FUNGAL KERATITIS: IMMUNE RECOGNITION, NEUTROPHIL-HYPHAE INTERACTIONS, AND FUNGAL ANTI-OXIDATIVE DEFENSES by SIXTO MANUEL LEAL JR. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Eric Pearlman, Ph.D. Department of Pathology CASE WESTERN RESERVE UNIVERSITY August, 2012 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication I dedicate this cumulative work to the invisible hand that has blessed my personal and academic life with incredible people, guidance, talent, courage, perseverance, and productivity. 3 Table of Contents List of Figures 7 List of Tables 9 Acknowledgements 10 List of Abbreviations 12 Abstract 14 Chapter 1. Introduction Fungi in their natural environment 16 Fungi and human disease 18 Fungi that cause human corneal infection 21 Fungal keratitis- Clinical characteristics and outcome 22 Anti-microbial Defenses at the Ocular Surface 23 Immune Recognition of Fungi 27 β2 integrins and complement receptor 3 32 The neutrophil and its microbicidal arsenal 36 Neutrophil NOX activation and role in killing hyphae 40 Fungicidal potential of neutrophil-derived ROS 44 Fungal anti-oxidative stress defenses 44 Chapter 2. Characterization of the immune response in human corneal tissues infected with the filamentous fungi Aspergillus and Fusarium Summary 50 Introduction 52 Materials and Methods 53 Results 56 Discussion 66 Chapter 3. Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus keratitis Summary 70 Introduction 71 Materials and Methods 72 Results 82 Discussion 106 Chapter 4. Thioredoxin and superoxide dismutase enhance survival of hyphae against CD18-dependent neutrophil NADPH oxidase activity Summary 119 Introduction 120 Materials and Methods 122 Results 137 Discussion 169 4 Chapter 5. Discussion and Future Directions 175 Data summary Abstract 176 Working Model - Immune recognition of fungi during corneal infection 177 Working Model- Oxidative stress at the neutrophil-hyphae interface 179 Future focus Inflammatory responses to fungi during infection CR3 or LFA-1 mediated ICAM-1 interaction and extravasation 181 CR3 or LFA-1 mediated migration through the cornea 182 CXCR1/2-mediated β2-integrin activation 183 ELR+ chemokine mediated CXCR1/2 activation 183 Pattern recognition receptor-mediated fungal recognition 184 Distinctions between Dectin-1 and CR3-mediated signaling 185 CR3 and complement-mediated neutrophil recruitment 186 IL-1 family-mediated neutrophil recruitment 186 Inflammasome-mediated IL-1β and IL-18 processing 187 Inflammasome-independent IL-1β and IL-18 processing 188 Role of T-cell and neutrophil-derived IL-17-in fungal infection 189 Role of IFNγ in early and late stage fungal infection 189 Activation of neutrophil fungicidal mechanisms Priming neutrophil NOX during fungal infection 190 CR3-mediated signaling and NOX activation 191 β2-integrin-mediated neutrophil NOX activation and fungal killing 192 CR3-mediated NOX activation and fungal killing during infection 193 CR3 interaction with complement fragment iC3b during infection 194 CR3 and Dectin-1 dependent NOX activation and phagocytosis 195 CR3 and Dectin-1 dependent recognition of fungal β-glucan 196 TLR4-mediated fungal killing during infection and DAMPs 197 Neutrophil degranulation during infection 199 The neutrophil fungicidal arsenal- one granule at a time 200 Tertiary granules- tissue migration and revving the engines 200 Secondary granules- the give and take of iron and oxidation 201 Primary granules- tissue destruction and the last stand 202 Fungal defenses against neutrophil fungicidal mechanisms Genetic dissection of fungal thioredoxins 202 The role of fungal peroxiredoxins in defense against oxidation 203 Sensitivity of fungal mitochondria to oxidative stress 204 Genetic dissection of SODs and therapeutic potential of SOD inhibitors 204 Role of fungal toxins during chronic fungal keratitis 205 Role of glutathione and HOG-MAPK in anti-oxidative stress defenses 205 5 Therapeutic implications Targeting inflammatory mediators during fungal infection 206 Inhibiting Dectin-1mediated neutrophil recruitment 206 Inhibiting Syk-mediated neutrophil recruitment 208 Inhibiting IL-1R1 and CXCR1/2 mediated neutrophil recruitment 208 Inhibiting β2-integrin and ICAM-1 mediated neutrophil recruitment 209 Inhibiting IL-17 mediated neutrophil recruitment 210 Targeting cell wall β-glucan and phagocyte fungicidal activity 210 Targeting fungal anti-oxidative stress responses 212 Concluding remarks 214 References 215 6 List of Figures Chapter 1 Figure 1.1 Clinical characteristics of fungal keratitis 24 Figure 1.2 Normal eye anatomy and cornea histology 25 Figure 1.3 TLR4 and TLR2 signaling 30 Figure 1.4 Dectin-1 signaling 31 Figure 1.5 CR3 I domain and lectin-like binding domains 33 Figure 1.6 CR3 inside-out signaling 34 Figure 1.7 CR3 outside-in signaling 35 Figure 1.8 Granulopoeisis and granule contents 37 Figure 1.9 NOX regulation and activation 42 Figure 1.10 Neutrophil adhesion and wrapping around hyphae 45 Figure 1.11 ROS derived from neutrophil NOX 47 Figure 1.12 Fungal anti-oxidative stress defenses 48 Figure 1.13 Possible role for toxins in human fungal keratitis 49 Chapter 2 Figure 2.1 Cellular composition of fungal keratitis ulcers 60 Figure 2.2 Expression of PRRs in fungal keratitis ulcers 62 Figure 2.3 Expression of cytokines in corneal ulcers 64 Figure 2.4 β-glucan and cellular infiltration in fungal keratitis ulcers 65 Chapter 3 Figure 3.1 Aspergillus fumigatus keratitis in cyclophosphamide treated mice 86 Figure 3.2 The role of macrophages in A. fumigatus keratitis 90 Figure 3.3 ß-glucan mediated Dectin-1 signaling in A. fumigatus keratitis 92 Figure 3.4 Role of Dectin-1 in keratitis caused by A. fumigatus Af293.1RFP 93 Figure 3.5 Role of Dectin-1 in keratitis caused by a clinical isolate 96 Figure 3.6 Role of Dectin-1 in activation of bone marrow-derived macs 100 Figure 3.7 The role of TLR2, TLR4, and MD-2 in A. fumigatus keratitis 103 Figure 3.8 The role of MyD88 in A. fumigatus keratitis 105 Figure 3.9 The role of TIRAP and TRIF in A. fumigatus keratitis 108 Figure 3.10 The role of IL-1R1 in A. fumigatus keratitis 109 Figure 3.S1 Construction of RFP-Aspergillus fumigatus 110 Figure 3.S2 Normal mouse cornea histology and eye 111 Figure 3.11 Working model of cornea inflammation 117 Chapter 4 Figure 4.1 Neutrophils are required to kill fungi during infection 141 Figure 4.2 Neutrophil NOX is required to kill A.fumigatus during infection 144 Figure 4.3 iNOS is not required to kill fungi during corneal infection 146 Figure 4.4 NOX but not iNOS or MPO is required for killing hyphae 149 Figure 4.5 CD18-dependent NOX activity is required for killing hyphae 152 Figure 4.6 Yap1 and SOD 1/2/3 mediate hyphae growth 155 Figure 4.7 Yap1 and SOD 1/2/3 mediate fungal growth during infection 157 7 Figure 4.8 Thioredoxin is required for hyphae survival during infection 160 Figure 4.S1 Quantification of corneal opacification 162 Figure 4.S2 NOX is required to control A.fumigatus in vivo 163 Figure 4.S3 NOX is required to control A.flavus in vivo 164 Figure 4.S4 NOX is required to control F.oxysporum in vivo 165 Figure 4.S5 Neutrophil NOX is required for ROS production 166 Figure 4.S6 Gliotoxin is not required for fungal infection 167 Figure 4.S7 TrxA is not required for fungal infection 168 Chapter 5 Figure 5.1 Working Model- Immune response to fungi during infection 178 Figure 5.2 Working Model-oxidative stress at the neutrophil-hyphae interface 180 Figure 5.3 Anti-inflammatory targets during fungal keratitis 207 Figure 5.4 Targeting the fungal cell wall 211 8 List of Tables Chapter 1 Table 1.1 PRRs that recognize fungi 29 Chapter 2 Table 2.1 Patient study characteristics 58 Chapter 3 Table 3.1 Fungal infection of TLR4-/- mice 104 Chapter 4 Table 4.1 Fungal strains utilized in this study 125 Table 4.2 Primers utilized in this study 126 9 Acknowledgements This work would not be possible without God’s will or the efforts and sacrifices of many individuals that enabled, guided, and assisted me on my path towards a doctoral education. My two brave grandfathers Jesus Leal and Pedro Martin made the first and largest sacrifices for my education having left communist Cuba for the USA positioning their offspring for better opportunities and access to high quality education. My parents Sixto Leal Sr. and Teresa Leal were exemplary in instilling upon me the importance of education. Through their guidance I took pride in completing assignments rapidly and to the best of my ability. This efficiency and pride has served me well throughout my education and I thank my parents for instilling these traits into the very core of me. At Florida International University, I discovered the world of biological sciences and in the words of my father, “Morphed from an academic caterpillar into a butterfly” under the tutelage of my mentor and great friend Dr. Kalai Mathee. This transition was crucial for my acceptance into the Case Western MSTP and I am greatly indebted to Clifford Harding and George Dubyak for this opportunity. I must also thank Eric Pearlman for being a great mentor and friend. He has provided endless support and guidance both personally and academically and has boosted my scientific knowledge, creativity, and confidence to the state needed for scientific independence. Under Eric’s tutelage, the academic butterfly morphed into a scientific falcon. For this I am greatly indebted. Throughout this journey, I must thank my loving family and the incredible people at Aravind Eye Hospital and the Mathee and Pearlman laboratories, whom I collectively call here the dream team.