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Development and Application of Immunoassays For FUNCTIONAL CHARACTERIZATION OF SPURT USING TRANSGENIC MOUSE MODEL by Lina Lukinskiene BS, Vilnius University, 1995 MS, Vilnius University, 1997 Submitted to the Graduate Faculty of Department of Environmental and Occupational Health Graduate School of Public Health in partial fulfillment Of the requirements for the degree of Master of Science University of Pittsburgh 2005 UNIVERSITY OF PITTSBURGH Graduate School of Public Health This thesis was presented by Lina Lukinskiene It was defended on 06/10/2005 and approved by Thesis Advisor: Yuan Pu Di, PhD, Assistant Professor Department of Environmental & Occupational Health Graduate School of Public Health University of Pittsburgh Committee Member: Bruce Pitt, PhD, Professor Department of Environmental & Occupational Health Graduate School of Public Health University of Pittsburgh Committee Member: Chad Steele, PhD Assistant Professor Division of Pediatric Pulmonology, Laboratory of Lung Immunology and Host Defense School of Medicine University of Pittsburgh ii Yuan Pu Di, PhD FUNCTIONAL CHARACTERIZATION OF SPURT USING TRANSGENIC MOUSE MODEL Lina Lukinskiene, MS University of Pittsburgh, 2005 Abstract The respiratory tract is the target of multiple infectious agents. Because the lungs are continually exposed to infectious pathogens in inspired air, natural defense mechanisms have devolved to prevent infection. These defense mechanisms coordinate with each other to provide efficient protection against infection. As a result, pulmonary infections can be viewed not just as a consequence of exposure to a virulent pathogen but as a result of a breakdown of natural host defenses. SPLUNC1 or SPURT (secretory protein in upper respiratory tracts) is small, secreted protein that is expressed in epithelial areas of the nose, mouth, pharynx and lungs. It may be involved in host defense because it is highly homologous to bactericidal/permeability-increasing protein (BPI); a protein that mediates LPS related bacteria killing. In this study, we established a constitutively overexpress CCSP-spurt transgenic mouse model to examine the biological function of spurt. We compared mRNA expression of CCSP- spurt in transgenic mice and their transgenic negative litermates. We determined that mRNA expression of CCSP-spurt was elevated in transgenic mice. The tissue distribution of overexpressed spurt in CCSP-spurt mice was confirmed to be only at trachea and lung and exist in no other tissues. Bronchoalveolar lavage (BAL) fluid from unchallenged CCSP-spurt mice iii had higher spurt concentration as was determined by ELISA and western blot. BAL from unchallenged CCSP-spurt mice also exhibited antibacterial activity. Furthermore, CCSP-spurt mice display enhanced bacterial clearance than wild-type mice after both groups of mice were challenged with aerosolized gram negative microorganisms Pseudomonas aeruginosa (PAO1) infection. BAL of CCSP-spurt mice had lower levels of proinflammatory cytokines than BAL of their wild-type littermates after the challenge with aerosolized P. aeruginosa. Results from our studies suggest that spurt is a novel BPI-like antibacterial protein that may play a critical role in airway specific innate immunity. Further studies of this protein might have potential public health significance in providing better understanding of natural defense processes in respiratory tract. iv TABLE OF CONTENTS 1. INTRODUCTION .................................................................................................................. 1 2. REVIEW OF THE RELEVANT LITERATURE .................................................................. 4 3. MATERIALS AND METHODS............................................................................................ 8 3.1. TRANSGENIC MICE GENERATION ......................................................................... 8 3.2. RNA ISOLATION AND RT-PCR ANALYSIS ............................................................ 9 3.3. REAL TIME RT-PCR ANALYSIS................................................................................ 9 3.4. DEVELOPMENT OF THREE ANTIBODY SANDWICH ELISA ............................ 10 3.5. THREE ANTIBODY SANDWICH ELISA................................................................. 11 3.6. SDS-PAGE ................................................................................................................... 12 3.7. WESTERN BLOT ANALYSIS ................................................................................... 12 3.8. BAL TESTING FOR ANTIBACTERIAL ACTIVITY ............................................... 13 3.9. NEUTRALIZATION OF ANTIBACTERIAL ACTIVITY BY ANTI-SPURT ANTIBODY.............................................................................................................................. 14 3.10. BACTERIAL CULTURE............................................................................................. 14 3.11. MICE EXPOSURE TO PSEUDOMONAS AERUGINOSA....................................... 15 3.12. BRONCHOALVEOLAR LAVAGE (BAL) ................................................................ 15 3.13. BACTERIAL ENUMERATION.................................................................................. 16 3.14. HISTOLOGY................................................................................................................ 16 v 3.15. IMMUNOHISTOCHEMISTRY................................................................................... 17 3.16. BIO-PLEX CYTOKINE ASSAY................................................................................. 17 4. RESULTS ............................................................................................................................. 18 4.1. ESTABLISHMENT OF TRANSGENIC MOUSE THAT OVER-EXPRESS HUMAN SPURT PROTEIN .................................................................................................................... 18 4.2. ENHANCED ANTIBACTERIAL ACTIVITY USING BAL FROM CCSP-SPURT MICE.. ...................................................................................................................................... 20 4.3. INCREASED ANTIBACTERIAL ACTIVITY OF CCSP-SPURT MICE AFTER AEROSOLIZED PSEUDOMONAS AERUGINOSA EXPOSURE ....................................... 24 4.4. DECREASED NEUTROPHILS INFILTRATION IN LUNGS OF CCSP-SPURT MICE AFTER AEROSOLIZED PSEUDOMONAS AERUGINOSA EXPOSURE............... 25 4.5. DECREASED PRO-INFLAMATORY CYTOKINE PRODUCTION IN CCSP- SPURT MICE AFTER AEROSOLIZED PSEUDOMONAS AERUGINOSA EXPOSURE . 28 5. DISCUSSION....................................................................................................................... 31 6. CONCLUSIONS................................................................................................................... 35 BIBLIOGRAPHY......................................................................................................................... 36 vi LIST OF FIGURES Figure 1 The human PLUNC locus ............................................................................................... 5 Figure 2 Generation and Assessment of CCSP-spurt Transgenic Mice..................................... 19 Figure 3 Over-expressed SPURT is observed in CCSP-spurt mice airway epithelium .............. 20 Figure 4 Enhanced antibacterial activity using BALF from CCSP-spurt transgenic mice. ........ 22 Figure 5 Neutralization of antibacterial activity by anti-plunc (spurt) antibody......................... 23 Figure 6 CCSP-spurt mice are more resistant to lung infection with P. aeruginosa................... 25 Figure 7 Total cell numbers of neutrophils were less in CCSP-spurt mice BAL than wild-type littermates.............................................................................................................................. 26 Figure 8 Histological view of wild type and CCSP-spurt transgenic mice before P. aeruginosa exposure. ............................................................................................................................... 27 Figure 9 Decreased neutrophils infiltration and lung injury in CCSP-spurt mice than wild type mice 4h after P. aeruginosa exposure................................................................................... 28 Figure 10 Decreased pro-inflammatory cytokine production in CCSP-spurt mice..................... 29 vii 1. INTRODUCTION The respiratory tract is the target of multiple infectious agents. Because the lungs are continually exposed to infectious pathogens in inspired air, natural defense mechanisms have evolved to prevent infection, which include mucociliary transport, innate immunity, cell-mediated immunity, and antibody production. These defense mechanisms coordinate with each other to provide efficient protection against infection. As a result, pulmonary infections can be viewed not just as a consequence of exposure to a virulent pathogen but as a result of a breakdown of natural host defenses. Upper respiratory tract infections affect the airways in the nose, ears, and throat. They can be caused by viruses, bacteria, or other microscopic organisms. Acute pharyngitis accounts for 1% to 2% of all visits to outpatient and emergency departments, resulting in 7 million annual visits by adults alone [1]. It is estimated that 31 million to 35 million US citizens have sinus disease [2]. About 12 million individuals are diagnosed with acute tracheobronchitis annually, accounting for one-third of patients presenting with acute cough [3]. The estimated economic impact of non-influenza-related upper
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