David B. Jacoby - US grants

This project deals with the effect of viral infection on the function of the airway epithelial cell, the primary target of most respiratory viruses. Alterations in both epithelial-smooth interactions and epithelial ion transport will be studied. I have demonstrated that decreased epithelial neutral endopeptidase is responsible for the increased airway smooth muscle response to substance P seen with viral infections. In this project I will examine the effect of viral infections on the response to other peptide mediators, both excitatory and inhibitory and the role of decreased neutral endopeptidase in these responses. Furthermore, I will examine the role of endogenous tachykinins (whose activities are increased in the absence of neutral endopeptidase) in causing the increased parasympathetic bronchoconstriction characteristic if viral airway infection. I will also determine the effect of viral infection on the release of epithelial mediators that increase or decrease smooth muscle contraction. In studying epithelial ion transport, which regulates water secretion, I will first determine the effect of viral infection on baseline (unstimulated) sodium absorption and chloride secretion, and the dependence of such changes on epithelial prostaglandin production. I will also examine the effect of viral infection on paracellular (between cells) and transcellular (through) ion permeability. Finally, I will determine the effect of decreased epithelial neutral endopeptidase on the ion transport response to tachykinins and other peptide mediators. These studies should lead to a greater understanding of the role of viral infections in airway hypersecretion and smooth muscle hyperresponsiveness, as well as insights into the pathophysiologic mechanisms of asthma. This may ultimately provide the basis for new therapeutic strategies.

DESCRIPTION (provided by applicant): Viral infections are a major cause of asthma attacks. Neural control of the airways is markedly abnormal in both humans and experimental animals with viral airway infections. Under normal circumstances, the release of acetylcholine from airway vagal fibers is limited by inhibitory M2 muscarinic receptors on the nerve endings. Loss of these receptors during viral infections increases bronchoconstriction. Host cells express interferons (IFNs) in response to viral infection. We have demonstrated that IFN-gamma causes M2 receptor dysfunction in cultured airway parasympathetic neurons. IFN-beta, as well as double-stranded RNA (a potent stimulus to IFN-alpha and beta production during viral infections) cause M2 receptor dysfunction and airway hyperreactivity in the absence of inflammation. We hypothesize that virus induced airway hyperreactivity and loss of M muscarinic receptor function are mediated by the production of IFNs. We propose the following specific aims: SPECIFIC AIM #1: To determine the effects of exogenous IFNs (alpha, beta, and gamma) on neuronal M2 receptors in vivo. SPECIFIC AIM #2: To investigate the role of IFNs in mediating hyperreactivity and loss of neuronal M2 muscarinic receptor function after viral infection and treatment with double-stranded RNA. Animals will be infected with parainfluenza virus or treated with dsRNA, and neutralizing antibodies to various IFNs and their receptors will be used to try to prevent hyperreactivity and M2 receptor dysfunction. SPECIFIC AIM #3: To investigate the effects of IFNs on M2 receptor function and gene expression in primary cultures of airway parasympathetic neurons and human neuroblastoma cells. Real time RT-PCR will be used to measure M2 receptor mRNA. Immunofluorescence will be used to measure M2 receptor protein. Stimulated release of acetylcholine and the ability of atropine to potentiate acetylcholine release (by blocking M2 receptors) will be measured to assess M2 receptor function. The neurons' repertoire of interferon regulatory proteins, and the requirement of new protein synthesis will be determined SPECIFIC AIM #4: To use M2 muscarinic receptor promoter reporter constructs to investigate the mechanisms by which IFNs decrease M2 receptor expression. Deletion constructs and site-directed mutagenesis will be used to identify specific promoter elements involved in IFN suppression of M2 receptor gene expression. Particular attention will be paid to IRF-E sites at -43 and -1147 BP, multiple C/EBPbeta sites, and a STAT-1 site at -2754 BP. Etectrophretic mobility shift assays will be used to detect activation of the relevant transcription factors and will allow correlation with functional effects on gene expression.

DESCRIPTION: Viral infections can be identified in as many as 80 percent of children during asthma attacks. While a variety of mechanisms may contribute to this effect, neural control of the airways is markedly abnormal in both humans and experimental animals under these conditions. Under normal circumstances, the release of acetylcholine from airway vagal fibers is limited by inhibitory M2 muscarinic receptors on the nerve endings. The negative feedback normally provided by these receptors is lost during viral infections, increasing acetylcholine release and reflex bronchoconstriction. M2 receptor dysfunction can occur via several mechanisms, some of which are dependent upon the inflammatory response to the virus. Although the inflammatory response to viral infections is typically characterized by neutrophils and mononuclear cells, this may vary depending on the atopic status of the host. As many asthmatics are also atopic, the inflammatory response to viral infection may involve an influx of eosinophils into the airways, as well as production of interleukin-5 by both CD4+ and CD8+ T-lymphocytes. It has been previously demonstrated that the eosinophil is responsible for M2 receptor dysfunction after inhalation of allergen. In contrast, in virus-infected animals, the eosinophil is not responsible for loss of M2 receptor function. This project will investigate the role of the eosinophil in M2 receptor dysfunction during viral infections in guinea pigs sensitized to a non-viral antigen (ovalbumin). It is hypothesized that in sensitized guinea pigs, viral infection will result in recruitment of eosinophils to airway nerves, eosinophil activation, release of major basic protein, loss of M2 receptor function, increased release of acetylcholine and hyperreactivity. The specific aims are designed to examine 1) whether loss of M2 receptor function in sensitized, virus infected animals is mediated via eosinophils, 2) what inflammatory mediators are required for recruiting eosinophils to the airway nerves in these animals, 3) what role CD4+ and CD8+ T-lymphocytes play in recruiting eosinophils, and 4) what effect eosinophil proteins have on neuronal M2 receptor functional.

[unreadable] DESCRIPTION (provided by applicant): Viral infections can be associated with activation of eosinophils, especially in the airways of patients with asthma. We have demonstrated that eosinophils in the airways have a pronounced antiviral effect. Our studies suggest that this is due to metabolism of bromide by eosinophil peroxidase (EPO). Eosinophil ribonucleases (eosinophil derived neurotoxin (EDN) and eosinophil cationic protein (ECP)) may also participate. We propose to investigate the mechanisms of the antiviral effects of eosinophils. In specific aim #1, we will test the ability of human eosinophils to kill virus (parainfluenza, influenza, rhinovirus, and respiratory syncytial virus) in vitro. The requirement for bromide will be tested, as will the role of eosinophil ribonucleases. We will determine whether direct contact of the eosinophil with the virus is required and, if so, whether interactions of virus attachment proteins with specific cellular receptors is involved. We will also isolate the eosinophils from wild type and EPO (-/-) mice, and test their antiviral properties in vitro. In specific aim #2, we will test the ability of purified human EPO, EDN, and ECP, as well as eosinophil major basic protein, to kill viruses in vitro. The bromide dependence of this reaction will be determined, and the potency of the antiviral properties of the various eosinophil proteins for the four viruses listed above will be determined. In specific aim #3, we will test the ability of eosinophils to kill virus in vivo in wild type, in EPO (-/-), in IL-5 transgenic mice, and in mice that are EPO (-/-) on an IL-5 transgenic background. The effects of reversing eosinophilia using monoclonal antibodies to IL-5 and to VLA4 will be determined. In specific aim #4, we will use an ex vivo preparation in which the lungs of antigen challenged mice will be infected with viruses in vitro under various bromide concentrations and in the presence or absence of ribonuclease inhibitors. These studies will advance our understanding of the beneficial role of eosinophils in the airways, and may suggest new therapeutic strategies for both viral infections and allergic airway disease. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Eosinophils cluster around airway nerves in patients with asthma. This physical interaction of eosinophils and airway nerves is central to virus induced dysfunction of inhibitory M2 muscarinic receptors and airway hyperreactivity in antigen sensitized hosts. We hypothesize that eosinophils are attracted to nerves by chemokines produced by the nerve cells, and that the eosinophils are anchored to the nerves by adhesion molecules, which facilitate their activation. We will investigate these interactions using an in vivo model of the interaction of antigen sensitization with viral infection, and will also undertake in vitro studies using primary cultures of human airway parasympathetic neurons, as well as guinea pig airway neurons and human neuroblastoma cells. We propose the following specific aims: SPECIFIC AIM #1: To determine the effects of blocking the CCR3 receptor, as well as ICAM-1 and VCAM, on recruitment of eosinophils to the airway nerves in vivo, and on the physiological responses to viral infection in naive and sensitized animals. SPECIFIC AIM #2: To determine the regulation of eotaxin as well as other CCR3 ligands), ICAM-1, and VCAM expression in primary cultures of human and guinea pig airway parasympathetic neurons and in human neuroblastoma cells. Regulation by TH2 cytokines IL4, IL5, and IL13 (expected to be important in the response to allergen sensitization) and by TNFa, and the interactions of these with interferon? (produced in response to viral infections) will be studied, as well as the effects of dexamethasone treatment. SPECIFIC AIM #3: To investigate the role of NF-?B and STAT-6 in regulating the expression of eotaxin by airway parasympathetic neurons. SPECIFIC AIM #4: To test the effects of blocking TNFa on the response to viral infections in sensitized animals. Effects on eosinophil recruitment to airway nerves, eosinophil activation, airway hyperreactivity, M2 muscarinic receptor dysfunction, and the neural expression of chemokines and adhesion molecules will be studied. This work is highly relevant to understanding how viral infections cause asthma attacks. In the course of these experiments, substances released by lung cells that contribute to asthma will be identified, and ways to block them will be tested. This will assist us in developing new treatments for asthma. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application proposes a Medical Scientist Training Program (MSTP) at Oregon Health & Science University (OHSU). The goal of the OHSU MSTP will be to prepare individuals for successful careers as physician-scientists by providing intensive focused training in laboratory-based, hypothesis-driven, basic or disease-oriented research, leading to a Ph.D. degree, coupled with broad training in the theory and practice of medicine, leading to an M.D. degree. The program is designed to be completed in seven to eight years, and will consist of the initial two years of medical school interspersed with laboratory research rotations, followed by graduate school (including course work, the preliminary examination and thesis research), and then by the clinical years of medical school. There is sufficient flexibility in the curriculum to allow highly individualized training in order to meet specific career goals. Both the Ph.D. and M.D. degrees will be awarded to successful candidates at the completion of both components of the program. Scientific training will take place within the interdisciplinary graduate programs in biomedical sciences at OHSU. Faculty available as mentors for M.D-Ph.D. students are members of the Departments of Behavioral Neuroscience, Biochemistry and Molecular Biology, Cell and Developmental Biology, Molecular and Medical Genetics, Molecular Microbiology and Immunology, and Physiology and Pharmacology. A rationale for the OHSU MSTP is that the need for skilled physician-investigators will continue to increase over the next decades as developments in "molecular-based medicine" have greater impact on the prevention, diagnosis, and treatment of disease. A robust and consistent institutional commitment, an enthusiastic and collegial research faculty, and a committed M.D-Ph.D. Committee actively involved in student selection, mentoring, and policy formation together will provide a strong foundation to ensure the success of this program.

Description (provided by applicant): This proposal describes our research training program in the Division of Pulmonary and Critical Care Medicine, Oregon Health &Science University. In this program, we offer state of the art, multidisciplinary research training in a wide range of disciplines including cell and molecular biology, whole organ pathophysiology, and both human based research and epidemiological research. Six post-doctoral fellows (primarily MD pulmonary fellows) and two graduate students will be offered funding for two years of research training each. Research will be supplemented by trainee participation in program wide research seminars and journal clubs. It is our belief that such program wide meetings and interactions are vital to providing young scientists with a broad perspective on research outside their own projects, avoiding early overspecialization and an inappropriately narrow research focus. Course work is also offered, and includes a mandatory course in Responsible Conduct of Research, as well as a wide range of courses appropriate to trainees embarking on a research career. These include statisics, experimental design, grant writing, manuscript writing, public speaking, and epidemiological methods, and are offered both in our graduate programs and in our Human Investigations Program. Research mentorship is provided by a faculty of seasoned scientists with extensive experience in training young scientists. About half the faculty conducts research primarily or principally involving the lung. The remainder have specific areas of expertise in cell and molecular biology that have either been applied to the study of lung disease or are easily applicable to it. Interactions and synergy among the laboratories in this program add to the rich and broad training environment. A well established system for regularly tracking the progress of trainees ensures their continued development, and broadens the sources of guidance and advice in this crucial stage of their careers. This approach will produce clinical and basic researchers prepared for the challenges of academic medicine and biomedical research, and will help fill the need in the next generation of scientists. These scientists will be prepared to address the public health issues raised by the increasing burden of asthma, chronic bronchitis, emphysema, acute respiratory distress syndrome, and other lung diseases.

DESCRIPTION (provided by applicant): While the addition of anticholinergics to (-agonists has been shown to be beneficial in acute asthma attacks, the use of anticholinergics in the management of chronic stable asthma has been disappointing, and seems to add little to treatment with other agents. We have recently made the surprising observation that treating ovalbumin sensitized animals with the anticholinergic atropine at the time of antigen challenge markedly increases vagally mediated hyperreactivity measured 24 hours later (by which time the atropine has worn off). Histological examination at that time shows markedly increased eosinophil activation, as reflected in increased extracellular deposition of eosinophil major basic protein. Thus the anticholinergic treatment made the airway disease worse. We hypothesize that anticholinergic medications block muscarinic receptors on eosinophils that normally function to limit eosinophil activation. Blocking these muscarinic receptors on the eosinophils increases eosinophil activation and worsens airway disease. Delineating the effects of acetylcholine on eosinophil function as well as the specific muscarinic receptor responsible will have substantial clinical implications. If the specific muscarinic receptor responsible for inhibiting eosinophil activation is different from the M3 receptor (which is responsible for smooth muscle contraction), then selective antagonism of the M3 receptor would allow bronchodilitation without potentiating eosinophil activation. Alternatively, if this is not possible, activation of eosinophils by anticholinergics might provide a rationale for combining anticholinergics with treatments aimed at limiting eosinophil activation, such as CCR3 antagonists. We propose two specific aims: Specific Aim 1: To use selective antagonists to determine which subtype of muscarinic receptor is responsible for the ability of anticholinergics to potentiate ovalbumin induced hyperreactivity to vagal stimulation. Specific Aim 2: To confirm the presence of muscarinic receptors on guinea pig eosinophils, to extend these observations to human eosinophils, and to determine the effects of stimulation of eosinophil muscarinic receptors on eosinophil activation in vitro. PROJECT NARRATIVE: Drugs that block substances released by nerves in the lungs are used to treat asthma. We have found that these drugs may activate white blood cells to make the asthma worse. In this project, we will find out how these drugs are making the asthma worse and, in so doing, find out how to design a treatment that won't have these negative effects.

DESCRIPTION (provided by applicant): Eosinophils cluster around airway nerves in patients with asthma. This physical interaction of eosinophils and airway nerves is central to virus induced dysfunction of inhibitory M2 muscarinic receptors and airway hyperreactivity in antigen sensitized hosts. We hypothesize that eosinophils are attracted to nerves by chemokines produced by the nerve cells, and that the eosinophils are anchored to the nerves by adhesion molecules, which facilitate their activation. We will investigate these interactions using an in vivo model of the interaction of antigen sensitization with viral infection, and will also undertake in vitro studies using primary cultures of human airway parasympathetic neurons, as well as guinea pig airway neurons and human neuroblastoma cells. We propose the following specific aims: SPECIFIC AIM #1: To determine the effects of blocking the CCR3 receptor, as well as ICAM-1 and VCAM, on recruitment of eosinophils to the airway nerves in vivo, and on the physiological responses to viral infection in naive and sensitized animals. SPECIFIC AIM #2: To determine the regulation of eotaxin as well as other CCR3 ligands), ICAM-1, and VCAM expression in primary cultures of human and guinea pig airway parasympathetic neurons and in human neuroblastoma cells. Regulation by TH2 cytokines IL4, IL5, and IL13 (expected to be important in the response to allergen sensitization) and by TNFa, and the interactions of these with interferon? (produced in response to viral infections) will be studied, as well as the effects of dexamethasone treatment. SPECIFIC AIM #3: To investigate the role of NF-?B and STAT-6 in regulating the expression of eotaxin by airway parasympathetic neurons. SPECIFIC AIM #4: To test the effects of blocking TNFa on the response to viral infections in sensitized animals. Effects on eosinophil recruitment to airway nerves, eosinophil activation, airway hyperreactivity, M2 muscarinic receptor dysfunction, and the neural expression of chemokines and adhesion molecules will be studied. This work is highly relevant to understanding how viral infections cause asthma attacks. In the course of these experiments, substances released by lung cells that contribute to asthma will be identified, and ways to block them will be tested. This will assist us in developing new treatments for asthma.

Our recent studies have demonstrated an entirely new and previously unrecognized effect of toll-like receptor 7 (TLR7) agonists in the airways, namely that they are potent bronchodilators, and prevent bronchoconstriction caused by methacholine, histamine, stimulation of parasympathetic nerves, and depolarization of smooth muscle by potassium chloride. Pharmacological studies using TLR7 antagonists, as well as studies in TLR7- deficient mice, demonstrate that part of the effect is TLR7 dependent and part is TLR7 independent. TLR7 dependent bronchodilation in guinea pigs and in human airways is mediated by production of nitric oxide. We have also shown that in human airways, stimulation of the other ssRNA receptor, TLR8, also causes bronchodilation, but that this pathway does not involve nitric oxide. In this application, we propose three specific aims: SPECIFIC AIM #1. To A) establish the role of the ssRNA receptors TLR7 and TLR8 in relaxation of human airway smooth muscle, B) test whether these receptors signal through nitric oxide, prostaglandins, and the large conductance, calcium activated potassium channel (BKCa) in human airways in vitro, and C) explore the role of nitric oxide and changes in intracellular calcium in rapid signaling by these receptors in primary cultures of human airway smooth muscle cells. SPECIFIC AIM #2. To identify which TLR7 mediated pathways are inhibited in antigen sensitized and in virus infected guinea pigs to cause the profound loss of TLR7 mediated bronchodilation that we identified in preliminary experiments (see figure 12). We will test whether a similar 2 log shift occurs in human airway tissues using passive sensitization and in vitro parainfluenza virus infection. SPECIFIC AIM #3: To determine the mechanisms of loss of TLR7/nitric oxide mediated bronchodilation after antigen sensitization or viral infection. The results of the experiments we propose will be important in establishing the potential of TLR7 agonists as treatments for asthma and other airway diseases. In addition, because these receptors respond to viral RNA, understanding the effects of stimulating TLR7 receptors in the airways, as well as the loss of these effects in models of asthma, will help us understand the pathophysiology of virus induced asthma attacks.

DESCRIPTION (provided by applicant): Eosinophil - Nerve Interactions in Mouse Models of Dermatitis PROJECT SUMMARY: The recruitment, accumulation, and/or activities mediated by eosinophils (e.g., degranulation) have been hallmark features of cutaneous allergic diseases. These events also correlate with the dominant symptoms associated with these patients, including histopathological changes in the skin and behavioral responses such as itching that together often lead to a breakdown in cutaneous barrier functions. In addition, this link between eosinophils and allergic skin inflammation is noted in the available mouse models of dermatitis, suggesting an underlying role for these granulocytes. Unfortunately, despite the strong correlative relationship, the definition of eosinophil-mediated events leading to changes in the skin that promote inflammatory symptoms such as itch responses have remained out of reach. The goal of this collaborative proposal is to bridge this gap by exploiting our extensive experience examining eosinophil activities using allergen provocation models of lung disease. Indeed, our preliminary studies using skin inflammatory models have already greatly benefited from the availability of eosinophil-specific antibodies and our transgenic line of mice congenitall deficient of eosinophils. Our objective in this proposal is to exploit these resources as well as the development of a next generation gene knock-in mouse model (iPHIL) that permits the inducible and selective loss of eosinophils. This collaborative effort will focus our collective experiences studying eosinophils using mouse models of inflammatory diseases to define causative events contributing to the itching associated with allergic dermatitis. In particular, through the selective use of our novel in vivo mouse models and ex vivo eosinophil - nerve co-culture studies we will test the central hypothesis that interactions between skin infiltrating eosinophils and cutaneous sensory nerves increases nerve growth and branching, as well as increased expression of tachykinins. In turn, these remodeling events contribute to the itch response associated with dermatitis. PUBLIC HEALTH RELEVANCE: Eosinophils are rare white blood cells whose destructive capabilities were assumed to contribute to the structural changes of the skin linked with allergic inflammation. Nonetheless, a growing literature suggests that this perspective is too narrow and that eosinophils may even also interact with sensory nerves in the skin to elicit behaviors associated with allergic inflammation such as itching. Our creation and use of genetically engineered mice targeting eosinophils and their activities provide a mechanism with which to test this possibility. In particular, our creation of mice that are capable of becoming eosinophil-less on an on demand basis (iPHIL) provides a needed opportunity to define unambiguously the specific roles of eosinophil- nerve interactions during inflammatory responses that lead to itch responses.

DESCRIPTION (provided by applicant): Viral single standed RNA is sensed by cells via toll-like receptor (TLR)-7 and TLR-8. We have recently shown that stimulating TLR-7 and TLR-8 relaxes airway smooth muscle and prevents broncho-constriction in response to a variety of stimuli. The effect is profound, and can lead to virtually complete relaxation of smooth muscle. We have demonstrated this effect in guinea pigs and mice, and in human airway tissues. TLR7 dependent broncho-dilation in guinea pigs and in human airways is mediated by production of nitric oxide, while TLR8 dependent broncho-dilation is not. Our preliminary data suggest that TLR8 mediated broncho-dilation is mediated by production of prostaglandins and opening of the large conductance, calcium activated potassium channel. We have also shown that both antigen sensitization and acute viral infection substantially impair TLR- dependent broncho-dilation. In this application, we propose three specific aims: Specific Aim #1. To A) use TLR7(-/-) mice, as well as newly generated TLR8(-/-) and TLR7(-/-)TLR8(-/-) mice, to more thoroughly investigate and establish the signaling pathways responsible for the effects of these receptors on airway neurons and smooth muscle, B) test whether these receptors signal through nitric oxide, prostaglandins, and the large conductance, calcium activated potassium channel (BKCa) in human airways in vitro, and C) explore the role of nitric oxide and prostaglandins and changes in intracellular calcium in rapid signaling by these receptors in primary cultures of human airway parasympathetic neurons and airway smooth muscle cells. Specific Aim 2: To test whether decreased TLR mediated broncho-dilation after antigen sensitization is mediated by decreased TLR7 and/or TLR8, testing signaling pathways in airway smooth muscle identified in Aim #1. Since these TLRs are on parasympathetic nerves, as well as on eosinophils recruited to the airway nerves, we will also test whether TLR7 and TLR8 change neural control in sensitized airways. SPECIFIC AIM #3: To test whether decreased TLR mediated broncho-dilation after viral infection is mediated by decreased TLR7 and/or TLR8 pathways, testing all second messenger signaling pathways in airway smooth muscle identified in aim one. We will also investigate the role of changes in parasympathetic function. The results of the experiments we propose will be important in establishing the potential of TLR7 AND TLR8 agonists as treatments for asthma and other airway diseases. In addition, because these receptors respond to viral RNA, understanding the effects of stimulating TLR7 receptors in the airways, as well as the loss of these effects in models of asthma, will help us understand the pathophysiology of virus induced asthma attacks.

DESCRIPTION (provided by applicant): Increased reflex bronchoconstriction is a characteristic of asthma. We have recently shown a doubling of airway epithelial sensory nerves in a mouse model of asthma. This increased reflex bronchoconstriction substantially. Eliminating eosinophils prevented the increases in both innervation and bronchoconstriction. We also showed that eosinophils increase the growth of cultured dorsal root ganglion sensory neurons, due to a soluble factor produced by the eosinophils. Thus eosinophils increase airway sensory innervation, and this may participate in both the bronchoconstriction and the cough that are common in asthma. We hypothesize that eosinophils promote airway sensory nerve growth and this increases reflex bronchoconstriction and cough. We propose three specific aims: SPECIFIC AIM #1: To define eosinophil-mediated effects on airway sensory innervation. In both an antigen challenge model and unique transgenic models of eosinophilic pulmonary inflammation, we will use our novel whole-mount nerve imaging [8] to define eosinophil-dependent changes in airway epithelial sensory innervation, as well as on substance P content, and TRPA1, TRPV1, TRPM8, and ASIC3 expression will be measured. These will be correlated with airway reflex bronchoconstriction. SPECIFIC AIM #2: To determine the reversibility of these pulmonary remodeling events by targeting eosinophils in mice with established disease. We will use our new strains of mice that allow inducible on demand ablation of eosinophils (the iPHIL mouse). We have also generated mice with eosinophils that lack the glucocorticoid receptor, allowing us to determine whether the effects of steroid treatment are mediated by suppressing eosinophils or via eosinophil independent pathways. SPECIFIC AIM #3: To characterize neural plasticity in human airway disease. Similar histological endpoints as in aims 1 and 2 will be measured in biopsies from well-characterized patients with 1) asthma (mild and severe), 2) cough-variant asthma, and 3) cough without asthma, comparing these with normal volunteers. Induced sputum and bronchoalveolar lavage eosinophil peroxidase will be measured as possible biomarkers.

? DESCRIPTION (provided by applicant): The MD/PhD Training Program at Oregon Health & Science University (OHSU) offers integrated medical and scientific training. Graduate programs include Biochemistry and Molecular Biology, Physiology and Pharmacology, Genetics, Cell and Developmental Biology, Molecular Microbiology and Immunology, Cancer Biology, Neuroscience, Behavioral Neuroscience, Biomedical Engineering and Biomedical Informatics. In addition, with the opening of our new School of Public Health offering PhDs in 1) Epidemiology, 2) Community Health, and 3) Health Systems and Policy. Substantial differences in curricula for both the MD and PhD programs (compared with those for straight MD or PhD students) are in place, and include elimination of several required classes and one clerkship in the MD curriculum, and elimination of the requirement for three research rotations for the graduate programs. In addition to several graduate school courses that our MD/PhD students are excused from, a system is in place to allow our students to challenge any required graduate school course by achieving a satisfactory score on the final exam. To tailor the graduate curriculum appropriately for the individual, a Scientific Oversight Committee of three faculty is assigned to each student. We have also added several courses specific to the MD/PhD program, including a required 5 - 6 week Clinical/Translational Research Clerkship (housed in our CTSA funded Oregon Clinical and Translational Research Institute) and a required Longitudinal Clinical Rotation, providing a formalized, mentored ongoing clinical experience two half days a month during the PhD years. Extensive student tracking and mentorship programs are in place. Our monthly MD/PhD Grand Rounds, presented by physician scientists on our faculty and taking the form of a case based introduction to a scientific talk, assures ongoing exposure to clinical and translational research, and the guidelines for our weekly MD/PhD Journal Club require that the article be placed firmly in a clinical context in the background presented. Our annual MD/PhD Research Retreat also features talks by OHSU scientists, many of whom have translational research projects, as well a keynote speaker of international reputation, selected for the clinical relevance as well as quality of his or her research. We foste public speaking skills, providing multiple opportunities for student presentations, with formal feedback from the Program Director. We also emphasize grant writing skills, providing multiple sources of assistance in crafting and submitting individual NRSA and similar grants from professional societies, with a very high rate of success in achieving funding. Our new medical school curriculum provides added flexibility with respect to the timing of reentry into the MD program, and should avoid students taking more than 7 - 8 years to complete the program except in exceptional cases. With the strong support of the Dean of the School of Medicine as well as the Oregon Clinical and Translational Research Institute and the Departments and Faculty as a whole, we provide an ideal environment for training the next generation of physician scientists.

The goal of the OCTRI KL2 program is to train a corps of clinical and translational scientists in the skills and competencies of clinical and translational research who can use cutting edge technologies to translate discoveries made in the laboratory to the patient and the community. Through this program, we will select outstanding early faculty who are committed to developing a research career; each of the scholars will participate in a mentored research experience for a minimum of 0.75 FTE. Scholars will develop a career development plan with the aid of a mentor team ? which will include a designated primary mentor ? to assist with career development, networking, manuscript writing, and grant development. All scholars will participate in activities that are specific to and that will enhance their research and career development needs: development of research competencies in clinical and translational research, professional activities, and experiential learning. Many scholars will enroll in the curriculum of the Master of Clinical Research (MCR) program; all scholars must participate in training in qualitative methods, scientific communication, and research leadership. Professional development includes opportunities through the OCTRI Scholar program for peer networking, Design Studio, and access to research consultation. Community engagement will be emphasized in research development, with all scholars gaining experience with stakeholders. Scholars will also gain experience with grant writing and grant review. They will have opportunities to participate in specific experiences, such as working with centers for health policy and evidence-based practice; allowing scholars to learn how to write a systematic review or to develop an evidence-based guideline. The goal of this program is to produce an independent, transdisciplinary clinical and translational researcher. The expectation is that scholars will move to an independent K award at the end of the KL2 funding period; exceptional scholars may move directly to an RPG.

The goal of the OCTRI KL2 program is to train a corps of clinical and translational scientists in the skills and competencies of clinical and translational research who can use cutting edge technologies to translate discoveries made in the laboratory to the patient and the community. Through this program, we will select outstanding early faculty who are committed to developing a research career; each of the scholars will participate in a mentored research experience for a minimum of 0.75 FTE. Scholars will develop a career development plan with the aid of a mentor team ? which will include a designated primary mentor ? to assist with career development, networking, manuscript writing, and grant development. All scholars will participate in activities that are specific to and that will enhance their research and career development needs: development of research competencies in clinical and translational research, professional activities, and experiential learning. Many scholars will enroll in the curriculum of the Master of Clinical Research (MCR) program; all scholars must participate in training in qualitative methods, scientific communication, and research leadership. Professional development includes opportunities through the OCTRI Scholar program for peer networking, Design Studio, and access to research consultation. Community engagement will be emphasized in research development, with all scholars gaining experience with stakeholders. Scholars will also gain experience with grant writing and grant review. They will have opportunities to participate in specific experiences, such as working with centers for health policy and evidence-based practice; allowing scholars to learn how to write a systematic review or to develop an evidence-based guideline. The goal of this program is to produce an independent, transdisciplinary clinical and translational researcher. The expectation is that scholars will move to an independent K award at the end of the KL2 funding period; exceptional scholars may move directly to an RPG.

We have recently shown that there is a fundamental difference in airway physiology between wildtype mice born to wildtype mothers and wildtype mice born to IL5 overexpressing mothers or housedust mite (HDM) sensitized mothers. The airways of adult wildtype offspring of these mothers are much more responsive, an effect that requires fetal eosinophilia that develops as the result of maternal IL5 crossing the placenta. Subsequent sensitization and challenge with housedust mite yields much more severe bronchoconstriction. The central hypothesis of this project is that high circulating IL5 during pregnancy induces fetal eosinophilia, and that this causes permanent changes in airway innervation that increase bronchoconstriction. In this project, we propose to determine the mechanisms of airway hyperreactivity in these WT offspring of IL5 transgenic mothers. We will characterize changes in airway nerve structure, transmitters, and receptor expression. We propose three specific aims: SPECIFIC AIM #1: Test the effects of maternal IL5tg and maternal HDM challenge on reflex bronchoconstriction, parasympathetic nerve function, and smooth muscle function. We will determine the role of maternal fetal transfer of IL5 in the maternal HDM challenge model, and test the role of maternal and fetal eosinophilia in these effects. We will also dissect the mechanisms of severe, lethal bronchoconstriction and potentiated airway inflammation when these adult offspring are antigen challenged. SPECIFIC AIM #2: Test whether exposure to IL5 in utero alters the architecture or neurotransmitter content of sensory and parasympathetic nerves. We will use our novel imaging method to determine epithelial and smooth muscle innervation, and to quantify changes in neurotransmitter expression in sensory and parasympathetic nerves. SPECIFIC AIM #3: To determine the role of airway epithelial neurotrophins in 1) heightened response to antigen challenge, 2) severe airway hyperresponsiveness, and 3) maintenance of airway nerve remodeling in adult offspring of IL5tg mothers and in adult offspring of HDM sensitized and challenged mothers. We will extend our preliminary studies of neurotrophin expression to include the different mouse models and treatments in Aim #1, and determine the roles of neurotrophins that are elevated by blocking with antibodies and treating animals with receptor antagonists.

We have recently shown that there are substantial changes in airway innervation in patients with severe asthma. In both these patients and in several mouse models of asthma, inflammatory changes lead to significant changes in nerve structure and phenotype. We hypothesize that structural and phenotypic changes in airway innervation are central to asthma pathophysiology. In order to characterize these changes functionally, we propose to develop an optogenetic approach to stimulating or silencing two populations of airway neurons: 1) cholinergic efferents, and 2) substance P containing afferents. We propose two specific aims: SPECIFIC AIM #1: We will apply optogenetic methods to stimulate or silence specific populations of airway neurons. We will express the light sensitive cation channel channelrhodopsin (ChR2) in two populations of neurons, by driving expression of the ChR2 with the promoters for choline acetyltransferase (for cholinergic parasympathetic neurons), and for substance P (Tac1, the promoter for the preprotachykinin gene, for substance P containing sensory neurons). This will allow us to activate these neurons selectively, and study the effects on airway function. Conversely, we will express halorhodopsin (a light-sensitive anion channel that hyperpolarized cells) in the same populations of neurons, allowing us to silence these neurons SPECIFIC AIM #2: We will apply these optogenetic methods to defining functional changes in the sensory and parasympathetic control of airway smooth muscle in a mouse model of eosinophilic asthma, using transgenic mice expressing IL5 in airway epithelial cells (causing intense airway eosinophilia and hyperinnervation). These mice display markedly increased reflex bronchoconstriction in response to inhaled serotonin. As both afferent (sensory) and efferent (parasympathetic) nerves are abnormal in asthma and reflex bronchoconstriction is increased in these animals, we will test the effects of selectively stimulating or silencing cholinergic neurons, and of stimulating or silencing substance P containing sensory neurons. At the completion of this project, we will have established a new method for investigating the neural control of the airways, and for defining the role of subpopulations of neurons as well as their changes in models of airway disease. Going forward, we will apply this method, as well as optogenetic stimulation or silencing of other populations of neurons, to the study of other models of asthma, including viral infections, antigen challenge, and ozone inhalation.