Beverly H. Koller - US grants

The long term objective of this proposal is the generation of a mouse model for cystic fibrosis (CF). Mutations known to cause CF in humans will be introduced into the murine CFTR gene of pluripotent embryonic stem (ES) cells. Mouse lines will be derived from ES cells carrying these mutations and evaluated for the presence of disease similar to that characteristic of CF in humans. The extent to which the pathophysiology of the disease in mice mimics that in humans will define whether the mouse model will be useful for testing therapeutic protocols, including gene therapy. We have six specific aims: 1. We will generate a mouse line carrying a CFTR gene in which a termination codon has been introduced into exon 10 by gene targeting. 2. We will generate a mouse line carrying the most common mutation found in human CF patients, the deletion of three bases in exon 10. 3. We will generate mice carrying the A117H mutation in exon 4, which is known to result in a mild form of CF in humans. 4. During the course of generating these three animals we will define factors which affect the targeting of DNA to the CFTR locus. 5. We will generate mice in which the expression of the CFTR gene can be detected with a chromogenic assay to assist in the analysis of disease in these animals. 6. We will evaluate the disease caused by the three CFTR mutations to determine whether the initiation and progression of the disease are similar to those in humans. This will determine to what extent these animals can be used for in vivo analysis of the pathophysiology of CF and for testing new approaches to the treatment of CF.

The long term objective of this proposal is the generation of a mouse model for cystic fibrosis (CF). Mutations known to cause CF in humans will be introduced into the murine CFTR gene of pluripotent embryonic stem (ES) cells. Mouse lines will be derived from ES cells carrying these mutations and evaluated for the presence of disease similar to that characteristic of CF in humans. The extent to which the pathophysiology of the disease in mice mimics that in humans will define whether the mouse model will be useful for testing therapeutic protocols, including gene therapy. We have six specific aims: 1. We will generate a mouse line carrying a CFTR gene in which a termination codon has been introduced into exon 10 by gene targeting. 2. We will generate a mouse line carrying the most common mutation found in human CF patients, the deletion of three bases in exon 10. 3. We will generate mice carrying the A117H mutation in exon 4, which is known to result in a mild form of CF in humans. 4. During the course of generating these three animals we will define factors which affect the targeting of DNA to the CFTR locus. 5. We will generate mice in which the expression of the CFTR gene can be detected with a chromogenic assay to assist in the analysis of disease in these animals. 6. We will evaluate the disease caused by the three CFTR mutations to determine whether the initiation and progression of the disease are similar to those in humans. This will determine to what extent these animals can be used for in vivo analysis of the pathophysiology of CF and for testing new approaches to the treatment of CF.

Description: (Taken directly from the application): Leukotrienes (LTs), potent lipid mediators derived from arachidonic acid (AA), can be isolated from virtually all inflammatory lesions and thus have been implicated in the pathogenesis of both acute and chronic inflammatory diseases, including allograft rejection. However, as response to inflammatory stimuli results in the activation of numerous mediators with overlapping activities, defining the role of LTs in the initiation, perpetuation and resolution of a particular inflammatory lesion in vivo has been difficult. To address this question directly we have generated mice deficient in the synthesis of these lipid. Specifically we have generated mice deficient in 5-lipoxygenase (5LO) and 5-lipoxygenase activating protein (FLAP) proteins essential for the production of all biologically active LTS, and in LTA4hydrolase, an enzyme required for the synthesis of LTB4-Using these mouse lines we have defined a role for these lipids in models of acute inflammation. These models provide simple in vivo systems for defining the contribution of these lipid mediators to inflammatory responses. In addition they provide systems for; identification of specific cell types that produce and are affected by these lipids, for the study of the regulation of the LT pathway in vivo, and for defining the interaction of LTS with other inflammatory mediators. Using these models, studies completed during the preceding grant period have identified powerful effects of background genes on the relative contributions of LTS to inflammation. Based on these data, we hypothesize that genetic factors have a profound influence on the actions of LTS in inflammatory responses including allograft rejection. In this application we will define the mechanisms by which genetic factors modify the role of leukotrienes in acute inflammation. In addition we will determine whether these same genetic factors determine the contribution of LTS to the initiation and progression of allograft destruction. Finally, we will determine the relative anti-inflammatory effects of ablating LT receptor signaling compared to inhibition of LT synthesis in both acute inflammation and in allograft rejection. We will determine whether, similar to the effects of inhibition of LT synthesis, the impact of this lesion can also be altered by modifier genes.

DESCRIPTION (Taken directly from the application) Extensive analysis has revealed that CFTR deficient mice do not develop the type of life threatening respiratory disease that characterizes human cystic fibrosis. This suggests that factors in addition to the loss of normal CFTR function may play a role in the pathogenesis of human CF. Two factors that we hypothesize may play such a role are mucus secretion and chloride secretion through channels other than CFTR. To test whether these factors due indeed play a role in the development of respiratory disease subsequent to loss of CFTR function, we will selectively alter chloride and mucus secretion in the airways of CFTR deficient mice. With regard to chloride secretion, we hypothesize that the damage to a particularly tissue caused by loss of CFTR function can be related to the extent to which the epithelium of that tissue is dependent on CFTR for chloride secretion. With regard to mucus secretion, we hypothesize that the damage caused by loss of CFTR function in a particular tissue can be correlated with the amount of mucin secreted by the epithelium in that tissue. In addition to testing these two hypotheses separately, we will also determine whether the pathological changes associated with loss of CFTR function may result from a strong reliance on CFTR for chloride secretion in combination with the secretion of relatively large amounts of mucus. The final aim of this application is to develop new techniques that will extend our ability to evaluate the role of particular gene products in the pathogenesis of CF in vivo. More specifically, we propose to develop a system that will allow us to modify the expression of genes in mouse airways in a tissue and developmentally specific manner. In addition to providing a better understanding of the pathogenesis of human CF, such a system should also aid in the identification of potential targets for therapeutic intervention in the disease.

The Mouse Core is designed to establish the transgenic mouse lines required by the PPG investigators. It is also responsible for the crossing of these various lines with mice deficient in various genes as a result of targeted mutations. The Core will also be responsible for all breeding and genotyping required for supplying the animals described in this proposal. Specifically, the Mouse Core will be responsible for the following: 1. Identification of founder animals after pronuclear injection of mouse embryos with the following constructs: CCSP-hP2U-R; CCSP-(beta, gamma) rENaC; CCSP-tri-cistronic (alpha, beta, gamma) rENac; CSSP(alpha, beta/T, gamma) rENaC. Breeding of the above founders in order to establish lines homozygous for each of the transgenes. Breeding sufficient numbers for the experiments outlined in the projects described in this application. 2. Breeding sufficient numbers of mice carrying mutations introduced by homologous recombination into the P2U-R, and ENaC loci for the experiments described. 3. Breeding to obtain mice with the following combination of mutations: (1) breeding for mice carrying transgenes for all sub-units (alpha, beta, gamma) of the sodium channel e.g., breeding CCSP-alpha rENaC animals with CCSP-(beta, gamma) rENac animals, CCSP (alpha, beta, gamma) rENaC; (2) breeding of these animals with CFTR(-/-) animals to obtain animals which overexpress the Na channel and do not express the CFTR gene; (3) breeding of P2U-R(-/-) animals with CFTR(-/-). 4. Introducing transgene mutations onto an inbred mouse stain. 5. Providing pregnant animals for projects.

DESCRIPTION (Adapted from applicant's abstract and specific aims): A key factor in the normal function of the respiratory tract is the regulation of mucociliary transport by the airway epithelium. A number of factors appear to influence this process, and the regulation of these factors is complex. Prostaglandins (PGs) have characteristics that suggest they could be important mediators controlling physiological parameters that affect mucociliary clearance (MCC), including ion transport, ciliary beat frequency (CBF) and perhaps even mucin release by goblet cells. Airway surface liquid (ASL) is a 15-30 um thick layer of low viscosity fluid that covers the mucosal surface of the epithelium lining the conducting airways. This fluid layer represents the medium in which ciliary beating occurs, propelling the overlaying layer of thicker, more viscous mucus and entrapped debris out of the lung. The efficiency of this process is highly dependent upon the thickness of the ASL, and this in turn is influenced by the rate of salt and water transport across the airway epithelium. Changes in the electrolyte composition of the ASL may also affect other aspects of mucociliary action. These include not only physical properties of the overlying mucous layer but also the CBF. The composition of the ASL may be critical for the antimicrobial activity of molecules such as defensins, which are secreted by cells of the airway epithelium and serve as a first line of defense against bacterial infection of the respiratory tract. The overall hypothesis tested in this application is that PGs play a central role in regulating those properties of ASL that are crucial for efficient MCC. The goal is to define and quantitate the contribution of PGs to regulation of MCC and to determine the underlying mechanism by which PGs contribute to this process. The focus will be on the prostaglandin PGE2 and its actions mediated through four cell surface receptors. The specific aims are: 1) Generation of mouse lines deficient in each of the PGE2 receptors; 2) Examination of PG production, receptor expression and signal transduction in normal and inflamed mouse airways; 3) Characterization of the role of PGE2 in regulation of ion transport, CBF, and mucus secretion in mouse airway epithelium; and 4) Examination of the role of PGE2 in airway disease in mouse.

The overall goal of this proposal is to test the hypothesis that the co- transporter, NKCC1, in parallel with CFTR and other apical membrane CL- transporters is required to regulate airway surface liquid volume in response to irritation and concomitant increase in mucin secretion by airway epithelium. The epithelia of the fetus is primary secretory. We believe that the movement of fluid across the fetal airway epithelium in both mouse and humans occurs by a variety of pathways, some of which involve the CFTR protein in parallel with the co-transporter. After birth, a decrease in the secretory function of airway epithelial cells must occur. This adjustment is brought about by decreasing the expression of the CFTR and NKCC1 genes and increasing the capacity of the airways to absorb Na+. It is perhaps only in the submucosal glands that the secretory function of the airway epithelium remains unchanged. We speculate that CFTR and NKCC1 co-transporter expression throughout the remainder of the adult airways reflects a preservation of the secretory ability of this airway. This secretory function may provide a sensitive inducible mechanism for adjusting the airway surface liquid volume. This may be mediated by increased secretion of fluid by epithelial cells of the small airways or by localized increases in secretion throughout the airways. The adjustment of the airway surface liquid volume by activation of Cl- secretion facilitates the flushing of noxious agents from the surface of the epithelium, and thus is critical for maintaining the health of the airways. To test this hypothesis we propose to generate mouse lines in which the function of the NKCC1 has been compromised. This mice will be generated from embryonic stem (ES) cell lines carrying a NKCC1 gene into which mutations have been introduced by homologous recombination. The impact of loss of this Cl-pathway on ion transport by airway epithelia and on the development and health of the mouse airways will be determined.

BRCA1 is a nuclear phosphoprotein expressed in a broad spectrum of tissues during cell division. The inheritance of a mutant BRCA1 allele dramatically increases a woman's lifetime risk for developing both breast and ovarian cancer. The discovery of loss of heterozygosity at the BRCA1 locus in a significant number of breast and ovarian tumors has lead to the classification of BRCA1 as a tumor suppressor gene. However, the mechanism by which loss of BRCA1 function leads to increased tumor incidence has not been established. A variety of evidence has led to various proposals concerning the function of the BRCA1 gene, but the lack of a BRCA1 deficient cell line has made it difficult to directly test these hypotheses. To overcome this limitation, we have generated mouse cell lines that are deficient in the murine homologue of BRCA1 from three different tissues. First, we have isolated a Brac1 -/- embryonic stem (ES) cell line. We have used ES cells heterozygous for a mutant Brac1 allele to generate mice carrying the mutation. Some mice carrying a single copy of the Brac1 allele, as well as a mutant p53 allele, develop mammary tumors after exposure to gamma irradiation. Brac1 -/- cell lines have been established from these tumors. Finally, the survival of a small percentage of p53/Brac1 -/- mice has allowed us to obtain Brac1 -/- primary fibroblasts and mast cells. In this proposal we determine the contribution of BRCA1 to maintenance of genome integrity. Using these cell lines we examine the effect of loss of Brac1 function on cell growth and sensitivity to DNA damaging agents. We then determine whether these differences reflect a role for BRCA1 in maintenance of cell cycle checkpoints and/or a direct role in DNA repair. We believe that these studies will contribute to development of a model for the mechanism by which BRCA1 plays an essential role in normal cell growth and by which loss of BRCA1 function contributes to malignant transformation.

DESCRIPTION (the applicant's description verbatim): Pharmacological and clinical evidence has implicated prostanoids, arachidonic acid (AA) metabolites produced by cyclooxygenases 1 and 2 (COX-1 and COX-2), in the physiology of the blood vessel. In this proposal, we focus on the ductus arteriosus (DA) as a model for studying the role of prostanoids in vascular remodeling. The DA is an arterial connection in the fetus that directs blood away from the pulmonary circulation and towards the placenta where oxygenation occurs. Permanent closure of the DA depends on a series of structural changes that are independent of the reversible muscular contraction that initially reduces blood flow through this shunt after birth. It has been hypothesized that the DA has an intrinsic tone that is opposed by the dilatory effects of prostanoids such as PGE2 and that a postnatal decrease in levels of circulating prostanoids provides the signal that triggers DA closure. This idea was recently challenged by our observation of mice deficient in the EP4 receptor for PGE2. Contrary to our expectations, we found that the DA in EP4-/- mice remained patent throughout gestation but failed to close after birth, leading to perinatal death. To reconcile the results of previous pharmacological studies with those obtained through our genetic approach, we proposed a model in which EP4 expressed by the DA acts as a sensor that triggers closure of the DA in response to a perinatal drop in circulating levels of PGE2. We propose here to use a combined genetic, molecular, and pharmacological approach to develop a comprehensive molecular model for the mechanism by which the PGE2/EP4 pathway contributes to the events leading to vascular remodeling of the DA at birth. In addition, we propose to use a population of mice in which DA closure occurs in the absence of the EP4 receptor to identify other pathways that contribute to vascular remodeling of the DA. We believe that understanding these mechanisms and pathways will improve our general understanding of the way in which blood vessels are remodeled in specific disease states.

DESCRIPTION (provided by applicant): Eicosanoids, products of arachidonic acid (AA) metabolism, are potent modulators of the inflammatory response. A significant role for these lipid mediators in the pathogenesis of asthma has been demonstrated, and drugs targeting one branch of this pathway, the leukotriene pathway, have recently been developed and added to our therapeutic armamentarium against asthma. A number of studies have suggested that prostanoids, cyclooxygenase (COX) metabolites of AA, have a significant impact on inflammation in asthmatic patients. The most extensively studied of these is prostaglandin E2 (PGE2). A number of lines of evidence suggests that PGE2 plays a significant anti-inflammatory role in asthma, and might produce many of the same beneficial effects observed after treatment with corticosteroids. Moreover, PGE2 has been shown to have not only immunomodulatory actions, but also to oppose antigen induced bronchoconstriction. While all other prostanoids are thought to act through a single cell surface receptor specific for that prostanoid, four receptors for PGE2 have been identified and cloned, and have been termed EP1-EP4. Each receptor subtype binds PGE2 with equal affinity, but these receptors can trigger unique signal transduction pathways, which in turn can evoke opposing physiologic actions. Most cells express a unique combination of EP receptor subtypes, and the physiologic response of that cell to PGE2 is determined by the subset of receptors expressed. The therapeutic potential of PGE2 has not yet been exploited in part due to this complex physiology. We hypothesize that PGE2 plays a key role in shaping the pathogenesis of allergic airway disease and that its role in asthma is complex due to the diverse and sometimes opposing actions of the different EP receptor isoforms. As increased intracellular cAMP levels cause relaxation of smooth muscle cells and inhibition of inflammatory responses, we posit that stimulation of the Gs-coupled EP2 and EP4 receptors will attenuate disease. Conversely, stimulation of the PLC-coupled EP1 receptor might cause bronchoconstriction and our preliminary studies have identified potent pro-inflammatory actions of the EP3 receptor. Therefore, we suggest that stimulation of EP1 and EP3 receptors might promote allergic airway disease. By defining the precise role of each EP receptor along with maneuvers designed to enhance or reduce PGE2 production in the lung, we will determine the potential of PGE2 and its receptors as therapeutic targets in asthma.

(Applicant's Abstract) Eicosanoids, products of arachidonic acid (AA) metabolism, are potent modulators of the inflammatory response. A significant role for these lipid mediators in the pathogenesis of asthma has been demonstrated, and drugs targeting one branch of this pathway, the leukotriene pathway, have recently been developed and added to our therapeutic armamentarium against asthma. A number of studies have suggested that prostanoids, cyclooxygenase (COX) metabolites of AA, have a significant impact on inflammation in asthmatic patients. The most extensively studied of these is PGE2. A number of lines of evidence suggests that PGE2 plays a significant anti-inflammatory role in asthma, and might produce many of the same beneficial effects observed on treatment with corticosteroids. Moreover, PGE2 has been shown to have not only immunomodulatory actions, but bronchodilatory effects as well. While all other prostanoids are thought to act through a single cell surface receptor specific for that prostanoid, four receptors for PGE2 have been identified and cloned, and have been termed EP1-EP4. Each receptor subtype binds PGE2 with equal affinity, but these receptors can trigger unique signal tranduction pathways, which in turn can evoke opposing physiologic actions. Most cells express a unique combination of EP receptor subtypes, and the physiologic response of that cell to PGE2 is determined by the subset of receptors expressed. The therapeutic potential of PGE2 has not yet been exploited in part due to this complex physiology. The overall aim of this proposal is to evaluate the contribution of PGE2, through each of these the receptors, to the pathogenesis of allergic airway disease and to determine the potential of both PGE2 and the four EP receptors as therapeutic targets for the treatment of asthma.

DESCRIPTION (provided by applicant): Airways are protected from invading microorganisms by a highly efficient innate immune system. One defense mechanism available to the lung is the production of airway surface liquid and protective mucus, which ensnares viral and bacterial particles. This protective barrier coupled with the action of the ciliated airway cells, expels most foreign particles without engaging resident immune cells. In addition, most epithelial cells, including those of the airways, secrete microbicidal proteins into the biofilms that separate them from the external environment. One well-characterized family of antimicrobial peptides produced by mammals are the defensins. These are small cationic peptides capable of directly killing both gram positive and gram-negative bacteria as well as fungi and some enveloped viruses. They are also found in neutrophils where they are believed to be essential for non-oxidative killing of ingested microbes. The importance of this family of molecules is underscored by the fact that, not only have these genes been identified in all mammalian species studied, but it is likely that with more than 40 defensins in the human genome, there has been selection for redundancy in this system to ensure maximal protection against a broad spectrum of pathogens. More recently, evidence has emerged that suggest that these peptides might have a number of other important functions both in the innate immune response and in the transition of this response to one that engages lymphocytes. They may act both as natural antibiotics and as signaling molecules that activate host cells involved in immune defense and repair. We have identified a novel gene conserved between mouse and man, termed onzin, whose structure and sequence suggest that it is related to defensins. This gene is expressed by both the airway and the intestinal epithelia and is also expressed in high levels in neutrophils. In this application we test the hypothesis that onzin represents a novel defensive pathway that has a role in the innate immune response of the airways

DESCRIPTION (provided by applicant): NKCC1 is an electrolyte transporter localized to the basolateral membrane of airway epithelial cells that mediates electroneutral transport of chloride into the cell. The apical expulsion of CI- provides a driving force for the formation of airway surface liquid. Along with its high level of expression on airway epithelial cells, NKCC1 is widely expressed throughout the lung in airway smooth muscle cells as well as both resident and recruited immune cells. Less is known about its function in these non-epithelial cells where it has traditionally been considered to act simply as a regulator of cell volume. However, recent evidence suggests that NKCC1 might contribute to fundamental cellular functions including cell division, cell migration and optimal activity of a number of intracellular signaling pathways. The apparent importance of NKCC1 in this wide range of critical functions suggests that alterations in its expression level or activity might have direct consequences in the pathogenesis and pathophysiology of airway diseases. This view is supported by recent gene expression analyses from individuals with asthma revealing dramatic increases in the expression of NKCC1 in the asthmatic airway and by airway epithelial cells. This observation also suggests a molecular basis for previous reports showing that furosemide, an inhibitor of NKCC1, might be useful in the treatment of asthma. To further define the role of NKCC1 in airway disease we have recently examined the development of allergic airway disease in mice lacking NKCC1. We found that the loss of NKCC1 significantly attenuated inflammatory cell infiltration and airway reactivity. Based on these findings indicating a non-redundant role for NKCC1 in allergic airway disease, we propose to test the hypothesis that NKCC1 contributes to airway inflammation by: 1. modifying secretory functions of the airway epithelia; 2. increasing airway smooth muscle reactivity; and 3. by optimizing the migration and activation of inflammatory cells.

Leukotrienes (LTs) have been implicated in the regulation of immune responses, including inflammation and allograft rejection. These molecules belong to a family of biologically active lipids called eicosanoids that are derived from arachidonic acid (AA). The eicasanoids include both the cylooxygenase products(prostaglandins and thromboxanes) and the 5-lipoxygenase (5lo) products, the leukotrienes. The conversion of AA to leukotriene A4 (LTA4) by the enzyme 5lo is the first step in the synthesis of all leukotrienes. LTA4 can either be converted to leukotriene B4 by LTA4 hydrolase or be conjugated with reduced glutathione by LTC4 synthase to form the peptidyl leukotrienes, LTC4 and LTD4. We have generated mouse lines deficient in the synthesis of all leukotrienes and a line defective in the production of only LBT4. We have used these mice to define the contribution of lipids to various acute and chronic inflammatory responses including allograft rejection. We have found that this role can differ significantly depending on the sex of the mice and on the strain of the animals. One or more of these factors could be shown to be variables in both acute and chronic inflammatory responses. Initial studies suggest that the contribution of leukotrienes to allograft rejection may also be dependent on the strain combination examined. The overall aim of this proposal is to define the mechanism(s) by which the genetic background of host and graft determine the contribution of leukotrienes to inflammatory responses including acute and chronic allograft rejection. We will do this by testing the following possible hypotheses. a. Leukotriene synthesis by either the infiltrating leukocytes or the somatic cells of the graft is higher in strains in which ablation of this pathway has the greatest impact. b. Expression of leukotriene receptors is qualitatively or quantitatively different in these strains. c. Related pathways such as the cyclooxygenase pathway or lipoxin pathway are differentially modulated in various strains when 5lo pathway is blocked.

DESCRIPTION (provided by applicant): Prostaglandin E2 is an important modulator of airway physiology. During episodes of allergic inflammation such as in asthma, PGE2 may affect disease pathogenesis through 2 distinct pathways: by direct effects on airway tone and by modulating the intensity of the inflammatory response. These actions of PGE2 are mediated by 4 different receptors (EP1-4). In the previous funding period, we showed that activation of the EP1 and EP3 receptors by PGE2 leads to airway obstruction, while activation of the EP2 receptor protected against methacholine-induced bronchoconstriction. Moreover, EP1 and EP3 receptors are pro-inflammatory while the EP2 and EP4 receptors constrain inflammatory and immune responses. As a lipid mediator with a short half-life, regulation of PGE2 levels within specific pulmonary microenvironments could provide a mechanism to control these apparently disparate actions. PGE2 is synthesized from arachidonic acid by the sequential actions of phospholipases, cyclo-oxygenases, and PGE synthases. To date, 3 putative PGE synthases have been identified that generate PGE2 from endoperoxides. At least 1 enzyme responsible for in vivo metabolism of PGE2, 15-prostaglandin dehydrogenase (PGDH), has been identified. However, the roles of these various pathways for synthesis and metabolism of PGE2 in controlling its actions in the airways are not known. Our central hypothesis is that PGE2 primarily exerts a protective effect in allergic airway. We posit that the mechanism of this effect is: constraint of inflammation, protection against development of hyper reactive airways, and attenuation of airway remodeling. These protective effects are dependent on expression of the PGE2 EP2 and EP4 receptors. We will test this hypothesis using mouse models, some of which were developed in the previous funding period. Our preliminary studies suggest that the protective actions of PGE2 may diminish with age due to age-related increase in the pro-inflammatory PGE2 pathways. We hypothesize that the mechanism for this shift in the actions of PGE2 in older animals reflects a more prominent contribution of mast cells to promote airway inflammation. Defining the mechanisms used by PGE2 to control inflammation in the airways and developing strategies to enhance its anti-inflammatory effects should provide new approaches for attenuating the development of asthma.

[unreadable] DESCRIPTION (provided by applicant): Many of the mediators that regulate airflow in healthy individuals and that contribute to altered airway physiology in patients with pulmonary diseases signal through G protein coupled receptors. These receptors include not only the acetylcholine responsive muscarinic receptors of the parasympathetic pathways but also receptors for a number of inflammatory mediators. Increased production of eicosanoids, lipid mediators derived from arachidonic acid, is observed in virtually all airway diseases. In particular, the inflamed lung produces substantial quantities of thromboxane A2 (TXA2). Thromboxane activates a single GPCR Tp receptor that is broadly expressed in the lung. The mechanism(s) by which inflammatory mediators such as thromboxane contribute to altered airway physiology, in particular the development of hyperresponsive airways, is not well understood. Even less information is available concerning synergism between these inflammatory mediators and neuronal pathways that regulate airway smooth muscle tone in the healthy airway. TXA2 is a potent mediator of both bronchoconstriction and bronchial hyperreactivity in humans. The mechanism by which TXA2 mediates these actions is not well understood. The prevailing view is that TXA2, similar to other eicosanoids, regulates bronchial tone through direct, receptor-mediated effects on smooth muscle. However, based on our preliminary experiments, we suggest an alternative hypothesis: TXA2 regulates airway resistance in a complex manner that integrates direct actions on smooth muscle and indirect actions mediated by stimulating or amplifying specific cholinergic pathways or by altering mediator release by epithelial cells. In this application, we will test these hypotheses and determine the relative contributions of these distinct pathways for regulating airway resistance in the normal lung and during inflammation. Understanding of the interactions between the inflammatory Tp activated pathways and the cholinergic pathways that regulate airway tone will help define the events that lead to increased airway hyperresponsiveness and airway obstruction [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The neurotransmitter serotonin (5-HT) is produced by a two step enzymatic process from the amino acid tryptophan, primarily by neurons located in the raphe nuclei. These neurons project too many regions of the brain including the cortex, thalamus, hypothalamus, and amygdala. Released serotonin can act on over 14 different receptors, expressed by both presynaptic and postsynaptic neurons. Following vesicular release, the action of 5-HT is terminated by its removal from the synapse by the serotonin transporter (5-HTT, SERT, SLC6A4). Deregulation of serotonergic pathways has been implicated in many psychiatric and affective disorders including depression, autism, and obsessive compulsive disorder. These associations are supported by the demonstration that 5-HTT is the major site of action of many antidepressants. 5-HTT inhibitors (SRIs) are effective, not only in treating depression, but also in the treatment of other disorders including anxiety, obsessive compulsive disorder (OCD), and some aspects of autism. Genetic studies have reported association of both common and rare polymorphisms in the 5-HTT gene with increased risk for psychiatric and affective disorders. These results not only provide a rationale for targeting 5-HTT in the treatment of these illnesses, but also suggest that altered expression and activity of the transporter may contribute to the development of the illness itself. It has proved difficult to directly test either the contribution of polymorphisms to disturbances in serotonin homeostasis in vivo or the ability of these disturbances to manifest as measurable changes in behavior. To begin to address these questions, we propose the generation of novel mouse lines. These lines are designed to test the hypothesis that polymorphisms in the 5-HTT promoter alter expression of this gene and that the resulting changes in serotonin metabolism are sufficient to cause measurable changes in behavior. To accomplish this goal we have developed a unique two step method for exchange of the locus encoding a specific mouse gene with its human counterpart. Because the entire mouse gene, including the promoter and 5'and 3'flanking regions, is exchanged with the corresponding human sequence, it is possible using this method to address not only the functionality of non-synonymous SNPs but also non- coding polymorphisms in introns and regulatory regions of the gene. Using this vector system we propose to generate mice in which the endogenous 5-Htt gene is excised and replaced with a human 5-HTT gene with either the long (LA), the long (LG), or the short (S) allele of the 5-HTT linked polymorphic region (HTTLPR). The expression of 5-HTT, the activity of the serotonin pathway, and the response of the mice in behavioral tests will provide information concerning the functionality of the three promoter polymorphism. The replacement of the entire mouse gene with the syntenic human DNA will not only facilitate future testing of additional 5-HTT SNPs and variants, but also simplify the evaluation of gene-gene interactions by the intercross of these mouse lines to lines expressing disease related polymorphisms at other loci. Genetic studies are identifying an ever increasing number of polymorphisms linked to risk for affective and psychiatric illness. Determining the functionality of these genetic variations has proved difficult, particularly when no change in the primary structure of the encoded protein is predicted by the polymorphism. In this application, we present the development of a new strategy for the exchange of mouse genes with their human orthologues. We show that after this exchange SNPs can be introduced into the human DNA segment of the mouse genome. As this genetic manipulation is carried out in mouse embryonic stem cells, it is possible to generate mouse lines that differ only in a particular polymorphism associated with risk for disease in the human population. These mice can be used to study the impact of this polymorphism on gene expression, protein function, development, and on the behavioral tests/models known to be sensitive to alterations in this specific pathway in rodents.

A large number of studies indicate that environmental stimuli impact the pathogenesis of asthma both by triggering acute events and by molding the developing adaptive immune responses characteristic of this disease. Much of our study of the immune response of the asthmatic lung to innate stimuli such as endotoxin has focused on the role of the Toll receptors, cell surface molecules which through pattern recognition alert the cell in response to the presence of foreign agents. Recent studies by a number of labs, including that of our collaborator Dr. Ting, have identified a novel family of genes, initially termed CATERPILLER genes, which play a critical role in the response of cells to environmental insults. These genes encode cytoplasmic proteins characterized by pyrin, nuclear binding, and leucine-rich domains, similar to those found in Toll receptors. In response to various stimuli, a number of these proteins have been shown to assemble into complexes termed "inflammasomes" which help to orchestrate the response of the cell to the environmental threat. In the case of complexes formed with three of the CATERPILLER genes, Nlrpl, cryopyrin/Mrp3, and Nlrc4/lpaf1, this response includes the production of cytokines and the initiation of events that lead to necrosis or apoptosis. Previous studies by our group have shown that ATP is required for the maturation and release of IL-1(3 from endotoxin primed mouse macrophages and, furthermore, that this is mediated by ATP activation of the ion channel, P2X7. A model has emerged in which activation of P2X7 leads to alteration in intracellular K+, which in turn leads to activation of some inflammasomes. More recently, it has been suggested that P2X7 recruits the hemichannel pannexin-1 and that this complex mediates the passage of bacterial molecules from the endosomal compartment to the host cytosol leading to inflammasome activation. The overall hypothesis of this application is that the activation of the P2X7/inflammosome pathway plays an important part in the response of cells to environmental stimuli, regulating both the maturation and release of the family of IL-1 cytokines and modulating the apoptotic response of the lung to these stimuli. This pathway, therefore, can contribute both to the pathogenesis of asthma as well as to disease exacerbations triggered by environmental stimuli.

DESCRIPTION (provided by applicant): It is well recognized that there is a genetic component conferring risk for developing atopy and asthma. Linkage studies of human populations throughout the world have identified a large number of regions associated with these diseases. Candidate genes and polymorphisms within these regions have been associated with atopy and asthma. However, identifying a causative role for these alleles in the pathogenesis of these polygenic disorder(s) has proven difficult. The overall goal of this proposal is to develop mouse lines for testing causality of human genetic variants in functional screens of allergic airway disease. Specifically, we will test the hypothesis that individual substitution polymorphisms in genes of the IL13/IL4 pathways associated with atopy and asthma in various human populations are causative, altering the function of the protein in a manner that contributes to the development of allergic disease. We will also test the hypothesis that there is gene-gene interaction in atopy and asthma and that synergy between these disease associated alleles of the IL13/IL4 pathway further increases risk for disease. To test these hypotheses, we develop a novel panel of mouse lines which allows the consequence of inheritance of atopy associated IL13 and IL4R1 alleles to be evaluated both in primary cells ex vivo and during development of allergic lung disease in the mouse. PUBLIC HEALTH RELEVANCE. Asthma remains a major cause of morbidity in the US, with an enormous impact on health care costs and the economy. Particularly troubling is the lack of new treatments for this disease. The NIH has sponsored a large number of clinical trials investigating the genetics of asthma, and a number of candidate loci have been identified. However, there is no in vivo experimental data confirming the importance of these loci in the pathophysiology of asthma. This application plans to evaluate asthma candidate genes in the mouse, using genetic approaches.

DESCRIPTION (provided by the applicant): This application addresses the broad Challenge Area (15) Translational Science and specific Challenge Topic: 15-MH-104 Mouse models containing human genes implicated in mental disorders Differences in gene expression patterns between individuals are common, and it has been suggested that the polymorphisms responsible for these differences may account for the majority of human phenotypic variability. Gene expression is likely determined by the combined impact of environmental factors and inherited polymorphisms in regulatory regions of genes that modulate transcription or affect mRNA processing. Abundant polymorphisms in cis-acting sequences have been identified in genes implicated in psychiatric disorders. However, the elucidation of the contributions of these polymorphisms, both alone and when inherited in combination with polymorphisms at other loci, has been extremely difficult. The lack of progress in this area contrasts sharply with the progress that has been made in the evaluation of polymorphisms that alter protein structure. Mouse lines have been generated in which coding variants identified in patients have been introduced into the orthologous mouse gene using what are now standard genetic engineering techniques. These lines have provided important information regarding the functional importance of these polymorphisms. However, this method can rarely be used to study regulatory variants, as generally these polymorphisms are in regions that are less well conserved between mouse and human. In this application we propose a novel approach for evaluation of the functionality of polymorphisms in non-coding regions of genes believed to act in cis to regulate gene expression. We propose the generation of mouse lines in which the mouse ortholog of the human polymorphic gene is excised and then replaced with the syntenic human locus carrying either the disease associated or protective allele. The impact of the polymorphisms on gene expression during all stages of development, both during normal rearing and in response to imposed environmental stresses, can then be monitored. In addition, these lines will provide an opportunity to follow epigenetic changes at the human loci and to determine whether expression changes manifest as alterations in the behavior of the mouse. A large body of evidence indicates that both inherited and genetic components contribute to the risk for development of behavioral and psychological disorders. Numerous genetic studies have examined association between the development of these illnesses and the inheritance of alleles of genes in the serotonin and dopamine pathways. However, these studies are often inconclusive. This limits the ability of this information to be used in a clinical setting. In this application we propose a new method for validation of the function of variations in gene structure which are believed to confer risk for disease. Additionally, we propose methods for examination of the mechanism by which subtle differences in the primary structure of a gene can modify individuals'susceptibly for developing psychiatric and behavioral disorders. The mouse lines we propose to generate will help to define gene-gene interactions by allowing the study of multiple defined polymorphisms in mouse models related to these illnesses. This will help to define the risk conferred, not only by individual polymorphism but also the risk to the individual when multiple polymorphisms are inherited. We believe that these studies will, therefore, provide important information helpful in both the diagnosis and treatment of psychiatric illnesses.

DESCRIPTION (provided by applicant): GST alleles and oxidant pollutants in the pathogenesis of asthma. It is generally acknowledged that the etiology of many lung diseases involves a complex interplay between the genetics of the individual and his or her exposure to multiple environmental stimuli. Inhaled pollutants can contribute to the risk for development of lung disease through a number of pathologic processes, including inflammation of the airways. It is therefore reasonable to believe that individual differences in the activity of genes which function to detoxify and thus limit exposure to noxious agents can impact, not only the risk for disease, but also pathogenesis of disease and particularly the severity of the disease in affected individuals. Glutathione S-transferases (GSTs) are a superfamily of enzymes responsible for the detoxification of a wide range of xenobiotics and also for the response of cells to oxidative stress. The polymorphic nature of this family of genes and their expression in epithelial cells of lung and gut, cells continually exposed to environmental agents, has made them the focus of numerous human genetic studies. Many of these studies support a role for polymorphism in this gene family in determination of risk of developing asthma, COPD, chronic bronchitis and occupational lung disease. Here we test the hypothesis that the combination of GST alleles expressed by an individual determines his/her response to oxidant pollutant exposures and that this contributes to his or her risk for development of severe lung disease. PUBLIC HEALTH RELEVANCE: Genetic studies are identifying an ever increasing number of polymorphisms and environmental factors linked to risk for development of lung diseases. Determining the functionality of these genetic variations has proved difficult, particularly when no change in the primary structure of the encoded protein is predicted by the polymorphism. Furthermore, understanding which genetic variants are essential when combined with environmental risk factors to affect the development of lung diseases remains a largely unanswered question. In this application, we present the development of a new strategy for the exchange of mouse genes with their human orthologues. We show that after this exchange SNPs can be introduced into the human DNA segment of the mouse genome. As this genetic manipulation is carried out in mouse embryonic stem cells, it is possible to generate mouse lines that differ only in a particular polymorphism associated with risk for disease in the human population. These mice can be used to study the impact of this polymorphism on gene expression, protein function, and development, as well as whether combinations of genetic variants may act additively in development of lung disease or whether additional exposure to environmental risk factors are necessary for disease development.

DESCRIPTION (provided by applicant): Food allergies affect approximately 6% of young children and 4% of adults in the United States. Clinical allergies, including food allergies, impact the quality of life of many individuals, with symptoms ranging from mild hives and vomiting to life threatening anaphylaxis. Multiple determinants, including genetic variation, environmental factors and gene-environment interactions, contribute to the development of allergies. While it is generally acknowledged that the risk for developing allergies is influenced by genetic factors, and numerous candidate genes have been associated with this increased risk, direct evidence supporting a role for these variants in the pathogenesis of food allergies has yet to be demonstrated. Immunoglobulin E (IgE) clearly plays a role in the development of allergies, including food allergies, in many individuals, and genes that play a role in switching the immunoglobulin produced by B cells to IgE are prominent in the list of genes identified in human genetic studies of allergy. This includes polymorphisms in the genes encoding the cytokines IL-4 and IL-13, as well as their receptor IL4RA and the downstream signaling molecule STAT6. More recent genome wide association studies have also implicated variants of the gene encoding the alpha-chain of the high affinity IgE receptor, FceR1, in determining serum IgE levels. In this application we test the function of these polymorphisms, examining their impact on FCER1A expression, total and allergen specific IgE and the response to food allergens using mouse models. Individuals, especially children, with food allergies often display high levels of IgE. Studies show that total serum IgE and allergen specific IgE levels are determined by both genetic and environmental factors. Recent studies have identified an important role for polymorphisms in the gene encoding the receptor for IgE in determining serum IgE levels. These studies also suggest that this polymorphism may impact the production of allergen specific IgE levels. We propose to test the role of these polymorphisms using mouse models of allergic disease, including a mouse model of peanut allergy.

DESCRIPTION (provided by applicant): The renin-angiotensin system (RAS) plays a critical role in regulation of arterial pressures and sodium homeostasis. Disturbances in this system contribute to the etiology of hypertension, a major risk factor for cardiovascular and renal disease including diabetic nephropathy. As a well established genetic component contributes to the risk of developing hypertension, it is not surprising that polymorphisms in genes encoding the various components of the RAS have been intensely scrutinized both in genetic studies of blood pressure and in studies of conditions in which elevated blood pressure is believed to contribute to disease pathogenesis. Some of the most extensively studied polymorphism(s) of the RAS system are in the gene encoding angiotensin converting enzyme (ACE). This enzyme not only converts angiotensinogen to the biologically active product of the RAS, angiotensin II, but also degrades bradykinin a peptide with vasodilatory activity. While many genetic studies have reported a positive association between polymorphisms in this gene and risk for hypertension and related disorders, an almost equal number have failed to demonstrate such associations. These conflicting results likely reflect the fact that hypertension is a complex genetic disorder resulting from inheritance of a combination of small genetic variations that individually have modest impact. The contribution of a specific variant to hypertension may be difficult to isolate in human populations with varied genetic backgrounds and unique clinical and environmental histories. To address this limitation, we propose to test the hypothesis that polymorphisms in ACE can impact development of hypertension and related diseases using mouse lines which express various alleles of ACE in the absence of the mouse orthologue. Mice homozygous and heterozygous for diseases associated haplotypes will be evaluated under defined environmental conditions for differences in the activity of the RAS, development of hypertension, and development of secondary disease including kidney disease. PUBLIC HEALTH RELEVANCE: Hypertension is a common, chronic disease, and represents an important risk factor for stroke, heart disease and end stage renal disease. With a prevalence of approximately 27% world wide this disease places an enormous economical, health care and social burden on society. There is general consensus that genetic factors contribute to individual susceptibilities to hypertension, and many candidate genes and polymorphisms have been identified through human genetic studies. However, determination of which of these polymorphism(s) is functional and delineation of the mechanism by which the polymorphisms confer risk for disease has been difficult. In this application, using polymorphisms in the ACE gene as an example, we propose a new method for approaching this problem.

DESCRIPTION (provided by applicant): Addiction is a chronic, often relapsing illness characterized by persistent and compulsive use of drugs. This maladaptive behavior ultimately leads to physical and psychological dependence. Changes in brain function result from continued exposure, and these lead not only to increased tolerance to the drug but also to physiological changes that can extend for long periods of time after drug use ceases. Both genetic and environmental factors contribute to all aspects of addiction, including initiating use of addictive substances, the vulnerability to become addicted to the drug, the successful discontinuation of drug use. The genetic contribution to addictions, including nicotine dependence, alcoholism and addiction to drugs such as cocaine, has been well established in studies of cohorts of twins. Linkage mapping and association mapping, including recent genome wide association studies, have identified susceptibility loci for addiction related phenotypes. These and the studies of twins suggest that both common and substance specific genetic factors can contribute to risk for addiction. Recent genetics studies support the involvement of a number of loci encoding nicotinic acetylcholine receptors (nAChRs) in addiction genetics. nAChR are ligand gated ion channels, composed of various combinations of five subunit proteins, encoded by 11 different genes, the cholinergic nicotinic receptor subunit (CHRN) genes in mammals. These receptors are also the physiological targets of nicotine activating the mesolimbic dominergic reward and pleasure pathways. In its most simplistic form, treatment of nicotine dependence (ND) simply supplies this stimulus in the absence of the other components of tobacco smoke. Recent genetic data suggest that the nACh receptors may play a more complex role in the psychopathology of addiction, and that development of novel therapeutic agents directed at these receptors could be useful for the treatment of other addiction disorders as well as ND. Variants in the nACHRs are reported to increase risk of development of dependence on cocaine and alcohol. However, it is not clear how the variants in these genes alter the vulnerability to addiction, especially as the majority of the variants do not alter the amino acid composition of the encoded receptor. This has proven an obstacle for exploitation of the results from genetics studies directed at the identification of new therapeutic targets; nor has it been possible to use the presence of these variants as predictors for improved treatment and/or prevention. In this application we propose to develop a series of reagents that will address this limitation and accelerate research on addiction in many different venues. We propose to generate mouse lines that express human nAChR genes. These animals will be developed using a novel series of vectors that facilitate the excision and replacement of mouse loci with their human ortholog. Because these lines will express humanized nAChRs, they will provide an important tool for preclinical evaluation of therapeutic agents targeting this gene family. In the case of loci for which disease association has been demonstrated, the mouse gene can be replaced with various human haplotypes, either those believed to be protective or those associated with increased vulnerability to addiction. The phenotype of the mouse lines carrying the different human alleles can be compared, evaluating the impact of these SNPs on gene expression, gene-environment interactions, chromatin architecture, and behavior in paradigms that model addiction.

DESCRIPTION (provided by applicant): CFTR plays a critical role in chloride transport across airway epithelial cells. Loss of function mutations in this gene result in cystic fibrosis (CF). Because of the high carrier frequency of the gene encoding CFTR and the severe disease caused by mutations in this gene, this protein has been the focus of extensive study. It is arguably the best understood membrane protein associated with human disease. CFTR belongs to the ATP binding cassette (ABC) transporter superfamily and consists of two membrane spanning domains, two nucleotide binding domains (NBD1, NBD2) and a unique R region. Ninety percent of CF patients carry at least one CFTR allele with a three base pair deletion that results in the absence of a phenylalanine (F) in the first nucleotide binding domain of the CFTR protein. This ?F508 mutation results in misfolding of the protein, targeting it for proteosomal degradation. Little of the mutated protein reaches the cell surface. Experimental conditions that allow transport of the mutant protein to the cell surface reveal that the loss of F508 from NBD1 has additional consequences. Electrophysiological studies showed that ?F508-CFTR has much greater closed times, resulting in a large decrease in open probability, thus altering its capacity to transport Cl- across the epithelial surface. The stability of the small amount of the mutant protein that reaches the cells surface is also compromised, resulting in a shortened cell surface half life. Characterization of the biochemical basis for retention of CFTR in the ER and attempts to improve stability and transport of the mutant CFTR are important for identifying new strategies for the treatment of CF. In addition, information concerning CFTR transport may also aid in the treatment of other diseases caused by deficiencies in protein trafficking and altered protein folding. A number of studies have examined the effects of physical conditions, small molecules and second site mutations on the trafficking and stability of the ?F508 protein. A major limitation in the interpretation of these studies is that most have been carried out in vitro, many using non polarized epithelial cell lines. In most cases, it is unknown whether the information gleaned from these in vitro experiments will translate directly to the transport and function of ?F508-CFTR in vivo. Furthermore, it is uncertain if the maneuvers that increase ?F508 -CFTR function in vitro will result in a similar increase in apical ?F508 CFTR in vivo and, if so, whether this will result in sufficient CFTR function to be of physiologic consequence to the organism. In this application we address this limitation. We propose to develop an animal model that will allow the effects of both chemical interventions and second site mutations on ?F508-CFTR transport to be tested rapidly in vivo. PUBLIC HEALTH: Cystic fibrosis is a common, severe, genetic disease with a carrier frequency of approximately 1/2500 in Caucasians. It is caused by mutations in a gene name CFTR. In 90% of individuals a three base pair deletion, which results in the loss of a single amino acid (?F508 mutation), leads to the development of disease. Morbidity and death are primarily the result of loss of lung function secondary to the accumulation of viscous mucus in the lung and subsequent colonization of the airways with microorganisms. Recently studies carried out in vitro in cell lines suggest that it may be possible to treat this disease with small molecules that improve the function of the CFTR protein with the ?F508 mutation. In this application we propose to develop models that will not only test the feasibility of this approach, but that can also be used for the rapid screening for such therapeutic agents.

DESCRIPTION (provided by applicant): Oxidative stress has been implicated in the pathogenesis of many acute and chronic lung diseases including asthma and chronic obstructive pulmonary disease (COPD). The source of this stress can be either extrinsic/environmental or intrinsic originating from cellular processes. Oxygen derived free radicals and other reactive oxygen species (ROS) are normal byproducts of cellular metabolism, but are also produced at high levels during metabolism of environmental pollutant such as ozone (O3). Because of the potential of these molecules to react with and damage major components of the cell, including lipids, proteins and DNA, extensive and redundant mechanisms have evolved to regulate their levels. These include both antioxidant enzymes and non-enzymatic scavenger molecules. An imbalance resulting from either excess production of ROS or deficiencies in these antioxidant mechanisms results in oxidative stress. Oxidative stress can result in cell damage leading to activation of inflammatory pathways which can perpetuate the redox imbalance in the lungs. Defense mechanisms capable of detoxification and reduction of ROS and neutralizing the deleterious effects of oxidative stress are particularly important in the lung, because it is an important point of contact with environmental pollutants that can add significantly to the intrinsic ROS burden. Given the potential impact of alterations in the antioxidant capacity of the lung on risk for lung disease, it is not surprising that polymorphisms in genes that contribute to redox balance and encode enzymes that efficiently metabolize reactive oxygen species in the lung have been the focus of numerous genetic studies. The glutathione S transferase (GST) family includes enzymes whose ability to metabolize a wide range of oxidative stress substrates suggests they are likely to play an important role in maintaining cellular integrity. These enzymes, which conjugate hydrophobic and electrophilic compounds with reduced glutathione, can be grouped into eight different families, with the majority of genetic studies focused on polymorphisms in GSTP1, a member of the pi family and GSTM1 and GSTT1, members of the mu and theta families. Polymorphisms in these genes have been associated with increased response to O3, risk for asthma, and decline in lung function in COPD patients. However, not all studies have consistently observed these associations, and in some cases contradictory results have been reported, leading many to conclude that gene-gene and/or gene-environment interactions may confound elucidation of the contribution of these polymorphisms to disease risk. In this application we propose to address the role of the human polymorphisms in response to oxidative stress using a unique panel of mice expressing either the protective or disease associated GST alleles. PUBLIC HEALTH RELEVANCE: The lungs are constantly exposed to oxidants both from environmental pollutants and from normal cellular metabolism. Genetic variation between individuals in the genes that encode enzymes and proteins critical in protecting the lung from damage caused by excessive levels of oxidants are believed to influence the pathogenesis of many lung diseases, including asthma and COPD. Consistent with this, a number of genetic studies suggest that polymorphisms in glutathione S-transferase (GST) may in part influence either the development or severity of lung disease. In this application, we propose experiments designed to determine whether the polymorphisms in GSTM1, GSTP1 and GSTT1 identified in these association studies, either alone or when inherited in combination, are sufficient to alter the response of the lung to oxidative stress, specifically stress associated with ozone and allergen exposure.

DESCRIPTION (provided by applicant): Genetic studies, both association studies and more recently genome wide association studies (GWAS), have identified a large number of DNA variants that potentially confer risk for the development of atopy, asthma and COPD. However, in the majority of these studies the functional variant has not been identified. This largely reflets the fact that, in most cases, the polymorphism(s) identified do not alter the structure of the proteins encoded by genes located in these regions; rather the variants are generally found in non-coding DNA. While these regions often include sequences that could play an important role in regulation of gene expression, including promoters, enhancers or insulator regions, limited tools are currently available to assess the importance of these DNA variations in determining the expression of a given gene during normal development and, perhaps more importantly, in determining the impact of these variations on risk for disease, disease progression and/or the response of the patient to specific therapeutic intervention(s). Furthermore, in most cases it is clear that DNA variants at multiple loci, together with environmental factors, determine risk for disease: these interactions remain particularly difficult to define. In this application we propose to test the hypothesis that mouse models can be developed for testing the impact of disease associated non-coding DNA variants. These mouse models will allow testing of risk associated haplotypes as well as provide a means of resolving the contribution of an individual DNA variation on gene expression. The impact of the change in gene regulation conferred by the risk associated haplotype or individual variant can be evaluated in combination with environmental factors and also in combination with disease associated DNA variants at other unlinked loci. PUBLIC HEALTH RELEVANCE: One in 15 Americans suffer from asthma and in 50% of the cases disease is associated with allergies. The health care costs associated with both asthma and other lung diseases such as COPD are enormous. COPD costs exceed $23 billion dollars a year and the annual cost of asthma is estimated to be nearly $18 billion http://www.aafa.org. Genetic studies over the past ten years have identified numerous candidate genes that carry DNA variants associated with increased risk for asthma and COPD. The purpose of the studies described here are to develop a method for identifying which of these many polymorphisms uncovered in human genetic studies play an important role in either risk for developing asthma or in the progression of the disease. These studies will not only help to identify individuals at risk, thus allowing early environmental and therapeutic intervention, but they will also help in th identification of new targets for disease intervention.

DESCRIPTION (provided by applicant): All multicellular organisms have developed the ability to mount an innate immune response to invading microbes, and this system provides the first line of defense against these agents. In addition, differences in the innate response triggered by different pathogens provide essential early information to the organism concerning the nature of the threat at hand, thus ensuring proper tailoring of the adaptive immune response to efficiently eliminate the infectious agent. Cells of the immune system detect pathogen-associated molecular patterns, or PAMPs, expressed by the microbes, and this in turn leads to dramatic changes in gene expression and cell function, changes which together act to initiate a coordinated protective response to the pathogen. Exposure of macrophages to a broad spectrum of PAMPs results in increased production and release of cytokines, notably the processing and release of the pleiotropic, pro-inflammatory cytokine, IL-1b. Maturation and release of IL-1b is dependent on the assembly of cytoplasmic protein complexes referred to as inflammasomes. The inflammasome is composed of an NLR protein that oligomerizes in response to changes in the cell triggered either directly or indirectly by the encountered PAMPs or DAMPs (pathogen or danger-associated molecular patterns). The oligomerized NLR proteins recruit additional proteins to the complex via their CARD and PYRIN domains, including ASC (PYCARD), and pro-caspase-1. The recruitment of two pro-caspase-1 proteins leads to autocatalysis and release of active enzyme, which is then available for cleavage of pro IL-1b into its biologically active form. The NLRP proteins have emerged as critical regulators of the activity of the inflammasome and thus of the innate immune response in mammals. The most extensively studied of these is NLRP3. This protein is mutated in individuals suffering from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological cutaneous articular syndrome/neonatal-onset multisystem inflammatory diseases (CINCA/NOMID). Individuals heterozygous for missense mutations in this gene suffer from excessive inflammation characterized by increased serum IL-1b. More recently, common variants in the NLRP3 locus have been associated with an increasing number of chronic diseases including Crohn's disease and arthritis. In this application we propose a novel approach for study of the molecular pathophysiology of CAPS and for defining the functionality of disease associated polymorphisms in this gene. PUBLIC HEALTH RELEVANCE: The NLRP proteins have emerged as critical regulators of the activity of the inflammasome and thus of the innate immune response in mammals. The most extensively studied of these is NLRP3. This protein is mutated in individuals suffering from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurological cutaneous articular syndrome/neonatal-onset multisystem inflammatory diseases (CINCA/NOMID). Individuals heterozygous for missense mutations in this gene suffer from excessive inflammation characterized by increased serum IL-1b. More recently, common variants in the NLRP3 locus have been associated with an increasing number of chronic diseases including Crohn's disease and arthritis. In this application we propose a novel approach for study of the molecular pathophysiology of CAPS and for defining the functionality of disease associated polymorphisms in this gene.

DESCRIPTION (provided by applicant): The UDP glucuronosyltransferase isoenzymes (UGT) are critical for the detoxification and elimination of structurally diverse lipophilic molecules including products of normal cellular metabolism, environmental pollutants, and xenobiotics including therapeutic agents. These enzymes catalyze the conjugation of their substrates with glucuronic acid, a process that acts to both inactive the potentially toxic molecule and also to increase its water solubility and subsequent excretion. Despite the importance of this pathway in protection against accumulation of endobiotics and environmental toxins and its role in metabolism of therapeutic agents, many questions regarding the function of individual UGT enzymes remain, particularly in the case of the seven members of the UG2B subfamily. One of the reasons for this is that there are enormous differences between members of the human and mouse UG2B genes. Based on sequence homology it has not been possible to define the mouse ortholog of the seven human UGT2B genes. This has prohibited the use of null mouse lines in extrapolating the role of particular human UGT2B genes in the glucuronidation of specific substrates. In this application we propose a new strategy that we believe will generate mouse lines useful for determining the function of the UGT2B genes, and in particular of UGT2B15 and UGT217. We expect that these lines will express these genes in a manner predicted by studies of human tissues and that these genes will be critical, not only in steroid metabolism, but also in protection against environmental toxins. Furthermore, we will test the hypothesis that mice carrying different UGT2B17/UGT2B15 haplotypes will differ in these responses.

DESCRIPTION (provided by applicant): Antibodies regulate immune responses, not only by recognition of foreign agents but also through the modulation of effector cell function. This latte action is mediated through binding of the antibody constant region, the Fc region, to cell surface receptors. Fc receptors (FcRs), which are expressed by diverse populations of immune cells, can be grouped based on the antibody class they bind. The receptors that bind IgG antibodies, the Fc? receptors, represent the largest family and are of particular interest because antibody binding can either stimulate or attenuate the immune response, depending on the receptor activated. Understanding the function of these receptors and the variation between individuals in their structure and expression is important, not only because of their ability to regulate immune responses, but also because of the increased therapeutic use of therapeutic antibodies, especially in the treatment of cancer. Species differences in the mouse and human have hampered the use of the mouse in translational studies examining the engagement of these receptors by therapeutic human antibodies. In addition, these differences have limited the ability to examine the impact of polymorphisms in these receptors on the pathogenesis of immune diseases and on the responsiveness of patients to therapeutic antibodies. To address this problem we propose to generate a mouse line which expresses the low affinity family of human FCGR genes in place of the endogenous mouse genes. These lines will be easy to breed and thus will be appropriate not only for basic research studies, but also for preclinical evaluation o new therapeutic antibodies.

Assembly of disease-relevant pathways in the mouse The mouse continues to provide important information concerning the role of genes in both normative biology and pathogenesis of disease. Mouse models have been identified and developed for many diseases, including Alzheimer's, autoimmune diseases, cardiovascular diseases and cancer. These include complex models unique to specific inbred mouse lines such as the diabetic NOD mouse line and models that have been generated by manipulation of the mouse germline. These manipulations include the introduction of transgenes, the removal of genes and the introduction of point mutations into mouse genes by targeted mutagenesis. Often, the development of models requires the breeding of mice to generate animals carrying multiple mutations. While mouse models have been enormously useful in understanding the pathogenesis of disease, in many cases they are not amenable to the evaluation of new therapeutics. For example, few of the therapeutic antibodies in development cross react with the orthologous mouse gene, and the distribution of Fc receptors between species and differences in antibody clearance makes evaluation of these drugs difficult. Similarly, differences between human and mouse in the metabolism of small molecules limit the usefulness of many mouse disease models for the study of the efficacy of this class of therapeutics. Furthermore, in the majority of cases, the genetic architecture of most mouse disease models developed to date does not resemble that of individuals at risk for disease. Perhaps more importantly, the models are not amenable to further rapid genetic manipulation as new genetic factors influencing disease pathogenesis are identified. In addition, when novel disease associated polymorphisms are discovered, few of the models allow easy testing of the functional implications of these variants. In this application we propose the development of strategies and methods for the rapid generation of mouse models useful for: 1) the functional evaluation of disease associated polymorphisms, 2) the study of gene-gene interactions in disease pathogenesis, and 3) testing of therapeutics directed against these disease associated genes. Specifically we propose to develop cell and mouse lines useful for such evaluation of the ¿-secretase complex. This multi-subunit protease complex mediates intramembranous cleavage of a number of important molecules including amyloid precursor protein (APP). Cleavage of APP by ¿-secretase yields ¿-amyloid, a primary component of plaques characteristic of Alzheimer's disease.

PROJECT SUMMARY Over 100 million people worldwide drink water and eat food containing unsafe levels of inorganic arsenic (iAs). iAs is a potent human carcinogen. Chronic iAs exposure is also associated with increased risk of cardiometabolic, neurological, and respiratory diseases. In addition, gestational and early-life exposures to iAs are linked to low birth weight, infant mortality, and developmental neurotoxicity in children. In spite of extensive research over the past 20+ years, the mechanisms underlying the adverse effects of iAs exposure remain largely unclear, partly due to the lack of an adequate animal model. Many health effects reported in humans have not been reproduced in the laboratory because iAs metabolism in laboratory animals differs from that in humans. Strong evidence from both population and laboratory studies suggests that the differences in the structure and function of human arsenic methyltransferase (AS3MT) and mouse As3mt are responsible for the differences in iAs metabolism between humans and mice which, in turn, determine the differences in the susceptibility of these species to the adverse effects of iAs exposure. The ultimate goal of this project is to generate a mouse model in which the metabolism and adverse effects of iAs exposure described in humans can be reproduced and studied. We have developed vector systems for manipulation of the AS3MT/As3mt locus in both mouse and human pluripotent stem cells. We have used these systems to replace the As3mt locus in mouse embryonic stem cells (ES) with the syntenic segment of human DNA using CRISPR/cas9 augmented homologous recombination. The specific aims of this project are: 1. To compare the metabolism of iAs in mouse ES cells expressing human AS3MT and in primary cells (endodermal cells and hepatocytes) derived from these ES cells with iAs metabolism in human ES cells and differentiated cells. 2. To generate and characterize a mouse strain expressing human AS3MT. Mouse ES cells expressing AS3MT will be injected into blastocysts to generate mice expressing human AS3MT in the place of the mouse gene. To characterize the humanized mouse strain, we will compare AS3MT expression and iAs metabolism in the humanized mice with those described for humans. We will also characterize metabolic phenotype of the humanized mice exposed to iAs in drinking water at the levels that were associated with adverse metabolic phenotypes in human studies. Creation of a humanized mouse model represents a major innovation that will allow us to reproduce and study in the laboratory the adverse effects of iAs exposure found in population studies. This project has a potential to move forward the entire field of As toxicology and contribute significantly to our understanding of the disease associated with iAs exposure, providing key information for efficient treatment or prevention of this disease.

The importance of interactions between the fragment of crystallization (Fc) region of various IgG isoforms and the array of Fc?Rs expressed by effector immune cells in establishment of broad immunity to influenza viruses is increasingly recognized. However, the translational value of studies of these pathways in mice is limited in part by major species differences in the number, structure and expression pattern of the Fc?Rs, particularly the low affinity receptors clustered on chromosome 1. Similarly, although both the mouse and the human IgG locus encode four IgG constant region genes and thus produce four IgG isotypes, divergence between the species has made it difficult to assign mouse orthologs to human IgG constant region genes. These species differences have also limited the use of the mouse as a preclinical tool for evaluating reagents such as vaccines and humanized monoclonal antibodies (mAbs).To address this, we have generated mice in which the three loci encoding mouse IgG receptors, Fc?RII/III/IV, Fc?R1a, and FcRn (the IgG transporter), are humanized by syntenic replacement. Mice humanized for the FC?Rs and derived lines expressing only one of the three low affinity FCGR activating receptor genes, FCGR2A, FCGR2C or FCGR3A, will be used to evaluate the role of these receptors in antibody-mediated protection against influenza. The contribution of human Fc receptors would ideally be studied in animals in which the IgG isotypes produced in response to virus have human Fc regions, ensuring that the Fc-FC?R interactions mimic those observed in humans. We address this limitation by humanization of the ~200 kb mouse IgH constant region, as well as the constant region for the kappa light chains. As embryonic stem cells from mice humanized for the FCGR genes are used for this genome engineering, the mice generated will not only produce human IgG isoforms, but these isoforms will interact with human effector FC?Rs on effector cell populations.These animals will provide a model for evaluation of the effectiveness of human mAbs as well as for defining Fc-Fc?R pathways whose engagement modulates the pathogenesis of disease after viral exposure and/or improves immunity in vaccinated animals.

PROJECT SUMMARY Contamination of drinking water and foods with inorganic arsenic (iAs) represents a major public health risk in the U.S. and worldwide. Exposure to iAs has been linked to cancer, diabetes, cardiovascular, respiratory and neurological diseases. Humans and most other mammalian species have developed mechanism for detoxification of iAs, which involves a two-step conversion of iAs to methyl-As (MAs) and dimethyl-As (DMAs) and excretion of the methylated metabolites in urine. In mammals, iAs methylation is catalyzed by orthologs of a single enzyme, arsenic methyltransferase (AS3MT). An impaired capacity to methylate iAs, e.g., due to AS3MT polymorphism, has been linked to increased risk of diseases associated with iAs exposure. Mechanisms underlying the adverse effects of iAs exposure have been extensively studied using laboratory models. However, laboratory research has been hindered by substantial differences between laboratory animals and humans in their capacity to metabolize iAs. In particular, laboratory mice have been shown to methylate and detoxify iAs much more efficiently than humans, making it difficult to reproduce in mice some of the adverse phenotypes reported in population studies, specifically cancer and diabetes. The ultimate goal of the proposed research is to develop novel mouse models, in which iAs metabolism resembles that in humans and in which iAs-associated diseases can be studied at environmentally relevant iAs exposure levels. We have recently generated a new mouse strain in which the Borcs7/As3mt locus was humanized by syntenic replacement. AS3MT expression in tissues of the humanized (Hs/Hs) mice resembles that in human tissues and differs from expression of mouse As3mt: AS3MT expression is lower in livers and much higher in adrenals. Notably, the different pattern of AS3MT expression in tissues of Hs/Hs mice is associated with low efficiency of iAs detoxification and with the profiles for iAs and its methylated metabolites in tissues and excreta that are consistent with those reported in humans. The goals of this project are: (1) To characterize susceptibility of Hs/Hs mice to adverse effects of iAs exposure, focusing on the diabetogenic effects, (2) to generate a new mouse strain expressing AS3MT haplotype that has been linked to impaired iAs methylation and risk of iAs-induced diseases in human cohorts, and (3) to explore association between AS3MT expression in adrenals and adrenal function. The proposed research will generate and validate unique mouse models for iAs toxicology. These models will make it possible to study adverse effects of iAs at environmentally relevant exposure levels and in context with human-like metabolism of iAs and AS3MT polymorphism. Using these models will markedly improve translatability and impact of laboratory studies focusing on iAs induced diseases.

Abstract The COVID-19 pandemic caused by SARS-CoV-2 has resulted in swift and catastrophic losses of human lives globally. Acute respiratory distress syndrome (ARDS) is one of the most detrimental outcomes of COVID-19 infection that can lead to the rapid deterioration and death of patients. ARDS is primarily caused by the cytokine storm which unleashes a plethora of inflammatory cytokines during the late stages of COVID-19. The master cytokines that are thought to be responsible for much of the damage are interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor (TNF). Currently several clinical trials have already been initiated to test the efficacy of biologic inhibitors to target these pathways. However, in many cases, the mechanism and impact of these cytokines during ARDS are poorly understood. An indepth mechanistic understanding of cytokine induction is important because this understanding will significantly impact the design and success of ARDS treatment. This application focuses on the role and mitigation of the inflammasome complex which leads to the proinflammatory cytokine, IL-1?, in ARDS. The inflammasome is a protein supramolecular structure that leads to caspase 1 activation, which then cleaves pro-IL-1? and pro-IL-18 to mature IL-1? and IL-18. In addition to the release of IL-1? and IL-18, caspase 1 cleaves gasdermin D to cause inflammatory pyroptotic cell death, thus leading to a cascade of cell death and inflammation. The inflammasome is comprised of a receptor or sensor, with the most prominent ones represented by NLRP1, NLRP3, NLRP6, NLRC4 and AIM2. It also includes an adaptor molecule ASC (apoptosis- associated speck-like protein containing a CARD), and the effector caspase-1. Each receptor or sensor can be activated by specific pathogen products called PAMPs or cell damage associated molecules called DAMPs. NLRP3 is the most studied member since it is activated by a large list of stimulators. Studies of other coronavirus such as SARS show inflammasome activation by key viral proteins. Expression data from COVID-19 patients also show dramatic increases of inflammasome sensors in the bronchial alveolar lavage of COVID-19 patients. However the mechanism of inflammasome activation by SARS-CoV-2, especially in the human system, remains unknown. This proposal will identify the viral protein that activates human inflammasome, and further define the specific human inflammasome sensor/receptor that mediates the response. We will then design ways to reduce inflammasome activation during SARS-CoV-2 infection using established therapeutics as well as new approaches to broadly attenuate inflammatory cytokines.