Airway remodeling characterized by increased airway smooth muscle (ASM) mass is a hallmark of obstructive diseases of airways such as asthma. Current asthma medications control inflammation and reverse airway obstruction effectively, yet have very limited effects in deterring airway remodeling. We identified the expression of bitter taste receptors (BTRs) on human ASM cells and activation with known agonists resulted in ASM relaxation. Aerosol challenge in normal and allergen sensitized- and challenged- mice resulted in a robust bronchodilation. These studies suggest that BTRs can be used as new therapeutic targets in asthma. To further establish their effectiveness as anti-asthma medication, we have proposed studies to determine their effects on ASM growth. Preliminary studies demonstrate that known BTR agonists inhibit mitogen-induced growth of healthy and asthmatic ASM cells. Based on these observations we hypothesize that BTR agonist inhibit ASM growth through novel anti-mitogenic signaling in ASM. Aim 1 studies will determine effects of BTR agonists on mechanisms that are involved in the growth (proliferation, size and survival) of normal and asthmatic human ASM. To characterize the signaling mechanism(s) studies in Aim 2 will determine effects of BTR agonists on mitogenic signaling pathways, and identify those effectors through which BTRs mediate their anti-mitogenic effects. Studies in Aim 3 will ascertain in vivo efficacy of BTR agonists in inhibiting ASM remodeling using house-dust mite challenged mouse model of allergic inflammation. Collectively, the findings from the proposed studies will identify a novel class of GPCRs agonists and signaling pathways that can mitigate ASM remodeling.

Immunological phenotyping plays an important role in the development of approaches for personalized treatment of asthma. The objective of this project is to develop a point-of-care system that would allow automated, on-chip processing of induced sputum samples from asthmatic patients and analysis of immune cells present in sputum samples to establish a patient’s immunological phenotype. The specific aims are: Aim 1: Demonstrate a multi-color flow cytometry chip that can perform accurate, eight-parameter analysis of immune cells on samples of peripheral blood. Aim 2: Develop a microfluidic chip for liquefying sputum samples . Aim 3: Demonstrate an integrated microfluidic system for sputum processing and multi-parametric analysis of induced sputum samples from asthmatic patients, including the identification of neutrophils, lymphocytes, monocytes, eosinophils, epithelial cells, and macrophages. The proposed microfluidic system have the following advantages: 1) eliminate the need for highly trained personnel; 2) assure consistency, accuracy, and reproducibility of the processing and analysis of induced sputum samples; 3) reduce time and cost for sputum processing and analysis; and more importantly 4) require a much smaller amount of sample for accurate analysis than current benchtop flow cytometry or other existing methods. The last feature is extremely important because the amount of cell samples available from some asthma patients is often not enough to run the conventional flow cytometry. This feature fills an unmet need in the field. With these advantages and unprecedented features, the proposed microfluidic flow cytometry system is expected to have a transformative impact on both asthma research and clinical care.

Our long-tern goal is to understand the molecular mechanism underlying the pathogenesis of asthma and seek for novel therapeutic strategies. In this proposal, we propose to study the mechanism by which the transcription factor Miz1 controls the allergic response during asthma and its potential role in prevention and treatment of asthma. Overwhelming evidence shows that Toll-like receptor 4 (TLR4)-mediated inflammatory response in airway epithelial cells (ECs) plays a central role in the development of allergy to a variety of environmental allergens. Recently, we discovered that Miz1 plays a critical role in modulating lipopolysaccharide (LPS)-induced, TLR4-mediated inflammation in lung epithelial cells. Loss of the transcriptional repression activity of Miz1 in lung ECs results in hyper-inflammation in intratracheal LPStreated mice, through transcriptional repression of C/EBPδ, an amplifier of LPS-induced inflammation. Most importantly, loss of the transcriptional repression activity of Miz1 promotes airway inflammation in a murine model of asthma induced by house dust mites (HDM). Based on this observation, we propose the studies to test whether Miz1 plays a critical role in regulation of the allergic response during asthma. This proposal is novel, as it will study how Miz1 controls the allergic sensitization during asthma and how Miz1 itself is regulated during the pathogenesis of asthma. These studies will put forward a new paradigm regarding the molecular mechanism underlying the allergic sensitization during asthma, which has significant clinical implications.

High conductance voltage and calcium activated potassium channels (BK channels) control airway smooth muscle contractility by suppressing increases in intracellular calcium through negative feedback on voltage activated calcium channels ((1);(2);(3);(4)). It is known that the action of bronchodilators commonly used to treat asthmatic patients, such as β2 agonists and phosphodiesterase inhibitors, is suppressed when BK channels are inhibited (2). This suggests that BK channel activation is required to achieve the full therapeutic effect of these agents. In addition, human genetic studies link reduced BK channel activity to a familial form of asthma (5). Thus, every indication points to BK channel activators as a potential new therapy for asthma. Currently, the pharmaceutical industry relies on a cell-based assay using a thallium dye to identify BK channel active compounds. We have developed a new optical assay that is based on the expression and purification work originally supported by the AAF grant. Our new assay is not cell based but rather is analogous to an enzyme assay: therefore compounds identified must act directly on the channel and not a linked process within the cell. We have adapted the assay to 384 well plates for high-throughput analysis using a Hamamatsu 384 well plate reader, have identified our first novel activators of the BK channel, and have confirmed their activity in electrophysiological assays. Funding is requested to carry out a more extensive library screen and initial evaluation of compounds using electrophysiological and smooth muscle mechanics measurements, to begin structure-function chemistry on compounds already identified, and ultimately to attract the attention for further development by the pharmaceutical industry.

We have discovered that activation of the serotonin 5-HT2A receptor with the agonist (R)-DOI super-potently prevents the development of allergic asthma in a mouse model. This includes prevention of the development of airway hyperresponsiveness, eosinophilia, pulmonary inflammation, mucus overproduction, and inflammatory cytokine production from Th2 and innate cells within the lung. In this work we will investigate three aims towards bringing this novel therapeutic strategy to the clinic. In Aim 1, we will test the therapeutic efficacy of (R)-DOI to treat already established asthma, using a chronic OVA challenge mouse model. In Aim 2, we will test the efficacy of psilocybin to block the development of allergic asthma symptoms in an OVA challenge and sensitization rat model. If psilocybin is found to block asthma related symptoms similar to (R)-DOI, then moving forward to human clinical trials with psilocybin will be much more rapid because this drug is already in clinical trials for other indications. In Aim 3, we will perform a genetic association study between defined polymorphisms within the 5-HT2A receptor gene and multiple symptoms and variables of asthma. These results will inform personalized treatment strategies in the clinic to identify patients that may be more (or less) responsive to treatment with (R)-DOI or psilocybin. When all three aims are successful, we will have demonstrated the effectiveness of (R)-DOI and/or psilocybin to treat pre-existing asthma, be able to determine which patients may be more responsive to this treatment, and perhaps move quickly to clinical trials with psilocybin.

Asthma is one of the most common chronic illnesses affecting children in the U.S. and is increasing in prevalence worldwide. Genome wide association studies have implicated genes that regulate TH2 differentiation and function as well as genes that are involved in recognizing or responding to gut microbiota. E3 ubiquitin ligases regulate both of these processes. We have identified an E3 ubiquitin ligase adaptor, Nedd4-family interacting protein 1 (Ndfip1) that activates E3 ubiquitin ligases to dampen TH2 differentiation and limit proinflammatory cytokine production by macrophages. Importantly, we have identified a region of Ndfip1 that promotes ubiquitin charging of the E3 ubiquitin ligase Itch, thus triggering its activity. We hypothesize that therapeutic mimics of Ndfip1 will activate E3 ubiquitin ligases such as Itch and thus limit proinflammatory cytokine production in T cells and macrophages. When these regulatory mechanisms are defective, such as in mice lacking Ndfip1, severe inflammation develops in the lung that resembles asthma, with goblet cell hyperplasia, accumulation of mucous in the airways and infiltration of TH2 cells and eosinophils. Additionally, we have identified polymorphisms in Ndfip1 that occur more commonly in patients with asthma. Our Aims for this proposal are to (Aim 1) reveal the underlying biochemistry of how Ndfip1 activates Itch, and test whether Ndfip1 peptides can be used to dampen cytokine production by T cells (Aim 2) and macrophages(Aim 3) and thus limit inflammation. These studies will determine whether Ndfip1 peptide mimics have the potential to be used therapeutically for treating asthma.

Identifying points of control in inflammation is essential to discover innovative anti-inflammatory medicines. The fatty acid ethanolamides (FAEs) are a family of endogenous anti-inflammatory lipid mediators. They are hydrolyzed by the enzyme N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages. Funding from AAF allowed us to discover the first potent NAAA inhibitor, (S)-OOPP, and show that this compound increases FAE levels in macrophages and, by doing so, blunts inflammatory responses. These results suggest that NAAA might provide a novel target for anti-inflammatory medicines. Because (S)-OOPP has severely limited plasma stability, we modified its chemical scaffold and identified a novel class of potent, selective and drug-like NAAA inhibitors. In the present application, we propose to explore the utility of the prototype of this class, ARN726, as a therapeutic in asthma. Specifically, we will: Aim 1: Characterize the anti-inflammatory properties of ARN726 in models of lung inflammation. Initial studies have shown that ARN726 is potent at inhibiting carrageenan-induced inflammation in mouse lungs. We will examine the consequences of oral administration of ARN726 in three additional mouse models of lung inflammation: (a) LPS-induced injury; (b) ovalbumin-induced inflammation; and (c) house dust mite-induced inflammation. Aim 2: Evaluate the pharmacokinetic properties of ARN726. We will characterize the pharmacokinetic profile of oral and intravenous ARN726. The proposed experiments will allow us to determine whether to continue our development work on ARN726 for asthma. If successful, they will provide preliminary data necessary to apply for an NIH STTR grant (Phase 2) to support such work.

Asthma is an inflammatory airway disorder primarily associated with T helper type 2 (TH2) cell-mediated production of IgE antibodies and recruitment of mast cells and eosinophils. Interleukin (IL)-33 is a key activator of TH2 inflammatory responses in airways – when IL-33 binds its exclusive receptor ST2 on the surface of TH2 cells, it triggers a cascade of additional cytokines (e.g., IL-5, -9 and -13) that induce B cell isotype switching to IgE, as well as maturation and survival of mast cells and eosinophils. The genes for IL-33 and ST2 have been linked to asthma by genome-wide association studies and depletion of either of these proteins in model systems attenuates airway inflammation. Accordingly, targeted inhibition of IL-33-mediated TH2 responses represents an attractive, yet unrealized, therapeutic approach for the control of asthma. In the proposed studies, we will define the molecular mechanisms by which IL-33 and related cytokines agonize and antagonize cell signaling in order to rationalize our engineering efforts to develop a diverse set of IL-33 signaling inhibitors. Specifically, we aim to: (1) to elucidate the molecular basis of agonist and antagonist cytokine signaling through the IL-36 receptor by X-ray crystallography, mutagenesis and functional analyses (IL-33, an ST2 agonist, has no known antagonist counterpart); (2) to determine the structure of the IL-33 signaling complex and to engineer extracellular protein-based inhibitors of IL-33; and (3) to define the physical basis of cytoplasmic protein assembly initiated by IL-33 signaling complex formation and to develop intracellular peptide-based IL-33 inhibitors.