Terry L. Noah, MD - US grants

Infection of the airways with respiratory syncytial virus (RSV) or human influenza virus is associated with acute airway inflammation in normal children, and may trigger exacerbation of lung disease in children with cystic fibrosis (CF). Little is currently known about the role of epithelial mediators in the interaction between airway epithelium, infectious agents, and inflammatory cells. The in vitro study of airway epithelia has led to the discovery of cellular and molecular mechanisms of airway disease. Preliminary data in this laboratory show that epithelial cells in culture can be infected with RSV, and produce platelet-activating factor (PAF), surface adhesion protein, and inflammatory cytokines as a result. The proposed research is designed to test the hypothesis that airway epithelial cells infected with RSV or human influenza virus produce factors which promote or modulate airway inflammation, and that these factors are more strongly expressed in epithelial cells from populations at risk for severe viral disease. The specific goals of the project are to (1) Establish optimal conditions for infection of airway epithelial cells by RSV and influenza virus. Markers of infection will include virus replication, virus antigen-specific immunofluorescence, and viral mRNA. (2) Measure the effect of viral infection on epithelial production of pro- inflammatory factors including IL-1, IL-6, IL-8, GM-CSF, and platelet- activating factor (PAF). (3) Measure the effect of viral infection of epithelia on production of neutrophil chemotactic factors and factors promoting adherence of neutrophils to epithelial cells, including ICAM-1, GMP-140, and LEC-CAM. (4) Compare the above responses in transformed epithelial cells to those in airway epithelial cells in primary culture, and compare epithelial cells of normal adult origin to those of CF or pediatric origin. The results of these experiments will suggest mechanisms responsible for the initiation and maintenance of airway inflammation by respiratory viruses, in normal airways and in CF or pediatric airways. Hypothetical mechanisms will be readily testable using the model system described, and understanding these mechanisms will ultimately suggest specific anti-inflammatory therapies for these patients.

Respiratory syncytial virus (RSV)-induced acute wheezing syndromes produce great morbidity among children. Inflammation is probably a key factor in the pathogenesis of wheezing, but the inflammatory pathways resulting in virus-induced wheezing are unknown. RSV induces production of the chemokines IL-8 and RANTES by respiratory epithelial cells in culture. These chemokines are potent chemotactic or priming agents for leukocytes which may cause bronchospasm, edema, and airway hyperreactivity. We hypothesize that individuals who wheeze during RSV infection have greater respiratory epithelia chemokine production than those who do not wheeze during viral infections. Nasal epithelium is susceptible to RSV infection and can be sampled noninvasively in children, and available evidence suggests that nasal inflammation reflects or influences the lower airway in humans. We therefore propose a series of studies which will determine nasal epithelial chemokine responses to RSV and their relationship to inflammation, memory T cell responses, and wheezing. The specific aims of this proposal are to determine (1) the time course of, cellular sources of, and effects of antiinflammatory agents on epithelial chemokine production during human RSV respiratory illnesses; (2) the nasal epithelial subtypes and infection status of the cells producing chemokines during RSV infections; (3) if the nasal chemokine and/or memory T lymphocyte cytokine responses to RSV differ between children who do vs. Do not manifest wheezing with RSV infection; and (4) if RSV-induced chemokine responses by nasal epithelial cells are greater in cells cultured from subjects with vs. Without recurrent wheezing. Studies will include serial nasal lavage and mucosal biopsies obtained from children with naturally-acquired RSV infections and from adults with experimental RSV infections. Chemokine production and cell types of origin in respiratory mucosa will be determined using a combination of ELISA, RT-PCR, and immunohistochemistry. In experimental RSV infections of adults, the effects of topical antiinflammatory agents on nasal inflammation and chemokine production will be determined. Production of cytokines by cultured T lymphocytes stimulated with RSV antigens will be measured by RT-PCR, in situ hybridization, and ELISA. Nasal epithelial cell cultures established from subjects with and without recurrent wheezing will be infected with RSV for comparison of chemokine induction responses. These studies will establish the kinetics of RSV-induced epithelial chemokine induction in the upper respiratory tract, its relationship to common acute childhood wheezing syndromes, and the relative degree to which epithelial and T lymphocyte cytokine responses correlate with susceptibility for wheezing with acute RSV infection. Definition of specific epithelium-driven inflammatory pathways triggered by RSV infections should lead to identification of future therapies or preventive strategies for RSV-induced respiratory illnesses.

Previous studies have demonstrated that the incidence and severity of respiratory virus infections is greater in smokers than in non-smokers, but the mechanisms mediating these responses are currently not well understood. Our preliminary data demonstrate that cultured nasal epithelial cells from smokers are more susceptible to influenza virus infections, shed more virus, and have decreased expression of type 1 interferons. This in vitro model thus provides an important tool to investigate the cellular and molecular basis for enhanced susceptibility to influenza virus seen in smokers. In addition, our preliminary data demonstrate that nasal administration of live attenuated influenza virus (LAIV) offers the possibility of studying influenza virus infections safely in humans in vivo. Using tightly linked human in vitro and in vivo approaches, this proposal is designed to test the hypothesis that chronic exposure to cigarette smoke alters epithelial antiviral and inflammatory responses to influenza virus infection via two potentially related mechanisms: decreased expression of phase II (antioxidant) enzymes and suppression of type 1 interferon (antiviral) pathways. We further hypothesize that upregulation of phase II enzymes via nutritional supplementation with SFN is a potential therapeutic strategy to mitigate these effects. Specific aim 1 will use an in vitro model of differentiated human nasal epithelial cells to determine mechanisms that modify influenza-induced antiviral defense responses in smokers, initially focusing on the role of type I IFN antiviral defense responses and the potential role of cigarette smoke-induced gene silencing. Specific aim 2 will use our existing protocol of administration of LAIV vaccine as a model for influenza virus infections to confirm mechanisms that mediate enhanced susceptibility to influenza infections in smokers in vivo. LAIV-induced viral replication and antiviral defense responses will be assessed in smokers and non-smokers using endpoints measured in nasal biopsy tissue and lavage fluids. Outcomes within each study cohort will be grouped based on changes in innate immune defense gene expression found in specific aim 1. Specific aim 3 will use both the in vitro and in vivo models to determine the relationships between antioxidant gene expression, antiviral pathways, and virus-induced inflammation in smokers and non-smokers. We will assess how upregulation of HO-1 as a result of supplementation with SFN can improve key abnormalities in antiviral pathways and inflammatory/immune response changes associated with smokers, as identified in SA1+2. Data derived from these studies will yield insights into the mechanisms that enhance the susceptibility to influenza virus infections in smokers and explore potential therapeutic interventions using a translational research design.

Abstract This renewal for years 41-45 of the Program in ?Multidisciplinary Training in Pulmonary Diseases? at The University of North Carolina at Chapel Hill supports six postdoctoral trainees with M.D., M.D./Ph.D. or Ph.D. degrees for research training in Respiratory Medicine, emphasizing a joint training program for Medicine and Pediatric trainees. The Program provides multidisciplinary training in basic, translation and clinical research within the pulmonary divisions of the Departments of Medicine and Pediatrics and our three Centers devoted to understanding lung health and disease. The breadth of training provided by the Program is expanded by faculty from 12 clinical (3) and basic science (9) departments in the Schools of Medicine and Public Health and the College of Arts and Sciences. M.D. trainees will enter a 3-5 year clinical and research training experience designed to provide them with skills required for a career in academic pulmonary medicine. Ph.D. trainees will typically enter in the second year of their post-doctoral fellowship, and they will be well integrated into the translational and clinical research components, as well as the basic science. Each trainee will have a scholarly oversight committee that includes trainers from varying disciplines who will facilitate their scientific growth. Each area of lung research offered by the Program is multidisciplinary in nature and emphasizes a knowledge of the basic, translational, clinical and impact implications for each trainee's question, even though a trainee's research focuses on one aspect. Areas of research include the genetic basis of airways diseases, particularly cystic fibrosis, primary ciliary dyskinesia, and idiopathic bronchiectasis, cell and molecular biology of airway epithelia in health and disease, inflammatory and innate immune responses during bacterial and viral infections, endothelial cell biology and vascular permeability in infection and ARDS, outcomes research and clinical trials in critical care, the control of airway inflammation, basic and translational proteomics of airways, the airway microbiome in health and in cystic fibrosis and COPD, airway function and leukocyte kinetics in COPD, comparative effectiveness research in COPD, the responses to injury by physical, chemical, and microbial environmental agents, and clinical and basic studies in asthma. Novel programs that exemplify the multi- disciplinary nature include the Virtual Lung Project, ARDS and Critical Care Research, and the COPD-Lung Cancer Working Group, each of which require cross-disciplinary interactions amongst a very wide range of expertise to accomplish their goals. Clinical studies will be integrated with basic observations using translational physiologic, biochemical, molecular and genetic technologies and Omics approaches and will provide training in state-of-the-art bioinformatics, database design, use and analyses, and statistical interpretation. Emphasis is on mechanisms underlying common and rare lung diseases in pediatric and adult populations and development of novel therapeutic strategies. Our Program provides an opportunity for trainees to develop as scientists and become independent investigators within the national community.