Awardees and Abstracts

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2010 Awardees

Senior Awards

Daniel J. Dumont , Ph.D.
Sunnybrook Research Institute
Molecular and Cellular Biology
Manipulating the Angiopoietin Signaling Axis to Treat Asthma

Evan E. Eichler , Ph.D.
University of Washington
Genome Sciences
Comprehensive Analysis of the Effects of Copy Number Variation on Asthma

Stuart B. Hooper , Ph.D.
Monash University
Monash Institute for Medical Research
Imaging Lung Motion for Studying the Dynamics of Asthma and Its Treatments

Richard M. Maizels , Ph.D.
University of Edinburgh
Institute of Immunology and Infection Research
Suppression of Airway Allergy by Products of Helminth Parasites

Andrew N. McKenzie , Ph.D.
Medical Research Council
Laboratory of Molecular Biology
The Role of Nuocytes in the Regulation of Asthma

Kevin J. Tracey , M.D.
Feinstein Institute for Medical Research
Laboratory of Biomedical Science
Targeting Nicotinic Acetylcholine α7 Receptors in Allergic Lung Inflammation

Dario A. Vignali , Ph.D.
St. Jude Children's Research Hospital
Immunology
Interleukin-35, iTr35 and Allergic Asthma

H. Eric Xu , Ph.D.
Van Andel Research Institute
Structural Sciences
Molecular Mechanism of Dissociated Glucocorticoids

Early Excellence Awards

Brian A. Cobb, Ph.D.
Case Western Reserve University
Pathology
Commensal Antigen-induced T Regulatory Cells in Airway Tolerance

Andre Levchenko , Ph.D.
Johns Hopkins University
Biomedical Engineering
Integrative Biology-based Investigation of Interplay between Inflammatory Signaling and Single-cell Mechanics of Human ASM

Carla M.P. Ribeiro , Ph.D.
University of North Carolina, Chapel Hill
Medicine
IRE1β-dependent Airway Mucin Production and ATP Release: A New Pathway in Asthma

Thaddeus S. Stappenbeck , M.D., Ph.D.
Washington University, St. Louis
Pathology and Immunology
Role of Microbial Driven Autophagy within Respiratory Airways in the Pathology of Asthma

Extension Award

Sven-Eric Jordt, Ph.D.
Yale University
Pharmacology
Sensory Chemoreceptors in Asthma and Airway Hyperresponsiveness

2010 Awards Project Abstracts

Brian A. Cobb, Ph.D. — 2010 Early Excellence Award

Case Western Reserve University

Commensal Antigen-induced T Regulatory Cells in Airway Tolerance

Chronic airway hyperresponsiveness and chronic inflammation are hallmarks of asthma. While Th2 cells and a number of innate immune cells are generally responsible for the induction of asthmatic airway inflammation, the production of IL-10 by T regulatory (Treg) cells is thought to play a role in blocking inflammation. Epidemiologic studies have shown that immunologic events early in life can have a strong impact on the development of asthma, where early exposure to commensal bacteria is linked to protective regulatory pathways that reduce the risk of developing asthma later in life. These observations provide a link between exposure to commensal bacteria and the induction of IL-10+ Treg cells that can maintain airway homeostasis. Carbohydrate antigens expressed by some commensal bacteria have been shown to down-regulate inflammation in murine models of inflammatory bowel disease. These T cell-dependent antigens also induce human Treg cells in vitro, characterized by suppressive activity against local inflammation via IL-10 secretion. Since exposure to commensal antigens is a beneficial factor in asthma, these observations fit well into the "hygiene hypothesis" whereby exposure to these antigens could provide a protective immune response (Tregs) that reduces the risk of asthma. This proposal therefore seeks to explore the Treg population as a function of exposure to commensal bacteria and how these cells may ameliorate airway inflammation in a murine model of asthma. If successful, these studies could shed light upon the commensal antigens that contribute to airway homeostasis and how they protect against asthma-associated inflammation.

Daniel J. Dumont , Ph.D. — 2010 Senior Award

Sunnybrook Research Institute

Manipulating the Angiopoietin Signaling Axis to Treat Asthma

Asthma is an inflammatory disease of the airways that can lead to vascular remodeling and ultimately decreased lung function. The exact underlying cause of this response remains unclear; however, the development of animal models that recapitulate several of the key attributes of asthma provides a fertile platform from which to uncover new therapeutics. One class of growth factors, the Angiopoietins, are known to affect both the endothelial and inflammatory response to allergens. We (Drs. Dan Dumont and Paul Van Slyke) have developed a completely synthetic Angiopoietin-ligand mimetic, called Vasculotide, and have shown that it recapitulates the cellular signaling properties of Angiopoietin 1. Based on the findings that Angiopoietin 1 is known to protect animals from airway inflammation and hyperreactivity in asthma, we propose to test the following hypothesis: “Vasculotide, a fully synthetic Angiopoietin-1 ligand mimetic, will ameliorate experimentally induced asthma.” If these proof-of-principle studies show efficacy, they will provide a platform from which to further develop Vasculotide as a therapeutic for asthma.

Evan E. Eichler , Ph.D. — 2010 Senior Award

University of Washington

Comprehensive Analysis of the Effects of Copy Number Variation on Asthma

Asthma has a strong genetic component. Genome-wide association studies of asthma with hundreds of thousands of common single nucleotide polymorphisms (SNPs) have revealed some asthma-susceptibility variants. However, these loci explain very little of the genetic risk of developing this disease, suggesting that other types of genetic variation and/or other models of disease may play a role. Copy-number variants (CNVs) are large insertions, duplications, or deletions of DNA sequence. Interestingly, several lines of evidence suggest that CNVs could be important in the development of asthma. First, many associations between common CNVs and disease have been with immune-related phenotypes, such as lupus, Crohn’s disease, and psoriasis. Furthermore, immune genes are over represented in CNV regions. Given the unexplained genetic risk for asthma and the data implicating CNVs in immune-related phenotypes, we hypothesize that common CNVs are an important source of genetic risk for asthma. The broad objectives of this proposal are to perform a thorough association analysis of common CNVs with asthma and to identify genetic variants that influence this disease. We will perform initial association studies in a cohort of Hutterites. The Hutterites have a communal lifestyle, which greatly reduces the environmental differences among individuals and makes this population ideal for studies of complex traits. We will also conduct a thorough search for rare copy-number variants by performing whole-genome sequencing in asthmatic and non-asthmatic Hutterite individuals. Discovery of CNVs associated with asthma will lead to a better understanding of the biology of this disease and potential therapeutic targets.

Stuart B. Hooper , Ph.D. — 2010 Senior Award

Monash University

Imaging Lung Motion for Studying the Dynamics of Asthma and Its Treatments

Although most lung diseases adversely affect airflow during breathing, little is known about normal lung motion and how it is altered by disease. Current imaging techniques provide little or no information on lung motion and components of lung function, mainly because they require the lung to be still to avoid movement artefacts. We have recently shown that X-ray velocimetry can track lung tissue movement to reconstruct velocity fields that define speed and direction of regional lung motion throughout a breath. Regional maps of expansion and average time of expansion are generated which reveal lung regions with abnormal movement caused by disease. We have demonstrated the capability of this technique to detect and quantify motion within both diseased and healthy regions of lung tissue. Importantly, this technique has a higher certainty of detection of lung disease in its earliest and most subtle stages than either plethysmography or histological sections. This proposal aims to (1) exploit the regional nature of this imaging capability to gain a more detailed, regional understanding of the pathology of asthma and (2) gain previously unavailable, real-time, regional, highly-sensitive data on the efficacy of pharmacological treatment strategies as a function of both the pharmacological agent and delivery method.

Sven-Eric Jordt, Ph.D. — 2010 Extension Award

Yale University

Sensory Chemoreceptors in Asthma and Airway Hyperresponsiveness

In our AAF-funded research we identified the sensory neuronal ion channel TRPA1 as a major determinant of the asthmatic phenotype in mice. In the murine ovalbumin (OVA) asthma model, TRPA1-deficient mice showed dramatically reduced levels of eosinophilia, goblet cell hyperplasia, cytokines and airway hyperreactivity. These effects were recapitulated by treatment of OVA wild-type mice with a TRPA1 antagonist, HC-030031. We also demonstrated efficacy of HC-030031 in the murine aspergillus asthma model. Mechanistic analysis of TRPA1-deficient mice showed diminished airway release of inflammatory neuropeptides such as CGRP. Thus, we identified TRPA1 antagonists as new anti-asthmatic agents with efficacy against multiple allergens.

The class of TRPA1 antagonists represented by HC-030031 has little oral bioavailability and lacks the specificity desired for a drug development candidate. In 2009 a novel chemical class of TRPA1 antagonists was identified, represented by A-967079 that is orally available in rodents. For our extended studies, we propose to (Aim I) examine the efficacy and potency of A-967079 in murine models of asthma. Proof of anti-asthmatic activity of two separate chemical classes of TRPA1 antagonist should provide a solid incentive for further development of TRPA1 antagonists for the treatment of asthma.

Based on our mechanistic studies we also propose to (Aim II) study the effects of novel CGRP antagonists in murine asthma models. Recently, phase III clinical trials showed promising efficacy and potency of CGRP antagonists for inflammatory pain. We hope to spur further development and clinical efforts by showing that asthma is an additional indication for CGRP antagonists.

Andre Levchenko , Ph.D. — 2010 Early Excellence Award

Johns Hopkins University

Integrative Biology-based Investigation of Interplay between Inflammatory Signaling and Single-cell Mechanics of Human ASM

Stiffening and higher contractility of airway smooth muscle (ASM) cells can dramatically contribute to the symptomatic changes in acute asthma attacks and chronic asthma progression. The main hypothesis underlying this study is that inflammation can influence the ASM mechanics in two ways: indirectly, through fibroblast-mediated ECM remodeling. and directly, by affecting the pathways controlling ASM contractility/mechanics. These effects can be synergistic, requiring a combined treatment affecting both fibroblasts and smooth muscle cells. For instance, a combination treatment might utilize the drugs affecting not only the inflammatory stimuli, but also the signaling pathways in ASMs, including those leading to modification of cytoskeleton organization, e.g., those targeting the RhoA pathway, if these drugs can be delivered specifically. We plan to test this hypothesis by using a combination of novel techniques, relying on our experience in micro- and nano-fabrication of high-throughput and quantitative cell analysis platform, with precise delivery of biochemical and biomechanical inputs and precise measurement, analysis and modeling of the resulting data. In particular, we will mimic the ECM re-organization by nano-structuring, in a bio-mimetic fashion, the cell adhesion substrata. We will also test a battery of inflammatory stimuli for their ability to affect the biomechanical state of ASM cells and their subsequent interaction with different ECM-like structures. We will also use these platforms to screen the ability of pharmacological agents to reverse the potentially deleterious signaling and biomechanical changes in ASM cells. We anticipate that this novel, bioengineering-integrative biology approach can provide new and anticipated insights into asthma biology.

Richard M. Maizels , Ph.D. — 2010 Senior Award

University of Edinburgh

Suppression of Airway Allergy by Products of Helminth Parasites

Asthma is on the rise in the developed world, while in developing countries its prevalence remains low. Epidemiological studies have shown that asthma prevalence negatively correlates with parasitic infection, leading to the hypothesis that parasites can suppress allergic immune responses. We (and others) have previously shown that parasitic infection can suppress pathology in a mouse model of asthma. Our preliminary data show that this suppression of asthma pathology by parasite infection can be replicated by administration of the excretory/secretory products of Heligmosomoides polygyrus (HES). Therefore we hypothesize that HES contains immunomodulators that could be novel therapeutics for the treatment of asthma in humans. This research proposal aims to confirm the asthma-protective functions of HES and to dissect the mechanism of the suppression. We will examine the effects of HES on cytokine production, dendritic cells, basophils, eosinophils, macrophages and regulatory T and B cells during the initiation of asthma in vivo. Following the results of our initial experiments, we will focus on in vitro assays of affected mouse and human immune cell subsets to identify the target pathways of immunomodulatory activity in HES. We will then fractionate HES by column chromatography, and test HES fractions for immunomodulatory effects, using in vitro screens. Molecules within the immunosuppressive fractions will be identified by mass spectrometry. We will then express each candidate immunosuppressive protein in a range of transgenic systems to replicate the effects of HES with recombinant products. Successful mediators would then be available to be developed as therapeutics for human disease.

Andrew N. McKenzie , Ph.D. — 2010 Senior Award

Medical Research Council

The Role of Nuocytes in the Regulation of Asthma

Innate immunity provides the first line of defence against invading pathogens and provides important cues for the development of adaptive immunity. Type-2 immunity – responsible for protective immune responses to helminth parasites and the underlying cause of the pathogenesis of allergic asthma – consists of responses dominated by the cardinal type-2 cytokines interleukin (IL)-4, IL-5 and IL-13. T cells are an important source of these cytokines in adaptive immune responses, but innate cell sources of type-2 cytokines also exist and remain to be fully elucidated. Using novel Il13eGFP reporter mice, we have identified and functionally characterised a new innate type-2 immune effector leukocyte that we have named the nuocyte. Nuocytes expand in vivo in response to the type 2-inducing cytokines IL-25 and IL-33, and they represent the predominant early source of IL-13 during helminth infection with Nippostrongylus brasiliensis. Using the helminth infection model we have shown that, in the combined absence of IL-25 and IL-33 signalling, nuocytes fail to expand, resulting in a severe defect in worm expulsion that is rescued by the adoptive transfer of in vitro cultured wildtype, but not IL-13-deficient, nuocytes. We believe that this newly identified cell population is likely to play important and previously unappreciated roles in asthma responses by producing substantial levels of IL-13 and IL-5, and by enhancing T cell-mediated responses. We propose to investigate the roles of nuocytes in experimental asthma and so provide a foundation for our understanding of these cells in human asthma.

Carla M.P. Ribeiro , Ph.D. — 2010 Early Excellence Award

University of North Carolina, Chapel Hill

IRE1β-dependent Airway Mucin Production and ATP Release: A New Pathway in Asthma

Asthmatic airways exhibit mucin overproduction and higher levels of ATP. This application proposes a novel mechanism for mucin overproduction and tests whether it contributes to the higher ATP levels in asthma. Airway inflammation induces endoplasmic reticulum stress mediated by activation of Inositol Requiring Enzyme 1 (IRE1β or β). IRE1β is functionally relevant for Th1 cytokine secretion by airway epithelia. Our data now suggest that IRE1β is functionally important in asthma by mediating allergic airway inflammation-triggered mucin overproduction. IRE1β was only found in gut and respiratory tissues expressing mucous cells. Only Clara cells and mucous cells, but not ciliated or alveolar type II cells, expressed IRE1β. Ovalbumin-up-regulated airway mucin production was blunted in IRE1β-/- mice. A strong correlation between IRE1β and mucin production/glycosylation genes was revealed by a gene expression database. Knocking-down IRE1β in mucin-producing Calu-3 cells blunted interleukin-13-induced mucous cell metaplasia. Notably, mucin secretion and ATP release are coupled in Calu-3 cells, and ATP is localized within mucin secretory granules. Thus, IRE1β-dependent increased mucin production/storage may couple to increased ATP storage in mucin granules, and the higher ATP levels in asthmatic airways may derive from mucin granule exocytosis. Our Specific Aims will test the hypothesis that IRE1β is required for mucin production by airway mucous cells, it stimulates mucin transcription and/or regulates genes involved in mucin production or glycosylation, its over-expression potentiates mucin production, and it regulates airway ATP release by regulating the ATP content in mucin granules. These studies may reveal IRE1β as a new therapeutic target for asthma.

Thaddeus S. Stappenbeck , M.D., Ph.D. — 2010 Early Excellence Award

Washington University, St. Louis

Role of Microbial Driven Autophagy within Respiratory Airways in the Pathology of Asthma

Abnormal mucus and mucus secreting cells are an important aspect of the pathology of asthma as mucus plugging is associated with fatalities in this disease. My lab studies pathways that maintain secretory cell function in the intestinal epithelium. Our studies in this organ have uncovered a novel role for indigenous intestinal microbes that stimulate the process of autophagy in the epithelium during injury. We also found that the process of autophagy is critical for highly secretory cells such as colonic goblet cells to maintain their normal function. Thus, we have identified an interesting pathway involving microbial-driven autophagy that can counteract signals during injury that would otherwise alter mucus and mucus secreting cells. The overall goals of this proposal are to apply these ideas to the respiratory epithelium and asthma. Mucus secreting cells in the lung are distinct from intestinal goblet cells, and careful examination of the role of autophagy in respiratory mucus and comparison to the role in the intestine will be informative. We will also determine the role of autophagy in models of asthma and evaluate the role of indigenous airway microbes in their ability to stimulate autophagy in mucus secreting cells. If our overall hypothesis is supported by these studies, we will be in a position to study the mechanism by which autophagy is inhibited during lung injury and the manner in which microbes stimulate autophagy. By understanding this process, we hope to develop new ideas for therapies that can improve mucus cell function during asthma.

Kevin J. Tracey , M.D. — 2010 Senior Award

Feinstein Institute for Medical Research

Targeting Nicotinic Acetylcholine α7 Receptors in Allergic Lung Inflammation

Allergic lung inflammation is induced by exposure to environmental agents that elicit allergic inflammation and transient airway obstruction. The allergic immune response leads to inflammation characterized by infiltration of the airway mucosa with eosinophils, mast cells and lymphocytes. Persistent allergic inflammation is a hallmark of this disease. In the proposed studies we hypothesize that activation of α7 nicotinic acetylcholine receptors (α7nAChR) will attenuate allergen induced lung inflammation. In our preliminary studies we discovered that a selective agonist of the α7nAChR suppresses inflammation in standardized preclinical models including collagen induced arthritis, colitis, endotoxemia, and ischemia-reperfusion injury. To date this approach has not been studied in allergic lung inflammation. In Specific Aim 1, we will study the protective effects of a highly selective α7nAChR agonist in allergic lung inflammation. In Specific Aim 2, we will use α7nAChR knock out mice to determine the influence of α7nAChR on pathogenesis. In Specific Aim 3, using adoptive transfer experiments in knock out mice, we will determine the contribution of bone marrow derived cells to the protective effects of administering α7nAChR agonists. The proposed studies will provide important knowledge that may be used to identify new therapeutic targets for treating inflammatory lung diseases.

Dario A. Vignali , Ph.D. — 2010 Senior Award

St. Jude Children's Research Hospital

Interleukin-35, iTr35 and Allergic Asthma

Regulatory T cells (Tregs) can repress allergic asthma but the molecules and mechanisms used remain unknown. We have shown that interleukin-35 (IL-35: Ebi3/p35) is a potent inhibitory cytokine that is required for maximal Treg activity, can block Th2 proliferation, and has the capacity to generate Foxp3–induced regulatory T cells (iTr35) that have inhibitory activity in vivo. In this project, we will assess the capacity of IL-35 and iTr35 to control allergic asthma and will evaluate their potential as therapeutic modalities. We will utilize our extensive experience in T cell biology to pursue our first asthma research project. We will determine if allergen-specific iTr35 can protect mice from disease, thereby determining if a nature source of IL35 is sufficient to prevent and/or ameliorate allergic asthma. We will also use directed evolution by yeast surface display mutagenesis to generate highly stable IL-35 mutants that retain inhibitory activity and could be evaluated as potential therapeutics.

H. Eric Xu , Ph.D. — 2010 Senior Award

Van Andel Research Institute

Molecular Mechanism of Dissociated Glucocorticoids

Asthma is a chronic inflammatory lung disease that affects 7% of the U.S. population. Glucocorticoids are the most effective and widely used anti-inflammation drugs for the prevention and treatment of asthma, but the long-term use of glucocorticoids can cause diabetes, osteoporosis, and other unwanted side effects. While the anti-inflammation effects of glucocorticoids are mediated through the transrepression activity of the glucocorticoid receptor (GR) on major proinflammatory factors, the undesired side effects are mainly attributed to the transactivation activity of GR. Therefore, the designing of dissociated glucocorticoids that only retain the transrepression activity of GR has immense importance for the treatment of asthma, arthritis, and other inflammatory diseases. The molecular basis of how dissociated glucocorticoids work remains unclear. A lack of structural information of dissociated glucocorticoids bound to GR has hampered the discovery of the next generation of glucocorticoid drugs. Guided by new structural insights from the GR ligand binding domain (LBD), we have identified a novel compound that has the promising properties of a dissociated glucocorticoid. In this project, we propose to use molecular, structural, and chemical approaches to determine the fundamental mechanism of dissociated glucocorticoids. Our specific aims are 1) to use GR LBD mutations to dissociate transrepression from transactivation; 2) to crystallize and determine the structure of the dissociated conformation of GR that favors only transrepression; and 3) to use molecular docking to design a better dissociated glucocorticoid. Achieving these specific aims will provide a rational basis for designing the next generation of anti-inflammation drugs for treating asthma.