This application will examine the potential of a unique pathway in modulating asthma. Our laboratory has discovered that glutamate signaling through the kainic acid receptor (KAR) leads to enhanced ADAM10 activity and as a result leads to increased production of soluble CD23 and IgE production. This finding is critical because it is one of the first to highlight the importance of a neurotransmitter receptor on B cells and that signaling by this receptor may play a role in IgE mediated disease. Importantly, a specific antagonist of the KAR, NS-102, blocks the KAR and may serve as an innovative pharmacological target for asthma treatment. Two aims are proposed. The immune reactivity of KAR knockout mice will be tested with respect to their B cell proliferative capacity and IgE production. Secondly, the mice will be examined in mouse lung inflammation models. NS-102 or related derivatives will be examined with respect to whether KAR antagonism will modulate lung inflammation. In the second aim, we will look at the role of ADAM10 in lung pathology. Our data indicate that the KAR activity is at least partially explained by ADAM10 and that mice which lack ADAM10 in their B cells or wild type that are treated with an ADAM10 inhibitor have greatly reduced pathology especially in an IgE-mast cell dependent model. In this aim, we will determine if the enhanced immunogenicity is due to exosome incorporation of the IgE complexes and test the hypothesis that ADAM10 blockade inhibits inflammation by blocking Th2 activation.

New therapeutic approaches and strategies are needed for patients with severe asthma. Asthma is generally thought to be a Th2 cell-associated inflammatory disease, and the Th2-type cytokines Interleukin-4 (IL-4) and IL-13 are both thought to play major roles in driving asthma pathology in patients. In particular, IL-13 acts on a receptor expressed on lung tissue to induce mucous secretion and a hyper-reactive airway state. Given that IL-4 is also an important regulatory cytokine whose antagonism would likely have adverse effects on immune homeostasis, we are focusing on specific disruption of the IL-13 branch of this signaling axis in asthma. In this proposal we take advantage of existing structural and functional data on the complex receptor biology of IL-13 signaling to engineer this cytokine by selecting variants from combinatorial libraries that will possess both receptor-selective and antagonistic properties. We are taking a very direct route to engineering a high affinity IL-13 ‘dominant-negative’ cytokine for potent blockade of IL-13 signaling. Currently, IL-13 activity as a therapeutic is limited by two factors: 1- IL-13 has a relatively low affinity for its signaling receptor IL-13Rα1, and 2- IL-13 is scavenged from the circulation by the very high affinity decoy receptor IL-13Rα2. Informed by the crystal structure of the IL-13 receptor complexes we determined during the term of our prior AAF award, we will take a mechanism and structure-based approach to increase the affinity of IL-13 for IL-13Rα1, and decrease or eliminate IL-13 binding to IL-13Rα2, while at the same time disrupting the interaction with IL-4Rα. We believe that this affinity maturation can be obtained through mutation of only a few amino acids, and that subverting the hijacking effect of IL-13Rα2 is essential for an effective antagonistic cytokine. The methodologies combine combinatorial biology (yeast display), cell signaling (FACS bar-coding and multi-plexed CyTof), and animal studies in collaboration with the AAF core and other labs.

Significance: Asthma is a prevalent, often refractory disease: cures could yield major human benefits.

Hypothesis: Asthma is an airway smooth muscle (ASM) dysrhythmia, that like cardiac dysrhythmias has triggers (e.g. allergens in asthma), but which is curable by ASM pacemaker ablation.

Overall aim: To develop electrophysiological diagnosis and curative therapy for asthma

Rationale:
1. Prenatal airways feature rhythmic peristalsis from large airway pacemakers.
2. Postnatal airways continually modulate their caliber: this homeokinesis is abnormal in asthma.
3. Homeokinesis may provide the background for catastrophic shifts to airway closing in acute attacks.
4. Bronchial thermoplasty is a promising therapy delivered to large and medium sized airways for refractory disease (despite asthma being a small airway problem.
5. We postulate prenatal airway pacemakers persist to generate homeokinesis and bronchoconstriction, and thermoplasty renders distal airways quiescent by ablating these.

Specific Aims:
1. To determine normal airway electrophysiology (EP) and thermoplasty’s impact in vivo.
2. To use airway EP to detect and modulate airway contractility in vivo.

Approach: We will characterize the ‘electropulmonogram’ (EPG) in porcine airway in vivo using intraluminal catheters and extraluminal implanted ones, and assess the impact of thermoplasty and relationship to contractility.

Expected results: We expect to define the EPG and identify pacemakers, whose ablation renders distal airway quiescent.

Caveats: It is unlikely airway electrophysiology is unrecordable given surrounding electrically-active ASM and modern catheters’ reduction of extraneous artifact. If pacemakers cannot be identified, we will, like cardiology, ablate circumferentially around chosen airways to isolate ASM proximally and distally and compare EPGs.

Fungi have always been associated with asthmatic diseases, for example fungi are recognized as the most serious environmental trigger of childhood asthma in New Orleans post-Katrina. However, as of yet, the exact mechanism(s) by which fungi induce asthma is not known. Our laboratory has recently established a major paradigm shift in fungal/plant signaling where fungi and plants have been found to induce, intercept and recognize each other’s oxylipin signaling molecules. We propose here that a similar ‘stealth by mimicry’ is of consequence in asthma development. Arachidonic acid oxylipins, specifically 5-lipoxygenase derived leukotrienes (LTs), are recognized as critical molecular inducers of inflammation, mucus secretion, vasodilation and bronchial constriction associated with asthmatic diseases; indeed current asthma therapies target both 5-lipoxygenases and LT receptors. This LT induced asthma is presumed to be of human origin. We suggest that in addition to – and possibly in lieu of – human sources, fungal LT play a major role in asthma development. The major hypothesis of this proposal is that fungal lipoxygenases and LTs initiate and/or exacerbate inflammatory asthma. Our three specific aims are to:

1. Elucidate the consequence of Aspergillus fumigatus lipoxygenase(s) entry into host immune cells and determine what LTs they produce,
2. Determine the effect of fungal oxylipins on human cells and recapitulate the fungalasthma relationship in a novel microfluidic in-vitro model, and
3. Assess in vivo models to detect the role of fungal oxylipins in asthma.

Asthma patients have elevated levels of the cytokines IL-4 and IL-13 in their airways, which result in mucus production, airway hyperresponsiveness (AHR), eosinophil recruitment, Thelper cell 2 (Th2) activation, resulting in immunoglobulin class switching to IgE, and inflammation. These two cytokines signal through a common receptor, IL-4Rα, which recruits Stat6 leading to asthma phenotype.

Hypotheses and Specific Aims: The overall hypothesis of this research is that blocking the SH2 domain of Stat6 with cell-permeable, phosphatase-stable peptide mimetics will decrease symptoms of asthma. Such compounds are expected to prevent recruitment of Stat6 to IL-4 and IL-13 receptors and therefore inhibit phosphorylation of Tyr641. This will disrupt the formation of Stat6 dimers, translocation to the nucleus, and expression of downstream genes leading to asthma symptoms. The specific aims are as follows:

Aim 1. Develop high affinity phosphopeptides to the SH2 domain of Stat6.
Aim 2. Study the binding of the peptides to Stat6 using biophysical methods such as isothermal calorimetry and X-ray crystallography. This information will help guide inhibitor development.
Aim 3. Test for ability to inhibit Stat6 phosphorylation in cells stimulated with IL-4 or IL-13. High affinity phosphopeptides will be converted to prodrugs. Dose response and time course will be assessed. Selectivity for the SH2 domain of Stat6 versus the other Stats, p85P13K, and Src, and the inhibition of expression of downstream genes will be determined by western blots of cell lysates.
Aim 4. Test for inhibition of asthma phenotype in mouse models.

While the role of Th2 cytokines in asthma has been established, it is clear that asthma is a heterogeneous disease involving the complex interplay of both cells of the acquired and innate immune system. In particular, though atopic/allergen-induced asthma appears to be driven by Th2 cells, it has been suggested that steroid–resistant-disease is mediated byTh17 cells. Our laboratory has identified a central role for mTOR in integrating signals leading to CD4+ T cell effector differentiation. We have determined that mTORC1 signaling controls Th1 and Th17 differentiation while mTORC2 signaling selectively controls Th2 differentiation. Most recently we have identified the downstream mTORC2 substrate SGK1 as a novel mediator of Th2 differentiation. The overall hypothesis of this proposal is that mTORC1 and mTORC2 signaling selectively regulate the generation of atopic and steroid resistant asthma and that pharmacologically targeting these pathways will lead to novel therapeutic interventions. To this end we will test the following specific aims: First we propose that mTORC1 deficient T cell mice will be resistant to Th17-mediated disease, and that pharmacologic inhibition of mTORC1 will prevent/treat this form of asthma. Likewise that mTORC2 deficient T cell mice will be resistant to the development of atopic/allergen-mediated disease and that pharmacologic inhibition of mTORC2 will prevent/treat this form of asthma. In the second Aim we seek to demonstrate that SGK1-/- T cell mice will be resistant to the development of atopic asthma and that pharmacologic inhibition of SGK1 represents a novel means of treating this form of the disease.

Our current work funded by the AAF has identified both a cellular and a molecular target for further exploration. First, our work demonstrates that the airway epithelial cells play a critical role in establishing tolerance versus airway inflammation. Specifically, the normal function of epithelial cells is a critical tolerance versus airway inflammation. Specifically, the normal function of epithelial cells is a critical determinant when allergens such as the house dust mite are first encountered via the nasal route (as likely to occur in humans). Second, we have identified that the cytokine interleukin-10 level is severely reduced in mice with defective epithelial cell function leading to airway inflammation. Although the source of this IL-10 is still being defined, remarkably, intranasal administration of exogenous IL-10 could almost completely ‘rescue’ the airway inflammation induced by the house dust mite allergen, even when epithelial cell function remains impaired. Reduced IL-10 levels in the BAL fluid of human asthmatics have been observed, but therapies using IL-10 to reverse or alleviate the symptoms have not been reported. Phase I trials with systemic administration of IL-10 do not seem to show short-term toxicity and the half-life of IL-10 in tissues (hrs) also seem reasonable. In this AAF extension award, we propose to test the feasibility of using IL-10 as an aerosol (either alone or with corticosteroids) to limit airway inflammation in the context of specific allergens, first in mice and subsequently in humans. Should aerosol administration of IL-10 prove useful in reversing airway inflammation, this may lead to the rapid development of new therapeutics for asthma.

Antigen presenting cells (APCs) are fundamental in maintaining immune homeostasis - adroitly keeping the critical balance between immune reaction and immune tolerance. The inappropriate activation of APCs has been proposed to be a key factor in the pathogenesis of asthma. Yet, the mechanisms underlying APC hyperactivation remain unknown. We hypothesize that the TAM (Tyro3, Axl and Mer) receptor tyrosine kinase signaling pathway is triggered in APCs to prevent chronic airway inflammation. This proposal seeks to address the function of TAM signaling in mouse models of asthma. Specifically, we propose (1) to determine whether the TAM receptors function as anti-inflammatory agents in vivo and (2) to identify the mechanism of TAM receptor activation in mouse models of asthma. Our studies will constitute the foundation for future efforts centered on investigating the association of deficiencies in this pathway with human chronic inflammatory diseases of the airways and the pharmacological activation of the TAM pathway in the treatment of asthma.

Asthmas are diseases wherein the patients hyper-respond to allergens, resulting in chronic inflammation of the bronchi leading into the lungs. Animal (mainly murine) models suggest that respiratory tract dendritic cells stimulated by particulate pollutants and microbes secrete cytokines that evoke maturation of airway-homing effector T cells and trigger infiltration by macrophages that release NO, provoking bronchial smooth muscle contraction, thereby inducing airway narrowing. Smooth muscle cells can exacerbate bronchial inflammation by themselves secreting various mediators that recruit and activate additional inflammatory cells (including mast cells). Nonetheless, the molecular bases for the hyper-responsiveness of the immune mechanisms involved in induction and regulation of these processes, which underlie asthma disorders, are not well understood. In the case of childhood (early onset) asthma, however, analysis of genome-wide human sequence variation has linked predisposition for this condition to mutations that up-regulate expression of the ORMDL3 gene, presumably increasing ORMDL3 protein. Recently, the yeast (Saccharomyces cerevisiae) orthologs (Orm1 and Orm2) were shown to bind directly to and negatively regulate serine palmitoyltransferase, which catalyzes the first step in sphingolipid biosynthesis. Thus, the hyper-responsiveness underlying childhood asthma may arise from a reduction in the plasma membrane sphingolipid-to-glycerophospholipid ratio. The specific aims of this proposal are, first, to explore whether experimental treatments and conditions that compromise sphingolipid production affect standard read-outs of immune response and, second, to examine whether these same perturbations specifically cause defects in endocytosis and/or down-modulation of TLRs, cytokine receptors, or other classes of cell surface molecules implicated in asthma pathogenesis.

Dr. Tsien is the recipient of the 2011 American Lung Assosciation/American Asthma Foundation Senior Investigator Award, given to a non-pulmonologist conducting novel and innovative research on asthma.
Many extracellular protease activities play crucial roles in acute asthma and subsequent chronic airway remodeling. However, little is known about precisely where and when different proteases become enzymatically active in intact animal models, let alone patients. Previous techniques to molecularly image protease activities in asthma have been limited to assessing pooled matrix metalloproteinase (MMP) activity by fluorescence dequenching, an imaging modality with limited depth penetration. We propose to apply a new class of probes for protease activity, activatable cell-penetrating peptides (ACPPs) and their macromolecular conjugates, for which we already have varieties responsive to MMPs, elastase, thrombin, and plasminogen activators. We will start by imaging fluorescent ACPPs for MMPs, elastase, and plasminogen activators during both acute asthma and chronic induction of airway remodeling in animal models, correlating against time, type of stimulation, disease intensity, and genetic background including suppression of individual proteases. Noninvasive epifluorescence and fluorescence molecular tomography will be validated by postmortem histology and biochemical quantitation of probe cleavage and retention. New ACPPs with selectivities for mediators such as MMP-12, ADAM33, chymase, and H₂O₂ will be developed and analogously tested in these models. These studies may help clarify controversies over where and when different proteases exacerbate vs. ameliorate disease. We will also develop ACPPs for nonoptical imaging modalities such as SPECT, PET, and MRI, whose greater depth penetration makes them more suitable for noninvasive molecular imaging in humans. Finally, we will attempt to use ACPPs to target drug-loaded nanoparticles to sites of high protease activity to increase their therapeutic index.

G-protein coupled receptors (GPCRs) are important regulators of multiple cell types involved in asthma, and drugs targeting specific GPCRs are used to treat asthma. Our long-term goal is to determine whether Regulator of G-protein Signaling (RGS) proteins, intracellular modulators of GPCRs, provides new and more effective targets for treatment of asthma. Recently, we found that asthmatics had reduced expression of RGS2, a member of the RGS family that selectively regulates bronchoconstrictor GPCR signaling. RGS2 knockout mice are predisposed to have AHR, the pathophysiologic hallmark of asthma. In addition, interleukin-13 (IL-13), a key mediator of AHR in allergic asthma, induces a selective RGS2 down-regulation and enhanced contraction of airway smooth muscle (ASM). Since excessive ASM contraction is directly responsible for AHR, we hypothesize that RGS2 is a key regulator of ASM contraction and that IL-13-induced RGS2 repression plays a crucial role in the AHR of asthma. We will test this hypothesis by studies that integrate molecular, cellular, tissue, and animal models. Aim 1 will define the RGS2-regulated signaling pathways mediating ASM contraction; Aim 2 will elucidate mechanisms of IL-13-induced RGS2 repression in ASM cells; Aim 3 will determine the pathological importance of RGS2 repression in ASM in a murine model of asthma. Completion of this project will identify specific aberrant signaling pathways and molecules involved in the enhanced ASM contractility and AHR in asthmatic patients, leading to increased understanding of asthma pathogenesis. The aberrant signaling molecules identified may serve as future therapeutic targets and/or biomarkers for controlling the AHR of asthma.

While the contribution of adaptive immunity and Th2 cells in allergic airway inflammation and acute hyper-reactivity has been extensively studied, the role of innate cells has so far received little attention. Emerging data suggest that macrophages play an active role in inducing airway inflammation and contribute to the development of asthma. Macrophages are a heterogeneous population of immune cells, with distinct phenotypic characteristics and specific roles in promoting T cell lineages. However, transcription factors that underlie macrophage subsets remain largely undefined. This proposal builds on our recent discovery of the master regulator of pro-inflammatory macrophage polarization, the transcription factor IRF5, and investigates its functional role in the pathogenesis of allergy and asthma. Our research may lead to the development of new therapeutic strategies comprising the administration of a therapeutically effective amount of IRF5, or an agent that induces the expression of IRF5 in macrophages.