2016 Progress Report: Environmental Determinants of Host Defense

EPA Grant Number: RD834515C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant RD834515
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Denver Children’s Environmental Health Center - Environmental Determinants of Airway Disease in Children
Center Director: Schwartz, David A.
Title: Environmental Determinants of Host Defense
Investigators: Schwartz, David A. , Yang, Ivana , Huber, Jonathan , Oakes, Judy , Eyring, Ken
Institution: National Jewish Health , University of Colorado at Denver
EPA Project Officer: Hahn, Intaek
Project Period: September 1, 2009 through August 31, 2014 (Extended to June 21, 2017)
Project Period Covered by this Report: June 22, 2015 through June 21,2016
RFA: Children's Environmental Health and Disease Prevention Research Centers (with NIEHS) (2009) RFA Text |  Recipients Lists
Research Category: Children's Health , Health

Objective:

The overall goal of this project is to understand how and why air pollution alters lung host defense. Although the proposed research is focused on mice, we believe that the discoveries we make in mice will prove to be relevant to basic mechanisms of lung host defense in children. In fact, our findings in mice will be tested in Project 1 of this program. The environmental, clinical, and biological significance of this project is supported by the following observations. First, air pollution accounts for substantial morbidity and mortality throughout the world, including lung infections and preventable deaths in children. Second, endotoxin is ubiquitous in the environment and is associated with the development and progression of asthma and other forms of airway disease. However, the relationship between endotoxin and asthma is not simple because early childhood exposure to endotoxin, at least in certain populations, appears to protect children from developing asthma and atopy. Air pollution is contaminated with endotoxin, so this pathogen associated molecular pattern (PAMP) or other PAMPs may play a role in the pathophysiology of air pollution. Third, the ability of the host to respond to lipopolysaccharide (LPS; a specific form of endotoxin) and other PAMPs is highly variable in mice and humans, yet polymorphic host defense genes only account for a portion of this variable response. Fourth, innate immunity provides a first line of host defense against microbial pathogens that is conserved over a wide variety of species from flies to mammals. Indeed, innate immune signaling mechanisms in mice are almost identical to those in humans. Finally, the innate immune system is biologically dynamic and is responsive to both ozone and PAMPs. We have recently found that the expression of innate immune receptors on macrophages can be enhanced by ozone or PAMPs. Moreover, others have reported that some innate immune cells avoid excessive inflammation by selectively downregulating proinflammatory genes while continuing to transcribe antimicrobial genes. Thus, the overall hypothesis of this project is that the expression of toll-like receptors (TLRs) in the lung are influenced by environmental (ozone and/or PAMPs) and genetic factors, and the dynamic expression of TLRs has profound effects on lung host defense and, consequently, the development of lung infections and allergic airway disease.

Progress Summary:

During the past year, we completed characterization of the effect of in vivo ozone exposure on lung innate immune response to Pam3CYS, a TLR2/TLR6 agonist. Ozone pre-exposure resulted in (1) increased whole lung lavage (WLL) cell influx, (2) increased IL-6 and KC, and decreased MIP-1α and TNF-α and (3) increased cell surface expression of TLR4, TLR2 and TLR1 on macrophages as a result of ozone alone or in combination with Pam3CYS. In addition, we demonstrated that ozone followed by Pam3CYS resulted in a large increase in phosphorylation of both p44/42 (Erk1/2) MAPK and JNK kinases and significant reduction in nonphosphorylated p44/42 MAPK at 4 hours post Pam3CYS. This enhanced signal associated with ozone/Pam3CYS treatment was not present at 24 hours. To further characterize the priming effect of ozone on innate immunity at the molecular level in an unbiased manner, we performed gene expression profiling on lung tissue of mice from the four exposure groups. Ozone exposure has the strongest effect on gene expression at the 4-hour time point with the effect diminishing by the 24-hour time point. Ozone pre-exposure prior to Pam3CYS treatment (O3/Pam3CYS vs. FA/Pam3CYS) enhanced induction of Trmt5 at the 4 hours and Cck at 24 hours. Expression of Ttk (4 hour timepoint), Pbp2, Gjb4, Ncapg, and Pbk (24-hour time point) were also increased in the O3/Pam3CYS vs. FA/Pam3CYS but significantly less than in the O3/saline vs. FA/saline comparison. The most significant result among downregulated genes is downregulation at 24 hours of killer cell lectin-like receptors (Klra3, Klra8, Klra9, Klra10, Klra15, Klra21, Klra22, Klra23, Klrb1a, and Klrk1). Our study demonstrates that expression of TLRs on macrophage surface is a dynamic process that is influenced by ozone and that this process is associated with differential expression of a number of previously unexplored genes. This dynamic nature of TLR expression is likely more general and could be influenced by other components of air pollution and in cell types other than macrophages. This priming effect of air pollution and genes that are associated with the process also represent potential therapeutic targets for air pollutant exposure in the context of pulmonary infection or allergic airway inflammation.

To understand how epigenetic mechanisms alter dendritic cell function and contribute to the etiology of allergic airway disease, we have developing a line of investigation that examines epigenetic marks and transcriptional profiles in distinct lineages of DCs that exist within the lung and recruited to the draining lymph nodes in response to allergic sensitization. Following intratracheal (i.t.) administration of FITClabeled HDM or cockroach(CR) allergen, antigen-bearing DCs (CD103+ and CD11+ subsets) have been purified by FACS sorting and the transcriptional profiles have been analyzed. This analysis has identified 100 genes in the CD103+ subset and 23 genes in the CD11b+ subset that are differentially regulated by both allergens. Current work is focusing on epigenetic regulation of specific targets identified in this transcriptional analysis to better understand the early processes of the innate response that lead to activation of adaptive immunity.

To follow up on our published findings on the role of methyl donor diet in the development of allergic airway disease in mice, we compared allergic airway disease phenotypes between methylenetetrahydrofolate reductase (MTHFR) deficient mice on a C57/Bl6 background to wild-type (WT) C57/Bl6 mice using a house dust mite (HDM) allergen model. In brief, mice received an intraperitoneal (i.p.) sensitization of 10μg HDM or saline on days 0 and 7 followed by an intratracheal (i.t.) challenge of 5μg HDM or saline on days 14 and 15. Forty-eight hours after the final challenge, total cells and eosinophils in the bronchoalveolar lavage were 5.76 fold and 7.87 lower, respectively, in HDM-treated MTHFR deficient compared HDM-treated WT mice. Furthermore, HDM-treated MTHFR KO mice demonstrate a 1.64 fold (p < 0.05) reduction in lung resistance compared to HDM-treated WT mice in response to inhaled methacholine. These results suggest that allergic airway disease may be suppressed through the loss of MTHFR. Over the past year, we have characterized epigenetic (DNA methylation) and transcriptional response of MTHFR deficient mice to HDM sensitization and challenge to determine molecular mechanisms by which these mice are protected from developing allergic airway disease. There were 1,031 transcripts differentially expressed in HDM-treated MTHFR KO compared to HDM-treated WT mice with an adjusted p-value < 0.05. Forty-eight transcripts with an adjusted p-value < 0.05 differed between the two saline-treated groups. Pathway analysis identified several enriched pathways. As to be expected with MTHFR KO mice, a number of biochemical pathways as loss of MTHFR shifts the balance of other folate forms. There also are a number of immune signaling and developmental pathways identified. In the HDM-treated mice, 146 differentially methylated regions (DMRs) were identified between MTHFR KO and WT mice with an adjusted p-value < 0.10. 28 DMRs were hypermethylated leaving 118 hypomethylated in MTHFR KO mice. Seven genes were significant after multiple testing correction in both the expression and methylation data set; these genes represent high priority candiates for future functional validation.

Future Activities:

To follow up on our published findings on the role of epigenetics in regulation of expression of the scavenger receptor MARCO on LPS tolerance, we have performed transcriptional profiling on both in vitro and in vivo tolerized macrophages from three different compartments – alveolar, peritoneal and bone marrow. We are currently analyzing these data to understand commonalities and differences in LPS tolerance in the three macrophage populations. This analysis will identify targets that will be the focus of further investigation into epigenetic regulation of their expression.


Journal Articles on this Report : 11 Displayed | Download in RIS Format

Other subproject views: All 23 publications 14 publications in selected types All 14 journal articles
Other center views: All 51 publications 30 publications in selected types All 30 journal articles
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Journal Article Das R, Subrahmanyan L, Yang IV, van Duin D, Levy R, Piecychna M, Leng L, Montgomery RR, Shaw A, Schwartz DA, Bucala R. Functional polymorphisms in the gene encoding macrophage migration inhibitory factor are associated with gram-negative bacteremia in older adults. Journal of Infectious Diseases 2014;209(5):764-768. RD834515 (2013)
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  • Journal Article De Arras L, Seng A, Lackford B, Keikhaee MR, Bowerman B, Freedman JH, Schwartz DA, Alper S. An evolutionarily conserved innate immunity protein interaction network. Journal of Biological Chemistry 2013;288(3):1967-1978. RD834515 (2013)
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  • Journal Article Gao Z, Dosman JA, Rennie DC, Schwartz DA, Yang IV, Beach J, Senthilselvan A. NOS3 polymorphism, lung function, and exposure in swine operations: results of 2 studies. Journal of Allergy and Clinical Immunology 2014;134(2):485-488. RD834515 (2013)
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  • Journal Article Henao-Martinez AF, Agler AH, LaFlamme D, Schwartz DA, Yang IV. Polymorphisms in the SUFU gene are associated with organ injury protection and sepsis severity in patients with Enterobacteriacea bacteremia. Infection, Genetics and Evolution 2013;16:386-391. RD834515 (2013)
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  • Journal Article Jing J, Yang IV, Hui L, Patel JA, Evans CM, Prikeris R, Kobzik L, O'Connor BP, Schwartz DA. Role of macrophage receptor with collagenous structure in innate immune tolerance. Journal of Immunology 2013;190(12):6360-6367. RD834515 (2013)
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  • Journal Article Kelada SN, Wilson MS, Tavarez U, Kubalanza K, Borate B, Whitehead GS, Maruoka S, Roy MG, Olive M, Carpenter DE, Brass DM, Wynn TA, Cook DN, Evans CM, Schwartz DA, Collins FS. Strain-dependent genomic factors affect allergen-induced airway hyperresponsiveness in mice. American Journal of Respiratory Cell and Molecular Biology 2011;45(4):817-824. RD834515 (2013)
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  • Journal Article Lai PS, Hofmann O, Baron RM, Cernadas M, Meng QR, Bresler HS, Brass DM, Yang IV, Schwartz DA, Christiani DC, Hide W. Integrating murine gene expression studies to understand obstructive lung disease due to chronic inhaled endotoxin. PLoS One 2013;8(5):e62910. RD834515 (2013)
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  • Journal Article Long H, O'Connor BP, Zemans RL, Zhou X, Yang IV, Schwartz DA. The Toll-like receptor 4 polymorphism Asp299Gly but not Thr399Ile influences TLR4 signaling and function. PLoS One 2014;9(4):e93550 (10 pp.). RD834515 (2013)
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  • Journal Article Oakes JL, O'Connor BP, Warg LA, Burton R, Hock A, Loader J, LaFlamme D, Jing J, Hui L, Schwartz DA, Yang IV. Ozone enhances pulmonary innate immune response to a Toll-like receptor-2 agonist. American Journal of Respiratory Cell and Molecular Biology 2013;48(1):27-34. RD834515 (2013)
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  • Journal Article Warg LA, Oakes JL, Burton R, Neidermyer AJ, Rutledge HR, Groshong S, Schwartz DA, Yang IV. The role of the E2F1 transcription factor in the innate immune response to systemic LPS. American Journal of Physiology-Lung Cellular and Molecular Physiology 2012;303(5):L391-L400. RD834515 (2013)
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  • Journal Article Yang IV, Alper S, Lackford B, Rutledge H, Warg LA, Burch LH, Schwartz DA. Novel regulators of the systemic response to lipopolysaccharide. American Journal of Respiratory Cell and Molecular Biology 2011;45(2):393-402. RD834515 (2013)
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  • Supplemental Keywords:

    Endotoxin, exposure, children, asthma, risk, health effects, susceptibility, sensitive populations, genetic pre-disposition, genetic polymorphism, indoor air, dose-response, ozone, remediation, human health,health, health effects, biology, health risk assessment, children's health, allergens/asthma, asthma indices, intervention, Health, Scientific Discipline, HUMAN HEALTH, Health Risk Assessment, Allergens/Asthma, Health Effects, Biology, asthma, sensitive populations, asthma triggers, endotoxin, children, airway inflammation, allergic response

    Progress and Final Reports:

    Original Abstract
  • 2010
  • 2011
  • 2012
  • 2013
  • 2014 Progress Report
  • 2015
  • Final

  • Main Center Abstract and Reports:

    RD834515    Denver Children’s Environmental Health Center - Environmental Determinants of Airway Disease in Children

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    RD834515C001 Endotoxin Exposure and Asthma in Children
    RD834515C002 Environmental Determinants of Early Host Response to RSV
    RD834515C003 Environmental Determinants of Host Defense