Asthma is a chronic inflammatory disorder of the airways involving interaction of cells and mediators, which result in high levels of reactive oxygen and nitrogen species (ROS, RNS). Increase of intracellular glutathione is a response to ROS/RNS, and a critical determinant of cellular tolerance to oxidizing environments. Our preliminary data show that glutathione is increased in asthmatic airways. Here, we hypothesize that assessment of glutathione using the radiopharmaceutical 99mTc-HMPAO (Technetium99m-hexamethylpropylene amine oxime) and single photon emission computed tomography (SPECT), will identify and localize regions of inflammation in asthma. The lipophilic 99mTc-HMPAO is quantitatively converted to a hydrophilic nondiffusible form in the presence of GSH and thus retained within tissues. In aim 1, we optimize methods for SPECT-HMPAO scanning and determine whether HMPAO uptake is related to GSH levels in a murine model of asthma. In aim 2, we identify whether or not 99mTc-HMPAO uptake is different among asthmatics and healthy controls, and determine the temporal change(s) in 99mTc-HMPAO uptake following an acute asthmatic response to allergen. In aim 3, we quantitate inflammation (and glutathione levels) in bronchoscopic samples of lung regions that have high vs. low HMPAO uptake in order to validate that the SPECT-HMPAO image parallels sites of inflammation. Overall, our goal is to develop an innovative and scientifically sound noninvasive method for evaluation of regional inflammation in asthma. Inflammation imaging would be a significant advance highly relevant for asthma research and potentially the clinical care of asthmatic patients.

The soluble epoxide hydrolase (sEH) has recently been demonstrated as a novel therapeutic target for treating inflammation. Control of airway inflammation is critical in asthma treatment, and we propose to investigate sEH as a target for asthma management. We hypothesize that 1) sEH contributes to the development of pulmonary inflammation in a well-established murine ovalbumin (OVA)-induced asthma model and 2) inhibition of sEH can attenuate this response. 3) Metabolomics, especially oxylipin profiling, is an efficient tool to monitor the asthma pathogenesis progress and crosstalk between the metabolic branches of arachidonate cascade. 4) The anti-inflammatory effects of NSAIDs or FLAP inhibitors can be synergized by co-administration with sEH inhibitors in the murine model. This innovative investigation will uncover basic mechanisms and enhance current knowledge of the pathogenesis of asthma through the use of novel metabolomic analyses resulting in quantitative data on arachidonate and linoleate oxylipin metabolites, in addition to traditional analyses of inflammatory cytokines, pulmonary immune cell recruitment, lung function, and other pathological events associated with asthma. Moreover, these data will shed light in the role of the little studied P450 branch of the arachidonate cascade as it interacts with the COX and 5-LOX branches in asthma. From a clinical standpoint, testing the above hypotheses may lead to new biomarkers of asthma diagnosis and treatment. The data also may determine if subsequent clinical trials for sEH inhibitors (sEHI) in asthma are warranted.

Asthma has shown an alarming increase in incidence in the West but this has not been mirrored in developing countries. This is particularly striking, as IgE levels are often greatly elevated in the latter countries due to non-specific induction by universally present parasitic helminths. These observations have led to the idea that helminths may protect against asthma and this is supported by both human epidemiological studies and work investigating the effects of helminths on asthma development in rodents. Consistent with this, we have recently described a helminth-derived molecule – ES-62, that is able to directly inhibit FcεRI-induced degranulation and release of mediators of allergy from human mast cells and also protect mice against FcεRI-mediated mast cell-dependent hypersensitivity in the lungs. ES-62 works by forming a complex with TLR4, leading to sequestration and degradation of PKC-α an important component of the signaling pathway that facilitates degranulation. We now wish to employ ES-62 to address three questions: (i) how does ES-62 subvert PKC-α trafficking to inhibit mast cell degranulation? (ii) Does the effect of ES-62 on mast cells impact on the inflammatory responses associated with chronic asthma? (iii) Can we develop drugs based on ES-62 that target asthma? Overall, the work will increase our understanding at the molecular level of how helminths protect against asthma, provide new information pertinent to understanding the pathogenesis of asthma and finally, develop and test small molecule analogues of ES-62 in models of asthma with the ultimate aim of developing a novel therapeutic.

Eczema (atopic dermatitis) is a common allergic skin inflammation that has a particularly high prevalence among children. Importantly, a large percentage of patients suffering from eczema go on to develop asthma later in life, demonstrating a strong clinical link between these two allergic disorders. Although the susceptibility of eczema patients to asthma is well documented, the mechanism that mediates progression from eczema to asthma is unclear. Using genetic engineering to generate mice with deletion of Notch signaling in their skin, we created mice with chronic skin-barrier defects and subsequent eczema-like disorder. When challenged with inhaled allergen, these mice develop sever asthma, an observation that enabled us to investigate how a skin-specific defect predisposed the lungs to allergic asthma. We identified thymic stromal lymphopoietin (TSLP), a cytokine secreted by barrier-defective skin keratinocytes into the systemic circulation, as the agent sensitizing the lung to allergens. We demonstrated that high systemic levels of skin-derived TSLP were both required and sufficient to render lung airways hypersensitive to allergens even in the absence of skin pathology. Thus, these data suggest that an early treatment of skin-barrier defects to prevent TSLP overexpression and systemic inhibition of TSLP may be crucial in preventing progression from eczema to asthma in patients. Because blocking NfκB and vitamin D signaling is not practical as a preventive strategy, we propose to investigate in detail how the skin senses barrier defects and how it up-regulates and secretes TSLP. Any of these mechanisms (barrier defect sensing, expression, secretion) may contain targets amenable to therapeutic intervention in progression from AD to asthma.

Antigens are inhaled into the complex filigree of the lung, to be phagoctyosed by one of a selection of antigen-presenting cell (APC) types that reside in the airways or along the parenchyma. Asthma does not result in allergies to all environmental antigens that enter the lung, but only some. A high variability in APC phenotype in the lung suggest the possibility that antigens may be broadly specified toward populations which could in turn serve as ‘conduits’ of those towards T cells mediating tolerance or activation. This in turn suggests a high-risk/high-reward study encompassing a ‘screening’ theme; toward identifying features of particulate antigens, which play to divergent features of the APC subsets.

The proposal will use and elaborate real-time imaging assays together with a combinatorial panel of targeting regimens to probe, with high specificity, the capacity of APC subsets to pull antigens out of the airspace in lungs in situ. As the connection between APC uptake and presentation to specific subsets is still not well understood, we will subsequently seek to establish a direct link between uptake populations and the ensuing activation of regulatory versus pathogenic T cell activation. Without a presupposition of being able to further modify the intrinsic capacity of APCs to present to T cell subsets (e.g. anti-inflammatory therapies), which represent another future layer of therapy, we aim to exploit variations in strategies to deliver antigens into the lymph.

Drugs to treat asthma fall largely into one of two major categories. The first is beta agonists that provide urgent symptomatic relief by reducing bronchoconstriction. The second strategy is to attenuate the patient’s immune response. The most common treatment involves some form of steroids, though there have been suggestions that targeting specific cytokines or pathways should be considered1. Here, we seek to explore a third strategy. We aim to blunt the effects of cytokines on the endothelium, epithelium and smooth muscle cells of the lung. This strategy is based on preliminary studies demonstrating that the endothelial-specific receptor Robo4 stabilizes the endothelium of the lung and blocks the effects of multiple cytokines by enhancing cell-cell contacts. We suspect that different Robo receptors expressed in the epithelium and the smooth muscle cells might have analogous functions. As all Robo receptors share common cognate ligands, Slit proteins, we will explore whether activating the Robo receptors on the central cells types of the lung offers a new strategy for treating asthma. We propose two Specific Aims: 1) Determine whether Slit-Robo signaling blunts the effects of cytokines on non-endothelial cells of the lung, and 2) Define the role of Slit-Robo signaling in animal models of asthma. These studies take a new approach to asthma by taking advantage of self-defense mechanism encoded in lung cells that protect them from the disruptive influences of the body’s own inflammatory response.

Asthma has become an epidemic affecting more than 155 million people in the world including ~22 million people in the United States. The chronic inflammation in asthma is due largely to the persistence of Th2 lymphocytes and Th2 cytokines/chemokines produced by both the structural cells of the lung as well as the infiltrating CD4+ lymphocytes, eosinophils, and basophils and the resident mast cells, leading to progressive loss of lung function. Recent studies have shown that IL-25 functions as an important mediator of Th2 responses and lies upstream of the classical Th2 cytokine responses. We recently reported that Act1, a novel E3 ubiquitin ligase, is an essential signaling molecule for IL-25 signaling, recruited to IL-25R upon ligand stimulation. Act1 deficiency in epithelial cells reduced IL-25-mediated allergic pulmonary inflammation, while Act1 deficiency in T cells also resulted in diminished Th2 responses and lung inflammation. Based on these findings, we hypothesize that the IL-25 induced Act1-mediated signaling pathway plays essential roles in Th2 cell responses and allergic pulmonary inflammation through the distinct impact on epithelial and T cell compartment. To test this hypothesis, we propose two Specific Aims: (1) Investigate the mechanistic role of IL-25-induced Act1-mediated signaling pathway in Th2 responses and allergic pulmonary inflammation; (2) Elucidate the molecular mechanism by which Act1 mediates IL-25 signaling. The proposed research will provide significant insight into the events that both initiate and maintain the asthmatic phenotype, leading to increased potential to develop specific inhibitors of these pathways and novel therapeutics for the treatment of asthma.

Reports of asthma among children in the United States continue to increase, with urban children particularly prone to the disease. Despite the alarming statistics, it has become clear that the two most critical components to alleviating the disease are early diagnosis and identification of triggers. The development of an inexpensive, portable, highly sensitive and selective molecular sensor could serve a dual purpose for early indication and mediation of asthma. First, by sensing oxidative byproducts of the disease from exhaled human breath, such a sensor could promote accurate and noninvasive diagnoses in children, who are unable to perform traditional airway assessments. Second, via continuous, real-time detection of environmental triggers, such a device could serve as a portable “molecular shield.” Biomimicking smart materials which integrate chemical recognition moieties with sensitive transducers could provide a general platform for highly specific analyte sensors. Oligopeptides are robust substrates for the selective recognition of a variety of chemical and biological species. Likewise, semiconducting nanowires are extremely sensitive gas sensors. Here we propose the clinically-directed detection of asthmatic indicators by synthetically linking selective peptides to silicon nanowire sensors. By utilizing a hierarchical assembly of peptide/nanowire arrays, these sensors can serve the twin objectives of targeting molecular triggers present in complex chemical environments, as well as fingerprinting the disease from human breath. Finally, we anticipate that, via the integral, real-time sensing of environmental triggers with disease diagnosis, this program may reveal fundamental understandings of the relative contributions of environmental and genetic factors in the development of asthmatic symptoms.

Calcium activated chloride channels (CaCCs) play a major role in epithelial mucus secretion and airway biology. Until recently, the CaCC molecular identity remained unknown. The identification of TMEM16A as the founding member of a conserved set of eukaryotic membrane proteins found from yeast to humans that recapitulate native CaCC functional properties constitutes a new opportunity for understanding the molecular basis of CaCC activity. TMEM16As are a unique ion channel class. Thus, developing structural knowledge of TMEM16As should prove indispensable for unraveling how this family of channels function and how they are related to human diseases such as asthma and chronic obstructive pulmonary disease (COPD). As eukaryotic membrane proteins, TMEM16As are among the most challenging subjects for structural studies principally due to difficulties in sample production. Therefore, we plan a multifaceted approach to elucidate CaCC structure that includes: Biochemical, structural and functional investigation of the CaCC calcium-sensing apparatus and the development of expression systems for the production, purification, crystallization, and structure determination of TMEM16A family members. As the eukaryotic membrane protein expression problem is a general one, we anticipate that the technologies and knowledge developed here should have a general impact. Further, the elucidation of TMEM16A structural elements should provide an important framework for understanding how CaCCs function and for the development of new pharmaceutical agents that can control CaCC function and that may prove useful for the treatment of asthma and COPD.

Platelet-activating factor (PAF) is one of the most potent mediators of allergy and inflammation, and several studies have involved PAF in asthma. In spite of its importance in asthma pathogenesis, we know little about how PAF is synthesized and moves in cells, how it is released from producing cells, and what is the best strategy to inhibit PAF in asthma. Answering these questions would be greatly aided by being able to measure PAF levels accurately and to visualize PAF in cells with high resolution.

PAF is a phospholipid that bears a choline head group. We recently devised a novel chemical method for microscopic detection of choline-containing phospholipids in cells. We noticed that a refinement of our method based on the unique chemical structure of PAF offers a novel strategy to detect PAF in vivo with high specificity and sensitivity. The present proposal uses chemical biology, cell biology and small molecule screening to accomplish the following aims:

1. To develop chemical methods to measure PAF levels and to visualize PAF in cells

2. To use the novel PAF visualization method to determine the genes involved in PAF synthesis, trafficking and release from cells

3. To identify small molecule inhibitors of PAF by high-throughput, cell-based screening

These studies will advance our understanding of PAF metabolism and function, will identify new targets for the inhibition of PAF by drugs, and will discover small molecule inhibitors of PAF production and release, which will be used as leads to develop powerful new therapies in asthma.

Asthma is a chronic inflammatory disease with hyper-responsive bronchoconstriction and airway remodeling, leading to extensive airway narrowing. Among unsettled challenges in asthma management are the cellular and molecular mechanisms for bronchospastic asthma, early detection and monitoring prognosis, and novel therapeutic avenues with more effectiveness and selectiveness. We hypothesize that inhibited endogenous H₂S production in airway and the lung contributes to bronchoconstriction in asthma; and that application of H₂S help lessen bronchoconstriction and airway remodeling in asthma. Study 1 will determine the distribution of 2 H₂S-generating enzymes, cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) in different lung tissues and production profile of endogenous H₂S. Study 2 will examine H₂S-induced relaxation of airway smooth muscle and the effect of H₂S on KATP channels. Study 3 will examine the effect of H₂S on airway smooth muscle proliferation and airway remodeling in asthma. Study 4 focuses on the therapeutic value of H₂S in regulation of bronchoconstriction under healthy and asthma conditions. Asthma will be induced in wide-type mice and CSE deficient mice (KO) by ovalbumin challenges. The availability of CSE KO mice to our team makes it possible to establish the role of CSE in asthma. It is anticipated this research will shed light on the mechanisms for bronchoconstriction in asthma and identify nasal H₂S level as a novel biomarker for early diagnosis and monitoring prognosis of asthma. Intranasal administration of H₂S would also prove to be an effective new prevention and therapeutic means for asthma.

Asthma is a chronic inflammatory disorder of the airways that is characterized by impaired airflow and sudden constriction of the bronchioles commonly known as asthma attacks. The disease starts with an allergic response that progresses to a permanently activated immune system in the lungs. At an advanced stage, asthma is associated with changes in airway architecture and a dysregulation of the autonomous nervous system (ANS). The ANS is composed of two opposing branches, the parasympathetic and the sympathetic system. In healthy individuals, parasympathetically controlled airway contraction and sympathetically controlled airway dilation are delicately balanced. In asthmatics, an exaggerated parasympathetic reflex without an effective sympathetic response causes asthma attacks. Although ANS plays an important role in the pathology of asthma, the relationship between ANS and asthma remains relatively unexplored. We propose to investigate the cellular and molecular basis of the neuronal defects in asthma using mice in a perfused preparation. This preparation provides unequaled access to the ANS for direct monitoring of sympathetic and parasympathetic activity, as well as lung properties and heart function. By using mice, we also have access to genetic tools to selectively manipulate cell activity and gene expression. We believe that the use of mouse models in a perfused preparation is an excellent platform to investigate ANS defects in asthma and the role of ANS in general lung function.