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A Thesis Entitled the Role of Antimicrobial Peptide Murine Beta A Thesis entitled The Role of Antimicrobial Peptide Murine Beta Defensin-3 in Protection against Oropharyngeal Candidiasis by Bemnet G. Mengesha Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology ________________________________________ Dr. Heather Conti, Committee Chair ________________________________________ Dr. Malathi Krishnamurthy, Committee Member ________________________________________ Dr. Jianyang Du, Committee Member ________________________________________ ________________________________________ Dr. Amanda C. Bryant-Friedrich, Dean College of Graduate Studies The University of Toledo December, 2017 Copyright 2017, Bemnet G Mengesha This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of The Role of Antimicrobial Peptide Murine Beta Defensin-3 in Protection against Oropharyngeal Candidiasis by Bemnet G. Mengesha Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology The University of Toledo December, 2017 Oropharyngeal candidiasis (OPC, thrush) is an opportunistic infection caused mainly by Candida albicans. C. albicans is a commensal of oral mucosal surfaces, but can be pathogenic when antifungal immune defense mechanisms are impaired. There is a high prevalence of OPC in patients with human immunodeficiency virus (HIV), implicating CD4+ T helper (Th) cells in protection against OPC. IL-17 is an important pro- inflammatory cytokine produced by the Th17 subset, and studies in experimental models show increased susceptibility in mice with impaired IL-17 signaling. Deficiencies in IL-17 signaling lead to defects including the production of antimicrobial peptides such as β- Defensins (BDs). In OPC susceptible IL-17RA knockout mice, expression level of murine β-defensins-3 (mBD3) is decreased in infected tongue tissue compared to wild type mice suggesting the involvement of mBD3 in protection against OPC. mBD3 has direct antifungal properties against Candida, but can also modulate the immune response through potential interactions with chemokine receptors such as CCR6 necessary for immune cell trafficking and recruitment. Moreover, mDefb3-/- mutant mice expressing low level of iii murine β-Defensin-3 show high susceptibility to OPC suggesting the involvement of mBD3 in protection against OPC. Gene expression profile of mDefb3 -/- mice show impaired expression of IL-17 inducing cytokines such as IL1- β and IL-6 and inflammatory cytokine IL-17 following Candida infection showing mDefb3 -/- mutant mice have defects in IL-17 signaling pathway. In addition, mDefb3 regulates expression of beta defensins mDefb1 (BD1), mDefb2 (BD2), chemokine CXCl2 but not chemokine CXCL1. Loss of expression of Defb3 in mDefb3 -/- mice was compensated by high expression of Defb14, and increased expression level of CCL20 early during infection (day 2 post infection) but was not enough to offset susceptibility to OPC. Using mouse model of OPC, we show that mDefb3 regulate innate immune response against OPC via regulation of AMPs (mDefb1 and mDefb2) and proinflammatory cytokines (IL-17, IL-6 and IL1β). iv Acknowledgements My utmost thanks and sincere gratitude go to my advisor, Dr. Heather Conti for the guidance, encouragement and support during the development and execution of this work. I would like to express my gratitude to my committee members: Dr. Malathi Krishnamurthy and Dr. Jianyang Du for serving in my thesis committee and their valuable insight and suggestions. I would like to thank my family: my wife Mrs. Elizabeth Senbetu and my daughters Mihret and Saron Gashawbeza for their everlasting love and support, and my parents: my father, Mr. Gashawbeza Mengesha and my mother, Mrs. Bayush Nicola for supporting me spiritually. Finally, I thank members of Conti lab for the support and congenial work environment. v Table of Contents Abstract .............................................................................................................................. iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Figures…………………………………………………………………………...viii List of Abbreviations ...........................................................................................................x List of Symbols…………………………………………………………………………...xi 1. Literature Review………………………………………………………………….1 1.1 Introduction ...................................................................................................….1 1.2 Pathogenicity mechanisms of C. albicans .........................................................2 1.3 Innate recognition of microbial pathogens…………… ....................................3 1.4 Antifungal immunity ..........................................................................................5 1.5. Effector mechanisms of antiCandida immunity...............................................7 1.5.1 The role of neutrophils and chemokines in antifungal immunity...........8 1.5.2 The role of antimicrobial peptides in antifungal immunity…………..10 1.6. Hypothesis and Objectives………………………………………………….13 2. Materials and Methods--------------------------------------------------------------------15 2.1 Preparation of C. albicans culture…………………………………...15 2.2 Induction of OPC…………………………………………………….15 2.3 Candida killing assays .........................................................................16 2.4 Experimental mice ...............................................................................16 2.5 RNA extraction, cDNA synthesis and gene expression studies ……..17 2.6 Quantification of fungal burden……………………………………...17 vi 2.7 Microscopy ..........................................................................................17 2.8 Neutrophil count .............................................................................….18 2.9 Data analysis------------------------------------------------------------------18 3. Results ………........................................................................................................19 3.1 rMBD3 and rhBD2 exhibit candidacidal activity in vitro ....................19 3.2 mDefb3-/- mice are susceptible to OPC .................................................20 3.3 mDefb3 regulates expression of mDefb1 and mDefb2 during oral Candida infection..................................................................................21 3.4 mDefb3 regulates IL-17 and Th17 inductive cytokines IL1β and IL-6 ........................................................................................................23 3.5. mDefb3 regulates CCL20 and CXCL2 but not CXCL1 ......................24 3.6. Immunohistochemical analysis show Defb3-/- mice do not elicit defects in neutrophil extravasation ----------------------------------35 4. Discussion……………………………………………………………………………..42 4.1 rMBD3 and rhBD2 show potent candidacidal activity………………42 4.2 mDefb3 is necessary for innate response to OPC………………………44 4.3 mDefb3 contributes to immunity against OPC via regulation of 4.4. AMPs, pro-inflammatory cytokines and chemokines……………….45 References………………………………………………………………………………..52 vii List of Figures 3-1 Candidacidal activities of recombinant mBD3 and recombinant hBD2 ................20 3-2 mDefb3 is important for protection against OPC ...................................................22 3-3 Gene expression changes during OPC show the involvement of Defb3 in Candida clearance ...............................................................................................25 3-4 Gene expression changes during OPC show mDefb3 regulates mDefb1 during OPC. ..........................................................................................................26 3-5 Altered gene expression during OPC show Defb3 regulates Defb2 during OPC ..27 3-6 Gene expression changes during OPC show Defb14 upregulation in mDefb3-/- mice during OPC ..................................................................................29 3-7 Altered gene expression during OPC show Defb3 regulate IL-17 expression during OPC ............................................................................................................30 3-8 Gene expression changes during OPC show Defb3 regulate IL1b expression during OPC ............................................................................................................32 3-9 Gene expression changes during OPC show Defb3 regulate IL-6 expression during OPC ..........................................................................................33 3.10 Gene expression changes during OPC show CCL20 compensates Defb3 deficiencies during OPC .......................................................................................34 3-11 mDefb3 does not regulate CXCL1 during OPC .....................................................36 3-12 Altered gene expression during OPC show mDefb3 regulate CXCL2 during OPC .........................................................................................................................37 3-13 Altered gene expression during OPC show mDefb3 regulate Csf3 during OPC ...38 viii 3.14 mDefb3 in oral mucosa does not regulate neutrophil migration during OPC........39 3.15 mDefb3 in oral mucosa does not regulate
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