TH2 cells play a key role in asthmatic pathogenesis. While current studies of TH2 differentiation are mainly focused on cytokine signaling and transcription factors, emerging evidence highlights that coordination of T cell metabolic programs with fate decisions is a fundamental issue in adaptive immunity. However, how the metabolic programs intersect with immune signals in T cell fates and functions is poorly defined. Our recent studies have revealed an indispensable role for mTORC1 signaling in TH2 cell differentiation and asthmatic pathogenesis. Mechanistically, mTORC1 coordinates multiple metabolic programs including glycolysis and lipid/cholesterol synthesis. These findings therefore implicate mTORC1 as a prototypical pathway to link immune signaling and cell metabolism. Our overarching hypothesis is that the interplay between mTORC1 and metabolic programs orchestrates a novel checkpoint for TH2 cell differentiation and asthmatic pathogenesis. Specifically, we will: (1) Establish the interplay between mTORC1 signaling and metabolic reprogramming in TH2 cell differentiation. (2) Determine the metabolic signature and requirement for TH2 cell differentiation. Insights from this study may impact our understanding of metabolic reprogramming and TH2 cell biology and manifest new therapeutic opportunities for asthma.

With major unmet therapeutic needs to ameliorate disease, discovery of a novel medication for asthma, preferably a mast cell specific stabilizer, remains a central goal of research in this area. Our previous studies established that the lysyl- tRNA synthetase (LysRS) is a critical upstream regulator of mast cell activation–in fact, through an on/off switch. Specifically, our studies have defined a mechanism by which phosphorylation changes the structure and function of LysRS, which ultimately leads to the activation of gene transcription of MITF (microphthalmia-associated transcription factor)-target inflammatory mediators, that are responsible for both the early and late-phase allergic responses. The goals of this proposal are to determine the role of this important LysRS-MITF pathway of mast cell activation in asthma, and to develop small molecular probes that function as LysRS-specific inhibitors for potential asthma therapy, particularly in late-phase asthmatic responses such as airway remodeling. In pursuit of these important goals we will (1) Determine the role of the LysRS-MITF pathway in airway mast cells. (2). Develop novel LysRS allosteric inhibitors for blocking LysRS-MITF activation in mast cells. (3) Characterize the LysRS inhibitors in reducing the burden of asthma. The proposed work seeks to break new ground by applying knowledge outside the field of immunity and using instead our expertise in tRNA synthetases to the study of asthma and of novel therapeutics. With the support of research labs and HTS the high throughput screening center at Scripps Florida, we are the best situated to carry out this exciting new research project and rapidly translate it into clinic.

Asthma is a chronic inflammatory disease mediated by cysteinyl leukotrienes (cys-LTs) and characterized by a T helper type 2 (Th2) cell-dependent inflammatory responses leading to airflow obstruction and airway hyperresponsiveness (AHR). Our previous studies demonstrated that inhibition of soluble epoxide hydrolase (sEH), the enzyme that converts anti-inflammatory epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and the resulting increase in EETs, leads to down-regulation of a number of cytokines and enzymes induced during inflammation, such as cyclooxygenase 2 (COX-2) and lipoxygenase 5 (5-LOX). In this AAF extension award, we propose to test the feasibility of using sEHI in an aerosol to treat airway inflammation in the murine asthmatic model. We are currently funded by an SBIR from NIEHS to continue IND enabling studies specific for pain relief. Extension funding from AAF will allow us to leverage those on-going safety studies and generate additional data needed to submit a second IND application for the treatment for asthma. The specific aims are: Aim 1: Test the safety of sEHI via inhalation at high doses in a rat pulmonary toxicity study. This study will provide preliminary data for developing IND-enabling toxicity protocols for asthma. Aim 2: Test the efficacy of three sEHIs in attenuating pulmonary inflammation when administrated by inhalation in an OVA-induced mouse asthma model. Investigate (i) target engagement by monitoring plasma and tissue levels of inhibitors and EpFAs and (ii) mechanism of action by measuring inflammatory cytokine profiles, pulmonary immune cell recruitment, lung function

Recent studies have suggested that the intestinal microbiota may play a role in asthma pathogenesis. To address this, we performed a pilot study of fecal bacteria in pediatric asthma patients. Using 16S rDNA next-generation sequencing to profile the microbiota, we observed decreased diversity of fecal bacteria in asthma patients. We also performed flow cytometry to assess the binding of endogenously secreted immunoglobulin A (IgA) to fecal bacteria. Interestingly, we found that the commensal intestinal bacteria bound to IgA are different in asthma patients, suggesting that immune responses to intestinal bacteria may play a role in asthma. Here, we propose to solidify these preliminary human studies by expanding the cohort of patients and assessing longitudinally whether these changes are associated with disease activity (Aim 1). We will also begin to assess how the intestinal immune response to commensal bacteria is associated with asthma. First, we will ask whether airway and intestinal bacteria are related in these patients (Aim 1). Second, we will characterize the intestinal bacteria associated with asthma and address whether they have unique genomic features and whether they can modulate murine asthma models (Aim 2). Finally, we will test the hypothesis that T cell trafficking between the lung and intestine explains the association between gut bacteria and asthma, by using high throughput analysis of the T cell receptor repertoire (Aim 3). Thus, these proposed studies will establish whether immune responses to intestinal commensal bacteria are associated with human asthma and will address potential mechanisms by which intestinal bacteria affect immune responses in the lung.

A key feature of asthma is airflow limitation, often characterized by hyperactive contraction of airway smooth muscle (ASM) cells. Consequently, a major therapeutic goal for asthma is to reduce ASM constriction. We have recently discovered that the peptide hormone cholecystokinin (CCK) and its receptor CCKAR, which are best known for regulating food intake and functions of the gastrointestinal tract, are expressed in and induce the contraction of primary human ASM cells. Our preliminary studies further showed that CCK expression in ASM cells is induced by both β-agonists and fatty acids. Moreover, CCK expression is elevated in the lungs of obese mice. We hypothesize that autocrine activation of CCKAR by elevated CCK in the ASM reduces airway relaxation by β-agonists and contributes to obesity-associated airway hyperresponsiveness (AHR). To test this hypothesis, we will further characterize the function and regulation of CCK/CCKAR signaling in human ASM cells (aim 1), investigate the role of CCK/CCKAR in inhibiting β-agonist-mediated bronchoprotection in mice (aim 2), and determine whether antagonizing CCK/CCKAR attenuates the innate AHR associated with obesity (aim 3). Results from this study will provide critical pre-clinical evidence required for developing CCK/CCKAR antagonists—most of them initially identified for treating gastrointestinal diseases--into new therapeutics for asthma. Furthermore, given that obesity is increasingly recognized as an important risk factor for asthma and that obese asthmatics respond poorly to existing therapies, our research may lead to much-needed therapeutic options for obese asthmatics.

Asthma is a highly heterogeneous inflammatory disease of the airway comprised of distinct clinical, immunologic, and genetic phenotypes. Nonetheless, an allergen-induced inflammatory response is one of the leading causes of asthma. Regulatory T cells (Tregs) are fundamental for the maintenance of immune homeostasis. The balance between Tregs and pathogenic T cells can play a decisive role in the development and progression of allergic asthma. Thus, a strategy that can efficiently mount allergen-specific Tregs in patients has great value for the treatment of allergic asthma. Dendritic cells (DCs) are major immune inducers and controllers. DCs in the airways play a central role in the pathogenesis of allergic asthma. However, DCs can also control allergic asthma by the induction of Tregs. A key remaining question is how to manipulate in vivo DCs to mount Tregs in asthma patients. We have recently discovered that activation of DCs via DC-asialoglycoprotein receptor (DC-ASGPR) with αDC-ASGPR antibody (mAb) can efficiently mount IL-10-producing antigen-specific Tregs. In this study, we therefore propose to test three aims: Aim 1: To investigate the effects of αDC-ASGPR mAb on asthma patient CD4+ T and B cell responses in vitro. Aim 2: To investigate the effects of αDC-ASGPR mAb on human lung DC functions to induce and activate CD4+ T cell responses in vitro. Aim 3: To investigate the effectiveness of αDC-ASGPR mAb on an animal model of allergic asthma. Data from this study will be fundamental for testingαDC-ASGPR mAb in a phase I trial in the near future.

5-Oxo-ETE, which acts via the G-protein-coupled OXE receptor (OXE-R), is the most potent eosinophil chemoattractant among lipid mediators. We postulated that a selective OXE-R antagonist (none of which were available) might be a useful therapeutic agent in asthma. To test this hypothesis, we prepared selective OXE-R antagonists, which, with the support of the AAF, we modified to a series of “2nd generation” antagonists, culminating in S3, which has low picomolar in vitro potency and excellent pharmacokinetic properties. Unlike our earlier antagonists, which are extensively metabolized, S3 is converted to a single major plasma metabolite, which is as active in vitro as S3. With help from the AAF, Lisa Miller at UC Davis, and the venture capital company AmorChem, we have completed skin and lung studies in monkeys that have generated a large number of samples. Encouraged by the preliminary data, in the coming year we plan to analyze these samples and extend our in vivo experiments, which we hope will pave the way for future clinical studies. The specific aims of the present study are to: 1)Complete the analysis of samples generated from our aerosol challenge study designed to determine whether S3 can inhibit allergen-induced airway inflammation, including soluble markers in BAL fluid, eosinophils and neutrophils in lung tissue, and tissue and plasma levels of S3. 2)Perform additional preclinical studies to pave the way for future clinical trials, including a dose-response study on allergen-induced dermal inflammation and toxicology testing. 3)Perform the total synthesis and pharmacokinetics of the main S3 metabolite (α-hydroxy-S3).

A growing body of evidence has linked alterations in dietary zinc levels to the incidence of asthma and the protective effects of zinc are modeled to be due to its antioxidant properties. We have previously discovered that calprotectin is one of the most abundant proteins during lung inflammation, and calprotectin exhibits potent zinc binding activities, suggesting that this protein is a key contributor to zinc distribution during inflammation. Notably, oxidized calprotectin has been identified at high levels in the lungs of asthmatic patients, and calprotectin has been modeled to have a protective role as an oxygen scavenger. Based on these observations, we hypothesize that calprotectin has a dual function as an antioxidant in the lung whereby its zinc binding properties contribute to airway zinc distribution while its oxygen scavenging properties protect against oxidative damage. Further, we hypothesize that the protective role of calprotectin during asthma is affected by dietary zinc levels. This model will be tested through a series of three integrated specific aims. In Aim 1 we will elucidate the impact of altered dietary zinc on asthma pathogenesis. Aim 2 studies will determine the contribution of calprotectin to metal distribution during asthma. Finally, experiments in Aim 3 will define the abundance and distribution of oxidized calprotectin in the asthmatic lung. Taken together, the results of these experiments will reveal the importance of dietary zinc and calprotectin in the pathogenesis of asthma and will lay the groundwork for the development of therapeutics and interventions for the treatment of asthma.