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1.  Strain-Dependent Genomic Factors Affect Allergen-Induced Airway Hyperresponsiveness in Mice 
Asthma is etiologically and clinically heterogeneous, making the genomic basis of asthma difficult to identify. We exploited the strain-dependence of a murine model of allergic airway disease to identify different genomic responses in the lung. BALB/cJ and C57BL/6J mice were sensitized with the immunodominant allergen from the Dermatophagoides pteronyssinus species of house dust mite (Der p 1), without exogenous adjuvant, and the mice then underwent a single challenge with Der p 1. Allergic inflammation, serum antibody titers, mucous metaplasia, and airway hyperresponsiveness were evaluated 72 hours after airway challenge. Whole-lung gene expression analyses were conducted to identify genomic responses to allergen challenge. Der p 1–challenged BALB/cJ mice produced all the key features of allergic airway disease. In comparison, C57BL/6J mice produced exaggerated Th2-biased responses and inflammation, but exhibited an unexpected decrease in airway hyperresponsiveness compared with control mice. Lung gene expression analysis revealed genes that were shared by both strains and a set of down-regulated genes unique to C57BL/6J mice, including several G-protein–coupled receptors involved in airway smooth muscle contraction, most notably the M2 muscarinic receptor, which we show is expressed in airway smooth muscle and was decreased at the protein level after challenge with Der p 1. Murine strain–dependent genomic responses in the lung offer insights into the different biological pathways that develop after allergen challenge. This study of two different murine strains demonstrates that inflammation and airway hyperresponsiveness can be decoupled, and suggests that the down-modulation of expression of G-protein–coupled receptors involved in regulating airway smooth muscle contraction may contribute to this dissociation.
doi:10.1165/rcmb.2010-0315OC
PMCID: PMC3208613  PMID: 21378263
asthma; airway hyperresponsiveness; inflammation; house dust mite; Der p 1
2.  Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice 
PLoS ONE  2012;7(7):e40789.
Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.
doi:10.1371/journal.pone.0040789
PMCID: PMC3397936  PMID: 22815821
3.  c-Kit Is Essential for Alveolar Maintenance and Protection from Emphysema-like Disease in Mice 
Rationale: Previously, we demonstrated a candidate region for susceptibility to airspace enlargement on mouse chromosome 5. However, the specific candidate genes within this region accounting for emphysema-like changes remain unrecognized. c-Kit is a receptor tyrosine kinase within this candidate gene region that has previously been recognized to contribute to the survival, proliferation, and differentiation of hematopoietic stem cells. Increases in the percentage of cells expressing c-Kit have previously been associated with protection against injury-induced emphysema.
Objectives: Determine whether genetic variants of c-Kit are associated with spontaneous airspace enlargement.
Methods: Perform single-nucleotide polymorphism association studies in the mouse strains at the extremes of airspace enlargement phenotype for variants in c-Kit tyrosine kinase. Characterize mice bearing functional variants of c-Kit compared with wild-type controls for the development of spontaneous airspace enlargement. Epithelial cell proliferation was measured in culture.
Measurements and Main Results: Upstream regulatory single-nucleotide polymorphisms in the divergent mouse strains were associated with the lung compliance difference observed between the extreme strains. c-Kit mutant mice (KitW-sh/W-sh), when compared with genetic controls, developed altered lung histology, increased total lung capacity, increased residual volume, and increased lung compliance that persist into adulthood. c-Kit inhibition with imatinib attenuated in vitro proliferation of cells expressing epithelial cell adhesion molecule.
Conclusions: Our findings indicate that c-Kit sustains and/or maintains normal alveolar architecture in the lungs of mice. In vitro data suggest that c-Kit can regulate epithelial cell clonal expansion. The precise mechanisms that c-Kit contributes to the development of airspace enlargement and increased lung compliance remain unclear and warrants further investigation.
doi:10.1164/rccm.201007-1157OC
PMCID: PMC3136992  PMID: 21471107
genetic; tyrosine kinase; SASH; chronic obstructive pulmonary disease; aging
4.  Using Mouse Genomics to Understand Idiopathic Interstitial Fibrosis 
Idiopathic interstitial pneumonia represents a broad category of lung disorders characterized by scarring or fibrosis of the lung accompanied by varying degrees of inflammation. A number of important hypotheses based on clinical observations have substantially contributed to our understanding of the pathogenesis of the most insidious and devastating of the idiopathic interstitial pneumonias, idiopathic interstitial fibrosis (IIF). Patients with IIF usually present late in the course of their illness; thus, animal models of the early, preclinical stage of these diseases are needed. Although no model faithfully recapitulates the clinical course of disease or the histopathology observed in humans, all result in scarring of the lung and may therefore be used to understand the biological processes that contribute to this scarring. The purpose of this article is to summarize the application of mouse genetic and genomic tools to these models to advance our understanding of IIF and to describe emerging agnostic approaches to identifying genes important to the fibroproliferative component of IIF.
doi:10.1513/pats.200607-147JG
PMCID: PMC2647620  PMID: 17202297
fibrosis; QTL; genomics; microarray
5.  The role of Scgb1a1+ Clara cells in the long-term maintenance and repair of lung airway, but not alveolar, epithelium 
Cell stem cell  2009;4(6):525-534.
Summary
To directly test the contribution of Scgb1a1+ Clara cells to postnatal growth, homeostasis and repair of lung epithelium, we generated a Scgb1a1-CreERTM “knock-in” mouse line for lineage tracing these cells. Under all conditions tested the majority of Clara cells in the bronchioles both self-renew and generate ciliated cells. In the trachea, Clara cells give rise to ciliated cells but do not self-renew extensively. Nevertheless, they can contribute to tracheal repair. In the postnatal mouse lung it has been proposed that bronchioalveolar stem cells (BASCs) which co-express Scgb1a1 (Secretoglobin1a1) and SftpC (Surfactant Protein C), contribute descendants to both bronchioles and alveoli. The putative BASCs were lineage labeled in our studies. However, we find no evidence for the function of a special BASC population during postnatal growth, adult homeostasis or repair. Rather, our results support a model in which the trachea, bronchioles and alveoli are maintained by distinct populations of epithelial progenitor cells.
doi:10.1016/j.stem.2009.04.002
PMCID: PMC2730729  PMID: 19497281
6.  Chronic LPS Inhalation Causes Emphysema-Like Changes in Mouse Lung that Are Associated with Apoptosis 
Lipopolysaccharide (LPS) is ubiquitous in the environment. Recent epidemiologic data suggest that occupational exposure to inhaled LPS can contribute to the progression of chronic obstructive pulmonary disease. To address the hypothesis that inhaled LPS can cause emphysema-like changes in mouse pulmonary parenchyma, we exposed C57BL/6 mice to aerosolized LPS daily for 4 weeks. By 3 days after the end of the 4-week exposure, LPS-exposed mice developed enlarged airspaces that persisted in the 4-week recovered mice. These architectural alterations in the lung are associated with enhanced type I, III, and IV procollagen mRNA as well as elevated levels of matrix metalloproteinase (MMP)-9 mRNA, all of which have been previously associated with human emphysema. Interestingly, MMP-9–deficient mice were not protected from the development of LPS-induced emphysema. However, we demonstrate that LPS-induced airspace enlargement was associated with apoptosis within the lung parenchyma, as shown by prominent TUNEL staining and elevated cleaved caspase 3 immunoreactivity. Antineutrophil antiserum-treated mice were partially protected from the lung destruction caused by chronic inhalation of LPS. Taken together, these findings demonstrate that inhaled LPS can cause neutrophil-dependent emphysematous changes in lung architecture that are associated with apoptosis and that these changes may be occurring through mechanisms different than those induced by cigarette smoke.
doi:10.1165/rcmb.2007-0448OC
PMCID: PMC2574529  PMID: 18539952
7.  Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping 
BMC Genomics  2008;9:598.
Background
The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK), a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci.
Results
Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05) after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes.
Conclusion
The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models of complex human diseases in a mixed cell population and might be a valuable technology to determine whether environmental exposures can lead to epigenetic changes.
doi:10.1186/1471-2164-9-598
PMCID: PMC2621211  PMID: 19077247
8.  In utero supplementation with methyl donors enhances allergic airway disease in mice 
The Journal of Clinical Investigation  2008;118(10):3462-3469.
Asthma is a complex heritable disease that is increasing in prevalence and severity, particularly in developed countries such as the United States, where 11% of the population is affected. The contribution of environmental and genetic factors to this growing epidemic is currently not well understood. We developed the hypothesis, based on previous literature, that changes in DNA methylation resulting in aberrant gene transcription may enhance the risk of developing allergic airway disease. Our findings indicate that in mice, a maternal diet supplemented with methyl donors enhanced the severity of allergic airway disease that was inherited transgenerationally. Using a genomic approach, we discovered 82 gene-associated loci that were differentially methylated after in utero supplementation with a methyl-rich diet. These methylation changes were associated with decreased transcriptional activity and increased disease severity. Runt-related transcription factor 3 (Runx3), a gene known to negatively regulate allergic airway disease, was found to be excessively methylated, and Runx3 mRNA and protein levels were suppressed in progeny exposed in utero to a high-methylation diet. Moreover, treatment with a demethylating agent increased Runx3 gene transcription, further supporting our claim that a methyl-rich diet can affect methylation status and consequent transcriptional regulation. Our findings indicate that dietary factors can modify the heritable risk of allergic airway disease through epigenetic mechanisms during a vulnerable period of fetal development in mice.
doi:10.1172/JCI34378
PMCID: PMC2542847  PMID: 18802477
9.  CD44 Regulates Macrophage Recruitment to the Lung in Lipopolysaccharide-Induced Airway Disease 
LPS from bacteria is ubiquitous in the environment and can cause airway disease and modify allergic asthma. Identification of gene products that modulate the biologic response to inhaled LPS will improve our understanding of inflammatory airways disease. Previous work has identified quantitative trait loci for the response to inhaled LPS on chromosomes 2 and 11. In these regions, 28 genes had altered RNA expression after inhalation of LPS, including CD44, which was associated with differences in both TNF-α levels and neutrophil recruitment into the lung. It has previously been shown that CD44 can modulate macrophage recruitment in response to Mycobacterium tuberculosis, as well as clearance of neutrophils after lung injury with both bleomycin and live Escherichia coli bacteria. In this study, we demonstrate that the biologic response to inhaled LPS is modified by CD44. Macrophages failed to be recruited to the lungs of CD44-deficient animals at all time points after LPS exposure. CD44-deficient macrophages showed reduced motility in a Transwell migration assay, reduced ability to secrete the proinflammatory cytokine TNF-α, reduced in vivo migration in response to monocyte chemotactic protein-1, and diminished adhesion to vascular endothelia in the presence of TNF-α. In addition, CD44-deficient animals had 150% fewer neutrophils at 24 h and 50% greater neutrophils 48 h after LPS exposure. These results support the role of CD44 in modulating the biologic response to inhaled LPS.
doi:10.1165/rcmb.2006-0363OC
PMCID: PMC1976546  PMID: 17446529
lung; environment; tlr4; hyaluronan; endotoxin
10.  Leukocyte-Derived IL-10 Reduces Subepithelial Fibrosis Associated with Chronically Inhaled Endotoxin 
Endotoxin (LPS), a Gram-negative cell wall component, has potent proinflammatory properties. Acute LPS exposure causes airway inflammation; chronic exposure causes airway hyperreactivity and remodeling. IL-10 is an important antiinflammatory cytokine, which is decreased in patients with airway disease, such as asthma and cystic fibrosis. To examine the physiologic and therapeutic role of IL-10 in acute and chronic LPS-induced airway disease. Mice were exposed to aerosolized LPS once or daily for 4 wk. Endpoints were airway inflammation, airway reactivity to methacholine, extracellular matrix protein expression, and histologic analysis. IL-10–deficient mice developed significantly enhanced airway cellularity and remodeling when compared with C57BL/6 mice after chronic LPS inhalation. However they demonstrated less airway hyperreactivity associated with higher inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), and lung lavage fluid nitrite levels. In a bone marrow transplantation model, the IL-10 antiinflammatory effect was dependent on the hematopoietic but not on the parenchymal IL-10 expression. Induced epithelial human IL-10 expression protected from the LPS effects and led to decreased collagen production. IL-10 attenuates chronic LPS-induced airway inflammation and remodeling. Physiologically, the antiinflammatory effect of IL-10 is mediated by hematopoietic cells. Therapeutically, adenovirus-driven expression of human IL-10 in airway epithelia is sufficient for its protective effect on inflammation and remodeling. The role of IL-10 on airway hyperreactivity is complex: IL-10 deficiency protects against LPS-induced hyperreactivity, and is associated with higher eNOS, iNOS, and airway nitrate levels.
doi:10.1165/rcmb.2006-0055OC
PMCID: PMC2643294  PMID: 16809636
airway hyperreactivity; airway remodeling; endotoxin; IL-10
11.  Integrating Murine Gene Expression Studies to Understand Obstructive Lung Disease Due to Chronic Inhaled Endotoxin 
PLoS ONE  2013;8(5):e62910.
Rationale
Endotoxin is a near ubiquitous environmental exposure that that has been associated with both asthma and chronic obstructive pulmonary disease (COPD). These obstructive lung diseases have a complex pathophysiology, making them difficult to study comprehensively in the context of endotoxin. Genome-wide gene expression studies have been used to identify a molecular snapshot of the response to environmental exposures. Identification of differentially expressed genes shared across all published murine models of chronic inhaled endotoxin will provide insight into the biology underlying endotoxin-associated lung disease.
Methods
We identified three published murine models with gene expression profiling after repeated low-dose inhaled endotoxin. All array data from these experiments were re-analyzed, annotated consistently, and tested for shared genes found to be differentially expressed. Additional functional comparison was conducted by testing for significant enrichment of differentially expressed genes in known pathways. The importance of this gene signature in smoking-related lung disease was assessed using hierarchical clustering in an independent experiment where mice were exposed to endotoxin, smoke, and endotoxin plus smoke.
Results
A 101-gene signature was detected in three murine models, more than expected by chance. The three model systems exhibit additional similarity beyond shared genes when compared at the pathway level, with increasing enrichment of inflammatory pathways associated with longer duration of endotoxin exposure. Genes and pathways important in both asthma and COPD were shared across all endotoxin models. Mice exposed to endotoxin, smoke, and smoke plus endotoxin were accurately classified with the endotoxin gene signature.
Conclusions
Despite the differences in laboratory, duration of exposure, and strain of mouse used in three experimental models of chronic inhaled endotoxin, surprising similarities in gene expression were observed. The endotoxin component of tobacco smoke may play an important role in disease development.
doi:10.1371/journal.pone.0062910
PMCID: PMC3652821  PMID: 23675439

Results 1-11 (11)