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Transcriptome Response Associated with Protective Immunity in T and B Cell Deficient Ebrz Afish

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Transcriptome Response Associated with Protective Immunity in T and B Cell Deficient Ebrz Afish Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2013 Transcriptome Response Associated with Protective Immunity in T and B Cell Deficient ebrZ afish Aparna Krishnavajhala Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Krishnavajhala, Aparna, "Transcriptome Response Associated with Protective Immunity in T and B Cell Deficient ebrZ afish" (2013). Theses and Dissertations. 4770. https://scholarsjunction.msstate.edu/td/4770 This Dissertation - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Automated Template B: Created by James Nail 2011V2.01 Transcriptome response associated with protective immunity in T and B cell deficient zebrafish By Aparna Krishnavajhala A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Immunology in the College of Veterinary Medicine Mississippi State, Mississippi August 2013 Copyright by Aparna Krishnavajhala 2013 Transcriptome response associated with protective immunity in T and B cell deficient zebrafish By Aparna Krishnavajhala Approved: _________________________________ _________________________________ Lora Petrie-Hanson Stephen B. Pruett Associate Professor Professor Basic Sciences Basic Sciences (Major Professor) (Committee Member) _________________________________ _________________________________ Xiu-Feng (Henry) Wan Larry Hanson Associate Professor Professor and Graduate Coordinator Basic Sciences Basic Sciences (Committee Member) (Committee Member) _________________________________ Kent H. Hoblet Dean & Professor College of Veterinary Medicine Name: Aparna Krishnavajhala Date of Degree: August 17, 2013 Institution: Mississippi State University Major Field: Immunology Major Professor: Lora Petrie-Hanson Title of Study: Transcriptome response associated with protective immunity in T and B cell deficient zebrafish Pages in Study: 124. Candidate for Degree of Doctor of Philosophy RAG1-/- mutant zebrafish lack T and B lymphocytes. However, when re-exposed to homologous bacteria, these fish mount a response that provides specific protection. To further define this response, we utilized microarray analyses to determine the mechanisms underlying innate immune system memory in zebrafish. We also analyzed interferon (IFN) gamma by qRT-PCR. It is produced by activated NK cells and could indicate if this cell mediates the protective response seen in lymphocyte deficient zebrafish. Pathological studies and in situ hybridizations were performed to observe tissue changes and location of the cells that produced IFN gamma. Following bacterial re- exposure, zebrafish transcripts in cell receptor activation, cell proliferation and cytotoxic function categories were differentially expressed. We found high expression of IFN gamma in the lymphocyte like cell population after bacterial exposure and this was induced to a higher level in fish that had been vaccinated. The phagocytic cell population showed no induction of INF gamma. Over-all, the pathological response was much less severe in the vaccinated (48 hps) fish. Our microarray and pathological findings indicate that the primary immune response of mutant zebrafish is not impaired, and they demonstrate an enhanced innate immune response following secondary bacteria exposure. Following homologous secondary exposure, mutant zebrafish have a cell population that is undergoing upregulated cell receptor activation, cell cytotoxic functions and cell proliferation. This cell population expresses INF gamma. Activated T cells, NK-T cells and NK cells express INF gamma. Since RAG1 deficient zebrafish do not have T or NK-T cells, this cell population is most likely NK cells. DEDICATION This work is dedicated to my beloved father and mother Krishnavajhala Mallikharjuna Vara Prasad Sarma,and Sumithra Devi without whose support and encouragement it would not have been possible. ii ACKNOWLEDGEMENTS First and foremost I would like to thank Dr. Lora Petrie- Hanson for her invaluable support during these past five years of my life. Dr. Petrie is the most adorable person whom you will never forget once you meet her. As my major advisor, she gave me complete freedom in pursuing my objectives and we always had insightful discussions in answering the scientific questions. She taught me both consciously and unconsciously. I sincerely appreciate Dr. Petrie for all her contributions in terms of time, intellectual ideas, funding, advice, patience, friendship, and motivation that kept my PhD experience as productive and stimulating. I also remain indebted for her understanding and support during the times when I was really down and depressed due to personal problems. She contributed immensely to my personal and professional life at MSU. I am also thankful for the excellent example she has provided as a successful woman and professor. I would like to extend special thanks to my committee members Dr. Henry Wan , Dr. Larry Hanson, and Dr. Stephen Pruett who provided me encouraging and constructive feedback. Without great help from Dr. Hanson, I could not have finished quantitative real time PCR analysis. Dr. Wan immensely helped me in analyzing microarray data and providing me critical and constructive feedback. My sincere gratitude is reserved for Dr. Pruett for being a member in my advisory committee, for being a mentor in teaching me research ethics and extending financial assistantship in my hour of need. iii I would also like to take this opportunity to thank Assistant Professors Dr. Todd Pharr and Dr. Fiona McCarthy for their valuable comments and suggestions. Dr. Fiona guided me with my data analysis to find Gene Ontology in spite of her busy schedule. Dr. Pharr was kind and generous in providing me with his expertise or study materials. I received valuable guidance and support from my lab mate Dr. Claudia Hohn from the very beginning of my Ph.D. She was constant source of inspiration, feedback and support in and out of my academic process. Amongst my fellow graduate students in the department of Veterinary medicine, I thank Kamalakar Chatla and Ahmed Omar-Ali who helped me in all possible ways they could in performing my lab work. Ahmed shared his expertise in reading pathology slides. I have appreciated the expertise of Lori Ford for her help in zebrafish sampling and her advice in trouble shooting the RNA isolation to get better yields. I am grateful to our departmental administrative assistant Tony Roberson for her help and support in every single time I approached her. I personally thank Suzette Johnson for her relentless and timely supply of resources that I required in my work. The group has been a source of friendship as well as good advice and collaboration. I thank my friends Dr. Krishna Kumari yalavarthi, Dr. Gail Moraru and Dr. Mais Ammari for their moral support and being there to listen and comfort me during my tough times. Without the support of Dr. Phyllis Benjamin I would not have successfully overcame some obstacles in my life. She gave me enough energy and spirit to move forward when I was really in need of it. I am greatly indebted to Karin Lee for iv recognizing and providing me with opportunities to participate in all possible community service through Starkville Multicultural Lions Club. Finally, I would like to acknowledge my family and friends for their invaluable support. They extended all their support to fulfill my dream. Words cannot express the feelings that I have for my family members. Dr. Bindu Nanduri was more a family member rather a faculty to me. I will never forget her moral support. v TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES .............................................................................................................x CHAPTER I. INTRODUCTION .............................................................................................1 Zebrafish: Immunological model .......................................................................1 Generation of RAG1-/- mutant Zebrafish: Targeting Induced Local Lesions in Genomes (TILLING) a reverse genetic mechanism ............4 Role of RAG protein in VDJ recombination .....................................................5 Pathogen .............................................................................................................8 Microarray........................................................................................................10 Why zebrafish, Edwardsiella ictaluri and microarray technology were used in the present study ......................................................................12 References ........................................................................................................14 II. REVIEW OF LITERATURE ..........................................................................19 References ........................................................................................................34
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