IL-33 drives asthma in animal models and its expression is correlated with disease severity in humans with asthma and chronic obstructive pulmonary disease (COPD). Genome wide association studies have linked Il33 and Il1rl1 (the receptor for IL-33) to asthma susceptibility. IL-33 is released from airway epithelial cells where it activates hematopoietic cells to promote allergic inflammation via the production of IL-13 and IL-5. A wealth of information exists on the biological effects of IL-33, including T cell differentiation, mast cell survival and activation, eosinophil degranulation, dendritic cell maturation, macrophage production of CCL24 and CCL17, and the regulation of IL-33 by the soluble IL-33 receptor. However, major gaps exist in our understanding of the cell types and the biochemical mechanisms that regulate IL-33 processing and activity. This study focuses on approaches to limit the processing and hyper-activation of IL-33. IL-33 lacks a signal peptide but can be released from viable airway epithelial progenitor cells where it can be further processed to a highly bioactive cytokine by proteases including neutrophil elastase and cathepsin G. Activation of the receptor interacting protein kinase-3 (RIPK3)/mixed lineage kinase domain-like (MLKL)-dependent necroptotic pathway can contribute to the generation of bioactive IL-33 isoforms that are consistent with processing by neutrophil proteases. Our preliminary data reveals that necroptosis of neutrophils triggers the formation of neutrophil extracellular traps (NETs) composed of DNA, histones and proteases including neutrophil elastase. The observation of NETs in human endobronchial biopsy specimens from asthma patients suggests that neutrophils may contribute to IL-33-mediated IL-4/5/13 production via a propensity to engage the necroptosis pathway. In this model, proteases associated with NETs from necroptotic neutrophils will proteolytically cleave IL-33 and increase its activity on surrounding cell types. In this study, we will examine a role for neutrophil necroptosis in IL-33 hyperactivation in response to house dust mite allergen and pulmonary S.pneumoniae infection, which is known to increase the risk of wheeze and asthma.

Despite the well-documented impact of early life exposure on asthma risk, the mechanisms by which early life events increase asthma susceptibility are poorly understood. The discovery of a new class of immune cells, innate lymphocytes, as critical mediators of asthma pathology has advanced approaches to asthma treatment; however, limited understanding of the development of these cell subsets presents a barrier for defining the cellular origins of asthma susceptibility. In this proposal, we aim to capitalize on our recent discovery of a developmentally restricted hematopoietic stem cell (HSC) that gives rise to innate lymphocytes to establish a novel model for investigating the developmental origins of asthma susceptibility. We will define the role of this novel HSC in the generation of innate lymphocytes that mediate asthma pathogenesis, and identify the molecular regulators of their unique capability (Aim 1). We then aim to define how perinatal immune insult, modeled by acute treatment with the TLR agonist poly(IC), perturbs developmental hematopoiesis and innate lymphocyte establishment (Aim 2). Specifically, we will investigate whether perinatal insult triggers the abnormal persistence of the developmentally limited HSC and the effects of this persistence on the cellularity of developmentally regulated immune cells in adulthood. Lastly, we will determine whether perinatal immune insult influences asthma susceptibility and define the cellular drivers of increased asthma risk (Aim 3). Together, these experiments will yield critical information regarding the origin of the cells that drive asthma pathogenesis, and determine how adverse life events translate into increased risk of asthma later in life.

Sphingolipids and their derivatives have emerged in recent years as major autocrine and paracrine signals within epithelial tissues that strongly influence cell survival and inflammation. Human genome-wide association studies have established a robust asthma risk allele on chromosome 17q21 that impacts the expression of the gene ORMDL3, a protein that directly impacts sphingolipid homeostasis. Specific manipulations of sphingolipid levels in model organisms, moreover, induces an asthma-like phenotype. Together, these observations suggest that the enzymes governing sphingolipid synthesis may be novel therapeutic targets for asthma and other diseases because the ORMDL3 allele has also been linked to several heritable inflammatory disorders. ORMDL3 is an integral component of a poorly understood membrane protein complex, the SPOTS complex, which includes enzymes responsible for the first and rate-determining step of sphingolipid synthesis as well as a glycerolipid phosphatase. We have purified functionally active SPOTS complexes for structural and functional studies and propose to (1) determine the structural mechanisms by which ORMDL3 regulates sphingolipid homeostasis; and (2) to determine the molecular basis of cross-talk between glycerolipid and sphingolipid homeostasis with the SPOTS complex. We are poised to determine the atomic-resolution mechanisms governing the SPOTS complex and thereby to overcome the roadblock limiting further basic investigations and pharmaceutical developments for sphingolipid diseases.

Airway epithelia provide a protective barrier for our lungs, warding off air-borne toxins and pathogens. The cells comprising these epithelia renew at some of the fastest rates in the body by cell death and division. Yet, the number of cells that die must match those that divide to prevent barrier function diseases or tumors from arising. My lab has discovered that when cells become too crowded, some extrude and die, thereby mechanically matching the number of cells dying to those dividing. The stretch-activated channel, Piezo1, senses crowding and triggers cells to emit Sphingosine 1-Phosphate, which activates Rho-mediated actomyosin contraction to seamlessly squeeze cells out of the epithelium. While 1.6-fold crowding triggers cells to extrude and die during normal turnover, we found that excessive crowding from an asthmatic bronchoconstriction causes excessive extrusion that destroys the airway epithelial barrier. Disruption of the epithelial lining could lead to the typical weeklong inflammatory period and high infection susceptibility that follows an attack. Further, this inflammation could promote more asthma attacks. Because crowding-induced epithelial cell extrusion requires Piezo1, we discovered that the stretch-activated channel inhibitor gadolinium (an MRI contrast agent) prevents epithelial extrusion and denuding from bronchoconstriction. Here, we will test if gadolinium treatment can prevent the inflammatory period following an attack and avert future attacks using a pre-clinical mouse model, by addressing the following aims: 1. Does hyper-extrusion following bronchoconstriction cause inflammation? 2. Test if gadolinium prevents bronchoconstrictive airway denuding in a pre-clinical mouse model. 3. Does pre-treating with gadolinium prevent asthma attacks?

Numerous commensal microbes normally exist in the lung with no adverse effect on host health. During allergic asthma, excessive immune responses to environmental antigens, including inhalable microbes, results in disease. This disease can be exacerbated by environmental insults such as air pollution that may cause irritation and facilitate excessive immune responses. Heavy air pollution events with high levels of particulate matter have been shown to increase the abundance of inhalable microbes. The combination of increased environmental insults with increased microbial burdens due to air pollution has not been explored as a potential cause of allergic airway disease such as asthma. In this proposal we will test the hypothesis that alterations to the inhalable microbes and noxious particles during air pollution events results in increased allergic asthma type responses to otherwise innocuous environmental microbes. This increased immune response in the lung is mediated by the effects of air pollution causing an increased exposure in the intestines to inhaled microbial antigens due to increased intestinal permeability in the presence of air pollutants. We will test this hypothesis using human samples and animal models by addressing the following specific aims: Aim 1. Does air pollution alter the environmental airborne and host-associated airway microbiota and responses to microbes in allergic asthma? Aim 2. Do constituents of air pollution increase intestinal pathogenesis and immune responses to microbes in the lung?

Asthma is a chronic disease resulting from a dysregulated immune response to environmental, microbial or allergic stimuli in the lung. Basic and translational studies suggest that asthma is orchestrated by the priming, expansion and effector functions of antigen-specific CD4+ T cells. Despite tremendous advances in understanding the pathogenesis of asthma, there is currently limited knowledge on pathways that can directly limit pathologic CD4+ T cell responses to defined antigens. In recent studies we have identified a novel regulatory pathway at another mucosal surface of the mammalian body, the intestinal tract. Specifically, we identified that an innate immune cell population, termed group 3 innate lymphoid cells (ILC3), directly limits pathologic CD4+ T cell responses to microbes through expression of major histocompatibility complex class II (MHCII). Furthermore, we now identify that MHCII+ ILC3 are enriched in the lung-draining lymph node and limit Th2 and Th17 cell responses in the lungs of allergen-exposed mice. Strikingly, the mixed Th2 and Th17 cell responses have different antigen-specificities towards allergens and microbes. In this proposal we will systematically interrogate: (i) whether ILC3-intrinsic MHCII regulates allergen-specific CD4+ Th2 cells in the asthmatic lung, and (ii) how ILC3-intrinsic MHCII is regulating microbe-specific CD4+ Th17 cells in the asthmatic lung. Collectively, these studies will employ our expertise in mucosal immunology and host-microbe interactions to investigate the role and functional significance of this novel regulatory pathway in the lung. Further, these studies may inform ongoing efforts to develop effective therapies targeting either ILCs or CD4 T cells to treat asthma.

Adenylyl cyclases are enzymes that convert ATP into the second messenger cyclic AMP. Their activity is regulated by a variety of factors, and they play important roles in many physiological processes, including the control of airway smooth muscle tone. They mediate the therapeutic effects of the beta agonist class of bronchodilators, and direct activation by small molecule activators has been shown to cause bronchodilation. However, the lack of high-resolution structural information for a full-length adenylyl cyclase has limited our understanding of the molecular mechanisms that regulate their activity, and has also hindered their development as a therapeutic target. In this proposal, I aim to utilize biochemical and biophysical approaches to elucidate detailed mechanisms for regulatory control of this class of proteins. This will entail developing methods for the expression and purification of a full-length adenylyl cyclase and then structure determination of the adenylyl cyclase both by itself as well as in complex with its various regulators. The results will have a significant impact on our understanding of adenylyl cyclase structure and function, as well as the role these enzymes play in the pathophysiology of asthma, and may also lead to the development of compounds that directly target these proteins.

Glucocorticoids are the most effective treatment for asthma. However, their clinical applications are limited by low efficacy and unwanted side effects. Therefore, it is important to develop novel glucocorticoids that deliver high efficacy to the lungs with fewer side effects. Supported by previous AAF funding, we have gained structural insights into glucocorticoid potency and the molecular mechanism of dissociated glucocorticoids. We found that high potency can be achieved by designing ligands with a lipophilic group at the C-17α position of the steroid D ring. Further, the dissociation of transactivation from transrepression can be achieved by designing small molecules that interfere with the dimerization of the glucocorticoid receptor ligand-binding domain. Applying that knowledge, we have developed an exceptionally potent glucocorticoid, VSGC12, which efficiently represses the inflammation signals in a murine asthma model and outperforms the leading asthma drugs currently used in clinics. VSGC12 can deliver effective treatment at a low dose without eliciting major side effects such as insulin resistance and bone loss. We have also developed a partially dissociated glucocorticoid, VSG111, which has promise for lowering unwanted side effects. Our specific aims are 1) preclinical evaluation of VSGC12 through intranasal administration in a mouse asthma model; 2) drug metabolism and pharmacokinetics (DMPK) and toxicity studies of VSGC12; and 3) preclinical evaluation the efficacy of VSG111 in a mouse asthma model. Achieving these specific aims will provide the basis for developing new drug candidates for treating asthma, which would fulfil the mission of AAF extension award toward an impact on therapy.