Awardees and Abstracts
2004 Awardees
Senior Awards
Vivek Malhotra, Ph.D.University of California, San Diego (Now at Center for Genomic Regulation, Barcelona)
Biological Sciences
Molecular Basis of Mucin Secretion in Airway Goblet Cells
Daphne Preuss, Ph.D.University of Chicago/HHMI
Molecular Genetics and Cell Biology
Immune Responses to Pollen Surface Components: Implications for Allergy and Asthma
Klaus Rajewsky, M.D.CBR Institute for Biomedical Research (Now at Max Delbrueck Center for Molecular Medicine, Berlin)
Cellular and Molecular Dissection of the Roles of NF-kB Activation Pathways and NKT Cell Autoreactivity in Asthma
William C. Sessa, Ph.D.Yale University School of Medicine
Pharmacology
Role of Nogo Isoforms in Airway Hypersensitivity and the Pathogenesis of Asthma
Ann-Bin Shyu, Ph.D.University of Texas HSC at Houston
Biochemistry and Molecular Biology
Cytokine and Chemokine mRNA Turnover and Airway Inflammation
Junior Awards
Sean B. Fain, Ph.D.University of Wisconsin, Madison
Medical Physics
Non-Invasive Imaging of Airway Closure, Edema and Cellular Activation in an Animal Model of Asthma
Peter J. Murray, Ph.D.St. Jude Children's Research Hospital
Infectious Diseases
Role of Macrophage Arginase in Asthma
Troy D. Randall, Ph.D.Trudeau Institute (Now at University of Alabama)
BALT, the Hygiene Hypothesis and the Development of Asthma
Yong-Rui Zou, Ph.D.Columbia University (Now at Feinstein Institute for Medical Research)
Microbiology
The Pathophysiological Role of CXCR4 in the Adult Immune System and in Asthma
Extension Award
Michael R. Blackburn, Ph.D.University of Texas HSC at Houston
Biochemistry and Molecular Biology
Adenosine Deaminase Enzyme Therapy and Asthma Exacerbations
Michael R. Blackburn, Ph.D. — 2004 Extension Award
University of Texas HSC at Houston
Adenosine Deaminase Enzyme Therapy and Asthma Exacerbations
Asthma is an inflammatory disease of the airways that is associated with acute and resolvable bronchonstriction, as well as chronic airway remodeling. Whereas pathways that lead to initial inflammatory cascades in asthma have been elucidated, little is known about the pathways associated with asthma exacerbations or chronic airway remodeling. Similarly, current asthma treatments are effective at controlling initial inflammatory insults in the lung; however, treatment strategies to control aspects of ongoing disease are lacking. Adenosine is a signaling nucleoside that is generated during cellular stress and damage. Accordingly, adenosine levels are elevated in the lungs of asthma patients. Numerous studies have implicated adenosine as a pro-inflammatory signal in the asthmatic lung. Our studies in models of adenosine-induced and Th2 cytokine induced airway injury have demonstrated that adenosine deaminase (ADA) enzyme therapy is effective in lowering adenosine levels in the injured lung and can improve the status of airway inflammation and remodeling. However, the efficacy of ADA enzyme therapy in the treatment of allergen-induced airway disease has not been examined. The specific hypothesis that will be addressed in this project is that ADA enzyme therapy can prevent and reverse exacerbations of allergen-induced airway inflammation and remodeling. To address this hypothesis, we will utilize a chronic model of ovalbumin-induced airway inflammation in the mouse to test the benefit of ADA enzyme therapy in response to allergen in the lung. Given that adenosine is elevated in the asthmatic lung and that lowering lung adenosine can improve airway inflammation and remodeling in adenosine-dependent lung disease in mice, it seems reasonable to pursue the development of ADA enzyme therapy for the treatment of asthma. Testing the efficacy of this therapy in allergen-based models will greatly facilitate this goal.
David E. Clapham, M.D., Ph.D. — 2004 Senior Award
Children's Hospital of Boston/HHMI
TRP Ion Channels as New Targets in Asthma
The hypothesis of this proposal is that the airway smooth muscle Transport Receptor Potential (TRP) channels are activated in the inflammatory and mechanically mediated changes that result in asthma. Based on their tissue distribution and results from genetically targeted mice, TRP channels regulate airway smooth muscle as well as airway epithelia and blood-derived inflammatory cells. As newly identified Ca2+-permeable ion channels, TRPs have not been the target of previous asthmatic therapies. In the proposed studies we will determine the role of airway smooth muscle TRP channels in airway constriction and remodeling.
The specific Aims of this proposal are to: 1) Determine the subtypes of transient receptor potential (TRP) channels present in the human and mouse airway smooth muscle. 2) Test whether blockade or elimination of TRP channels alters airway smooth muscle contractility and/or cell morphology. 3) Determine the long-term consequences of the elimination of TRP channels in the airway smooth muscle of genetically targeted mice.
Sean B. Fain, Ph.D. — 2004 Junior Award
University of Wisconsin, Madison
Non-Invasive Imaging of Airway Closure, Edema and Cellular Activation in an Animal Model of Asthma
Non-invasive imaging tools have begun to reveal a complex regional patho-physiology of asthma not captured by traditional pulmonary function measures. Moreover, the fusion of functional images from multiple modalities can map the regional physiology during an allergic response. Previous work in our laboratory has combined hyperpolarized gas MRI with proton MRI, and micro-PET to measure regional ventilation, inflammation, and cell metabolism for an allergic inflammation model of asthma in Brown Norway (BN) rats. In preliminary experiments, regional physiology measured after segmental allergen challenge is consistent with the location and cell concentrations observed with histology. Our hypothesis is that imaging can quantify the location and severity of airway response on a regional basis for both single time-point and longitudinal studies in a chronic asthma model. We propose to refine and apply these functional imaging methods to measure the extent and severity of inflammation relative to regions of airway narrowing and closure in chronic asthma. The specific aims of this proposal are: (1) To develop noninvasive imaging tools for functional imaging of airway obstruction, inflammation, and cellular activation associated with asthma in an allergic inflammation model, (2) To validate the measurements derived from the imaging tools developed in Aim 1 with histology, and (3) To measure the severity, extent, and pattern of ventilation changes, gas trapping, and inflammation in a model of chronic asthma. These experiments will improve our understanding of the timing and role of inflammation and cell activation in the onset and location of airway narrowing and closure.
Vivek Malhotra, Ph.D. — 2004 Senior Award
University of California, San Diego (Now at Center for Genomic Regulation, Barcelona)
Molecular Basis of Mucin Secretion in Airway Goblet Cells
Mucus hyper-secretion is a major cause of pathology in asthma and in several respiratory diseases. It leads to plugging of small airways and a dramatic reduction of airflow. Nearly all cases of lethal asthma are associated with airway occlusion by mucus plugs. A pharmacological control of mucus secretion would greatly improve asthmatic symptoms and reduce long-term damages. At present, there is no such treatment available. Mucus is composed mostly of mucins, which are secreted largely by surface epithelial goblet cells. Goblet cells accumulate mucins in secretion granules and release their content upon stimulation. The molecular mechanisms controlling mucin granule formation and release are poorly understood at present. We plan to develop an airway goblet cell line where exogenous horseradish peroxidase enzyme is targeted to mucin granules, allowing for a simple, robust and quantitative assay of secretion. We will use this assay to study the formation and exocytosis of mucin granules. We will also use this system to screen specific gene families by siRNA and perform a high-throughput screen of chemical libraries. These approaches will give us a better understanding of mucin granules biology and reveal new regulatory genes of mucin secretion and new chemicals affecting this process. Furthermore, the chemical library screen could yield entirely new leads for treatment of obstructive pulmonary diseases in general and asthma in particular.
Tobias Meyer, Ph.D. — 2004 Senior Award
Stanford University
Mast Cell Signaling Networks
Mast cells are key players in acute allergic reactions and have also been strongly implicated in the pathogenesis of asthma. Mast cells express the FcεR1 receptors which bind IgE antibodies with high affinity. When multivalent antigens contact IgE bound to FcεR1 receptors, the receptors aggregate, triggering a signaling cascade that leads to the extracellular release of histamine, serotonin, proteases, cytokines, and other mediators from the mast cell's cytoplasmic granules. Many of these secreted mediators have been found to enhance airway inflammation and to cause bronchial and vascular smooth muscle contraction. The secretion response in mast cells occurs rapidly - within minutes - and is regulated by a complex network of potentially tens of second messengers and hundreds of signaling proteins. In this proposal, we will use novel high-throughput single-cell imaging and analysis techniques, together with comprehensive DNA expression and siRNA signaling protein libraries, to carry out large-scale primary and secondary screens of the ~2000 signaling proteins estimated to be in mast cells and to quantitate their effects on mast cell secretion and intermediate signaling. Our strategy will likely lead to two important results. First, we will identify new players in the mast-cell signaling system that can explain how FcεRI activation triggers histamine release. Second, the timecourse data obtained simultaneously at multiple points in the signaling network will be useful to identify the overall modular structures and cross-talk in the mast cell signaling system. Both results will provide new insights into weak spots in the mast cell signaling response that can then be exploited as potential drug targets.
Peter J. Murray, Ph.D. — 2004 Junior Award
St. Jude Children's Research Hospital
Role of Macrophage Arginase in Asthma
Recent progress in asthma research has identified dramatic increases in genes involved in arginine metabolism in inflamed lungs. Arginase I (Arg I) is one of the most highly increased genes and expression of Arg I is most prominent in the lung inflammatory cells, including macrophages. We have been elucidating the mechanisms that lead to Arg I induction in macrophages. The strongest signals to switch on Arg I expression are the Th2 cytokines IL-4 and IL-13 that dominate the immune response in asthmatic lungs.
Through the hydrolysis of arginine, Arg I can control tissue repair by promoting collagen biosynthesis and regulate nitric oxide (NO) levels. A gap in understanding Arg I function in asthma is linking the relationship between the increase in expression and the production of downstream metabolites that could influence different elements of asthma such as tissue repair and NO levels. To understand the role of macrophage Arg I in asthma, we have created mice that specifically lack, or overexpress, Arg I in macrophages. The proposed research will use these mice in asthma models to test the possibility that macrophage Arg I controls tissue repair and remodeling by controlling collagen biosynthesis (Aim 1), reducing NO levels in the inflamed lung and thereby reducing inflammatory damage (Aim 2) and potentially regulating the overall asthmatic response (Aim 3). We predict these studies will establish macrophage Arg I as an important element in asthma pathogenesis
Sem H. Phan, M.D., Ph.D. — 2004 Senior Award
University of Michigan
Bone Marrow Progenitor Cells in Airway Remodeling
Airway remodeling in asthma contributes to bronchial hyperreactivity and reversible airflow obstruction. The overall objective of this project is to elucidate the origin of the fibroblasts/myofibroblasts involved in airway remodeling. Published and preliminary studies suggest that bone marrow derived progenitor cells can repopulate a variety of distal organs, including the lung. For instance, bone marrow derived cells can give rise to lung endothelial cells and fibroblasts, as well as alveolar and bronchial epithelium. Thus the central hypothesis of the project is that certain subpopulations of fibroblasts/myofibroblasts involved in airway remodeling are derived from bone marrow progenitor cells recruited by chemokine signals emitted by activated airway cells. To test this hypothesis four Specific Aims are proposed. First, the extent and kinetics of recruitment of bone marrow derived progenitor cells into remodeling airway wall will be examined in a murine cockroach allergen-induced model of asthma using GFP expressing bone marrow chimera mice. Second, the identity of the bone marrow progenitor cells will be determined. Third, the identity of the chemokines responsible for cell recruitment and their cellular source will be determined. Fourth, the significance of these recruited cells will be examined by evaluating the effects of abrogating their recruitment on airway remodeling. In this manner using a combination of immunological, cell biological and molecular techniques, the role of bone marrow progenitor cells in airway remodeling and the mechanisms of their recruitment can be elucidated. If this is determined to be important, then potentially novel therapeutic approaches could be envisioned for future studies.
Daphne Preuss, Ph.D. — 2004 Senior Award
University of Chicago/HHMI
Immune Responses to Pollen Surface Components: Implications for Allergy and Asthma
Traditional searches for pollen allergens have identified cytoplasmic proteins encoded by pollen cDNAs, but have overlooked much of the extracellular pollen matrix, a region where allergens likely reside. The pollen extracellular matrix is encoded by cDNAs expressed in developing floral tissue, and its proteins contain hydrophobic domains and are typically absent from commercial pollen extracts. My research has focused on the pollen surface; we have identified Arabidopsis mutations that alter lipid content, examined protein functions and wall composition, identified the entire extracellular proteome, and used genomics to characterize pollen matrix components from several species. Our preliminary studies have shown that Arabidopsis pollen surface proteins elicit an immediate inflammatory response in naïve rabbits and cause non-hypersensitive animals to produce antisera that are ~200-fold more potent than those raised against typical cytosolic plant proteins.
This proposal has three specific aims: 1) Extracellular and intracellular pollen fractions will be prepared from several species and probed with sera from individuals living in urban and rural environments; SNP genotypes and pedigrees will be used to examine genetic correlations with IgE binding. 2) Fractions containing allergens will be purified; we will isolate non-protein allergens and clone genes corresponding to novel protein allergens. 3) In collaborations at the University of Chicago, purified extracellular allergens will be tested for their ability to alter pulmonary physiology in mice or tostimulate responses in dendritic cells. This research could enhance allergy and asthma therapy through improved diagnostics or therapeutics that mitigate symptoms or promote immune tolerance.
Klaus Rajewsky, M.D. — 2004 Senior Award
CBR Institute for Biomedical Research (Now at Max Delbrueck Center for Molecular Medicine, Berlin)
Cellular and Molecular Dissection of the Roles of NF-kB Activation Pathways and NKT Cell Autoreactivity in Asthma
In asthma, allergens induce a complex interplay between various cell-lineages and tissues, ultimately leading to airway inflammation, obstruction, hyperreactivity and remodeling. These processes are accompanied and sustained by extensive reprogramming of gene expression. NF-κB proteins are a family of transcription factors that activate transcription of multiple genes involved in inflammation. These transcription factors play diverse roles in individual cell-types; therefore it is of great interest to elucidate their cell-type specific role in conditions of allergic lung inflammation. Recently it was discovered that Natural Killer T cells, whose generation depends on two forms of NF-κB activation, are essential for the induction of asthma in various mouse models. To date it is not clear whether activation of these autoreactive T cells, normally kept in check through inhibitory mechanisms, relies on recognition of lipid-antigens by their TCR, or on signaling through engagement of other receptors in the context of the immune response. Conditional gene targeting offers spatial and temporal control of genetic manipulations in the mouse. We propose to utilize the unique features of this technology, to explore aspects of NF-κB activation and NKT cell autoreactivity in the context of asthma as follows:
Aim 1: To identify the role of individual NF-κB activation pathways in cell-types and tissues relevant for the induction of airway inflammation, hyperresponsiveness and remodeling.
Aim 2: To analyze antigen recognition, differentiation potential and autoreactivity mediated by a canonical NKT cell receptor switched on in conventional T cells, replacing their original TCR.
Troy D. Randall, Ph.D. — 2004 Junior Award
Trudeau Institute (Now at University of Alabama)
BALT, the Hygiene Hypothesis and the Development of Asthma
The incidence of asthma has dramatically increased over the last 20 years and coincides with improved hygiene and a reduced incidence of childhood infections. This has led to the hygiene hypothesis, which states that the immune system does not receive proper stimulation early in development and is subsequently unable to respond appropriately to aeroallergens. Recent observations suggest that neonatal exposure to pathogens or microbial products, such as endotoxin, substantially reduces the risk of developing asthma. However, it is unclear how this effect is achieved. We hypothesize that prior to the development of vaccines to many of the childhood infections, most children were exposed to multiple respiratory infections and that these infections triggered the development of Bronchus Associated Lymphoid Tissue (BALT). Unlike other lymphoid organs, BALT develops postnatally in response to inflammation or infection. However, once developed, BALT persists due the activities of homeostatic chemokines, such as SLC and BLC. Thus, we suspect that BALT is actually a "normal" component of the lymphoid system that is missing or underdeveloped in most people (and mice) due to the lack of the normal triggers that initiate its development. We also hypothesize that once developed, BALT allows the respiratory tract to respond to aeroallergens in a way that does not promote type II inflammation or lead to asthma. Therefore, the experiments in this proposal will determine the microbial triggers that initiate BALT development and test whether the presence of BALT alters immune responses to aeroallergens and reduces the incidence or severity of asthma.
William C. Sessa, Ph.D. — 2004 Senior Award
Yale University School of Medicine
Role of Nogo Isoforms in Airway Hypersensitivity and the Pathogenesis of Asthma
Using proteomics as an inductive approach for discovery, our lab has identified the protein Nogo-B highly expressed in pulmonary endothelial cells, airway smooth muscle cells and in pulmonary epithelial cells. The functional role of this Nogo isoform is unknown. In preliminary data, we show that the amino terminus of Nogo-B, unlike Nogo-A, promotes the adhesion of endothelial and smooth muscle cells, and serves as a chemoattractant for endothelial cells while antagonizing PDGF-induced smooth muscle cell migration. Moreover, in a paradigm of vascular injury, the loss of Nogo-A/B results in an exaggerated inflammatory response and neointimal proliferation, and therapeutic gene transfer of Nogo-B rescues these knockout phenotypes. Thus, goal of this proposal is to examine the role of Nogo-B as a newly found endogenous regulator of vascular and epithelial cell homeostasis in the lung. We surmise that the loss of Nogo-function contributes to both the vascular and inflammatory aspects of asthma. In this grant, we plan to generate transgenic models to overexpress soluble Nogo in the lung, examine the pathogenesis of asthma in both Th2 cytokine driven and allergic forms of asthma and to develop therapeutic strategies for delivering Nogo in different models of asthma.
Ann-Bin Shyu, Ph.D. — 2004 Senior Award
University of Texas HSC at Houston
Cytokine and Chemokine mRNA Turnover and Airway Inflammation
The conservation of an AU-rich RNA-destabilizing element (ARE) in the 3' non-translated regions of mRNAs coding for ~90% of cytokines and chemokines, including all Th2-type cytokines, suggests that regulation of cytokine and chemokine mRNA turnover via AREs is critical in determining the duration and level of cytokine and chemokine production. It is clear that ARE-containing cytokine mRNAs are differentially stabilized after lymphoid cell activation. Several ARE-binding proteins (ARE-BPs) are known to either accelerate or block ARE-mediated mRNA decay. We hypothesize that rapid decay of cytokine and chemokine mRNAs mediated by the AREs are compromised by changes in ARE-BP functions during allergic asthma, leading to persistently high levels of cytokines and chemokines. The specific aims are: 1) Develop an in vitro model to study the mechanism and regulation of chemokine and Th2-type cytokine mRNA turnover under conditions relevant to allergic responses in asthma; 2) Identify destabilizing and stabilizing ARE-BPs that regulate bronchial epithelial chemokine and Th-2 type cytokine mRNA stability during allergic inflammatory responses; 3) Characterize the dynamic interplay between destabilizing and stabilizing ARE-BPs in regulating chemokine and cytokine mRNA levels; 4) Identify cell type(s) critical to compromised ARE-mediated RNA decay, leading to development of allergic inflammation in an established mouse model. These studies should yield novel and crucial insights into major basic mechanisms by which asthma-related cytokine and chemokine mRNA stability is controlled, an important area of asthma research that is presently ill studied.
Yong-Rui Zou, Ph.D. — 2004 Junior Award
Columbia University (Now at Feinstein Institute for Medical Research)
The Pathophysiological Role of CXCR4 in the Adult Immune System and in Asthma
Asthma is an inflammatory disease characterized by polarized Th2 responses that leads to excessive IgE production and increased recruitment of leukocytes into the airways. An important chemokine involved in the progression of asthmatic inflammation has been shown to be CXCL12, as the treatment of allergen-sensitized mice with specific antagonists to its receptor, CXCR4, significantly reduces eosinophilia and suppresses airway hypersensitivity. We propose to analyze the mechanism underlying CXCR4-mediated pathogenesis in the mouse model of asthma.
CXCR4-/- mice die in uterus, thus preventing analysis of the later function of CXCR4 in the immune system under physiological and pathological conditions. We have generated several mouse strains in which the CXCR4 gene has been deleted respectively in granulocytes, macrophages, B-lineage cells or T lymphocytes. First, we will characterize whether the inactivation of CXCR4 affects development, localization and function of these cells. Second, we will examine asthmatic responses in allergen-sensitized mice in which CXCR4 is inactivated in individual leukocyte lineages. Lastly, using additional mouse strains that carry the CXCR4 gene mutated in sequences encoding different conserved signaling domains, we will determine the precise biological effects and pathogenic roles contributed by different signaling pathways downstream of CXCR4. Together, these studies will help to identify the key leukocyte lineage and the important signaling pathway that contribute to the progression of asthmatic responses. Results obtained from our studies could facilitate the development of new therapeutic strategies in immune disorders.