Our goal is to investigate angiotensin converting enzyme (ACE) activity as a key risk factor in asthma, leveraging mechanistic insights as a basis for improved therapies. Patients with asthma are plagued by chronic inflammation. Lung macrophages (LMs) are critical to the local inflammatory response. Inflammation in asthma causes bronchospasm, which can result in localized hypoxia. Hypoxia, in turn, causes increased angiotensin II (ANGII), a key inflammatory mediator generated by ACE. We found that LMs generate hypoxia-induced inflammation that is blocked by an ANGII receptor (AT1) blocker. Studies have identified a deletion polymorphism (D/D) in an intron of the ACE gene that causes increased ACE and is associated with asthma severity. We found increased membrane ACE on LMs from D/D subjects. The mechanism is unknown, but our data suggest that the deleted sequence encodes microRNAs, small RNAs that regulate proteins. Based upon these data, our central hypothesis is that the ACE D/D genotype increases ACE translation, leading to an exaggerated ANGII response during hypoxia, and thus increasing the severity of asthma. In Aim 1, we will determine the impact of the ACE D/D genotype on hypoxia-induced inflammation in asthma. In Aim 2, we will define the mechanism underlying the effect of the ACE D/D genotype on ACE levels in the asthmatic lung. These studies will define the role of ACE in modulating the severity of lung inflammation, and determine if AT1 receptor blockers may be useful anti-inflammatory agents in asthma, repurposing medications that are widely used to treat other conditions.

Lipid peroxidation may represent an important and pharmacologically addressable pathway to pathogenesis in asthma, however, this hypothesis has long remained unverified. We will employ an exceptionally potent small molecule antilipoperoxidant, peridinin, recently discovered in our lab as a probe to test this hypothesis. The current model proposes that lipid peroxidation, resulting from reactive oxygen species (ROS) released by inflammatory leukocytes, leads to the formation of electrophilic breakdown products and pro-inflammatory signaling molecules. Thus, a normally innocuous irritant initiates a self-propagating pathway of lipid peroxidation that helps drive asthma pathophysiology. In search of exceptional antilipoperoxidants, we recently completed the first stereocontrolled total synthesis of the highly complex carotenoid peridinin, a difficult to isolate natural product produced by the “red tide” dinoflagellate that thrives despite extensive exposure to ROS. Furthermore, we discovered that peridinin is an order of magnitude more potent than astaxanthin, the current gold standard for carotenoid antilipoperoxidants. To our knowledge, this makes peridinin the most potent antilipoperoxidant discovered to date. We herein propose to test the role of lipid peroxidation in asthma pathophysiology via completion of two specific aims. First, we will use peridinin to block lipid peroxidation in mouse models of acute and chronic asthma and determine whether the asthmatic responses are attenuated. Second, we will use peridinin to block lipid peroxidation in bronchial epithelial and smooth muscle cells to probe its role in asthma pathophysiology. These studies will determine the importance of lipid peroxidation in asthma pathogenesis and may enable a therapeutic strategy for addressing this disease.

The goal of our research is to use genome-wide, loss-of-function screens to identify novel human proteins that are essential for productive rhinoviral infection. These human proteins are potential targets for anti-viral therapy. Rhinovirus, a member of the Picornaviridae, is strongly associated with asthma exacerbations. Wheezing-associated illness with rhinovirus in infants is the most important risk factor for asthma development later in life. It is unclear whether rhinovirus-induced tissue damage directly causes these effects or if it is an effect of an aberrant (innate) immune response. Rhinovirus infection triggers activation of interferon-stimulated genes, but the role of only a few of these genes is well understood. New antivirals against rhinovirus would likely limit severity of respiratory distress in asthmatic individuals. This proposal aims to identify proteins critical to rhinovirus infection or critical to the innate immune response triggered by infection. Aim 1. Genome-wide knockout screens in human cells to identify genes required for rhinoviral entry and replication. Aim 2. Validation and mechanistic studies to characterize candidate human genes used by rhinovirus. Aim 3. Systematic probing of the role of each interferon-stimulated gene in the context of rhinoviral infection using CRISP/CAS9 technology. Results of the study will elucidate a distinct subset of genes that are crucial for rhinoviral infection or the host response to infection. This will provide valuable clues to host-pathogen interactions by rhinovirus. Importantly, these proteins provide new targets for drugs that can be tested for anti-rhinoviral activity and possibly used for treatment of infected individuals suffering from asthma.

An itchy throat is a common symptom of asthma. Itch in the skin and airway is initiated by sensory neurons in dorsal root and vagal ganglion, respectively. Interestingly, sensory neurons in both ganglion share many similarities, expressing the same group of receptors and channels and responding to the same type of stimuli. Therefore, the concurrence of asthma and itch suggest they may share common molecular signaling components, such as receptors and ligands. Recently, we identified members of the G protein coupled receptor family Mrgprs including MrgprC11 as novel itch receptors. Our preliminary data show that MrgprC11 is specifically expressed in vagal nerves innervating the airway and may play a role in bronchoconstriction, a hallmark feature of asthma. We therefore hypothesize that MrgprC11 in the vagal ganglion plays an important role in airway constriction and inhibition of its human ortholog MrgprX1 is a promising way to treat asthma. The proposal will further characterize the role of MrgprC11 in an asthma model. First, we will investigate whether Bam8-22, an endogenous peptide ligand for MrgprC11, evokes airway constriction in naïve and sensitized mice. Second, we will determine whether MrgprC11 and Bam8-22 are upregulated and if MrgprC11+ neurons become hyperactive after airway sensitization. Third, we will test whether the small molecule antagonists of human MrgprX1 can block airway constriction in sensitized mice. Since Mrgprs are only expressed in vagal nerves and DRG and not in other tissues, MrgprX1 antagonists may represent a new family of therapeutics to treat asthma with limited side effects.

Asthma is a respiratory disease characterized by Th2 airway inflammation with concomitant increases in mucus production and smooth muscle contraction, leading to airway obstruction. Inhibition of both inflammatory and structural components of the asthma response is needed for optimal control of asthma symptoms. Our preliminary data identify diacylglycerol kinase (DGK)ζ, an intracellular enzyme that regulates the diacylglycerol signaling pathway, as a novel target in asthma. Airway hyperresponsiveness in a mouse model of allergic asthma was abolished in DGKζ-deficient mice compared to wildtype mice. In addition to a reduction in Th2 inflammation, our data suggest that DGKζ deficiency protects against asthma also by affecting structural components. Thus, we hypothesize that the inhibition of DGKζ will block the asthmatic airway response by affecting function of both hematopoietic and structural cells in the airway. Specific Aim 1 outlines experiments that will test the role of hematopoietic and non-hematopoietic cell types that are responsible for the attenuated airway response in DGKζ deficiency. Moreover, we will specifically investigate how DGKζ deficiency might affect T cell, eosinophil, mast cell, type II pneumocyte, and smooth muscle cell function during asthma. The second aim will test the potential of DGK inhibition for the treatment of asthma using genetic and pharmacological approaches. Enzymes such as DGKs represent an attractive therapeutic target, because of the potential of small molecule inhibitors to block the active site of these enzymes. The experiments laid out in this proposal will help determine whether DGK serves as a novel target for asthma treatment.

Non-resolving inflammation is now widely acknowledged as a major driver of disease. Implicit in this discovery is that chronic disorders which fail to abate reinforce the importance of understanding resolution in those that do. For asthma, an ostensibly “self-limiting” disease with both acute and chronic endotypes, the process of resolution remains poorly understood. Here we focus on three members of a new IFN-inducible GTPase superfamily that dominates the Th1 transcriptional profile during resolution of experimental allergen challenge in the lung. As such they could promote control over Th2-induced asthmatic inflammation to aid its abatement. These proteins belong to group of 65-73kDa immune GTPases -- the Guanylate Binding Proteins (GBPs) -- widely expressed in immune and non-immune cells with activities relevant for asthma. Recent genetic evidence show they are critical for IFN-γ-induced defense against inhaled pathogens and stimulate inflammasome assembly that may feed-forward to elicit Th1 responses via IL-18-dependent IFN-γ production. We will test their involvement in asthma resolution by challenging newly-generated Gbp1-/-, Gbp2-/- and Gbp5-/- mice with natural and experimental allergens. Here they may impact T-cell, macrophage, mucosal and mast cell responses. Mechanisms of action will be dissected in situ and in primary cell cultures where GBP mutants coupled with high-resolution imaging and reporter-based probes should uncover new points of Th1 control during asthma. Such knowledge could serve as a basis to therapeutically uncouple the protective from pathologic effects of IFN-γ signaling to alleviate asthmatic inflammation.

Aberrant IL-4/IL-13/IL4-Rα/STAT6 signaling plays a key role in asthma pathology resulting in mucus production, airway hyperresponsiveness (AHR), eosinophil and other inflammatory cell recruitment, and immunoglobulin class switching to IgE. Upon cytokine binding, signal transducer and activator of transcription 6 (STAT6) transmits signals directly from IL-4Rα to the nucleus and transcribes genes that underlie expression of asthma-like disease. To block this pathway, our laboratory is developing small-molecule, cell-permeable phosphopeptide mimetic prodrugs that target the SH2 domain of STAT6. Our lead inhibitor, PM-242H, when administered intranasally (50 µg/dose), inhibited STAT6 phosphorylation and the development of AHR, Th2 cell recruitment to the lung, eosinophilia, and goblet cell metaplasia in an Aspergillus-induced allergic lung disease model. More importantly, in established disease, AHR was reversed accompanied by reduced goblet cell metaplasia and no increase in fungal burden, demonstrating that inhibition of STAT6 phosphorylation is potentially an ideal treatment modality for asthma. Next generation compounds inhibit STAT6 phosphorylation in human airway cells (IC50 ~100 nM) and are selective for STAT6 over STAT1, STAT3, STAT5, PI3K, and Src. Of the new inhibitors, PM-43I inhibited the development of disease at 0.5 µg doses, a 100-fold gain in potency. To demonstrate the feasibility of these compounds as potential asthma therapeutics, the aims are: (1) Evaluate ability of lower doses of PM-43I to inhibit induction and reverse established disease. (2) Perform lung and plasma pharmacokinetics. (3) Evaluate toxicity of PM-43I and monitor potential immune suppression distal from the lung, evaluating molecular markers such as serum IgE and lung pSTAT levels.

Foxp3+ regulatory CD4 T cells (Treg) play an indispensable role in regulating immunity and tolerance. Defects in Treg generation and/or function are directly associated with uncontrolled lymphoproliferative diseases, including allergic pathology; thus indicating that Tregs are important regulators in Th2 mediated inflammation. However, the precise mechanisms that Tregs express to regulate Th2 immunity remain unclear. There is growing evidence that Treg function is defective in patients with allergic disease. Treg infusion therapy has thus been conceptualized and being tested to treat inflammatory diseases. Yet, Tregs tend to lose optimal suppressor function under inflammatory conditions, limiting the clinical applicability of Treg therapy. Therefore, identifying a pathway(s) that enhances Treg function is of vital importance. From the preliminary studies we found that IL-27 signaling in Foxp3+ Tregs is essential for suppressor function and that Tregs prestimulated with IL-27 exhibit greater suppressor function. The main hypothesis is that IL-27 induced during allergic inflammation optimizes Treg suppressor function. We also propose that suppressor function of Tregs is enhanced by IL-27 pre-stimulation, attenuating ongoing inflammation in the lung. Two specific aims are proposed to test the hypothesis. Aim 1 will determine if IL-27 signaling in Tregs is essential for suppressor function during allergic inflammation. Aim 2 will examine a cellular mechanism by which IL-27 controls Treg suppressive function during allergic inflammation. The success of these studies will have an immediate impact on the development of novel approaches that improve the efficacy of Treg therapy in treatment of asthma.

Epithelial barrier defects and remodeling are hallmarks of asthmatic airway disease. Epithelial defects are evident before excessive inflammation can be observed, and are not completely reversed by anti-inflammatory treatment. Since asthma may originate from an epithelial rather than an immunological dysfunction, causative asthma therapies should not only target inflammation, but also epithelial barrier defects and aberrant repair responses. However, physiological repair mechanisms of wet epithelia remain largely unclear. Cell culture studies have implicated ATP as an important mediator of epithelial repair in vitro, but its in vivo role has been rarely studied. Extracellular ATP is pathologically increased in asthmatic lungs. Our preliminary data show that too much ATP triggers aberrant epithelial remodeling in vivo. Therapeutically, extracellular ATP could be modulated in two ways, by blocking ATP secretion, or blocking its action on cell surface receptors. Progress on the first approach has been hindered by lack of physiological knowledge of the secretory mechanism. In Aim 1, we propose to address this problem in the context of an in-vivo epithelium using the powerful zebrafish tail fin microinjury assay. Progress on the second approach has been hindered by lack of knowledge of relevant receptors. Most studies suggest that extracellular ATP exerts its physiological effects via P2X and P2Y receptors. However, antagonists of these families have had little impact on asthma treatment. Based on our preliminary data, we propose that an alternative, more disease- relevant receptor exists that mediates epithelial remodeling in vivo. In Aim 2, we suggest experiments to identify this receptor.

Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is the most abundant gene in polarized airway epithelia and is present at high levels in normal airway surface liquid (ASL). We and others have shown that SPLUNC1 levels are significantly reduced in murine airway epithelium exposed to a Th2 cytokine milieu. In this proposal, we demonstrate that ASL SPLUNC1 levels are diminished in allergic asthmatic patients as compared to healthy control subjects. The role of SPLUNC1 in modulating airway allergic inflammation remains unknown. However, we have now found that SPLUNC1 is secreted serosally from human airway epithelia and that SPLUNC1 prevents airway hyperresponsiveness in house dust mite (HDM)-challenged mice. In this proposal, we will (i) determine how SPLUNC1 is decreased in asthmatics and HDM-exposed mice: (ii) determine which region/domain of SPLUNC1 modulates smooth muscle contraction and (iii) identify how SPLUNC1 prevents airway hyperresponsiveness. These specific aims will enable us to test the hypothesis that SPLUNC1 is an epithelial-derived factor that directly modulates airway smooth muscle contractility. Furthermore, data obtained in this grant will enable us to develop SPLUNC1, or SPLUNC1-derived peptides as a novel therapy to prevent airway hyperresponsiveness in asthma.