Shyam Biswal - US grants

cell proliferation; gene expression; acrolein; apoptosis; glutathione; environmental toxicology; oxidation reduction reaction; genetic mapping; nucleic acid sequence; cell line; genetic techniques; transfection; polymerase chain reaction; antisense nucleic acid; tissue /cell culture;

DESCRIPTION (provided by applicant): Pulmonary emphysema, a devastating disease associated with chronic exposure to cigarette smoke (CS) and environmental pollutants, is characterized by abnormal inflammation, air space enlargement, and the loss of alveolar structures. Other than congenital genetic deficiencies (which contribute to less than 5% of cases), the genes that determine susceptibility to emphysema are unknown. Our goal is to elucidate genes and pathways that can act as "risk modifiers" for emphysema. Our preliminary studies have shown that redox-sensitive bZIP transcription factor NF-E2 related factor 2 (Nrf2), which regulates antioxidant pathways, could be an important modifier gene for CS-emphysema. Disruption of the nrf2 gene (in the emphysema-resistant ICR strain) results in early onset and severe CS-emphysema. In response to CS, the lungs of Nrf2-/-mice have enhanced oxidative stress and apoptosis, increased alveolar inflammation, and loss of alveolar structures. We hypothesize that Nrf2 plays a central role in preservation of alveolar structures by maintaining the balances of pulmonary oxidants/antioxidants and proteinases/antiproteinases during CS exposure. Reduced or absent Nrf2 response causes imbalances that lead to early and severe emphysema. We will test this hypothesis by comparing mice with high Nrf2 response (ICR), low Nrf2 response (C57BL/6J Nrf2+/+) and no Nrf2 response (C57BL/6J Nrf2 -/-). Our aims are (1) To determine the relationship between Nrf2 response and preservation of lung structure in the CS-emphysema mouse model. (2) To investigate the Nrf2-mediated oxidant/antioxidant balance in inhibition of CS-emphysema. (3) To determine the mechanism by which Nrf 2 response inhibits emphysema by maintaining the proteinase/antiproteinase balance. (4) To test if overexpression of Nrf2 can inhibit emphysema. These studies promise to shed new light on novel pathways that modulate susceptibility to emphysema.

DESCRIPTION (provided by applicant): Our long-term objective is to understand the host factors that regulate the systemic inflammation and pathogenesis of sepsis, which affects 750,000 persons every year in United States alone and causes high mortality worldwide. We recently discovered a novel host factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) that plays a critical role in determining susceptibility to sepsis by regulating a compensatory pathway that controls the induction of protective cellular antioxidants. Nrf2 is a basic leucine zipper transcription factor that regulates the expression of antioxidant genes including the glutathione pathway and heme oxygenase in response to oxidative and inflammatory stress. Global disruption of Nrf2 (Nrf2 -/-) dramatically decreased survival after cecal ligation and puncture (CLP) and endotoxin treatment relative to wild-type mice (Nrf2 +/+). We hypothesize that Nrf2 regulates a host compensatory mechanism that causes transcriptional induction of antioxidant genes, which determines survival during sepsis. Disruption of Nrf2-dependent compensatory antioxidant pathways increases mortality by exaggerating the innate immune response and predisposing lymphocytes to increase apoptosis. This proposal will shed light on the Nrf2-dependent regulation of immunopathogenesis of sepsis and survival after CLP and strives to develop an intervention strategy targeting Nrf2 with a novel small-molecule activator. Specific Aim 1: To test the hypothesis that Nrf2 determines survival after CLP by protecting against deregulation of innate immune response by maintaining cellular redox balance. Specific Aim 2: To test the hypothesis that Nrf2 improves survival after CLP by attenuating apoptosis of lymphocytes. Specific Aim 3: To test the hypothesis of intervening sepsis by increasing Nrf2 activity with a small-molecule activator. The immunopathogenesis of sepsis remains poorly understood. Nrf2 provides a novel link between the regulation of oxidative stress, the innate immune response.and survival during sepsis. These studies may lead to the development of novel strategies based on Nrf2 for intervening in sepsis and improving survival.

PROJECT 4: Even though cigarette smoke (CS) exposure is the most recognized risk factor for COPD, genetic, environmental and developmental factors certainly contribute to the pathophysiology of this debilitating disease. Substantial epidemiological data indicate that impaired alveolar growth during infant life may increase the risk of developing CS-induced COPD in adults. Individuals exposed to high levels of pollutants in early life are at increased risk for sub-optimal lung growth as adults and parental smoking in childhood is associated with poorer lung function in adulthood. Our long term objective is to understand the mechanisms that cause increased susceptibility of developing lungs to pollutants that manifests itself as impaired lung function in adult life. Our preliminary studies have shown that the Nrf2 transcription factor plays a central role in counteracting the oxidative insult of CS. Developing mice have marked decrease in pulmonary Nrf2 activity as well as greater inflammation even after shorter CS exposure, relative to adult mice. Decreased Nrf2 activity is associatedwith increased TGF beta activation, greater oxidative stress and alveolar cell apoptosis in response to CS in developing lungs. We hypothesize that developing lung are intrinsically susceptible to oxidative insult by environmental pollutants such as CS, due to the lower Nrf2 activity. These injuries in the developing lungs lead to an abnormal lung structure in adult life, with enhanced predisposition to the development of emphysema upon re-exposure to CS or pollutants. We will test this hypothesis using the Nrf2 +/+,-/- and Nrf2 over-expressing transgenic mice. Finally, we will determine the association of genetic variation in Nrf2 gene and its activity with development of COPD in humans. We will find polymorphisms of the NRF2 gene and determine if it is associated with susceptibility and progression of COPD. This project will interact closely with Project 1 on a joint aim regarding a-1 antitrypsin efficiency, and will receive human biological specimens from Projects 2 and 5. This project will make use of Core D for histology, and physiologic measurements and Core C for ADC measurements of terminal airspace dimension.

In the last 20 years the prevalence of asthma has doubled and it now affects 10% of the population in the United States. The susceptibility to allergic asthma varies greatly in the population exposed to allergens. Gene-environment interaction may play an important role in pathogenesis of asthma. Our long-term objective is to understand the host factors that regulate susceptibility to allergens and the pathogenesis of asthma. Such an understanding is vital for developing new intervention strategies. We recently reported a novel host factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), that may play a critical role in determining susceptibility to allergic asthma. Nrf2 is a basic leucine zipper transcription factor that determines the severity to asthma in mice models by regulating environmental stress response that includes cellular antioxidants. Disruption of Nrf2 in mice leads to severe asthma in response to sensitization and challenge by variety of allergens such as ovalbumin and ragweed extract. The exaggerated asthmatic response in Nrf2-disrupted mice in response to allergen involves increased oxidative stress and pronounced infiltration of eosinophils into the lungs, greater T helper 2 cells and cytokines (IL-4, IL-13), antigen-specific IgE, mucus cell hyperplasia, airway hyperresponsiveness. Thus, Nrf2 is a critical regulator of asthma pathogenesis.Our central hypothesis is that Nrf2 dependent compensatory transcriptional program regulate the environmental stress response that protects against allergen-induced asthma by decreasing oxidative stress in the airway epithelium and Th2 effector cells that in turn inhibits cytokine and chemokine expression. Conversely, disrupted or suboptimal Nrf2 activity causes increased oxidative stress and asthmatic response. Specific Aim 1: To investigate the regulation of asthma by Nrf2 activity in the airway epithelium. Specific Aim 2: To investigate the regulation of asthma by Nrf2 activity in T effector cells. Specific aim 3: To test the hypothesis that genetic variation in Nrf2 and its target antioxidant genes is associated with susceptibility to childhood asthma and atopy.Specific Aim 4: To test the hypothesis that enhancing Nrf2 activity can attenuate asthma.The proposal will focus on investigating Nrf2 dependent regulation of asthma pathogenesis and strive to develop a preclinical intervention strategy by targeting this transcription factor for decreasing asthma in the susceptible individuals in the population.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Chronic Obstructive Pulmonary Disease (COPD) that comprises emphysema and chronic bronchitis, is a major public health burden that afflicts >800 million people worldwide and will become the third leading cause of death by 2020. Cigarette smoking represents the major risk factor for COPD. Cigarette smoking causes chronic oxidative stress that induces- oxidative damage to biomolecules resulting in alveolar epithelial injury and apoptosis, oxidative inactivation of antiproteases (11-antitrypsin) and surfactants resulting in remodeling of extracellular matrix, inactivation of histone deacytelase-2 (HDAC2) that promotes inflammation and causes corticosteroid resistance and mucus hypersecretion resulting in bronchitis. However, it is unclear (1) if increased oxidative stress and inflammation in lungs of COPD patients is a result of suboptimal antioxidant enzyme expression and whether suboptimal expression of antioxidant defenses are associated with the severity of COPD. Our preclinical studies have demonstrated Nrf2 as master regulator of a network of cytoprotective genes that includes antioxidant defenses in response to cigarette smoke and collectively protect against oxidative damage to macromolecules and attenuate inflammation and lung tissue injury. In response to chronic cigarette smoke exposure, Nrf2-disrupted mice lungs develops early and sever emphysema that is mainly mediated by impaired antioxidant defenses, oxidative damage, apoptosis and inflammation. However the regulatory role of Nrf2 pathway in human COPD pathogenesis is not clear. We hypothesize that there is a significant and progressive decline in the NRF2 activity leading to decreased expression of its lung antioxidant transcriptional targets in COPD lungs when compared to non-COPD lungs. This is an exploratory research proposal with main objective of investigating status Nrf2 pathway and oxidative damage with severity of COPD in limited number of lung tissue samples procured from LTRC. The proposal will measure expression of Nrf2, its co-regulators, antioxidant defenses, oxidative damage in lung samples from non-COPD smokers, mild COPD smokers and sever COPD smokers. These studies will decipher the role of Nrf2 in COPD pathogenesis and may suggest a novel molecular target for drug development. (End of Abstract) [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: [unreadable] [unreadable] Public Health Relevance Statement COPD major public health burden that afflicts >800 million people worldwide and will become the third leading cause of death by 2020. Current therapies (corticosteroid and bronchodilators) treat the symptoms but faila to stop or reverse the disease progression. Therefore, there is an urgent need of new molecular targets for development of therapies. This application focuses on deciphering the regulatory role of Nrf2 in the pathogenesis of COPD. Positive results will help in development of therapies targeting Nrf2 pathway. [unreadable] [unreadable] [unreadable] [unreadable]

CBET-0837946
Biswal

Occupational exposure to nanomaterials in manufacturing plants may pose serious health risks, and the industrial production rate of small size particles is growing exponentially. An estimated 745,000 workers are potentially exposed to engineered nanoparticulate matter in the manufacturing sector alone; within 10 years, that number is expected to increase to more than 1 million workers. Both human and animal studies have associated exposure to metals with a range inflammatory diseases, especially within the lungs (asthma, fibrosis and COPD). Toxicity of metals has been studied for a long time, however, the toxic potential of nanometal oxides, that are being produced in large scale due to their use in a wide variety of industrial applications, is not clear. Exposure pathways, resultant bioavailability, and potential adverse health outcomes of these metal nanoparticles in humans are poorly understood. This proposal will focus on physicochemical characterization, transport, biological response and occupational exposure of engineered nanometal oxides. Our specific aims are 1) Characterization of biological response, transport and physicochemical properties of metal oxides and 2) Pilot assessment of exposure and characterization of physicochemical properties of metal oxides in exhaled breath of exposed workers. The proposal involves a strong interdisciplinary collaboration between experts on exposure assessment, aerosol science, nanotechnology, the protective mucus barrier, pulmonary molecular toxicology, and a company focused on the large scale production of nanometal oxides (NanoProducts Corporation). Overall these studies will help in understanding the potential risk of nanometal oxides to human health and also develop the knowledge base required for future studies on a larger populations exposed to nanometal particles.
Supplemental key words: Nanometal oxides, exposure assessment, physico-chemical properties, mucus transport, biological affects, human lung cells

DESCRIPTION (provided by applicant): The risk of civilians or emergency responders being exposed to ionizing radiation is high in scenarios of nuclear/radiological terrorism or accidents. Reactive oxygen species and electrophiles generated after radiation exposure is involved in impaired immune response due to depletion of hematopoietic immune cells and loss of mucosal barriers, predisposing an individual to secondary infection by opportunistic bacteria and exacerbate radiation-induced morbidity and mortality driven by sepsis. There is an urgent need to develop effective therapies to mitigate synergistic effects of radiation and infection, which can be translated to mass casualty response. Our studies have shown that redox sensitive transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (NRF2) protects against inflammatory disorders caused by environmental oxidants and bacterial infection by inhibiting oxidative stress. Dissociation of the transcription factor from its inhibitor, KEAP1 by electrophiles or oxidants causes nuclear translocation of NRF2 and transcriptional induction of antioxidant genes - glutathione pathway, thioredoxin pathway, heme oxygenase-1, several protective pathways that collectively protect against oxidative stress and macromolecular damage. Our research provides strong rational to hypothesize that "enhancing the Nrf2 pathway will mitigate multi-organ injury by attenuating oxidative stress and improve survival following radiation-combined infection injury". The R21 phase will investigate the strategy of enhancing the Nrf2 pathway to mitigate multi-organ injury and improve survival following radiation-combined bacterial infection injury in mice models by genetic approach (using inducible deletion of Nrf2 inhibitor, KEAP1) and small molecule approach (using a potent Nrf2 activator, CDDO-Me [methyl ester derivative of 2- cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO)] that has undergone phase I clinical trial). Based on a positive outcome of intervening radiation-combined injury using Nrf2 activator, the R33 phase will focus on development of optimal treatment regimen and prognostic biomarkers of Nrf2 based therapy using CDDO-Me in mice model and then perform proof of concept studies in nonhuman primate model focusing on post-radiation induced gastrointestinal injury. These studies promise to develop a novel strategy to mitigate radiation- combined injury that can be used as an effective countermeasure in target population. Page 1 of 1 Public Health Relevance Statement Novel therapy for mitigating and treating radiation as well as radiation combined infection injury is urgently warranted because they present a huge public health problem in radiological/nuclear war /accident scenario. Other than clinical management, currently there are no effective therapies that can be translated to mass causality during a nuclear attack. This application is focused on development of a novel strategy for treating radiation injury by targeting a host defense factor.

The pathogenesis of asthma is a complex problem where environmental factors determine susceptibility. It is often triggered in children by allergens (from rodents, pets, dust mites and plant pollens) and irritants, especially cigarette smoke and other indoor and outdoor air pollutants. Air pollutants can exacerbate preexisting airway inflammation leading to increased airway hyper-responsiveness. The pollutants may also augment or modify immune responses to inhaled antigens or intensify the effect of other pollutants in the respiratory tract. Recent epidemiological studies revealed that diet is also a major risk factor in the pathogenesis of asthma. Most recently, dietary factors have been linked to poorer asthma health, pointing to a potential role for diet in perpetuating asthma morbidity. Insufficient intake of vitamins and other dietary nutrients during pregnancy, for example, have been associated with a greater risk of development of asthma and wheezing symptoms in children. The

Chemoresistance remains the greatest challenge in improving survival of lung cancer patients. Lung cancer cells have greater expression than normal cells of proteins involved in detoxification of electrophiles and drugs detoxification and efflux pumps [Glutathione and Thioredoxin system and Multidrug resistance proteins] that provides intrinsic resistance against chemotherapy. Studies in project 3 have established that activation of the redox-sensitive transcription factor, Nuclear factor erythroid-2 related factor 2 (NRF2), in response to xenobiotic stress, positively regulates the expression of electrophile, xenobiotic detoxification enzymes and efflux pumps which confer cytoprotection against oxidative stress and apoptosis in normal cells. Kelch-like ECH-associated protein (KEAP1) negatively regulates NRF2 activity by targeting it for proteasomal degradation. Increasing NRF2 activity by small molecule activators might protect the lung epithelium of high risk subjects from oncogenic damage due to carcinogens. However, a surprising finding has emerged in the project that provoked re-thinking about the NRF2 pathway in terms of lung and other cancers. We have recently discovered that dysfunctional KEAP1 activity is a frequent alteration in lung adenocarcinomas which results in greater nuclear accumulation of NRF2 and enhances the transcriptional induction of electrophile, drug detoxification, and efflux proteins. Thus, unlike normal cells, lung cancer cells have unrestrained high NRF2 activity leading to therapeutic resistance. KEAP1 in the adenocarcinomas has deletions, insertions, and missense mutations in functionally important domains of the protein, and a very high percentage of loss of heterozygosity at 19p13.2, suggesting that biallelic inactivation of KEAP1 in these cancers is a common event. Mutations were detected in all stages of adenocarcinomas (including stage IA). Decreased KEAP1 activity, in-vitro, causes radio- and Chemoresistance. We have also identified mutations in KEAP1 in prostate tumors and recently similar mutations were reported in breast tumors.The new aims of this proposal are: Specific Aim 1: To determine the association between high NRF2 activity and Chemoresistance in non-squamous non-small cell lung cancer (NSCLC);Specific Aim 2: To determine whether targeted NRF2 inhibition can diminish Chemoresistancein preclinical models;and Specific Aim 3: To determine whether pharmacologically decreasing NRF2 activity in lung cancer can reverse Chemoresistance. Relevance to Public Health: The studies in this project have potential for both identifying biomarkers to predict drug sensitivity in patients with lung adenocarcinomas and to devise new means to reverse Chemoresistance in patients with drug-resistant tumors.

DESCRIPTION (provided by applicant): Lung cancer is the most common cause of cancer-related death worldwide and intrinsic therapeutic resistance of non small cell lung cancer (NSCLC) cells remains a major challenge in improving the effectiveness of cancer therapy. Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile/ xenobiotic detoxification enzymes and drug efflux proteins and confers cytoprotection against oxidative stress and apoptosis in normal cells. Kelch-like ECH-associated protein (Keap1) negatively regulates Nrf2 activity by targeting it for proteasomal degradation. We recently reported that biallelic inactivation of Keap1 results in constitutive activation of Nrf2 function in NSCLC. Preliminary studies indicate that constitutive activation of Nrf2 in lung cancer cells increases activity of central metabolic pathways, promotes in vivo tumor growth and contributes to chemoresistance. Thus, we hypothesize that the gain of Nrf2 function resulting from the loss of Keap1 activity promotes tumorigenesis and confers therapeutic resistance and wild type Keap1 functions as a tumor suppressor by inhibiting the activity of Nrf2. Specific Aim 1: To test the hypothesis that the gain of Nrf2 function promotes lung tumorigenesis (in the presence of an oncogenic signal). Specific Aim 2: To test the hypothesis that restoring wild type Keap1 function in NSCLC cells with high Nrf2 levels attenuates Nrf2 activity and suppresses tumor growth. Specific Aim 3: To test the hypothesis that the gain of Nrf2 function in lung cancer cells increases glucose flux through pentose phosphate pathway and Tricarboxylic acid cycle essential for promoting tumor growth. Specific Aim: To test the hypothesis that blocking the activity of Nrf2-dependent phosphate pathway enzymes (Glucose-6 phosphate dehydrogenase and Transketolase) in NSCLC cells with gain of Nrf2 function inhibits tumor growth and attenuates chemoresistance. These aims will help in understanding the regulation of lung tumorigenesis by a novel pathway and develop a strategy for targeting this pathway to circumvent therapeutic resistance. PUBLIC HEALTH RELEVANCE: The studies proposed in this project have potential for developing new means to inhibit tumor progression and chemoresistance. Successful completion of this project will develop a new therapeutic strategy for lung cancer treatment.

DESCRIPTION (provided by applicant): Chronic Obstructive Pulmonary Disease (COPD) is a major cause of morbidity and mortality in the United States and is a growing cause of chronic disease internationally. Presently, there are limited treatment options for this disease to modify the progression of airflow obstruction and decrease periodic exacerbations. Recent evidence has emphasized the central role of oxidative stress as a mechanism of COPD pathobiology. Evidence from our group has shown that COPD patients and animals exposed to cigarette smoke have impairment of antioxidant defenses which are caused by a defect in activity of Nrf2, a prolific regulator of anti-oxidant enzymes, glutathione homeostasis, and cytoprotective proteins. Activation of Nrf2 protects mice from developing emphysema with chronic smoke exposure, decreases oxidative stress, increases proteasomal anti-apoptotic cytoprotective responses, improves bacterial phagocytosis and killing, and reverses tobacco-smoke induced corticosteroid resistance. Similarly, in vitro Nrf2 activation in human COPD lung cells has shown improved cytoprotection, improved bacterial clearance, and restoration of steroid sensitivity. This trial focuses on sulforaphane, a derivative of cruciferous vegetables, which is a potent in-vitro and in-vivo stimulator of Nrf2 activity. We want to know whether ingestion of sulforaphane by COPD patients will increase Nrf2 activity and expression of downstream antioxidants in alveolar macrophages and bronchial epithelial cells. Accordingly, we are proposing a placebo-controlled randomized proof of principle trial of 25 and 150 micromoles of sulforaphane for 4 weeks in 90 COPD patients. Collections of alveolar macrophages by BAL, bronchial epithelial cells by endobronchial brushings will be performed at baseline and 4 weeks. Other biospecimens will include nasal epithelial cells, PBMCs, and expired breath condensate. If we can establish a safe and tolerable dose of sulforaphane and proof of concept that it improves in-vivo antioxidants via Nrf2, then we will have a novel candidate treatment for longer-term efficacy trials. Ancillary studies are proposed to explore the efficacy and mechanisms of sulforaphane to increase bacterial clearance and to restore steroid sensitivity in COPD lung cells. RELEVANCE: Treatments that modify the course of COPD is a major unmet health need in the US. Compelling preliminary evidence in animals and human tissues suggests sulforaphane, a chemical derived from cruciferous vegetables such as broccoli and cabbage, has beneficial effects to disrupt the progression of COPD. If this trial is successful, it will represent a major new avenue for treatment of this serious chronic disease. (End of Abstract)

DESCRIPTION (provided by applicant): Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. We and others have reported that loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small cell lung cancer, breast cancer, gall bladder cancer and more recently in prostate cancer. We recently showed that constitutive activation of Nrf2 in lung cancer and prostate cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression suppressed growth of lung cancer and prostate cancer cells, and resulted in increased sensitivity to chemotherapeutic drug-induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus we hypothesize that dysregulated Nrf2-Keap1 pathway is a novel determinant of chemoresistance/radioresistance and inhibition of Nrf2 signaling will enhance the efficacy of chemotherapeutic and radiotherapy. However, efficient delivery and sustained action of siRNA still remains a major challenge limiting its use in treatment of solid tumors and small molecule inhibitors of Nrf2 will overcome this hurdle and prove very beneficial in this scenario. Specific Aim: To develop small molecule inhibitors of Nrf2 for circumventing therapeutic resistance and enhancing the efficacy of chemotherapy and radiotherapy. A pilot screening of Sigma LOPAC library identified few putative inhibitors of Nrf2 with our optimized screening cell based assay but they were not very effective in inhibiting Nrf2 dependent signaling. We propose to screen MLSMR repository of small molecules, which contains more than 300,000 synthetic and natural compounds. Screening and identification of potent small molecule inhibitors of Nrf2 will provide novel opportunities to modulate Nrf2 activity in patients with tumors and increase the efficacy of cancer therapy. PUBLIC HEALTH RELEVANCE: The studies proposed in this project have potential for developing novel drugs which can be used as adjuvant to enhance the efficacy of chemotherapy and radiotherapy. Successful completion of this project will develop a new therapeutic strategy for cancer treatment.

DESCRIPTION (provided by applicant): Chronic Obstructive Pulmonary Disease (COPD) is a highly morbid disease, mainly caused by cigarette smoking in the United States, affecting 24 million people. COPD is characterized by an abnormal persistence airway inflammation, which is frequently amplified during fhe periods of acute exacerbation caused by bacteria, virus and other environmental pollutants. Abnormal airway inflammation is thought to contribute to chronic bronchitis, small-airway obstruction and emphysema. Corticosteroids are the most effective anti}} inflammatory drugs prescribed for treating multiple inflammatory disorders; however they show poor therapeutic benefit in COPD. Therefore, there is a compelling need to develop therapies to inhibit inflammation and improve corticosteroid responses in COPD patients. A growing body of evidence indicate that reduction in histone deacetylase 2 (HDAC2) contributes to corticosteroid resistance in COPD. Alveolar macrophages and peripheral lung tissue show significant decline in HDAC2 levels with progression of COPD. Oxidative and or nitrosative stress induced post-translational modifications of HDAC2 is primarily responsible for HDAC2 instability in COPD. Nuclear factor erythroid 2-related factor 2 (Nrf2) a bZIP transcription factor, protects from oxidative stress by upregulating a robust cytoprotective response that includes antioxidant defenses. We and others found a marked decline in Nrf2 pathway in peripheral lung tissue and alveolar macrophages in COPD. We hypothesize that increasing Nrf2-regulated antioxidant defenses will prevent HDAC2 inactivity and improve corticosteroid responsiveness in COPD. The goal of this application is two-fold: First, verify if defective Nrf2 signaling is associated with HDAC2 inactivity and corticosteroid resistance in COPD; second test if pharmacological Nrf2 activators inhibit HDAC2 inactivity and improves corticosteroid responsiveness in lungs cells isolated from COPD patients or mouse models. Positive outcome of the proposal will support Nrf2 as a molecular target for improving corticosteroid responsiveness in COPD and will help in rapidly moving sulforaphane or other Nrf2 upregulators into clinical trials. RELEVANCE (See instructions); COPD is a morbid condition, primarily caused by tobacco smoking and is the fourth leading cause of death in the United States. Identification of drug targets that can improve efficacy of steroids to suppress ain/vay inflammation can significantly reduce morbidity and progression of the disease. This projects aims at preclinical development of a potential drug target that can be used in the clinical setting to improve steroid responsiveness in COPD nafients.

DESCRIPTION (provided by applicant): Inflammation is a central etiological component of numerous diseases, and anti-inflammatory drugs, such as corticosteroids, represent important therapies for combating a large number of diseases. However, many diseases do not adequately respond to current anti-inflammatory therapies. Thus new anti-inflammatory therapies are needed. The inflammasome is a recently identified arm of the innate immune response, which is becoming increasingly recognized as an important determinant of numerous inflammatory diseases. The inflammasome is a mult-protein complex that responds to numerous stimuli, including bacteria, viruses, silica, asbestos, cholesterol, and uric acid, to initiate a pro-inflammatory response that is mediated by the cytokines IL-1b, IL-18, and IL-33. Improper activation of the inflammasome has been implicated in multiple diseases, including gout, arthritis, diabetes, Alzheimer's, pulmonary fibrosis, and atherosclerosis. The role of the inflammasome in other disorders is still evolving, and inhibitors of this pathway will provide potential therapeutics for numerous diseases, and will provide mechanistic studies that will aid in dissection of this pathway. There are four inflammasome complexes identified thus far, and each inflammasome responds to a unique combination of molecular activators. However, a central component of all inflammasomes is its activation and secretion of the cytokines IL-1b, IL-18, and IL-33. Thus, secretion of IL-1b is a suitable marker for inflammasome activation, and identifying novel inhibitors of the inflammasome will provide potential therapies for a large number of inflammatory diseases. Secondary and tertiary studies will more directly measure activity of the inflammasome complex protein caspase-1. Specific Aim: To utilize a high throughput cell-based assay to identify small molecule inhibitors of the inflammasome. This proposal will focus on screening compounds to identify inhibitors of IL-1b, as a marker of inflammasome activity. We have developed a high throughput 1536-well assay using the AlphaLISA IL1b Kit (Perkin Elmer) to quantify IL-1b secretion from the human monocyte cell line THP-1. Our preliminary data demonstrates that we can readily detect IL-1b secretion from activated THP-1 cells and can quantify IC50 values of known inflammasome inhibitors. We have established a collaboration with the NIH Chemical Genomics Center (NCGC), and our chemical library will be derived from their Molecular Libraries Program. This library consists of an expansive set of small molecules totaling greater than 300,000 compounds. Drugs identified by this primary screen will be validated and characterized by secondary and tertiary screens to measure activity of caspase-1 and NF-kB, and secretion of other inflammasome-dependent and independent cytokines. Hit compounds will be validated in cells from mice deficient in various components of the inflammasome, and follow-up studies will examine efficacy in vivo. Successful completion of the proposed study will yield novel compounds that inhibit the inflammasome in cell-based systems.

Project Summary About 3 billion people, half the worldwide population, are exposed to smoke from biomass fuel compared with 1 billion people who smoke tobacco, which suggests that exposure to biomass smoke might be the biggest risk factor for respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections globally. There is a disproportionate increase in COPD and asthma among non-smokers in rural India who use biomass for cooking. Ambient air particulate matter (PM) is well studied and linked to increased pulmonary oxidative stress and inflammation and causes greater respiratory diseases in the urban population; however, the effects of indoor PM due to biomass fuel on lungs and innate immune defense in the rural population remains unclear. The PI and co-investigators from JHU are collaborating with the two key pulmonologists from the India team who have established a rural cohort in Vadu village of Pune district (Maharashtra, India) that uses biomass fuel as their sole source of cooking fuel with high indoor PM. The PIs have collected indoor air PM (from rural houses with non-smokers). The main goal of this proposal is to characterize and compare the indoor PM from various biomass sources (cow dung, wood, and crop residue), and perform controlled mouse model studies using indoor PM to dissect the effects on lungs and innate immune response without the confounding effects of ozone and other gaseous components. The outcome from this study will help to develop future strategies for intervention in the rural population to reduce lung diseases. This proposal is an extension of the recently awarded FIRCA Eligible Award Children's Environmental and Disease Prevention Research Center P01 ES018176-01 (Project 3, 9/1/2009 - 8/31/2014). Project 3 (Mechanisms of asthma-dietary interventions against environmental triggers) and will test the hypothesis that antioxidants such as sulforaphane present in the diet can cause chemoprotection by activating the Nrf2 pathway to counteract oxidative stress and inflammation and protect against the pulmonary asthmatic response following ambient air particle exposure in mice. The specific aims in our current proposal provide a unique opportunity to test our hypothesis and to study the biological effects of indoor PM due to exposure to biomass fuel: SA1: To collect indoor PM and determine physicochemical characterization of different sources of biomass collected in rural homes. Homes in rural India use either cow dung, wood, crop residue, or a combination of the three for cooking. We have identified homes that will use only one biomass fuel source. The size and concentration of indoor PM in rural homes that use these different sources of biomass are being collected, and we will determine protein, endotoxin, metal and organic concentration in these indoor PM samples. This data will be helpful for characterizing the relative toxicity of each fuel source. SA2: To compare the effects of various sources of biomass-derived indoor PM collected in rural homes on lungs and innate immune response. We will test the hypothesis that indoor PM causes (a) oxidative stress, inflammation, and airway hyper-reactivity, and (b) inhibits the innate immune response. We will perform pulmonary aspiration of different indoor PM samples in A/J mice and measure markers of oxidative stress, inflammatory cells in bronchoalveolar lavage and lungs, cytokines and chemokines, and airway hyper- reactivity. We will also determine whether exposure of macrophages to indoor PM suppresses phagocytosis of bacteria. Although an inhalation exposure model is more realistic, airway instillation has been demonstrated to be a very effective model for comparing relative toxicity in mice where samples are limited. A/J mice were chosen because of their heightened sensitivity to airway challenges.

DESCRIPTION (provided by applicant): Project Summary Bacterial exacerbations cause significant morbidity and mortality and account for 70-80% of COPD-related health care costs. COPD patients exhibit steroid resistance and therefore, inhaled corticosteroids show only modest effects in reducing COPD exacerbations. Currently, there are no effective therapies to prevent COPD exacerbations. Oxidative stress plays a central role in the pathogenesis of COPD exacerbations by impairing pulmonary anti-bacterial innate immune defenses and augmenting inflammatory responses in lung epithelial cells and alveolar macrophages of COPD patients by inducing epigenetic chromatin modifications via altered HDAC2 activity. Our laboratory has established that the transcription factor Nrf2 is a major regulator of an adaptive response that counteracts oxidative stress, inflammation and host defense in lungs of cigarette smoke exposed mice and COPD patients. Deficiency of the transcription factor, Nrf2, impairs host defense and augments oxidative stress and emphysema in CS-exposed mice, while lungs of COPD patients show diminished Nrf2 activity. On the contrary, augmenting Nrf2 by a small molecule attenuates pulmonary innate immune dysfunction, inflammation, and alveolar destruction and improves steroid sensitivity in CS-exposed mice. Broccoli sprouts contain sulforaphane, which is a potent activator of Nrf2. Evidence from several animal and human studies have demonstrated antioxidant, anti- inflammatory and anti-cancer properties of broccoli sprout extract (BSE) or sulforaphane, which is largely mediated by enhancing the Nrf2 pathway. We have completed testing of GMP-manufactured BSE on 65 COPD patients for determining safety and dose tolerance. In this application, we will evaluate whether dietary supplementation of BSE attenuates bacterial exacerbations in preclinical models. We will investigate the mechanisms by which BSE improves pulmonary anti-bacterial defenses and corticosteroid sensitivity and protects from alveolar destruction in mice exposed to chronic cigarette smoke. We will identify proof-of- mechanism PD biomarkers of Nrf2, which could be translated to clinical trial. Successful completion of our proposed research will help us in designing a definitive clinical trial for prevention of COPD exacerbations.

Project Summary: There is a critical gap in our knowledge about the toxicity and health effects of waterpipe tobacco smoke relative to other modified tobacco products, even though its prevalence has sharply risen in recent years. The social culture of waterpipes in cafés and restaurants and the perception that waterpipe smoking is less harmful than cigarettes, along with the distribution of flavored tobacco products, continues to drive its popularity among younger adults. A recent report from the CDC showed that waterpipe use among high school students rose sharply in 2014 and was even more popular than cigarettes. To provide data that directly informs and contributes to waterpipe regulatory action, we have assembled a multidisciplinary team of investigators with expertise in the measurement of harmful and potentially harmful constituents (HPHCs) in tobacco products identified by the FDA, exposure science, tobacco related biomarkers, in vitro and in vivo respiratory toxicology, ?omics? approaches and in silico modeling, and standardized water-pipe inhalation system. We will test our hypothesis that various waterpipe products and puffing regimens will generate unique profiles of harmful constituents that will yield a continuum of toxic health effects in in vivo and in vitro models. Recently, we developed a mouse model of waterpipe smoke exposure using a puffing regimen similar to the Beirut protocol, and our preliminary evidence demonstrates that acute exposure to waterpipe smoke induces airway inflammation. Furthermore, we have quantified PM2.5, CO, particle-bound PAHs, NNK, and nicotine in 46 waterpipe venues and collected biospecimens from 170 employees in those venues, which will enable us to model mainstream and secondhand exposures and compare our model data to human biospecimens. We have established an in vitro air-liquid interface (ALI) exposure system in combination with a human 3D organotypic model for comparing products for respiratory toxicology (MucilAir). Specific aim 1: To define the constituents of mainstream and secondhand waterpipe smoke using different puffing regimens and products. Specific aim 2: To conduct comparative in vitro and in vivo exposures to different waterpipe products and assess multiple effect endpoints of toxicity and genotoxicity in lungs and in vitro airway epithelia. Specific aim 3: To identify biomarkers from the in vivo and in vitro studies (SA1 and 2) and examine human urine samples from individuals with recent mainstream/secondhand exposure to waterpipe smoke. We will use bioinformatics approaches to integrate human in vivo and in vitro data from Aims 1-3 to develop an overall hazard index.

? DESCRIPTION (provided by applicant): Obesity and type II diabetes (T2D) have been increasing globally at epidemic proportions. Epidemiological studies indicate that air pollution is an environmental risk factor for development of insulin resistance (IR) and T2D. However, the mechanisms by which air pollution exposure causes progressive IR leading to T2D remain unclear. There is increasing evidence that IR, which often precedes the T2D by decades, may be modulated through epigenetic changes. Gestation and early childhood are periods of enhanced vulnerability to air pollution and also coincide with the stage at which epigenetic patterning is first established. We have previously established in mouse models that concentrated ambient pollution (CAP) exposure for >12 weeks potentiates the development of obesity, IR and T2D, and also induces a range of abnormalities that are prototypical for IR and T2D, including changes in immune function in systemic tissues (hypothalamus, circulation and adipose), reduced brown adipose tissue function, hepatic endoplasmic reticulum stress/steatosis and prototypical defects in insulin signaling in insulin response tissues such as liver, skeletal muscle and adipose. We have further demonstrated that early age is a period of vulnerability, as exposure to CAP during this period accelerates development of IR with overt changes in glycemic control developing in <10 weeks. Further, these defects are secondary to critical alterations in reactive oxygen species (ROS) pathways and inflammation in insulin responsive tissues. CAP exposure also results in genome-wide increases in in 5-mC. Hypothesis: Air pollution exposure results in global epigenetic alterations that result in metaboli re-programming in target tissues leading to obesity and insulin resistance. We posit that epigenetic programming during vulnerable periods of development (e.g. in utero or early childhood) is particularly prone to irreversible changes in the epigenome and the persistence of IR. This project will leverage access to state-of- the-art ambient exposure facilities, investigatie expertise in exposure science, disease pathways and epigenetics available at the University of Maryland and Johns Hopkins University to accomplish these specific aims. SA1: To characterize the epigenomic changes in response to air pollution that may either precede or overlap with the development of an insulin resistant phenotype. SA2: To define the IR and global epigenomic changes associated with early life CAP exposures. SA3: To determine whether cessation of air pollution exposure reverses insulin resistance and corresponding epigenomic changes. SA4: To integrate the epigenomic and transcriptomic perturbations that track with the development of the IR phenotype. These animal model studies with our well optimized controlled air pollution exposure that cause metabolic syndrome will reveal comprehensive epigenomic marks that precede the phenotype and are conserved between the target and surrogate tissues. The data will provide new leads for validation of mechanisms as well insight into interpretation of human studies.

? DESCRIPTION (provided by applicant): Electronic cigarette (EC) use has increased dramatically in recent years, especially among never smokers. We recently completed a survey of EC users in Baltimore and found that a surprising percentage of EC users are non-smoking young adults. The oral cavity is host to one of the most complex microbial floras in the human body, and disturbance in this equilibrium can lead to overrepresentation of pathogenic species. Alterations in oral host defense, including reductions in salivary flow and secretion of antimicrobial peptides and cytokines by salivary glands, epithelial cells, and immune cells, may create a hospitable environment for pathogenic bacteria, leading to oral diseases. Our recent report indicates that EC exposure increases oxidative stress and inflammation, hampers the innate immune response, and increases susceptibility to pulmonary bacterial infections in a mouse model, indicating a high potential for adverse effects to oral host defense. EC exposure also causes inflammation of the salivary gland ducts in this model. However, a mouse model for studying effects on oral health is not representative of human exposure because mice are obligate nose breathers. EC vapor exposure in cell culture is also not representative of human exposure. Furthermore, the development of oral disease requires many years of exposure. Thus, cross-sectional and longitudinal alterations to the host oral defense and microbiome, which are early markers of risk, as a result of chronic exposure to ECs in young adults needs to be deciphered. We hypothesize that chronic EC use in young adults impairs the oral host defense by increasing oxidative stress and cytokines, and reducing salivary flow, antimicrobial peptides and epithelial defensins, leading to oral microbial dysbiosis. Specific Aim 1. Community structure and functional characterization of the oral microbiome in young adult EC users. Using samples from a cohort of young adult EC users and controls (age, 18-34 years; N=100/group), structural and functional characterization of the oral microbiome will be performed by 16S rRNA sequencing and metatranscriptomics for (a) cross sectional comparison of EC users with non-users and (b) a longitudinal follow up of EC users at 0, 1, and 2 years. Data from this Specific Aim will demonstrate functional differences in oral microbiomes of EC users that correlate with mechanistic biomarkers of oral host defense, which may increase the risk for oral disease. Specific Aim 2. Mechanistic evaluation of oral host defense in EC users. Along with the microbiome assessment in Specific Aim 1, oral host defense will be assessed via (1) salivary gland function, including salivary flow rate and salivary gland-specific anti-microbial peptides; () epithelial/immune cell antimicrobial defenses, including secretion of anti-microbial peptides, cytokines, and oxidative stress; and (3) biomarkers of inflammation and oral health in saliva and buccal epithelial cells. The integrative analysis of Specific Aim 1 and 2 will fill a critical gap n our understanding of alterations in host oral defense and a potential microbial shift in EC users, which may predispose them to oral diseases; thus, providing the evidence-based foundation for future public health policies on EC use.

? DESCRIPTION (provided by applicant): Nearly 25% of lung adenocarcinomas (LuAD) have deletions or inactivating mutations of the gene for LKB1. In response to metabolic stress, wildtype LKB1 promotes catabolic reactions for generating ATP and conserving antioxidant (NADPH and GSH) levels. Since LKB1 counteracts ROS resulting from metabolic stress, the inactivation of this gene in cancer seems to be contrary to expectations, and indeed, LKB1-deficient cells are relatively resistant to oncogenic transformation and sensitive to metabolic stress. TCGA sequencing data revealed that ~ 50% of LKB1-mutant LuAD also harbor a Kelch-ECH associated protein 1 (KEAP1) mutation. Can the impaired ability of LKB1-deficient cells to adapt to nutrient and metabolic stress be overcome by parallel loss of KEAP1? Our preliminary studies revealed that combined loss of Lkb1 and Keap1 decrease ROS and dramatically enhances tumor growth (histologically, adenocarcinomas), and mortality in KrasG12D driven mouse model of lung cancer. Even in the absence of oncogenic Kras signaling, Keap1-/-Lkb1-/- animals form tumors in the lungs with long latency. Importantly, LKB1-deficient cells showed impaired ability to adapt to metabolic stress in the absence of Nrf2 pathway activation, but this defect was rescued by simultaneous loss of Keap1 signaling. KEAP1 mutations lead to gain of NRF2 function in NSCLC that drives antioxidant pathways and metabolic alterations. We hypothesize that KEAP1 mutations in LKB1 mutated lung adenocarcinoma cells causes gain of NRF2 for ROS detoxification and metabolic pathway alterations, which are critical for tumor cell survival. To exploit the vulnerability of this adaptation, we hypothesize that inhibiting NRF2 in tumors with loss of LKB1 and KEAP1 will decrease tumor growth due to metabolic stress. Specific aim 1 will determine if selective loss of Keap1 signaling in Lkb1 deficient lung cells maintains cellular redox homeostasis and promotes lung tumorigenesis. We have developed mouse models with selective deletion of Lkb1 and Keap1 (with or without oncogenic stress - KrasG12D), transgenic mice expressing Nrf2 with activating mutation combined with Lkb1 deletion as well as LKB1 mutant human lung adenocarcinoma cell lines with gain of Nrf2 or loss of Keap1 function. Specific aim 2 will determine the mechanisms by which loss of Keap1 signaling cooperates with Lkb1 signaling for metabolic alterations to provide survival advantage. Transcriptomic and stable isotope resolved metabolomics studies will be performed. Specific aim 3 will determine if disruption of Nrf2 signaling in our mouse models of SA 1 will decrease tumor growth and improve survival using genetic as well as small molecule approach. In addition to genetic knockout of Nrf2, our preliminary studies have shown the efficacy of a novel small molecule (developed in collaboration with NCATS) for inhibiting NRF2 in vitro and in which will be explored for its potential for targeting the Nrf2 pathway in treating LKB1-mutant cancers. These studies will (a) provide the molecular understanding of why 50% of LKB1 loss coexist with KEAP1 mutations in LuAD (b) create the preclinical knowledge essential for targeting this cooperation.

? DESCRIPTION (provided by applicant): Obesity and type II diabetes (T2D) have been increasing globally at epidemic proportions. Epidemiological studies indicate that air pollution is an environmental risk factor for development of insulin resistance (IR) and T2D. However, the mechanisms by which air pollution exposure causes progressive IR leading to T2D remain unclear. There is increasing evidence that IR, which often precedes the T2D by decades, may be modulated through epigenetic changes. Gestation and early childhood are periods of enhanced vulnerability to air pollution and also coincide with the stage at which epigenetic patterning is first established. We have previously established in mouse models that concentrated ambient pollution (CAP) exposure for >12 weeks potentiates the development of obesity, IR and T2D, and also induces a range of abnormalities that are prototypical for IR and T2D, including changes in immune function in systemic tissues (hypothalamus, circulation and adipose), reduced brown adipose tissue function, hepatic endoplasmic reticulum stress/steatosis and prototypical defects in insulin signaling in insulin response tissues such as liver, skeletal muscle and adipose. We have further demonstrated that early age is a period of vulnerability, as exposure to CAP during this period accelerates development of IR with overt changes in glycemic control developing in <10 weeks. Further, these defects are secondary to critical alterations in reactive oxygen species (ROS) pathways and inflammation in insulin responsive tissues. CAP exposure also results in genome-wide increases in in 5-mC. Hypothesis: Air pollution exposure results in global epigenetic alterations that result in metaboli re-programming in target tissues leading to obesity and insulin resistance. We posit that epigenetic programming during vulnerable periods of development (e.g. in utero or early childhood) is particularly prone to irreversible changes in the epigenome and the persistence of IR. This project will leverage access to state-of- the-art ambient exposure facilities, investigatie expertise in exposure science, disease pathways and epigenetics available at the University of Maryland and Johns Hopkins University to accomplish these specific aims. SA1: To characterize the epigenomic changes in response to air pollution that may either precede or overlap with the development of an insulin resistant phenotype. SA2: To define the IR and global epigenomic changes associated with early life CAP exposures. SA3: To determine whether cessation of air pollution exposure reverses insulin resistance and corresponding epigenomic changes. SA4: To integrate the epigenomic and transcriptomic perturbations that track with the development of the IR phenotype. These animal model studies with our well optimized controlled air pollution exposure that cause metabolic syndrome will reveal comprehensive epigenomic marks that precede the phenotype and are conserved between the target and surrogate tissues. The data will provide new leads for validation of mechanisms as well insight into interpretation of human studies.

It is estimated that while the majority of SARS-CoV2 infections in the ongoing coronavirus disease-2019 (COVID-19) pandemic are asymptomatic or have mild symptoms, hospitalizations and mortality largely occurs in patients with co-morbid conditions such as obesity, diabetes and COPD. Our understanding of the role of environmental exposures in modifying the response to SARS-CoV2 is emerging and air pollution; smoking and vaping have been associated with worst outcomes of SARS-CoV2 patients. There is a time sensitive urgent need to understand host defense mechanisms which are compromised due to environmental exposures and may increase susceptibility to SARS-CoV2 infection. This competing revision will forge collaboration with expert in SARS-CoV2 research to expand our horizon in this critical area. We will test the hypothesis of targeting a host defense pathway which is compromised in air pollution that may protect and modify the response to SARS-COV2 respiratory infection. Through the parent U grant, we have demonstrated that chronic exposure to PM2.5 has an overarching role in epigenetic reprogramming. Our studies have established that transcription factor Nuclear factor erythroid-factor 2 (Nrf2) is a key activator of anti-oxidative, anti-inflammatory, and innate immune defenses. We and others have demonstrated in human biospecimens and animal models that chronic exposure to PM2.5 causes a decline in Nrf2 activity that correlates with compromised innate immune defenses. In mice deficient for Nrf2 (Nrf2-/-), viral and bacterial infection causes oxidative stress, worsened lung inflammation, acute lung injury and greater mortality compared to wildtype mice. Genetic or pharmacological activation of Nrf2 pathway can rescue these effects. Disruption of Nrf2 pathway has been shown to cause upregulation of angiotensin-converting enzyme 2 (ACE2) which is the functional receptor for SARS-CoV2 entry into lung epithelial cells. Furthermore, hypomethylation in ACE2 gene has been demonstrated to increase ACE2 expression in immunocompromised patients. The goal for this project is to investigate the crosstalk of air pollution exposure, host defense and SARS-CoV2 infection. Preclinical testing of therapeutic efficacy of Nrf2 activators will provide proof of concept for further development a novel drug target for prevention and treatment of SARS-COV2 infection. The proposal will leverage expertise of our team on air pollution, respiratory diseases and an expert virologist with ongoing BSL-3 SARS-CoV2 research. Successful completion of this project will provide proof of concept for future studies directed towards development of a novel strategy of targeting host defense for prevention and treatment of SARS-CoV2 infection in susceptible populations.