica study.
Measurements and Main Results: Of the 1,776 differentially expressed genes between the pseudoglandular (gestational age: 7–16 wk) and the canalicular (gestational age: 17–26 wk) stages, we selected 5 genes in the Wnt pathway for association testing. Thirteen single nucleotide polymorphisms in three genes demonstrated association with lung function in CAMP (P < 0.05), and associations for two of these genes were replicated in the Costa Ricans: Wnt1-inducible signaling pathway protein 1 with FEV1 (combined P = 0.0005) and FVC (combined P = 0.0004), and Wnt inhibitory factor 1 with FVC (combined P = 0.003) and FEV1/FVC (combined P = 0.003).
Conclusions: Wnt signaling genes are associated with impaired lung function in two childhood asthma cohorts. Furthermore, gene expression profiling of human fetal lung development can be used to identify genes implicated in the pathogenesis of lung function impairment in individuals with asthma.
PMCID: PMC2822972  PMID: 19926868
asthma; lung development; lung function; genetic variation; gene expression
2.  Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels 
PLoS ONE  2013;8(11):e80294.
Airway branching morphogenesis in utero is essential for optimal postnatal lung function. In the fetus, branching morphogenesis occurs during the pseudoglandular stage (weeks 9–17 of human gestation, embryonic days (E)11.5–16.5 in mouse) in a hypercalcaemic environment (∼1.7 in the fetus vs. ∼1.1–1.3 mM for an adult). Previously we have shown that fetal hypercalcemia exerts an inhibitory brake on branching morphogenesis via the calcium-sensing receptor. In addition, earlier studies have shown that nifedipine, a selective blocker of L-type voltage-gated Ca2+ channels (VGCC), inhibits fetal lung growth, suggesting a role for VGCC in lung development. The aim of this work was to investigate the expression of VGCC in the pseudoglandular human and mouse lung, and their role in branching morphogenesis. Expression of L-type (CaV1.2 and CaV1.3), P/Q type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3), and T-type (CaV3.2 and CaV3.3) VGCC was investigated in paraffin sections from week 9 human fetal lungs and E12.5 mouse embryos. Here we show, for the first time, that Cav1.2 and Cav1.3 are expressed in both the smooth muscle and epithelium of the developing human and mouse lung. Additionally, Cav2.3 was expressed in the lung epithelium of both species. Incubating E12.5 mouse lung rudiments in the presence of nifedipine doubled the amount of branching, an effect which was partly mimicked by the Cav2.3 inhibitor, SNX-482. Direct measurements of changes in epithelial cell membrane potential, using the voltage-sensitive fluorescent dye DiSBAC2(3), demonstrated that cyclic depolarisations occur within the developing epithelium and coincide with rhythmic occlusions of the lumen, driven by the naturally occurring airway peristalsis. We conclude that VGCC are expressed and functional in the fetal human and mouse lung, where they play a role in branching morphogenesis. Furthermore, rhythmic epithelial depolarisations evoked by airway peristalsis would allow for branching to match growth and distension within the developing lung.
PMCID: PMC3840017  PMID: 24282533
3.  The Role of Hyaluronan and Hyaluronan Binding Proteins in Human Asthma 
The characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan (HA), a major extracellular matrix component, accumulates during inflammatory lung diseases including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that HA and its receptors would play a role in human asthma.
To investigate the role of HA and HA binding proteins in human asthma.
Twenty-one subjects with asthma and 25 normal control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage (BAL). Fibroblasts were cultured, HA and HA synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of HA binding proteins, CD44, TLR2 and TLR4 on BAL macrophages was determined by flow cytometry. IL-8 production by macrophages in response to HA fragment stimulation was compared.
Airway fibroblasts from asthma patients produced significantly increased concentrations of lower molecular weight HA compared to those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from asthmatic subjects showed an increase in responsiveness to low molecular weight HA stimulation, as demonstrated by increased IL-8 production.
HA homeostasis is deranged in asthma with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to HA fragments suggests a novel pro-inflammatory mechanism by which persistence of HA fragments could contribute to chronic inflammation and airway remodeling in asthma.
PMCID: PMC3149736  PMID: 21570715
Asthma; Hyaluronan; Cytokines; Fibroblasts; Macrophages
4.  A rhesus monkey model to characterize the role of gastrin-releasing peptide (GRP) in lung development. Evidence for stimulation of airway growth. 
Journal of Clinical Investigation  1994;94(4):1605-1615.
Gastrin-releasing peptide (GRP) is developmentally expressed in human fetal lung and is a growth factor for normal and neoplastic lung but its role in normal lung development has yet to be clearly defined. In this study we have characterized the expression of GRP and its receptor in fetal rhesus monkey lung and determined the effects of bombesin on fetal lung development in vitro. By RNA blot analysis, GRP mRNA was first detectable in fetal monkey lung at 63 days gestation, reached highest levels at 80 days gestation, and then declined to near adult levels by 120 days gestation; a pattern closely paralleling GRP expression in human fetal lung. As in human lung, in situ hybridization localized GRP mRNA to neuroendocrine cells though during the canalicular phase of development (between 63-80 days gestation) GRP mRNA was present not only in classic pulmonary neuroendocrine cells, but also in cells of budding airways. Immunohistochemistry showed that bombesin-like immunoreactivity was present in neuroendocrine cells, but not in budding airways, suggesting that in budding airways either the GRP mRNA is not translated, is rapidly secreted, or a related, but different RNA is present. RNase protection analysis using a probe to the monkey GRP receptor demonstrated that the time course of receptor RNA expression closely paralleled the time course of GRP RNA expression. In situ hybridization showed that GRP receptors were primarily expressed in epithelial cells of the developing airways. Thus GRP would appear to be secreted from neuroendocrine cells to act on target cells in developing airways. This hypothesis was confirmed by organ culture of fetal monkey lung in the presence of bombesin and bombesin antagonists. Bombesin treatment at 1 and 10 nM significantly increased DNA synthesis in airway epithelial cells and significantly increased the number and size of airways in cultured fetal lung. In fact, culturing 60 d fetal lung for 5 d with 10 nM bombesin increased airway size and number nearly to that observed in cultured 80 d fetal lung. The effects of bombesin could be blocked by specific GRP receptor antagonists. Thus this study demonstrates that GRP receptors are expressed on airway epithelial cells in developing fetal lung and that the interaction of GRP with the GRP receptor stimulates airway development.
PMCID: PMC295320  PMID: 7929836
5.  Epithelial N-cadherin and nuclear β-catenin are up-regulated during early development of human lung 
The aim of this study was to analyze the cell-specific expression of E- and N-cadherin and β-catenin in developing human lung tissues from 12 to 40 weeks of gestation.
Fortyseven cases of developing human lung including pseudoglandular, canalicular, saccular and alveolar periods were analyzed by immunohistochemisty for E- and N-cadherin and β-catenin and twentyone cases were also investigated by RT-PCR for E- and N-cadherin and β-catenin. For identifying the lung cells, the sections were also stained with antibodies against thyroid transcription factor-1 (TTF-1) and caveolin-1. Normal adult lung tissue was used as a control.
E-cadherin was strongly expressed in epithelium of bronchi and large bronchioles from week 12 onwards and it was also positive in alveoli in pretype II cells and type II cells. N-cadherin was present in most of the epithelial cells of bronchi and the largest bronchioles during the pseudo-glandular and canalicular periods. N-cadherin was not detected in epithelium of developing alveoli. β-catenin was strongly membrane-bound and positively expressed in bronchial epithelium from week 12 to week 40; it showed nuclear positivity in both developing airway epithelium and in the cells underneath the epithelium during pseudo-glandular period and to a lesser degree also in the canalicular period. β-catenin was positive in pretype II cells as well as in type I and type II pneumocytes within alveoli.
RT-PCR analyses revealed detectable amounts of RNAs of E- and N-cadherin and β-catenin in all cases studied. The amounts of RNAs were higher in early stages of gestation.
E-cadherin is widely expressed in bronchial and alveolar epithelial cells. N-cadherin exhibit extensive epithelial positivity in bronchial epithelial cells during early lung development. The presence of β-catenin was observed in several cell types with a distinct location in tissue and cells in various gestational stages, indicating that it possesses several roles during lung development. The expressions of protein and mRNAs of E- and N-cadherin and β-catenin were higher in early gestation compared to of the end. Moreover, the expressions of these factors were higher during the lung development than in the adult human lung.
PMCID: PMC2995473  PMID: 21080917
6.  Significance of the microbiome in obstructive lung disease 
Thorax  2012;67(5):456-463.
The composition of the lung microbiome contributes to both health and disease, including obstructive lung disease. Because it has been estimated that over 70% of the bacterial species on body surfaces cannot be cultured by currently available techniques, traditional culture techniques are no longer the gold standard for microbial investigation. Advanced techniques that identify bacterial sequences, including the 16S ribosomal RNA gene, have provided new insights into the depth and breadth of microbiota present both in the diseased and normal lung. In asthma, the composition of the microbiome of the lung and gut during early childhood development may play a key role in the development of asthma, while specific airway microbiota are associated with chronic asthma in adults. Early bacterial stimulation appears to reduce asthma susceptibility by helping the immune system develop lifelong tolerance to innocuous antigens. By contrast, perturbations in the microbiome from antibiotic use may increase the risk for asthma development. In chronic obstructive pulmonary disease, bacterial colonisation has been associated with a chronic bronchitic phenotype, increased risk of exacerbations, and accelerated loss of lung function. In cystic fibrosis, studies utilising culture-independent methods have identified associations between decreased bacterial community diversity and reduced lung function; colonisation with Pseudomonas aeruginosa has been associated with the presence of certain CFTR mutations. Genomic analysis of the lung microbiome is a young field, but has the potential to define the relationship between lung microbiome composition and disease course. Whether we can manipulate bacterial communities to improve clinical outcomes remains to be seen.
PMCID: PMC3578398  PMID: 22318161
7.  A genome-scale study of transcription factor expression in the branching mouse lung 
Mammalian lung development consists of a series of precisely choreographed events that drive the progression from simple lung buds to the elaborately branched organ that fulfills the vital function of gas exchange. Strict transcriptional control is essential for lung development. Among the large number of transcription factors encoded in the mouse genome, only a small portion of them are known to be expressed and function in the developing lung. Thus a systematic investigation of transcription factors expressed in the lung is warranted.
To enrich for genes that may be responsible for regional growth and patterning, we performed a screen using RNA in situ hybridization to identify genes that show restricted expression patterns in the embryonic lung. We focused on the pseudoglandular stage during which the lung undergoes branching morphogenesis, a cardinal event of lung development. Using a genome-scale probe set that represents over 90% of the transcription factors encoded in the mouse genome, we identified sixty-two transcription factor genes with localized expression in the epithelium, mesenchyme or both. Many of these genes have not been previously implicated in lung development.
Our findings provide new starting points for the elucidation of the transcriptional circuitry that controls lung development.
PMCID: PMC3529173  PMID: 22711520
lung; mouse; transcription factors; expression patterns; branching
8.  VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen 
Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.
PMCID: PMC4051183  PMID: 24519134
Congenital diaphragmatic hernia; VEGFR; Rat model; Nitrofen; Lung development
9.  Gene Expression Profiling in Human Asthma 
Asthma is a chronic inflammatory disease of the lungs, characterized by airway hyperreactivity, mucus hypersecretion, and airflow obstruction. Despite recent advances, the genetic regulation of asthma pathogenesis is still largely unknown. Gene expression profiling techniques are well suited to study complex diseases and hold substantial promise for identifying novel genes and pathways in asthma; however, relatively few studies have been completed in human asthma. The few studies that have been done have identified many novel candidate genes and pathways in asthma pathogenesis, including ALOX15 and serine proteinase inhibitors cathepsin C and G. The interpretation of results of these studies should be cautious, as limitations include small sample sizes and heterogeneity of study populations and tissues sampled. In the future, the promise of gene expression studies would be enhanced by the use of larger sample sizes and attempts to standardize phenotype, sample collection techniques, and analysis. As the field of expression profiling in asthma advances, we hope it will improve our understanding of critical questions about mechanisms involved in susceptibility to the disease, as well as help to personalize care by improving appropriate selection of patients for prevention and treatment strategies.
PMCID: PMC2647611  PMID: 17202289
airway; atopy; gene expression; inflammation; microarray
10.  Mechanisms of occupational asthma: Not all allergens are equal 
Asthma is a heterogeneous lung disorder characterized by airway obstruction, inflammation and eosinophil infiltration into the lung. Both genetics and environmental factors influence the expression of asthma, and not all asthma is the result of a specific immune response to allergen. Numerous asthma phenotypes have been described, including occupational asthma, and therapeutic strategies for asthma control are similar regardless of phenotype. We hypothesized that mechanistic pathways leading to asthma symptoms in the effector phase of the disorder differ with the inciting allergen. Since route of allergen exposure can influence mechanistic pathways, mice were sensitized by identical routes with a high molecular weight occupational allergen ovalbumin and a low molecular weight occupational allergen trimellitic anhydride (TMA). Different statistical methods with varying selection criteria resulted in identification of similar candidate genes. Array data are intended to provide candidate genes for hypothesis generation and further experimentation. Continued studies focused on genes showing minimal changes in the TMA-induced model but with clear up-regulation in the ovalbumin model. Two of these genes, arginase 1 and eotaxin 1 are the focus of continuing investigations in mouse models of asthma regarding differences in mechanistic pathways depending on the allergen. Microarray data from the ovalbumin and TMA model of asthma were also compared to previous data usingAspergillus as allergen to identify putative asthma ‘signature genes’, i.e. genes up-regulated with all 3 allergens. Array studies provide candidate genes to identify common mechanistic pathways in the effector phase, as well as mechanistic pathways unique to individual allergens.
PMCID: PMC2723297  PMID: 21432060
trimellitic anhydride; asthma; microarray; eosinophils; arginase
11.  Lung eQTLs to Help Reveal the Molecular Underpinnings of Asthma 
PLoS Genetics  2012;8(11):e1003029.
Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55×10−151). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.
Author Summary
Recent genome-wide association studies (GWAS) have identified genetic variants associated with lung diseases. The challenge now is to find the causal genes in GWAS–nominated chromosomal regions and to characterize the molecular function of disease-associated genetic variants. In this paper, we describe an international effort to systematically capture the genetic architecture of gene expression regulation in human lung. By studying lung specimens from 1,111 individuals of European ancestry, we found a large number of genetic variants affecting gene expression in the lung, or lung expression quantitative trait loci (eQTL). These lung eQTLs will serve as an important resource to aid in the understanding of the molecular underpinnings of lung biology and its disruption in disease. To demonstrate the utility of this lung eQTL dataset, we integrated our data with previous genetic studies on asthma. Through integrative techniques, we identified causal variants and genes in GWAS–nominated loci and found key molecular drivers for asthma. We feel that sharing our lung eQTLs dataset with the scientific community will leverage the impact of previous large-scale GWAS on lung diseases and function by providing much needed functional information to understand the molecular changes introduced by the susceptibility genetic variants.
PMCID: PMC3510026  PMID: 23209423
12.  The fetal sheep lung does not respond to cortisol infusion during the late canalicular phase of development 
Physiological Reports  2013;1(6):e00130.
The prepartum surge in plasma cortisol concentrations in humans and sheep promotes fetal lung and surfactant system maturation in the support of air breathing after birth. This physiological process has been used to enhance lung maturation in the preterm fetus using maternal administration of betamethasone in the clinical setting in fetuses as young as 24 weeks gestation (term = 40 weeks). Here, we have investigated the impact of fetal intravenous cortisol infusion during the canalicular phase of lung development (from 109- to 116-days gestation, term = 150 ± 3 days) on the expression of genes regulating glucocorticoid (GC) activity, lung liquid reabsorption, and surfactant maturation in the very preterm sheep fetus and compared this to their expression near term. Cortisol infusion had no impact on mRNA expression of the corticosteroid receptors (GC receptor and mineralocorticoid receptor) or HSD11B-2, however, there was increased expression of HSD11B-1 in the fetal lung. Despite this, cortisol infusion had no effect on the expression of genes involved in lung sodium (epithelial sodium channel -α, -β, or -γ subunits and sodium–potassium ATPase-β1 subunit) or water (aquaporin 1, 3, and 5) reabsorption when compared to the level of expression during exposure to the normal prepartum cortisol surge. Furthermore, in comparison to late gestation, cortisol infusion does not increase mRNA expression of surfactant proteins (SFTP-A, -B, and -C) or the number of SFTP-B-positive cells present in the alveolar epithelium, the cells that produce pulmonary surfactant. These data suggest that there may be an age before which the lung is unable to respond biochemically to an increase in fetal plasma cortisol concentrations.
PMCID: PMC3871449  PMID: 24400136
Glucocorticoid; liquid reabsorption; lung; preterm; surfactant
13.  IL-12-STAT4-IFN-γ axis is a key downstream pathway in the development of IL-13-mediated asthma phenotypes in a Th2 type asthma model 
Experimental & Molecular Medicine  2010;42(8):533-546.
IL-4 and IL-13 are closely related cytokines that are produced by Th2 cells. However, IL-4 and IL-13 have different effects on the development of asthma phenotypes. Here, we evaluated downstream molecular mechanisms involved in the development of Th2 type asthma phenotypes. A murine model of Th2 asthma was used that involved intraperitoneal sensitization with an allergen (ovalbumin) plus alum and then challenge with ovalbumin alone. Asthma phenotypes, including airway-hyperresponsiveness (AHR), lung inflammation, and immunologic parameters were evaluated after allergen challenge in mice deficient in candidate genes. The present study showed that methacholine AHR and lung inflammation developed in allergen-challenged IL-4-deficient mice but not in allergen-challenged IL-13-deficient mice. In addition, the production of OVA-specific IgG2a and IFN-γ-inducible protein (IP)-10 was also impaired in the absence of IL-13, but not of IL-4. Lung-targeted IFN-γ over-expression in the airways enhanced methacholine AHR and non-eosinophilic inflammation; in addition, these asthma phenotypes were impaired in allergen-challenged IFN-γ-deficient mice. Moreover, AHR, non-eosinophilic inflammation, and IFN-γ expression were impaired in allergen-challenged IL-12Rβ2- and STAT4-deficient mice; however, AHR and non-eosinophilic inflammation were not impaired in allergen-challenged IL-4Rα-deficient mice, and these phenomena were accompanied by the enhanced expression of IL-12 and IFN-γ. The present data suggest that IL-13-mediated asthma phenotypes, such as AHR and non-eosinophilic inflammation, in the Th2 type asthma are dependent on the IL-12-STAT4-IFN-γ axis, and that these asthma phenotypes are independent of IL-4Ralpha-mediated signaling.
PMCID: PMC2928926  PMID: 20592486
asthma; interferon-γ; interleukin-12; interleukin-13; respiratory hypersensitivity; Th2 cells
14.  Glutathione Peroxidase-2 Protects from Allergen-Induced Airway Inflammation in mice 
To identify and validate the biological significance of new genes/ proteins involved in the development of allergic airway disease in a murine asthma model.
Gene microarrays were used to identify genes with at least a 2-fold increase in gene expression in lungs of two separate mouse strains with high and low allergic susceptibility, respectively. Validation of mRNA data was obtained by western blotting and immunohistochemistry, followed by functional analysis of one of the identified genes in mice with targeted disruption of specific gene expression.
Expression of two antioxidant enzymes, glutathione peroxidase-2 (Gpx-2) and glutathione-S-transferase Omega (GSTO) 1-1 was increased in both mouse strains after induction of allergic airway disease and localized in lung epithelial cells. Mice with targeted disruption of the Gpx-2 gene showed significantly enhanced airway inflammation compared to sensitized and challenged wild-type mice.
Our data indicate that genes encoding the antioxidants Gpx-2 and GSTO 1-1 are common inflammatory genes expressed upon induction of allergic airway inflammation, independently of allergic susceptibility. Furthermore, we provide evidence to illustrate the importance of a single antioxidant enzyme, Gpx-2, in protection from allergen-induced disease.
PMCID: PMC2911780  PMID: 19897562
Airway hyperreactivity; asthma; Glutathione peroxidase; glutathione S-transferase
15.  Role of inducible nitric oxide synthase on the development of virus-associated asthma exacerbation which is dependent on Th1 and Th17 cell responses 
Experimental & Molecular Medicine  2010;42(10):721-730.
Asthma is characterized by airway inflammation induced by immune dysfunction to inhaled antigens. Although respiratory viral infections are the most common cause of asthma exacerbation, immunologic mechanisms underlying virus-associated asthma exacerbation are controversial. Clinical evidence indicates that nitric oxide (NO) levels in exhaled air are increased in exacerbated asthma patients compared to stable patients. Here, we evaluated the immunologic mechanisms and the role of NO synthases (NOSs) in the development of virus-associated asthma exacerbation. A murine model of virus-associated asthma exacerbation was established using intranasal challenge with ovalbumin (OVA) plus dsRNA for 4 weeks in mice sensitized with OVA plus dsRNA. Lung infiltration of inflammatory cells, especially neutrophils, was increased by repeated challenge with OVA plus dsRNA, as compared to OVA alone. The neutrophilic inflammation enhanced by dsRNA was partly abolished in the absence of IFN-gamma or IL-17 gene expression, whereas unaffected in the absence of IL-13. In terms of the roles of NOSs, dsRNA-enhanced neutrophilic inflammation was significantly decreased in inducible NOS (iNOS)-deficient mice compared to wild type controls; in addition, this phenotype was inhibited by treatment with a non-specific NOS inhibitor (L-NAME) or an specific inhibitor (1400 W), but not with a specific endothelial NOS inhibitor (AP-CAV peptide). Taken together, these findings suggest that iNOS pathway is important in the development of virus-associated exacerbation of neutrophilic inflammation, which is dependent on both Th1 and Th17 cell responses.
PMCID: PMC2966746  PMID: 20841959
asthma; interferon-γ; interleukin-17; neutrophils; nitric oxide synthase type II; RNA viruses; Th1 cells
16.  ADAM8: A new therapeutic target for asthma 
A proteinase with a disintegrin and a metalloproteinase domain-8 (ADAM8) has been linked to asthma.
To explore whether ADAM8 is a therapeutic target for asthma.
We reviewed literature on ADAM8’s function and expression and activities in lungs of humans and mice with allergic airway inflammation (AAI). We used these data to generate hypotheses about the contributions of ADAM8 to asthma pathogenesis.
ADAM8 levels are increased in airway epithelium and airway inflammatory cells in mice with AAI and human asthma patients. Data from murine models of AAI indicate that ADAM8 dampens airway inflammation. It is not clear whether ADAM8 contributes directly to structural remodeling in asthmatic airways. Additional studies are required to validate ADAM8 as a therapeutic target for asthma.
PMCID: PMC2784995  PMID: 19397475
ADAM; airway hyper-responsiveness; allergy; asthma; disintegrin; eosinophil; epithelium; inflammation; leukocyte; macrophage; metalloproteinase; proteinase; remodeling; signaling
17.  Interleukin-5 reduces the expression of uteroglobin-related protein (UGRP) 1 gene in allergic airway inflammation 
Immunology letters  2005;97(1):123-129.
Airway inflammation is thought to play a major role in the pathogenesis of bronchial asthma. The precise role of individual inflammatory cells, mediator and asthma related genes in allergic lung diseases is not completely understood. The uteroglobin-related protein (UGRP) 1 was proposed to be an asthma candidate gene and play a role in regulating lung inflammation, however its precise function in the airways remains obscure. In this investigation, we used a mouse model of allergic airway inflammation to establish a relationship between UGRP 1 and IL-5 in airway inflammation. Ovalbumin (OVA) challenged mice demonstrate eosinophilia in airway tissues and high levels of IL-5 in bronchoalveolar lavage (BAL) fluid analogous to that found in bronchial asthma. Interestingly, these “OVA-challenged” mice show down-regulation of Ugrp1 expression as compared with the control group. Regression analysis further demonstrates a significant negative correlation between Ugrp1 mRNA expression in the lung and IL-5 levels in BAL fluid with r = 0.948 and P < 0.0001 when IL-5 levels were normalized by log transformation. Intranasal instillation of IL-5 to mice revealed an inhibitory effect of IL-5 on the expression of Ugrp1 mRNA. Together, these results indicate an involvement of IL-5 in the down-regulation of Ugrp1 expression in airway inflammation such as allergic asthma disease.
PMCID: PMC1343456  PMID: 15626484
Uteroglobin-related protein 1 (UGRP 1); Interleukin-5 (IL-5); Airway inflammation; Allergic asthma; UGRP, Uteroglobin-related protein; CCSP, Clara cell secretory protein; OVA, ovalbumin; BAL, bronchoalveolar lavage; IL, Interleukin; BHR, bronchial hyperresponsiveness; SCGB, secretoglobin; MARCO, macrophage scavenger receptor with collagenous structure
18.  Apoptosis of mesenchymal cells during the pseudoglandular stage of lung development affects branching morphogenesis 
Experimental lung research  2008;34(8):481-499.
Apoptosis has been implicated as an important process in the development of several organ systems, including limbs, kidneys, and the heart. In developing murine lungs, we found that apoptosis was more predominant during the pseudoglandular stage of lung development than during the saccular stage with ninety-three percent of the apoptotic structures in the mesenchyme. Murine lung explants cultured in the presence of zinc chloride and aurintricarboxylic acid, two blockers of endonuclease function, showed decreased branching. These observations suggest that apoptosis predominates in mesenchymal cells during the pseudoglandular stage of lung development, and may be important for normal progression of lung branching morphogenesis.
PMCID: PMC2665177  PMID: 18850375
19.  Characterization of CD19+CD23+B2 lymphocytes in the allergic airways of BALB/c mice in response to the inhalation of Aspergillus fumigatus conidia 
Fungal sensitization in patients with asthma often indicates an unusual disease course in which traditional asthma treatments have little effect and in which morbidity is particularly severe. Airway hyperresponsiveness (AHR), inflammatory infiltrates, smooth muscle hyperplasia, and irreversible fibrotic remodeling of the bronchial architecture are features of allergic fungal asthma. The systemic production of IgE has long been associated with the immunopathogenesis of allergic asthma; however, the role of B lymphocytes and their products in the response to fungal allergens remains unclear. In the present study, we hypothesize that B lymphocytes are recruited to the allergic lung to impact the allergic response. Using a murine fungal aeroallergen model to mimic the human syndrome, we characterized the B cell population in the lung after fungal challenge and found that CD19+CD23+ B2 lymphocyte numbers are increased in the allergic lung in a dynamic process. IgA, IgG2a, and IgE were prominent in the serum and bronchoalveolar lavage fluid of allergic animals. It was evident that a tissue-centric production of these antibodies was possible. IgA-, IgG-, and IgE-producing cells from the allergic lung were identified by flow cytometry and immunohistochemistry. This study shows for the first time that CD19+CD23+ B2 lymphocyte numbers change in the lung in a dynamic process after inhalation of fungal conidia and their increase has a significant impact on the Ab production in the pulmonary compartment in the context of fungal allergy.
PMCID: PMC3541783  PMID: 23316260
fungal asthma; murine model; Ab production; B lymphocytes
20.  Immune Pathways for Translating Viral Infection into Chronic Airway Disease1 
Advances in immunology  2009;102:245-276.
To better understand the immune basis for chronic inflammatory lung disease, we analyzed a mouse model of lung disease that develops after respiratory viral infection. The disease that develops in this model is similar to asthma and chronic obstructive pulmonary disease (COPD) in humans and is manifested after the inciting virus has been cleared to trace levels. The model thereby mimics the relationship of paramyxoviral infection to the development of childhood asthma in humans. When the acute lung disease appears in this model (at 3 weeks after viral inoculation), it depends on an immune axis that is initiated by expression and activation of the high-affinity IgE receptor (FcεRI) on conventional lung dendritic cells (cDCs) to recruit interleukin (IL)-13-producing CD4+ T cells to the lower airways. However, when the chronic lung disease develops fully (at 7 weeks after inoculation), it is driven instead by an innate immune axis that relies on invariant natural killer T (iNKT) cells that are programmed to activate macrophages to produce IL-13. The interaction between iNKT cells and macrophages depends on contact between the semi-invariant Vα14Jα18-TCR on lung iNKT cells and the oligomorphic MHC-like protein CD1d on macrophages as well as NKT cell production of IL-13 that binds to the IL-13 receptor (IL-13R) on the macrophage. This innate immune axis is also activated in the lungs of humans with severe asthma or COPD based on detection of increased numbers of iNKT cells and alternatively activated IL-13-producing macrophages in the lung. Together, the findings identify an adaptive immune response that mediates acute disease and an innate immune response that drives chronic inflammatory lung disease in experimental and clinical settings.
PMCID: PMC3010226  PMID: 19477323
21.  Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model  
Airways inflammation is thought to play a central role in the pathogenesis of asthma. However, the precise role that individual inflammatory cells and mediators play in the development of airways hyperreactivity and the morphological changes of the lung during allergic pulmonary inflammation is unknown. In this investigation we have used a mouse model of allergic pulmonary inflammation and interleukin (IL) 5-deficient mice to establish the essential role of this cytokine and eosinophils in the initiation of aeroallergen-induced lung damage and the development of airways hyperreactivity. Sensitization and aerosol challenge of mice with ovalbumin results in airways eosinophilia and extensive lung damage analogous to that seen in asthma. Aeroallergen-challenged mice also display airways hyperreactivity to beta-methacholine. In IL-5-deficient mice, the eosinophilia, lung damage, and airways hyperreactivity normally resulting from aeroallergen challenge were abolished. Reconstitution of IL-5 production with recombinant vaccinia viruses engineered to express this factor completely restored aeroallergen-induced eosinophilia and airways dysfunction. These results indicate that IL-5 and eosinophils are central mediators in the pathogenesis of allergic lung disease.
PMCID: PMC2192412  PMID: 8551223
22.  Expression of Interleukin 9 in the Lungs of  Transgenic Mice Causes Airway Inflammation, Mast Cell Hyperplasia, and Bronchial Hyperresponsiveness  
The Journal of Experimental Medicine  1998;188(7):1307-1320.
Interleukin (IL)-9, a pleiotropic cytokine produced by the Th2 subset of T lymphocytes has been proposed as product of a candidate gene responsible for asthma. Its wide range of biological functions on many cell types involved in the allergic immune response suggests a potentially important role in the complex pathogenesis of asthma. To investigate the contributions of IL-9 to airway inflammation and airway hyperresponsiveness in vivo, we created transgenic mice in which expression of the murine IL-9 cDNA was regulated by the rat Clara cell 10 protein promoter. Lung selective expression of IL-9 caused massive airway inflammation with eosinophils and lymphocytes as predominant infiltrating cell types. A striking finding was the presence of increased numbers of mast cells within the airway epithelium of IL-9–expressing mice. Other impressive pathologic changes in the airways were epithelial cell hypertrophy associated with accumulation of mucus-like material within nonciliated cells and increased subepithelial deposition of collagen. Physiologic evaluation of IL-9–expressing mice demonstrated normal baseline airway resistance and markedly increased airway hyperresponsiveness to inhaled methacholine. These findings strongly support an important role for IL-9 in the pathogenesis of asthma.
PMCID: PMC2212487  PMID: 9763610
interleukin 9; transgenic mice; asthma; mast cells; eosinophilia
23.  Nicotine alters lung branching morphogenesis through the α 7 nicotinic acetylcholine receptor 
There is abundant epidemiological data linking prenatal environmental tobacco smoke with childhood asthma and wheezing, but the underlying molecular and physiological mechanisms that occur in utero to explain this link remain unelucidated. Several studies suggest that nicotine, which traverses the placenta, is a causative agent. Therefore, we studied the effects of nicotine on lung branching morphogenesis using embryonic murine lung explants. We found that the expression of α 7 nicotinic acetylcholine receptors, which mediate many of the biological effects of nicotine, is highest in pseudoglandular stage lungs compared with lungs at later stages. We then studied the effects of nicotine in the explant model and found that nicotine stimulated lung branching in a dose-dependent fashion. α-Bungarotoxin, an antagonist of α 7 nicotinic acetylcholine receptors, blocked the stimulatory effect of nicotine, whereas GTS-21, a specific agonist, stimulated branching, thereby mimicking the effects of nicotine. Explants deficient in α 7 nicotinic acetylcholine receptors did not respond to nicotine. Nicotine also stimulated the growth of the explant. Altogether, these studies suggest that nicotine stimulates lung branching morphogenesis through α 7 nicotinic acetylcholine receptors and may contribute to dysanaptic lung growth, which in turn may predispose the host to airway disease in the postnatal period.
PMCID: PMC2665174  PMID: 17545491
lung growth; nicotinic receptors
24.  Effect of aging on pulmonary inflammation, airway hyperresponsiveness and T and B cell responses in antigen sensitized and challenged mice 
The effect of aging on several pathologic features of allergic-asthma (pulmonary inflammation, eosinophilia, mucus-hypersecretion), and their relationship with airway hyperresponsiveness (AHR) is not well characterized.
To evaluate lung inflammation, mucus-metaplasia and AHR in relationship to age in murine models of allergic-asthma comparing young and older mice.
Young (6-week) and older (6-, 12- 18-month) BALB/c mice were sensitized and challenged with ovalbumin (OVA). AHR and bronchoalveolar fluid (BALF) total inflammatory cell count and differential were measured. To evaluate mucus-metaplasia, quantitative PCR for the major airway mucin-associated gene, MUC-5AC, from lung tissue was measured, and lung tissue sections stained with periodic acid-Schiff (PAS) for goblet-cell enumeration. Lung tissue cytokine gene expression was determined by qPCR, and systemic cytokine protein levels by ELISA from spleen-cell cultures. Antigen-specific serum IgE was determined by ELISA.
AHR developed in both aged and young OVA-sensitized/challenged mice (OVA-mice), and was more significantly increased in young OVA-mice than in aged OVA-mice. However, BALF eosinophil numbers were significantly higher, and lung histology showed greater inflammation in aged OVA-mice than in young OVA-mice. MUC-5AC expression and numbers of PAS+ staining bronchial epithelial cells were significantly increased in the aged OVA-mice. All aged OVA-mice had increased IL-5 and IFN-γ mRNA expression in the lung and IL-5 and IFN-γ protein levels from spleen cell cultures compared to young OVA-mice. OVA-IgE was elevated to a greater extent in aged OVA-mice.
Although pulmonary inflammation and mucus-metaplasia after antigen sensitization/challenge occurred to a greater degree in older mice, the increase in AHR was significantly less compared with younger OVA-mice. Antigen treatment produced a unique cytokine profile in older mice (elevated IFN-γ and IL-5) compared with young mice (elevated IL-4 and IL-13). Thus, the airway response to inflammation is lessened in aging animals, and may represent age-associated events leading to different phenotypes in response to antigen provocation.
PMCID: PMC2818115  PMID: 17845421
Aging; murine; asthma; airway hyperresponsiveness; eosinophil; inflammation
25.  Activating transcription factor 3 is a negative regulator of allergic pulmonary inflammation 
The Journal of Experimental Medicine  2008;205(10):2349-2357.
We recently demonstrated the pivotal role of the transcription factor (TF) activating TF 3 (ATF3) in dampening inflammation. We demonstrate that ATF3 also ameliorates allergen-induced airway inflammation and hyperresponsiveness in a mouse model of human asthma. ATF3 expression was increased in the lungs of mice challenged with ovalbumin allergen, and this was associated with its recruitment to the promoters of genes encoding Th2-associated cytokines. ATF3-deficient mice developed significantly increased airway hyperresponsiveness, pulmonary eosinophilia, and enhanced chemokine and Th2 cytokine responses in lung tissue and in lung-derived CD4+ lymphocytes. Although several TFs have been associated with enhanced inflammatory responses in the lung, ATF3 attenuates the inflammatory responses associated with allergic airway disease.
PMCID: PMC2556774  PMID: 18794337

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