FUNGAL KERATITIS: IMMUNE RECOGNITION, -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 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 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 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 during chronic fungal keratitis 205 Role of 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 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 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 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 117

Chapter 4 Figure 4.1 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. Luckily, my dream wife,

Rachael Leal, was also recruited to the dream team and divinely delivered to my

10 doorstep at the Institute of Pathology Room 102. Thank you for blessing every aspect of my life and giving me my beautiful daughter Ayva Mayrita Leal. This journey has been amazing and I thank all those whom cleared the path and have enabled and traveled along with me.

11 List of Abbreviations

CARD helicase caspase-recruiting domain kinase CAT-catalase; CR3- complement receptor 3; CXCL1/KC keratinocyte-derived chemokine CXCL8/IL-8 interleukin 8 DC dendritic cell Dectin-1- dendritic cell associated C-type lectin ERK extracellular signal-related kinase GR-glutathione reductase; GSH- glutathione; GSSG dimeric glutathione; HCEC human corneal epithelial cell line IFN interferon IKK inhibitor of NF-κ kinase IL-1R IL-1 receptor IL-17- interleukin 17 iNOS- inducible nitric oxide synthase; JNK c-Jun N-terminal kinase LPS LRR leucine-rich repeat MAL MyD88-adaptor-like MAP kinase mitogen-activated protein kinase M-CSF colony stimulating factor MD-2 Myeloid Differentiation-2 MPO- myeloperoxidase; ; MyD88 Myeloid differentiation factor 88 NF-κB nuclear factor kappa enhancer binding protein NOX- NADPH oxidase; PAMP associated molecular pattern PI3K phosphoinositide 3-kinase Poly(I:C) polyinosinic-polycytidylic acid PRR pattern recognition receptor PX-12- 2-[(1-methylpropyl)dithio]-1H- imidazole; ROS- ; SOD- superoxide dismutase; TIR Toll/IL1 receptor TLR Toll-Like receptor

12 TLR4 Toll-like receptor 4 TR- thioredoxin reductase; TRAM TRIF-related adaptor molecule TRIF TIR-domain-containing adaptor protein inducing

13 Fungal Keratitis: Immune Recognition, Neutrophil-Hyphae Interactions and

Fungal Anti-Oxidative Defenses

Abstract

by

SIXTO MANUEL LEAL JR.

Filamentous fungal infections of the cornea caused by Aspergillus and Fusarium species are an important cause of blindness and visual impairment worldwide accounting for > 65% of corneal ulcers in developing countries and an estimated 80,000 total cases in India alone. Tissue pathology is often severe leading to either extensive scar tissue formation, cornea transplantation (>10% ), or enucleation of the affected eye. The series of studies described herein delineate our multifaceted approach to investigate: 1. the mechanism by which neutrophils are recruited into fungal-infected corneas, 2. the effectors required by neutrophils to kill fungal hyphae, and 3. the microbial factors utilized by hyphae to resist attack.

In collaboration with Aravind Eye Hospital in Tamil Nadu, India, we first characterized the human immune response during fungal keratitis using ulcerative material from infected corneas as well as infected whole corneas from patients undergoing corneal transplantation. Analysis of these tissues identified neutrophils as the major cellular infiltrate with smaller populations of macrophages and T cells. In addition, utilizing qPCR and RNA extracted from fungal-infected human corneas we identified elevated expression of: 1. fungal-sensing cell-surface receptors (Dectin-1,

TLR2, and TLR4), 2. neutrophil-recruiting cytokines (IL-8, IL-1β, IL-17) , 3. proteins

14 involved in IL-1 β maturation (NALP3, ASC), and 4. cytokines associated with adaptive immune responses (IFN- ,IL-17, but not IL-4) .

Having characterized the human immune response during fungal keratitis, we next developed and utilized a novel mouse model of trauma-induced fungal keratitis and knockout mice to identify that ß-glucan on A.fumigatus germinating conidia activates Dectin-1 on resident macrophages to produce IL-1ß, and CXCL1/KC, which together with IL-1R1/ MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4 but not MD-2 dependent fungal killing. In the next series of studies we show that hyphae activate neutrophil NADPH oxidase via complement receptor 3 (CD11b/CD18) and utilizing a novel neutrophil adoptive transfer model that neutrophil NADPH oxidase is essential for killing hyphae in vivo.

In addition, utilizing mutant A.fumigatus strains, our mouse model , and a novel in vitro neutrophil-hyphae killing assay we show that the ROS-sensing

Yap1, the ROS detoxifying- superoxide dismutase, and the Yap1-regulated thioredoxin antioxidant pathway, but not fungal 2° metabolites such as gliotoxin are required for resistance to oxidation by neutrophils.

In conclusion, this work has significantly advanced our knowledge of the immune and microbial mediators of disease outcome during fungal keratitis. In this effort we identified: Dectin-1, IL-1R1, CXCR2, CD18, Syk, and MyD88 as potential anti-inflammatory targets to minimize excessive neutrophil recruitment during fungal keratitis. Lastly, we identify fungal superoxide dismutase and thioredoxin as potential anti-fungal targets to enhance fungal sensitivity to oxidative stress and neutrophils.

15 Introduction

Fungi in their natural environment

Fungi that cause human infections can be separated into three categories based on morphology: , filamentous fungi, or dimorphic fungi (1). Yeasts are small cells which replicate via cell division and are similar in size to the spores/conidia of filamentous fungi. In contrast, filamentous fungi are characterized by the formation of long and often branched multinucleated tubular morphologies referred to as hyphae(1).

Dimorphic fungi can exhibit either yeasts or hyphal morphologies (1, 2).

Filamentous and dimorphic fungi dominate the natural environment and constitute a large percentage of the total biomass on the planet performing a vital saprophytic role in ecological systems via the catabolism of vegetative matter into reusable forms utilized by (3). Fungi that are pathogenic to humans form hyphae within microbial communities consisting of nonpathogenic fungi, bacteria, and protozoans(3). In such diverse microbial communities, the struggle to acquire carbon sources and limited essential metals such as iron is fierce(2). As such, hyphae have evolved to utilize a variety of carbon sources, acquire essential metals via siderophores, react to oxidative stress, pH, dessication, and no doubt other environmental stressors making environmental fungi incredibly adaptive and durable(2). Traits such as responses to oxidative stress and iron acquisition are likely essential for hyphae to infect human tissues.

Despite the adaptability of hyphae to numerous environments, soil microenvironments are highly dynamic and temporally unpredictable(3). Given that hyphae are sessile, they can only colonize new environment via direct extension of their

16 cell wall and cytoplasm. This method of growth requires energy and if carbon sources are depleted in their local environment hyphae are trapped until the soil becomes fertile once again. This lack of motility is evolutionarily dangerous for hyphae and because of this selective pressure hyphae evolved to produce metabolically inert durable conidia which are readily dispersed through air currents into neighboring fertile soils(3).

Meteorological studies in the Netherlands have identified seasonal variation in fungal colony forming units (CFU) with 25-200 CFU per cubic meter of outside air and hospital air (Figure X)(4). The average human breathes in 0.005m2/breath and takes 12-

20 breaths/min. Since there are 1440 min/day, it is estimated that humans breathe in anywhere from 2160- 28800 fungal conidia/day. Approximately, 200 of these spores are A.fumigatus(4). In rural areas and construction sites conidial air density is increased further (5). Thus unless there is direct inoculation of hyphae into human tissues, hyphae do not contact their host directly but rather do so indirectly via conidia.

When conidia encounter a carbon source (fertile soil//cornea), metabolic activity is initiated and a protective outer hydrophobin protein coat is shed(2).

Hydrophobin-shedding results in the exposure of underlying cell wall components, including β-glucan (6). As metabolic activity continues, conidia begin to swell and unidirectional tubular extension of the and cytoplasm proceeds(2).

Fungi with a small tubular extension are called germ tubes, whereas fungi with larger extensions are called hyphae. Hyphae eventually branch and interconnect with neighboring hyphae to form large mycelia. However, if the is dimorphic, conidia can germinate into either hyphae or yeasts depending on the local environment and temperature (1). Upon exhaustion of carbon sources or exposure to significant

17 environmental stress, conidia are formed as described above completing the life cycle of filamentous and dimorphic fungi.

Fungi and human disease

The World Health Organization (WHO) estimates that approximately, 90% of the world’s visually impaired live in developing countries (7) and that ocular trauma and corneal ulceration results in 1.5 to 2 million cases of corneal blindness annually(8).

In developing countries fungal infections cause up to 60% of all microbial keratitis(9).

Taking into account the number of people blind from cornea ulcers, the percentage of the global blind living in developing nations, and the percentage of microbial keratitis caused by fungal infections in developing nations it is reasonable to estimate that globally 600,000-1,000,000 people acquire fungal keratitis annually. In the conference proceedings for the The Fungal Research Trust 20th Anniversary Meeting, it was estimated that in Asia and Africaalone there are approximately 1 million cases annually(10). The incidence in South America and other tropical regions of the world are mostly unreported. However, given the similarities in climate and rural lifestyle it is likely that the incidence of fungal keratitis is similar to those observed in Asia and

Africa. A recent study in Brazil, tracked prescriptions for topical medications to estimate that approximately 2,000 individuals in Brazil are treated for fungal keratitis annually(11). This number likely underestimates the total affected population because the patients most prone to fungal keratitis live in rural environments with limited access to health care. For this reason, visual health outreach programs to rural villages have been initiated by hospitals throughout the world, including the

18 Aravind Eye Hospital in South India, where patients with fungal keratitis are encountered oftenly.

Filamentous fungal infections of the cornea occur primarily in immunocompetent individuals in developing nations(12). In contrast to fungal keratitis, most other fungal infections occur in immunosuppressed individuals and as this human population increases so too does the incidence of fungal infections in these patients. It is estimated that worldwide A.fumigatus causes severe invasive pulmonary infection in

200,000 immunosuppressed patients per year and > 50% die, even with treatment (10).

In addition, the of approximately 3 million people are chronically infected with

A.fumigatus including patients with post-tuberculosis cavitary lesions or chronic granulomatous disease(10).

In addition the dimorphic fungus C. albicans causes approximately 300,000 cases of systemic infection annually with a mortality rate of 30-55%(10). Other infections caused by C.albicans include oral thrush in approximately 9.5 million people worldwide and vulvovaginal infection which affects 75 million women annually(10).

Globally, Cryptococcus lung infection is observed in approximately 300,000 individuals, Pneumocystis lung infections in 200,000 patients, Histoplasma lung infections in 500,000 patients, and dermatophyte infections in 1.5 billion people worldwide(10). In addition, fungi also contribute to the formation of severe allergies including 4 million individuals affected with allergic bronchopulmonary and 12 million people with allergic fungal sinusitis(10). Not all fungal infections are covered in this discussion and no doubt as humans expand further into fungal pathogen endemic lands new fungal will emerge to cause human disease.

19 HIV infection contributes significantly to the immunosuppressed human population, however, current antiretroviral therapy and the potential of a future cure or vaccine will hopefully eliminate 2° fungal infections due to HIV infection (13).

However, the success of iatrogenic to treat autoimmune disease, its requirement for stem cell transplantation, its unavoidable occurrence as a side effect, and the global diabetes pandemic will no doubt continue to result in an increased incidence of fungal infections in immunocompromised patients for the foreseeable future(14, 15).

Fungal infections of immunocompetent individuals such as fungal keratitis and dermatophyte infections will likely also continue. In the case of fungal keratitis, most infections occur via trauma associated with vegetative matter(12). Such transmission routes require a rural setting with high air conidia density or vegetative matter bearing fungal material. As the human population expands and industrializes and machines take over agricultural work and fungal-laden forest floors are paved over to build shopping malls the incidence of fungal keratitis will likely decrease. Evidence of this is observed by comparing tropical regions of developed nations to tropical undeveloped nations, such as South Florida and South India, respectively. In South Florida, fungal keratitis is observed primarily in agricultural migrant workers and the majority of the urban population is not affected or exposed (16, 17). In contrast, in South India everyone is exposed and the incidence is estimated at 80,000/year (18). As developing countries industrialize further, the incidence of fungal keratitis is likely to decrease over time until at the very extreme like the USA, the majority of the population of the world is unexposed and disease is restricted to poor agricultural workers that till the soil for the

20 greater masses. In contrast, the incidence of athlete’s foot will likely be maintained irrespective of industrialization so long as shoes are worn and humans sweat(19).

Fungi that cause human corneal infection

Fungal keratitis can be classified into 2 distinct clinical phenotypes based on geographical location, patient immune status, the infectious agent, and disease outcome

(12). In developed nations, the majority of fungal keratitis occurs in immunosuppressed individuals with increased susceptibility to infection by the dimorphic commensal fungus albicans (12). Fortunately, C.albicans keratitis is often readily treatable and since patients are often immunosuppressed there is minimal inflammatory damage to cornea tissue during infection(12).

In contrast, in developing nations and tropical regions of developed nations fungal keratitis occurs primarily in immunocompetent individuals with no preexisting conditions and is usually initiated by trauma with vegetative matter bearing conidia from filamentous fungi(12). Unfortunately, these infections are not readily treatable and inflammatory mediated tissue damage is often severe leading to >10% of infections requiring cornea transplantation (20).

Filamentous fungal infections are caused by numerous fungal genera including:

Aspergillus, Fusarium, Alternaria, Curvularia, , etc.(20). However, the vast majority of disease is caused by Aspergillus and Fusarium species including

A.fumigatus, A.flavus, F.solani, and F.oxysporum (20). A.fumigatus is a saprophyte commonly found in material and is often the culprit of moldy bread in urban kitchen environments (21). A.fumigatus conidia are very easily aerosolized due to their

21 small nature (2-3μM) and it is estimated that humans breathe in 100-200 of them daily(21). A.flavus and Fusarium species are major plant pathogens commonly found on vegetative matter in the environment (22, 23). A.flavus infamously infects peanuts contaminating them with a DNA-damaging agent called (23), whereas F.solani infects the fruits and roots of a variety of plant species including peas, soybeans, and potatoes. Likewise, F.oxysporum routinely damages tomato crops and like A.fumigatus is commonly found in urban kitchen environments(22). The ability of Aspergillus and

Fusarium species to colonize and evade plant defense mechanisms has likely provided positive evolutionary selection of virulence factors, which enhance their ability to cause infection (24). It is likely that these same virulence factors enable Aspergillus and

Fusarium species to cause infections in humans.

Fungal keratitis- Clinical characteristics and outcome

Aspergillus (A.flavus, A. fumigatus,) and Fusarium (F.solani,

F.oxysporum) species are the main etiologic agents of fungal keratitis (Figure 1.1) (9).

In the corneal stroma, conidia germinate into hyphae, which then penetrate throughout the stroma and the basement membrane, where they also infect the anterior chamber, causing severe pain, photophobia and vision loss(12). Topical natamycin or voriconazole are effective if given very early, but fungal keratitis is notoriously difficult to treat, especially after the hyphae penetrate deeper stromal layers (20, 25, 26).

Depending on the inoculum and the time until treatment, infected individuals will require corneal transplantation (10% of cases)(20). Following transplantation, the rejection or reinfection of transplanted cornea tissue can occur, and in severe cases the

22 only recourse is enucleation of the affected eye (12). In milder cases, resolution of infection is accompanied by fibrosis, resulting in visual impairment(20). In contrast to trauma – induced fungal keratitis, contact lens associated fungal keratitis is likely due to the hyphal stage. Airborne conidia settle in a lens case, germinate and form a biofilm on the lenses and the case (27, 28). Following contact with the ocular surface, hyphae penetrate into the cornea stroma through minor epithelial abrasions where they establish infection(20).

Anti-microbial Defenses at the Ocular Surface

Figure 1.2 illustrates a cross-sectional diagram of the human eye and cornea. The transparent cornea provides most of the refractive index that is essential for the accurate transmission of light through the pupil and to the retina, where photoreceptor cells transmit images to the visual cortex of the brain(29). Infection or inflammation disrupts the role of the cornea, as inflammation is associated with edema and a change in refractive index, resulting in impaired vision. The avascularity of the cornea is also important in maintaining corneal clarity(29). The cornea and ocular surface are protected from trauma and infection by physical and molecular defenses. Perhaps the simplest and most effective defense involves eyelid closure and blinking, which protects the cornea from physical trauma, and removes microbes from the ocular surface (29). In addition, ocular surface mucins and the tear film restrict pathogen interaction with the corneal epithelium (29). The tear film also contains β-defensins, calprotectin, and lysozyme (31, 32).Tears also contain high levels

23 Figure 1.1 Clinical characteristics of fungal keratitis

A. B.

A. Fungal morphology, eye image and slit-lamp microscopy of a patient with contact- lens releated infection with Fusarium oxysporum strain 8996 at the Cole Eye Institute, Cleveland, OH. B. Fungal morphology, eye image, and cornea transplant operation on a patient with trauma-induced A.fumigatus strain UH at University Hospitals, Cleveland , OH.

24 Figure 1.2 Normal eye anatomy and cornea histology

A. Diagram of human eye. B. Normal H&E stained human cornea histology section. C. GMS-stained cornea of a patient with A.flavus keratitis requiring corneal transplantation. (30) Reprint permission obtained from publisher.

25 of the iron-chelating protein lactoferrin (33) and the siderophore-binding protein lipocalin 1/tear lipocalin (34). Though their role in tears is not fully understood, it is likely that cation (Fe2+ Zn2+) sequestration in the ocular surface inhibits fungal germination and growth, which requires these essential metals .

Figure 1.2 B is a histological section of the normal human cornea, showing the main layers of cornea, which are the corneal epithelium, stroma, endothelium, in addition to the underlying anterior chamber. The major physical barrier against bacterial and fungal infection is the non-keratinized, stratified corneal epithelium, comprising three layers of epithelial cells with tight junctions that form a physical barrier preventing microbial access to the corneal stroma (35). Murine studies of fungal keratitis or Pseudomonas aeruginosa have shown that an intact corneal epithelium will restrict access of millions of live organisms to the corneal stroma even under stressful conditions such as long term contact lens wear(36, 37). However, the main site of infection is the corneal stroma, which comprises 90% of the tissue, and is a dense, highly organized matrix, with anti-parallel layers of collagen separated by keratan sulfate proteoglycans that are essential for corneal transparency (38).

When the integrity of the corneal epithelium is breached due to trauma, fungal conidia or hyphae can penetrate into the corneal stroma, where growth is uninhibited in the absence of leukocyte recruitment (36, 39, 40). During infection, hyphae penetrate through this dense matrix and also the basement membrane of the corneal endothelium.

In the absence of an effective host response, hyphae enter the underlying anterior chamber and in some cases can also invade the posterior eye vitreous causing blinding endophthalmitis (Inflammation in the posterior eye), at which point enucleation of the

26 infected eye is often indicated. Figure 1.2 C, shows heavy fungal burden and penetration throughout the corneal stroma and towards the anterior chamber in a post- transplant infected cornea from a patient with fungal keratitis (A.flavus) who underwent corneal transplantation. The stroma contains resident macrophages and dendritic cells

(41-46) which express pattern recognition receptors and can respond to fungal cell wall components by producing pro-inflammatory cytokines that mediate neutrophil recruitment from the peripheral, limbal blood vessels, and kill fungal hyphae (39, 47).

Immune recognition of fungi

At the cellular level, the initiation of an inflammatory response, in the absence of tissue damage, fundamentally relies upon recognition of the invading microbe as foreign. This is accomplished through the binding of various host-cell

Pathogen Recognition Receptors (PRRs) (Such as the toll like receptors (TLRs) and C- type lectins) to microbial products, with the subsequent activation of signaling cascades that mediate /chemokine production and inflammation(48). In the context of fungal infections, it is likely that the availability of any given microbial ligand to the extracellular domain of PRRs will differ between fungal species, depending upon the cell wall surface exposure of the ligand in question. Thus distinct fungal-specific PRRs are required for host protection against different fungal species (49). To date, the identity of fungal-specific microbial ligands for Host PRRs has been studied most extensively in Candida albicans (Table 1.1). In these studies the TLR2/6 heterodimer was shown to recognize phospholipomannan (50), TLR4 was shown to recognize O‑ linked mannans (Figure 1.3) (51), and TLR9 was shown to detect CpG fungal

27 DNA(52). Further studies showed that the C-type lectin Dectin 1 recognizes ß-glucan

(Figure 1.4) (53), while Dectin 2 (54) and Galectin 3 (55) recognize mannose, and

Complement Receptor 3 (CR3) (56) recognizes both ß-glucan and mannose. Yet another C-type lectin, MINCLE, was recently shown to recognize C. albicans, though its associated ligand is still undefined (57).

Despite the fact that Dectin 1-/- mice were more susceptible to Pneumocystis carinii (58) and in one of two reported studies, to C. albicans (58, 59), at the start of this project, no study had examined A. fumigatus infection in Dectin 1-/- mice in the cornea or elsewhere (58). However, experiments performed in vitro with macrophages from various tissue sources had shown a role for cytokine induction by Dectin 1 upon exposure to post-germination stages of A. fumigatus (Post-germination stages= ß-glucan surface exposure)(60, 61). Additionally, these same studies and others have hinted at a role for TLR2 and TLR4 in cytokine induction upon exposure to A. fumigatus.

However, some of these reports implicate only TLR2 (62), or only TLR4 (63), and yet others implicate both in A. fumigatus recognition (64). Some studies even implicate

TLR2 in the production of anti-inflammatory cytokines upon exposure to hyphae (65).

The ambiguous nature of these results and lack of in vivo demonstration of a role for

Dectin1, TLR2, or TLR4 during A. fumigatus infection stimulated the investigations described in Chapter 3 of this dissertation.

28 Table 1.1 PRRs that recognize fungi (47)

Reprint permission obtained from publisher.

29 Figure 1.3 TLR4 signaling

TLR4 can bind to LPS-bound MD2, fungal-derived o-linked mannans, or DAMPs such as HMGB1 and dimerize resulting in MAL/TIRAP-mediated recruitment of MyD88. MyD88 recruits IRAK-4 which phosphorylates and recruits IRAK1 and IRAK2 forming a complex that recruits the E3 ubiquitin ligase TRAF6. TRAF6 auto- K63polyubiquitinates itself and the polyubiquitin chain recruits the E2 ubiquitin ligase complex Ubc13/Uev1A. Ubc12/Uev1a polyubiquitinates the Tak1/TAB1/TAB2/3 complex resulting inMAPK activation and AP-1 translocation into the nucleus as well as phosphorylation of NEMO/IKKγ and activation of an IKK complex (NEMO/IKKγ/IKKα/ IKKβ), which phosphorylates IκB leading to polyubiquitination and proteasomal degradation. NFκb is then free to translocate into the nucleus. TLR4 is also endocytosed and within the endosome, TLR4/TRAM recruit and activate TRIF, which activates TRAF6/RIP1 resulting in AP-1 and NFκB translocation. TRIF also recruits TRAF3, which mediates TBK1/IKK activation and phosphorylation of IRF3 which tranlocates into the nucleus.(66) Reprint permission obtained from publisher.

30 Figure 1.4 Dectin-1 signaling

Dectin-1binds to both soluble and particulate β-glucan, however, only particulate β- glucan induces receptor dimerization and clustering. With significant receptor clustering, the inhibitory phosphatases CD45 and CD148 are physically excluded from the Dectin-1 phagocytic synapse enabling src-family kinases (Hck, Lyn, Fgr) to phosphorylate the hemi-ITAMs on the cytoplasmic tail of Dectin-1. The phosphorylated ITAMs recruit and activate spleen tyrosine kinase (Syk) which phosphorylates CARD9 resulting in the formation of a CARD9/BCL10/ MALT1 ternary complex that mediates NFκB translocation into the nucleus. There is also an unclear role for the guanine nucleotide exchange factor (GEF) Raf1 and Ras GTPases in NFκB translocation. In addition, Syk phosphorylates MAP kinases which mediate AP-1 translocation into the nucleus. Dectin-1 also activates PI3K through an unknown mechanism and Syk activates PLCγ resulting in an IP3-mediated rise in intracellular Ca2+ and diacylglycerol (DAG) mediated activation of protein kinase C (PKC). High intracellular Ca2+ induces a conformational change in calmodulin which activates the phosphatase calcineurin leading to NFAT translocation into the nucleus. Simultaneously, Syk activates the GEFs vav1/2/3, which activate Rho GTPases that mediate actin polymerization and phagocytosis of Dectin-1 with bound particulate β- glucan. (Figure Modified from Steven deJesus Carrion)

31 β2 integrins and complement receptor 3

CD18 also known as integrin β2 is expressed on the surface of macrophages and neutrophils where it is found in heterodimeric association with one of four alpha integrins: CD11a (αM β2-LFA-1), CD11b (αM β2-CR3/Mac-1), CD11c (αXβ2,

ITAX,CR4), or CD11d (αDβ2, ITAD)(67). Both CR3 and LFA-1 bind to endothelial- expressed ICAM-1and mediate neutrophil arrest and transmigration into inflamed tissues(67). TheI-domain of CR3 is unique amongst β2 integrins in that it can also bind to iC3b, fibronectin, fibrinogen, and heparan sulfate proteoglycans (67). In addition, only CR3 contains a lectin-like binding domain shown to bind to β-glucan (Figure 1.5)

(67, 68).

CR3 exists on the surface of resting leukocytes in a folded conformation termed low affinity, which masks ligand binding sites(67). Prior to ligand engagement, CR3 must first be activated by a 2nd signal in a process called inside-out signaling, shown in

Figure 1.6. Inside-out signaling induces a conformational change in CR3, termed intermediate affinity, which exposes both ligand-binding domains(67). The 2nd signal for CR3 activation is received upon neutrophil engagement of E and P selectins with

PSGL and ELR+ chemokine Pro-Gly-Pro (P-G-P) engagement with CXCR1 or

CXCR2(67). This interaction results in LFA-1 or CR3 binding to ICAM-1 and neutrophil extravasation into inflamed tissues. Upon entering inflamed tissues neutrophil CR3 is likely in the intermediate affinity conformation described above, which enables ligand engagement through either the I domain or the lectin-like domain(67). Ligand binding to CR3 induces a conformational change within the receptor and activation of intracellular signaling(67). This process termed outside-in

32 Figure 1.5 CR3 I domain and lectin-like binding domains

Complement receptor 3 consists of two ligand binding domains. The I-domain can bind to the inactivated complement component iC3b, fibronectin, ICAM-1, fibrinogen, and heparin sulfate on proteoglycans. The lectin-like binding domain binds to both soluble and particulate β-glucan(68). Reprint permission obtained from publisher.

33 Figure 1.6 CR3 inside-out signaling

Signaling through both selectin engagement and chemokine receptor activation results in PLCγ activation with production of IP3 and DAG which activates the guanine nucleotide exchange factor CalDAG-GEF1. CalDAG-GEF1 subsequently converts Rap1-GDP to RAP1-GTP which translocates to the membrane and helps recruit both the hematopoetic specific protein kindlin 3 and talin to the cytoplasmic tail of CD18. Talin subsequently disrupts the interactions between the CD18 and C11b cytoplasmic tails increasing the distance between them, inducing extension of the extracellular domains of CD18 and CD11b, and exposure of both the I domain and lectin-like domains. This conformation is termed intermediate affinity. In addition to enhancing ligand affinity, inside out signaling also mediates receptor clustering in lipid rafts which enhances avidity and localizes CR3 within membrane microdomains exhibiting high levels of signaling molecules. Under shear forces such as those experienced in the blood stream a third conformation termed high affinity enhances ICAM-1 binding affinity(67). Reprint permission obtained from publisher.

34 Figure 1.7 CR3 outside-in signaling

Following inside-out signaling CR3 receptors begin to cluster through an unknown mechanism into membrane microdomains called lipid rafts. The cytoplasmic tails of intermediate affinity CR3 exhibit bound inside-out signaling proteins (talin and Rap1), and via an unknown mechanism associate with low levels of Src family kinases (SFK- Hck, Fgr, and Lyn) prior to ligand engagement. Upon ligand binding, CR3 undergoes a conformational change which separates the cytoplasmic tails leading to SFK-mediated phosphorylation of the CD18 cytoplasmic tail. Jab-1 is recruited to this sight mediating the activation of C-fos resulting in AP-1-mediated transcriptional responses. In addition, SFKs phosphorylate focal adhesion kinase (FAK) and its homolog Pyk2 which phosphorylate PI3K, and subsequently, PI3K phosphorylates membrane-bound phosphatidylinositols. SFKs also phosphorylate the ITAM adaptors DAP12 and FcRγ, which recruit Spleen tyrosine kinase (Syk). Syk subsequently activates PLC which cleaves phosphorylated PtdIns into DAG and IP3 with activation of PKC signaling and elevation of cytoplasmic Ca2+. Syk also mediates phosphorylation of the adaptor protein SLP76 in association with the adaptor molecules ADAP and its myeloid specific homolog PRAM-1. SLP76 recruits the Rho gaunine nucleotide exchange factors Vav1/2/3 recruit PLCγ into a SLP76/Vav/PLCγ complex required for PLCγ activation. In addition, Vav proteins activated by CR3 signaling activate the RhoGTPase RhoA not Cdc42 or RAC1/2. In contrast, Vavs activated by Dectin-1 activate Rac1/2 not RhoA or Cdc42. GTP-RhoA also stimulates CR3-dependent phagocytosis(67). Reprint permission obtained from publisher. signaling is highlighted in detail in Figure 1.7 and results in cellular activation and phagocytosis in the absence of significant pro-inflammatory cytokine production.

35 The neutrophil and its microbicidal arsenal

The anti-microbial arsenal of neutrophils is contained with 1° azurophilic, 2° specific, 3° gelatinase, and secretory vesicles (69-72). The transcription of granule proteins is temporally regulated throughout neutrophil maturation in the bone marrow

(Figure 1.8). The 1st set of granule proteins produced in the ER shuttle to the golgi and are incorporated into the lumen of relatively large vesicles that bud from the golgi, termed 1° granules(71). As neutrophils mature, a 2nd set of transcripts encode proteins packaged in 2°granules followed by a 3rd set destined for 3° granules(71). In general, as neutrophils mature the transcription of one set of genes ceases and the next set starts, however, the process is fluidic and proteins destined for a certain type of granule can be incorporated into multiple granules(71). In addition, the neutrophil cytoplasm contains endocytic vesicles derived from the plasma membrane which contain mostly serum derived proteins such as albumin in the lumen and function primarily as easily- mobilized intracellular storage compartments for membrane proteins.

Exocytosis of secretory vesicles results in the release of soluble serum proteins such as albumin and surface expression of proteins involved in microbial defense: gp91phox/p22phox, CD11b, CR1, FcγRIII, & CD14 as well as proteins involved in neutrophil adherence (CD11b) and matrix degradation (MMP25 and

CD10/neprolysin)(71, 72). β2 integrin signaling results in the exocytosis of 3° granules, which contain high amounts of the secreted “gelatinase” MMP9 for which 3°/gelatinase

36 Figure 1.8 Granulopoeisis and granule contents

The neutrophil granule stage of formation, degranulation propensity, characteristic proteins, and other proteins stored within granule subsets(71). Reprint permission obtained from publisher.

37 granules are named and like secretory granules translocate gp91phox/p22phox, CD11b

(CR3), and MMP25/leukolysin to the plasma membrane(69, 70, 73). MMP9 and

MMP25 are thought to mediate collagen breakdown and penetration past the blood vessel basement membrane(73). In addition, 3° granule fusion releases both lysozyme

& arginase-1, both of which exhibit anti-fungal activity (74, 75)and they mobilize β2 microglobulin to the cell surface which is required for surface expression of both MHC-

I and the MHC-like HFE protein involved in transferrin receptor-mediated activation of the iron-regulating hormone hepcidin/liver expressed antimicrobial peptide

(LEAP)(76).

Within tissues, high cytokine levels and microbial ligands stimulate 2° granule release followed by 1° granule release(71). 2° granule exocytosis mobilizes the greatest amount of gp91/p22phox to the plasma membrane(71). In addition, 2° granules release several proteins involved in iron sequestration including: the iron chelator lactoferrin(77), the bacterial siderophore-binding protein NGAL/lipocalin 24p3(78), the hemoglobin (heme-iron) binding protein haptoglobin(79), the membrane-bound β2- microglobulin(76), and the secreted lipocalin-family α1-acid glycoprotein/orosomucoid with unknown function. 2° granule fusion also releases the fungal opsonizer pentraxin

3(80), the fungicidal antimicrobial peptide hCAP18-LL-37(81), the cobalamin chelating protein and folate pathway inhibitor vitamin B12 binding protein (B12BP), and low levels of fungicidal α-defensins (HNP1-4)(71, 82). 2° granules also release MMP8 which is required

38 for neutrophil migration into the central cornea and heparanase which degrades heparin sulfate proteoglycans(83). Lastly, CD11b is mobilized to the plasma membrane and more lysozyme is secreted(71).

Upon further stimulation, 1° granules fuse with the plasma membrane(70).

These granules characteristically harbor myeloperoxidase which converts H2O2 to highly reactive hypohalous acids, and elastase which cleaves collagen IV and elastin as well as several bacterial virulence factors (69, 70). MPO is required for defense against

C.albicans (82, 83) and elastase is required for optimal defense against A.fumigatus lung infection (84). In addition, 3° granule fusion results in the secretion of the proteases: cathepsin G and proteinase 3(85), and β-glucoronidase which cleaves heparin and keratin sulfate proteoglycans, which result in significant tissue digestion. 1° granule exocytosis also releases the fungicidal azurocidin/CAP37, α-defensins HNP1-4, and the bacterial cell-wall pore forming molecules BPI(71). Also both sialidase and β- glucoronidase are translocated to the neutrophil surface(71). Sialidase cleaves sialic acids on proteins on the neutrophil surface enhancing adhesion to the ECM and perhaps to microbes as well(86). Readers interested in further information on proteins within granule subsets are referred to two excellent studies performed by McLeish et al (69,

72). In addition neutrophil actvation results in transcription of pro-inflammatory genes including IL-8 and IL-1β (87). Although the majority of the neutrophil antimicrobial arsenal is contained within granules it is possible that upon stimulation neutrophils can transcribe, translate, and release newly synthesized antimicrobials during infection.

39 Neutrophil NOX activation and role in killing hyphae

NADPH oxidase (NOX) is a multi-enzyme complex which catalyzes the conversion of molecular oxygen to superoxide(88). A role for NOX in killing hyphae is suggested by the enhanced susceptibility of chronic granulomatous disease (CGD) patients to invasive Aspergillus pulmonary infection (15, 71) and by murine lung infections showing a role for NOX in controlling A.fumigatus growth (89, 90).

However, none of these studies have directly shown in vivo that neutrophil NOX is required to kill hyphae.

NOX activity is regulated by the physical separation of its subunits, with gp91/p22phox restricted to the plasma membrane, secretory vesicles, and 2°/3° granule membranes and p47/p67/p40phox localized in the cytoplasm (Figure 1.9) (71, 88).

Neutrophil NOX can either exist in a resting, primed, activated, or inactivated state(88).

Circulating blood neutrophils are in the resting state and exhibit 60-70% of gp91/p22phox in 2° granules, 20-25% on 3°granules, and the rest on the plasma membrane and secretory vesicles, while p47/p67/p40phox and Rac2 are restricted to the cytoplasm(88).

Neutrophil adhesion, exposure to pro-inflammatory cytokines, or antimicrobial agents can drive neutrophil NOX towards a primed state (88). Priming denotes a phenotypic change in which a stimulus does not activate neutrophil NOX directly, but rather increases the sensitivity and extent of NOX activation in response to a 2nd stimulus. Although NOX priming enhances microbial killing in vitro, the functional significance of neutrophil priming in vivo is questionable given the high levels of pro- inflammatory and microbial stimuli that neutrophils encounter at the infectious foci.

40 Given this caveat, the primed state of neutrophil NOX is believed to be caused by phosphorylation of serine 345 on p47phox (88). Phosphorylated S345does not in itself induce translocation of p47phox to the surface, however, through an unknown mechanism it enhances the phosphorylation of other serine residues in the carboxy terminus of p47phox(88). Dependent on the stimulus, different serine/threonine kinases including ERK1/2 and p38 phosphorylate S345(88). In addition, the activation that results in NOX priming can also simultaneously elevate intracellular Ca2+ levels which trigger secretory vesicle and 3° granule exocytosis and increased translocation of gp91phox/p22phox to the plasma membrane(71, 91). It is possible but unlikely that signaling cascades that mediate neutrophil priming but not activation will stimulate 2° granule release to the plasma membrane. These primed neutrophils now harbor more

NOX components on their plasma membrane and when given a 2nd stimulus can more readily activate the translocation of cytosolic NOX components to the plasma membrane(88).

Upon entering the inflammatory/infectious site “primed” neutrophils encounter high levels of cytokines and microbial stimuli which trigger 2° granule exocytosis and the additional mobilization of 60-70% of total cell gp91/p22phox to the plasma membrane(88). In addition,

41 Figure 1.9 NOX regulation and activation

NADPH oxidase is regulated by the physical separation of membrane-bound and cytosolic components. Upon phosphorylation of p47phox the cytosolic components translocate to the membrane forming the functional enzyme (More details in text)(88). Reprint permission obtained from publisher.

42 dependent on the stimulus, different protein kinases (PKC, PAK, ERK1/2, and p38) will be activated that can mediate phosphorylation of serine residues on the carboxy terminus of p47phox (92). Phosphorylation of at least three serine residues (S303,

S304, S328) induces a conformational change in p47phox exposing two cryptic SH3 domains and one pleckstrin homology (PX) domain(92). The two SH3 domains enable binding to proline-rich domains on membrane- bound p22phox and the PX domain targets p47phox to phosphatidylinositol (3,4) bisphosphate (PtdIns (3,4)P2) on the plasma membrane (88). The Rho GTPase Rac2 also translocates to the NOX complex forming the functional enzyme and extracellular or phagosomal superoxide production(88).

At the start of this project, the only report on neutrophil mediated killing of A. fumigatus hyphae had identified that prior exposure of neutrophils to TLR2 and TLR4 ligands primes them for more efficient killing of A. fumigatus hyphae (93). While interesting, this study failed to address the obligate endogenous interactions between

PRRs on neutrophils (TLR2, TLR4, Dectin 1, & CR3) and A. fumigatus hyphae and the subsequent activation of antifungal effectors including NOX. A second study, which did not look at fungal killing, identified that the fungal cell wall component β-glucan, is recognized by complement receptor 3 not Dectin-1 on human neutrophils (94, 95). In

Chapter 4 of this dissertation, we address the mechanism by which neutrophils recognize fungal hyphae, activate NOX, and ultimately kill hyphae.

43 Fungicidal potential of neutrophil-derived ROS

Neutrophils wrap themselves around hyphae and thus presumably release NOX-derived extracellular superoxide within close proximity to the fungal cell wall (Figure 1.10).

. This extra-cellular O2 can oxidize directly or react with H2O to form hydrogen peroxide (H2O2), which is more stable, uncharged, and thus more cell permeable (88,

96). In addition, extracellular H2O2 can be converted to hypohalous acids by myeloperoxidase, which oxidize cell wall components and are likely too short-lived to

- enter the cytoplasm (97). H2O2 can also be converted to peroxynitrite (ONOO ) upon reaction with iNOS-derived nitric oxide (NO), both of which can enter the cytoplasm

(97). Figure 1.11, illustrates the oxidative products released by neutrophils. Once in the cytoplasm, reactive oxidants can potentially block the activities of multiple proteins and pathways, leading to cell death (97). However, it has also been proposed that ROS microbicidal activity is due to indirect effects such as microbicidal protein activation within neutrophil granules (92, 98); and activation of microbicidal neutrophil extracellular traps (92, 99, 100). Experiments described in Chapter 4 aim to decipher how ROS kills hyphae.

Fungal anti-oxidative stress defenses

Also investigated in this study is the mechanism by which hyphae resists oxidation by neutrophils. Figure 1.12 illustrates the known anti-oxidative defenses used by

A.fumigatus hyphae to counter oxidative stress. Upon exposure to oxidative stress, the fungal transcription factor Yap1 translocates to the nucleus and up-regulates the expression of the secreted catalases (Cat1/2), which convert H2O2 to H2O (101).

44 Figure 1.10 Neutrophil adhesion and wrapping around hyphae

Human neutrophils were incubated with hyphae from an RFP-expressing A.fumigatus strain and imaged using time-lapse microscopy (1-min intervals). The blue arrow indicates one neutrophil adhering and enveloping hyphae. Within this neutrophil:hyphae interface, degranulation and extracellular release of superoxide likely mediates fungal killing.

45 Additionally, Yap1 upregulates the thioredoxin antioxidant system, including two peroxiredoxins, AspF3 and Prx, which reduce H2O2 to H2O and are themselves reduced by thioredoxin protein (101). Similarly, the glutathione antioxidant system, which is not

Yap-1 regulated and not examined in this study, neutralizes cytoplasmic oxidants(102).

A.fumigatus also expresses three superoxide dismutases (SODs) which catalyze the conversion of superoxide to H2O2, and two catalases which convert H2O2 to H2O (103,

104). SOD1, 2, and 3 are not secreted; SOD2 is on the mitochondrial membrane, whereas SOD1 and SOD3 localization is restricted to the cytoplasm (104). In contrast,

Cat1 and 2 are secreted, whereas CatA is cytoplasmic (103). In addition to catalases and

SODs, the LaeA transcription factor upregulates expression of 2° metabolites including gliotoxin and fumagillin, which are secreted and can inhibit NADPH oxidase and neutrophil fungicidal activity (105-109). Figure 1.13 shows evidence that fungal toxins may be involved in human fungal keratitis.

46 Figure 1.11 ROS derived from neutrophil NOX

Extracellular superoxide reacts with H2O to form H2O2 which can be converted to hypohalous acids via 1ºgranule-released myeloperoxidase or converted to reactive nitrogen species by iNOS-derived nitric oxide (NO)

47 Figure 1.12 Fungal anti-oxidative stress defenses

See text for details

48

Figure 1.13 Possible role for toxins in human fungal keratitis

The cornea of a patient infected with A.flavus was sectioned and stained with gomori- methanamine silver (GMS) to detect fungi and hematoxylin and eosin (H&E) to detect inflammatory infiltrates.High fungal burden is detected in the apical cornea but not the basal cornea. Inflammatory cells are detected in the basal cornea but not the apical cornea. It is possible that the failure of inflammatory cells to interact with fungi is due to fungal production of compounds that damage host cells (i.e. toxins). This hypothesis was tested in the current study.

49

Chapter 2.

Characterization of the immune response in human corneal tissues infected with the filamentous fungi Aspergillus and Fusarium

50 Summary

Background: Filamentous fungi of the genus Aspergillus and Fusarium are major causes of corneal ulcers in the USA and in the developing world, resulting in significant visual impairment and blindness. Methods: RNA was extracted from 110 corneal ulcers in southern India within one week of infection with either F. solani or A. flavus, and gene expression was determined by quantitative PCR. Post-transplant corneas from later stage disease (>2 weeks post infection) were also examined. Results: Expression of Dectin-1, TLR2, TLR4, TLR9, NLRP3 and ASC mRNA was elevated >1,000-fold compared with normal donor corneas, whereas Dectin-2 was constitutively expressed in normal corneas. Further, IL-1β expression was elevated >1,000 fold, whereas IL-1α expression was not increased. IL-8, IL-17 and TNF-α expression was also elevated.

CD3+ and CD4+ T cells were detected in infected, post-transplant corneas. IL-17 and

IFN- expression was elevated but not IL-4. There were no significant differences in the host response between Aspergillus and Fusarium–infected corneas at any time point. Conclusions: there is a common innate and adaptive immune response to these filamentous fungi, including generation of Th1 and Th17 cells.

51 Introduction

Fungal keratitis is an important cause of blindness and visual impairment worldwide. In the USA and other industrialized countries, contact lens wear is the primary risk factor for fungal keratitis, as illustrated by several hundred cases in the

USA, Western Europe and Singapore during the 2005/2006 outbreak of Fusarium keratitis (110-112). Although the incidence of fungal keratitis dropped after withdrawal of the lens care solution associated with the outbreak (111), the prevalence due to trauma or contact lens wear has remained consistent in the USA, especially in southeastern regions (113).

Contact lens related fungal keratitis is associated with biofilm formation (28), whereas in trauma related disease, corneas are infected as a result of ocular surface trauma caused by plant material, insects or branches contaminated with fungal spores

(18, 114, 115). Once the corneal epithelial barrier is breached, the conidia germinate and penetrate throughout the corneal stroma. Depending on the inoculum and the time between infection and the patient receiving anti-fungal treatment (usually topical natamycin or voraconazole), infected individuals will either recover, or will undergo corneal transplantation (12). In China and India, the major risk factor is agricultural work; consequently, the incidence of fungal keratitis increases during the harvest season, when there is greater exposure to airborne soil and contaminated plant material

(5, 18, 114-116). Once in the corneal stroma, Aspergillus and Fusarium conidia germinate, and hyphae penetrate the corneal stroma, causing corneal ulceration, severe pain and visual impairment.

In contrast to pulmonary and systemic fungal infections, which occur primarily

52 in immune compromised individuals such as stem cell recipients (117-120), patients with fungal keratitis are immunocompetent. In addition, fungal infections of the cornea present a unique opportunity to examine infected tissues at very early stages of infection as infected individuals experience severe pain and visual impairment, and generally know when they experienced ocular surface trauma which is the likely time of infection. As treatment failure is also common, patients undergo corneal transplant, thereby providing infected tissue from a later time point after infection.

In the current study, we therefore examined the host response to these pathogenic fungi in infected corneal ulcers within days of infection, and in infected post-transplant corneas at later time points after infection. Our findings demonstrate that infected individuals mount a profound host response to Aspergillus and Fusarium hyphae that is characterized by infiltration of neutrophils, macrophages and T cells, and by expression of pro-inflammatory, chemotactic and regulatory cytokines.

Materials and Methods

RNA Extraction, cDNA conversion and quantitative PCR analysis

Corneal scrapings from patient ulcers were homogenized in TRIzol (Invitrogen,

Carlsbad, CA), using a handheld homogenizer (Labware Scientific, USA), and total

RNA was extracted from corneal tissue samples according to the manufacturer's directions followed by DNase treatment (Invitrogen, Carlsbad, CA). The quality of

RNA was checked by agarose gel electrophoresis, and samples with a 260/280 ratio >

1.8 were used to generate cDNA.

cDNA was generated using the SuperScript First Stand synthesis system

53 (Invitrogen) using standard methods. cDNA samples were then digested with RNaseH

(Invitrogen) for 20 min at 37OC, and quantitative PCR was performed using the SYBR green system (Applied Biosystems, Carlsbad, CA). Primer sequences were designed using NCBI Primer BLAST or downloaded from Primer bank (Table 1), and synthesized either at Bioserve India Ltd, Hyderabad, India or at Integrated DNA

Technologies (San Diego, CA). Universal PCR conditions were utilized for cDNA amplification for all primer sets. Melting curve analysis was performed to confirm specific gene amplification, and PCR product sizes were confirmed by agarose gel electrophoresis.

The quantification cycle of the target gene was normalized using GAPDH (which was consistent for all samples), and the fold change with respect to non-infected, donor corneas was calculated using the 2-ct method. Data are therefore presented as log of relative gene expression (log(RQ)) as originally described by Livak and Schmittgen

(121).

Characterization of host cells in corneal tissues

Infected corneal ulcer material was spread on a standard microscope slide and stained with the modified Wrights Geimsa solutions (Diff-Quik). Infected corneas obtained after transplantation were fixed in 4% paraformaldehyde, paraffin-embedded, and 5µm sections were stained with Periodic Acid Schiff (PASH), and Gomori-

Methanamine Silver (GMS) following standard protocols. For immunohistochemistry, paraffin-embedded 5µm sections of infected corneas were deparaffinized with xylene and ethanol gradient methods, and incubated 10 minutes at room temperature with 3%

54 freshly prepared hydrogen peroxide solution to block endogenous peroxidase activity.

Antigen retrieval was performed by three cycles of microwaving with slides submerged in Citrate Buffer (Sodium Citrate Dehydrate 2.94 g/L, pH to 6.0) for 6 minutes at 50 % power. The slides were then washed with PBS-T (PBS with 0.05 % Tween-80), blocked with 5% horse serum in PBS-T for 30 min, and incubated with mouse monoclonal antibodies with specificity for human neutrophil elastase, macrophages CD68, or human CD3 or CD4 (Abcam, Boston, MA). All primary antibodies were used at a 1:50 dilution in PBS-T and incubated overnight at 4°C.

The sections were then washed, incubated 1 hour at room temperature with the secondary anti-mouse antibody (ImmPRESS Reagent kit; Vector labs, Burlingame,

CA), washed with PBS, and incubated with 3,3'-diaminobenzidine (DAB, Vector labs) in the dark for 2–10 min. The reaction was stopped with ddH2O, and sections were counterstained with hematoxylin. Sections were dehydrated in EtOH gradients, dipped in xylene, and mounted using DPX mounting medium (Merck, India) and dried overnight before examining. For anti-CD4 antibody staining, sections were incubated with Alexafluor-488 conjugated anti-mouse IgG antibody (Invitrogen), washed with

PBS-T, and VectaShield with DAPI (Vector Labs, USA) was used as the mounting medium.

To detect -glucan, corneal sections were incubated with primary mouse anti-fungal - glucan IgM (BF-Div; Biothera/Brown Univ.) for 1h at 37°C, followed by 1h at 37°C

1mg/ml alexafluor-488 conjugated rabbit-anti-mouse IgM (Invitrogen). Slides were washed in PBS-T, mounted with VectaShield and sections were imaged by fluorescence microscopy.

55 Statistical analysis

Differences in gene expression between normal, uninfected donor corneas and infected corneas was calculated by 1 way ANOVA, and differences between

Aspergillus and Fusarium infected corneal tissue were calculated by the unpaired t test using GraphPad Prism software (LaJolla, CA). Statistical significance was defined as p<0.05.

Results

Characteristics of the study population

Patients with corneal ulcers are routinely treated at the Aravind Eye Hospital, which is a primary care facility in Madurai, Tamil Nadu. These patients generally present with severe pain and visual impairment within days of experiencing ocular trauma. Although bacteria are frequently the cause of corneal ulcers, the majority of ulcers are caused by Aspergillus or Fusarium species and are associated with corneal injury from plant or soil material, or from other causes such as insects sticks and cattle tails (18, 122) (L.P. unpublished observations). In the current study, undertaken between January and March 2010, samples from patients with corneal ulcers but without systemic illness or immunosuppressive therapy were selected. Ethical clearance was obtained from the internal Institutional Review Board at Aravind Eye Hospital, and informed consent was obtained from all participants.

Corneal ulcer scrapings were cultured, and morphological analysis identified 55 patients infected with Fusarium species and 55 infected with (Table

2). Subsequent examination of these isolates by sequence analysis confirmed the

56 identity of A. flavus, and showed that Fusarium species were almost exclusively members of the F. solani family complex (123, 124)(D.G., unpublished studies).

Patients in Groups IA and IB (n=85) presented at the clinic between 3-7 days after infection, and corneal ulcer scrapings were used for analysis, whereas Group II patients (n=20) had been infected for longer than 2 weeks, and had undergone corneal transplantation; therefore, infected post-transplant corneas were used in the current study. All patients were between 46 and 53 years old, with slightly more males than females infected. The size and depth of ulcers in Groups IA and IB ranged from <5mm2 to >14 mm2 diameter, and the majority of patients showed hypopyon formation, which is accumulation of neutrophils in the anterior chamber. 7-38% of patients in Groups IA and IB did not heal, although 36-52% of patients did not appear for a follow-up examination. All patients in Group II were failed treatment. Control tissues were corneal donors aged 61.70± 6.40 who died of natural causes, and who had no history of corneal infection or other systemic disease

Clinical appearance and cellular infiltration in early-stage Aspergillus and Fusarium keratitis

Figure 1 shows representative corneal ulcers caused by either A. flavus (Figure

1A) or F. solani (Figure 1B). The A. flavus keratitis patient shown is a 47-year old woman with a central corneal ulcer and hypopion formation, who experienced a foreign body trauma to the ocular surface while working on a farm, and who presented at the

57 Table 2.1 Patient study characteristics

Reprint permission obtained from publisher.

58 clinic one week after infection. Her ulcer did not respond to treatment, and she successfully underwent corneal transplantation two weeks later. The F. solani keratitis patient shown is a 55 - year old woman with a perforated ulcer whose cornea was scratched by a sharp leaf and appeared at the clinic ten days later. She also had a corneal transplant two weeks later, although the transplanted cornea was rejected.

To identify infiltrating cell in the corneal ulcer, a scraping was stained with

Diff-Quik, and examined by light microscopy. As shown in, hyphae were readily detected in corneal ulcers infected with A. flavus (Figure 1A) and F. solani (not shown). Neutrophils comprised 95% of infiltrating cells, with 5% mononuclear cells, including epithelial cells and monocytes (Figure 1B,C).

Expression of pathogen recognition receptors and inflammasome proteins in early- stage Aspergillus and Fusarium keratitis

To determine the expression of pathogen recognition receptors in early stage fungal keratitis, RNA was extracted from 85 corneal ulcer scrapings within one week of trauma (Group I), reverse transcribed, and qPCR was performed. ∆Ct data were calculated using GAPDH (which was consistent for all samples), and the fold change with respect to the mean of 10 non-infected, donor corneas was derived using the 2-ct method (121). Figure 2 shows that Dectin-1 expression was elevated an average of 500 fold in Aspergillus and Fusarium infected corneas compared with donor corneas, TLR4 was elevated 500-fold, and TLR2, TLR9, ASC and NLRP3 were elevated >1,000 fold above normal corneas (Figure 2). There were no significant differences in expression of any of these genes between Aspergillus and Fusarium infected corneas.

59 Figure 2.1 Cellular composition of corneal ulcers from patients with fungal keratitis

Shown are representative corneal ulcers caused by Aspergillus flavus (A) or by Fusarium solani (B), with the arrow indicating accumulation of neutrophils in the anterior chamber (original magnification 310). Ulcerative tissue was fixed and stained with Diff-Quik, and representative images show A. flavus hyphae (C ) and the cellular infiltrate (D ) in infected corneas (original magnification3400). The inset in panel D shows the typical nuclear morphology of neutrophils. The percentages of neutrophils and mononuclear cells were determined by counting 100 cells from 10 patients with Aspergillus infection and 10 patients with Fusarium infection (E). Reprint permission obtained from publisher.

60 ∆Ct values for donor corneas and fungal infected corneal ulcer material are shown in

Figure S1. These data show low expression of these pathogen recognition molecules and inflammasome proteins in donor corneas (high ∆Ct), and significantly higher expression in corneal ulcer material (low ∆Ct) from infected corneas. Figure S1 also shows that Dectin-2 expression was elevated (low ∆Ct) in donor and infected corneas, with no significant differences, indicating constitutive expression of this receptor.

Differential expression of pro-inflammatory and chemotactic cytokines in early-stage fungal keratitis

Given the elevated expression of NLRP3 and ASC, we examined expression of

IL-1β and other cytokines associated with neutrophil recruitment and activation. As shown in Figure 3A, IL-1β transcripts in Group IA were elevated an average of 100- fold above normal corneas, whereas IL-1α expression was not upregulated. TNF-α and

IFN-γ expression were elevated 100-fold and 10-fold, respectively, whereas IL-8 was upregulated 1,000-fold.

Consistent with Dectin-1, we also found that IL-17 expression in Aspergillus and Fusarium - infected corneas was 2,000-fold elevated above control corneas (Figure

3B). In addition, IL-18 and IL-23 transcripts were 50 and 100-fold elevated, whereas

IL-4 expression was not increased above control corneas. As with pathogen recognition receptors, there were no significant differences in cytokine expression between Aspergillus and Fusarium - infected corneas, indicating that both genera of pathogenic fungi stimulate production of chemotactic and pro-inflammatory cytokines.

61 Figure 2.2 Expression of pathogen recognition receptors in corneal ulcers from patients with fungal keratitis.

RNA was extracted from corneal ulcerative tissue, reverse transcribed, and processed for quantitative polymerase chain reaction. Data points represent individual patients with Aspergillus or Fusarium keratitis, and the values are presented as the log of relative gene expression (log[RQ]) relative to uninfected donor corneas calculated using th ΔΔCt method described in the text. There were no significant differences in gene expression between Aspergillus-infected corneas (closed circles) and Fusarium-infected corneas (P<0.05) (open circles). TLR, Toll-like receptor. Reprint permission obtained from publisher.

62 Figure S1 shows ∆Ct values, confirming that IL-1α and IL-4 expression was low in donor and infected tissues, whereas expression of all other cytokines was significantly elevated infected compared with donor corneal tissues.

Expression of β-glucan and characterization of the cellular infiltrate in post- transplant corneas infected with Aspergillus or Fusarium

As Dectin-1 recognizes β-glucan on germinating conidia and hyphae (125), we next examined β-glucan expression in post-transplant, infected corneas. 5 µm corneal sections were either stained with GMS, or were immunostained with an antibody that recognizes 1,3 and 1,6 β -glucan. Figure 4A shows extensive fungal growth in GMS stained corneas infected with A. flavus, and β -glucan expression in infected corneas.

There was no expression of β -glucan in normal, donor corneas (data not shown).

To examine infiltrating cells in fungal infected post-transplant corneas, sections were immunostained with cell specific antibodies. We found that 65-75% of infiltrating cells were elastase+ neutrophils, 25-35% were CD68+ macrophages, and 3-8% were

CD3+ and CD4+, with no differences between Aspergillus and Fusarium infected tissues (Figure 4B). Representative images are also shown (Figure 4C).

To determine the cytokine response in later stage fungal keratitis, RNA was extracted from post-transplant corneas infected with Aspergillus or Fusarium, and gene expression was determined by qPCR. As shown in Figure 4D, expression of IL-17 and

IFN-γ in Aspergillus and

63 Figure 2.3 Expression of proinflammatory, regulatory, and chemotactic cytokines in corneal ulcers

Q-PCR analysis of corneal ulcerative tissue was performed as described in Methods. Data points represent individual patients infected with Aspergillus or Fusarium and values were calculated as described above. There were no significant differences in gene expression between Aspergillus- (closed circles) and Fusarium- (open circles) infected corneas (P<0.05). IFN, interferon; IL, interleukin; TNF, tumor necrosis factor; log(RQ); log of relative gene expression. Reprint permission obtained from publisher.

64 Figure 2.4 β-glucan and cellular infiltration in infected posttransplant corneas.

A. Five-micron sections of Aspergillus flavus–infected corneas stained with Gomori- Methanamine silver to identify fungal hyphae or immunostained with antibodies to β- glucan (Dectin-1 ligand) (original magnification 3100 in upper left panel, 3400 in upper right and lower left panels, and 31000 in lower right panel). Stroma and anterior chamber (ac) are indicated. B. Percentage of cell types (neutrophils [neuts] and macrophages [macs]) in Fusarium- and Aspergillus-infected corneas; 5 slides were examined for each stain, and 100 cells were counted on each section. C, Representative corneal sections of Aspergillus- and Fusarium-infected corneas immunostained with antibodies to neutrophils (elastase), macrophages (CD68), CD3, and CD4. DAPI (blue) showed the presence of cell nuclei in the CD4-stained sections, and 3,3- diaminobenzidine and hematoxylin were used to visualize cells in other sections (original magnification 3400). D. Results of quantitative polymerase chain reaction performed on corneal tissue as described in the legend to Figure 2. Data points represent corneas from individual patients infected with Aspergillus or Fusarium in relation to uninfected donor corneas as described in the text. There were no significant differences in gene expression between Aspergillus- and Fusarium-infected corneas (P <0.05). IFN, interferon; IL, interleukin; log(RQ), log of relative gene expression. Reprint permission obtained from publisher.

65 Fusarium infected corneas was elevated an average of 100-fold compared with normal donor corneas, whereas IL-4 expression was not increased. ∆Ct values show significantly elevated IL-17 and IFN-γ but not IL-4, between donor and infected corneas, and that IL-4 expression was low in donor and infected corneas (Figure S1).

Together with results from Figure 4, these findings are consistent with a Th1 and Th17 in infected corneas, but not a Th2 response.

Discussion

Studies of family members and stem cell transplant recipients with the Tyr238X

Dectin-1 early stop codon mutation are more susceptible to mucocutaneous fungal infections, and blood mononuclear cells from these individuals have an impaired cytokine response to Candida albicans infections (126). Similarly, susceptibility to mucocutaneous candidiasis is associated with a point mutation in CARD9 (127), which is downstream of Dectin-1. Further, stem cell patients with TLR4 polymorphisms were susceptible to invasive aspergillosis (128). In the current study, we examined the expression of Dectin-1 and TLR4 in human tissue infected with Aspergillus or

Fusarium.

We found that Dectin-1 expression is elevated in early stage disease, which is consistent with our studies using a murine model of A. fumigatus keratitis in which

Dectin-1 mediates cytokine production, neutrophil recruitment and fungal survival (39).

Murine models of pulmonary aspergillosis, and of mucosal and systemic candidiasis also indicate that Dectin-1 regulates fungal survival and the outcome of disease (129,

130), although one study found no role for Dectin-1 in candidiasis (58).

66 In contrast to Dectin-1, Dectin-2 recognizes cell wall high mannose residues, responds to fungal hyphae rather than , and functions together with Dectin-1 to induce IL-17 production (54, 131, 132). In the current study, we found that IL-17 expression was greatly elevated in early stage disease, and that Dectin-2 expression was elevated in donor and infected corneas. Functional studies using mouse models of keratitis are currently underway to ascertain the role of Dectin-1 and Dectin-2 in IL-17 production and in disease pathogenesis.

TLR4 expression was also elevated in Aspergillus and Fusarium infected corneas at early time points, and is consistent with the reported correlation between

TLR4 polymorphisms and susceptibility to Aspergillus infections (128), and with our findings that TLR4 regulates Aspergillus and Fusarium clearance from mouse corneas

(39, 40). Although the mechanism has yet to be determined, TLR4 recognizes n-linked mannans (133), and we showed that TLR4 – mediated fungal killing is independent of the LPS receptor, MD-2(39), suggesting that n-linked mannans may bind directly to

TLR4.

We also found elevated TLR2 expression in infected corneal ulcer material.

TLR2 contributes to inflammasome mediated production of mature IL-1β in oral and systemic candidiasis (127, 134). Furthermore, fungal activation of TLR2 on neutrophils induces an oxidative burst (135, 136), and contributes to fungal clearance in a murine model of Candida albicans keratitis (137). However, we found no role for

TLR2 in murine models of Fusarium or Aspergillus keratitis (39, 40); therefore, the significance of our current finding that TLR2 expression is elevated in infected human corneas has yet to be determined. TLR9 expression was also highly up-regulated in

67 Fusarium and Aspergillus infected human corneas, which may indicate a role for this receptor. In support of this notion, two recent reports demonstrated that TLR9 is recruited to phagosomes containing Aspergillus conidia, that Aspergillus DNA contains unmethylated CpG motifs, which activate TLR9 in vitro, and that TLR9-/- mice have an impaired response to Aspergillus in the lung (138, 139).

We anticipated that pro-inflammatory and chemotactic cytokines would be elevated, and we found >1000-fold elevated elective expression of the neutrophil chemokine IL-8, consistent with the presence of neutrophils in the corneal ulcer. In addition, the pro-inflammatory cytokines IL-1 β and TNF-α were elevated in infected corneas, whereas IL-1α expression was no elevated. In contrast to Dectin-2, this was not due to constitutively high expression in donor corneas, but to low expression in all tissues examined. Consistent with elevated IL-1 β, expression of the NLRP3 and ASC inflammasome proteins was greatly elevated, suggesting a role for inflammasomes in fungal keratitis. This finding is supported by reports showing an important role for IL-

1R in murine models of Fusarium and Aspergillus keratitis (39) (40). Although we found that TLR2, TLR4 and TLR5 are important in murine models of bacterial keratitis

(2, 140), we have yet to determine if these markers or inflammation are also upregulated in human bacterial keratitis.

The cytokine profile of late-stage disease showed elevated expression of IL-17 and IFN-γ but not IL-4, which together with the presence of 3-8% CD3 and CD4 cells, indicates that Th1 and Th17 cells are generated and recruited to the corneal cornea.

Although the presence of these subtypes in peripheral blood has yet to be determined, this finding is consistent with the predominant IFN-γ / T cell responses

68 reported for A. fumigatus in normal, asymptomatic individuals (141, 142), which likely occurs as a result of inhalation of these ubiquitous organisms (21). Fusarium is also ubiquitous in agricultural environments; therefore it seems reasonable to assume that most infected patients harbor memory T cell responses to both Fusarium and

Aspergillus prior to inoculation of spores into the corneal stroma, and that Th1 and

Th17- like responses contribute to corneal pathology.

In conclusion, the current study provides direct characterization of the host response to pathogenic fungi in infected human tissues at early and later stages of disease in an endemic population. These findings will allow us to examine specific host response genes using animal models of fungal keratitis, and may identify potential targets for immune intervention that could regulate the severity of the host response and its effect on blindness and visual impairment caused by these organisms.

69

Chapter 3.

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis

of Aspergillus fumigatus keratitis

70 Summary

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo , we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1ß and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that ß-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1-/- corneas have impaired IL-1ß and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high ß-glucan. In contrast to Dectin 1-/- mice, cellular infiltration into infected TLR2-/-, TLR4-/-, and MD-2-/- mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4-/- mice, but not TLR2-/- or MD-2-/- mice. We also found that TRIF-/- and TIRAP-/- mice exhibited no fungal-killing defects, but that MyD88-/- and IL-1R1-/- mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which ß-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1ß, and CXCL1, which together with IL-1R1/

71 MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.

Introduction

Fungal infections of the cornea (i.e. fungal keratitis) account for approximately

1% of corneal ulcers in temperate regions of industrialized nations (12). However, in tropical regions of developed countries, such as in the southeastern United States, fungal infections of the cornea account for up to 35% of all corneal ulcers resulting in severe visual impairment and blindness (12, 17, 143). Globally, the impact of fungal keratitis on visual health is even greater, with reports of up to 60% of corneal ulcers attributable to fungal infection in developing nations including China, Nepal, India,

Bangladesh, Ghana and Mexico (9, 18, 115, 144-148). The etiological agents of these corneal infections are most commonly filamentous Fusarium (F.solani, F.oxysporum)

(12, 111), and Aspergillus species (A.flavus, A. fumigatus, nomius (149), A.tamarii

(150), A. terreus, and A.tubingensis (151, 152)), which are prevalent in hot, humid climates, and where the predominant risk factor is traumatic injury associated with agricultural work.

As Aspergillus is ubiquitous in the environment, the population at large is constantly exposed to this opportunistic pathogen (14, 21, 153), and conidia are inhaled at an estimated 200 conidia per day (5, 21, 154). In areas of high endemicity, A. fumigatus can also be isolated from the conjunctival sac of healthy individuals (155).

Thus, in the setting of a disrupted corneal epithelium, there is broad opportunity for inoculation of A. fumigatus conidia (2-3 µm diameter) or conidiophores (>100

72 individual conidia per hyphal stalk) from airborne vegetative matter or the conjunctiva into the corneal stroma in association with traumatic injury (9, 156). Subsequently, fungal virulence factors that facilitate hyphal invasion of the tissue (e.g. and protease secretion) (105, 157-161), together with low efficacy of anti-mycotic therapy

(12, 26, 162), and the resulting inflammatory response all converge to induce destruction of corneal tissue. Treatment failure also occurs in up to 60% of patients, who may require at least one and sometimes repeated corneal transplantation, and in severe cases results in enucleation of the infected eye (12, 156). In contrast to pulmonary aspergillosis, which is associated with immune suppression, fungal keratitis occurs in otherwise healthy, immunocompetent individuals.

Despite the impact of filamentous fungal infections on global blindness, and a recent outbreak of contact lens-associated Fusarium keratitis in the USA (111), the pathogenesis of this disease is not well understood. We therefore developed a murine model of corneal infection using Aspergillus that constitutively express dsRed fluorescent protein at each stage of development, and are therefore readily detected in the transparent cornea. Our findings demonstrate that Dectin-1 mediates the initial wave of pro-inflammatory and chemotactic cytokine production and cellular infiltration into the cornea. We also show that the IL-1R1/MyD88 pathway further propagates cellular recruitment into the cornea during Aspergillus infection, and that in contrast to Dectin

1, TLR4 does not regulate cellular infiltration, but is essential for anti-fungal activity.

73 Materials and Methods

Source of mice

All animals were treated in accordance with the guidelines provided in the

Association for Research in Vision and Ophthalmology ARVO statement for the Use of

Animals in Ophthalmic and Vision Research, and were approved by Case Western

Reserve University IACUC. C57BL/6 mice (6–12 wk old), and IL-1R1-/- mice on a

C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME).

129SvEv mice were purchased from Taconic Farms (Hudson, NY). Dectin 1-/- mice on a 129SvEv background were provided by Dr. Gordon Brown, University of Aberdeen,

UK, MD-2-/- mice were provided by Dr K. Miyake, University of Tokyo, and TLR2-/-,

TLR4 -/-, TRIF-/-, and MyD88-/- mice on a C57BL/6 background were provided by Dr.

Shizuo Akira, Osaka University, Osaka, Japan, TIRAP -/- mice were provided by Dr

Ruslan Medzhitov, Howard Hughes Medical Institute, Yale University, NewHaven,

CT. MAcrophage Fas-Induced Apoptosis (Mafia) mice were obtained from Sandra

Burnett, Dept. of Microbiology and Molecular Biology, Brigham Young University,

Provo, UT (163).

Fungal Strains, Media, and Growth Conditions

Aspergillus fumigatus strains used in this study were cultured on Vogel’s

Minimal Media (VMM) w/wo 4% agar +/- supplementation with 10 mM uracil and 5 mM uridine at 37˚C / 5% CO2 unless otherwise stated. A. fumigatus Strain Af-BPis a fungal keratitis clinical isolate from a patient treated at Bascom Palmer Eye Institute

(Miami, FL) provided by Dr. Darlene Miller. A. fumigatus Strain B-5233 is a clinical

74 strain isolated from the lungs of a patient with severe neutropenia provided by Dr.

Kwon-Chung (NIAID) (164). The uracil auxotroph strain Af293.1 (∆pyrG1) (165) was a gift from Gregory May (M.D. Anderson Health Science Center, Texas).

Complementation of pyrG1 and constitutive RFP expression by strain Af293.1 was achieved via transformation with the plasmid pRG3AMA1-RFP forming strain

Af293.1RFP (Figure S1).

Construction of the dsRed Fluorescent A. fumigatus Strain Af293.1RFP

All primers used in this study are listed in Supplementary Figure S1A. The first step in the construction of Af293.1RFP, entailed PCR amplification of the glyceraldehyde 3 phosphate dehydrogenase promotor (gpdA) on the plasmid pDV2

(166) using a forward primer bearing a 5’ Kpn1 overhang (Kgpd-F) and a reverse primer bearing a 5’ complementary DNA sequence to the improved monomeric dsRed fluorescent protein- encoding gene (rfp), expressed on the plasmid pMT-RFP (Rgpd-R)

(167, 168). Next, the rfp gene on pMT-RFP was PCR amplified using a forward primer bearing a 5’ complementary sequence to the 3’ end of the gpdA promotor on pDV2 (Grfp-R) and a reverse primer harboring a 5’ Kpn1 cut site overhang (Krfp-R). Both PCR amplicons (gpdA promotor= 1084 Af-BP; rfp= 769 Af-

BP) underwent fusion PCR, using primers Kgpd-F and Krfp-R to yield a single fusion

PCR amplicon gpdA:rfp (Af-BP). Lastly, Kpn1-digested gpdA:rfp was ligated to Kpn1- digested pRGAMA-1 yielding an 11.8 kb plasmid (Figure S1B). The resulting plasmid, pRG3AMA1-RFP, was transformed into the uracil auxotroph strain, Af293.1∆pyrG according to the method described by May (169). Figure S1C shows that the resulting

75 strain, Af293.1RFP, constitutively expressed dsRED fluorescence (580 nm) at each morphological stage of the organism. Additionally, there was no difference in the growth kinetics of this strain with the parental Af293.1 strain (data not shown).

Development of a mouse model of Aspergillus keratitis

A. fumigatus strains were cultured for 2-3 days on VMM without uracil or uridine in 25 cm2 tissue culture flasks. Fresh conidia were disrupted with a bacterial L- loop and harvested in 5 ml PBS. Pure conidial suspensions were obtained by passing the culture suspension through sterile PBS-soaked cotton gauze positioned at the tip of a 10 ml syringe. Conidial suspensions were quantified using a haemacytometer, and adjusted to a final concentration of 5 x 104 conidia/ µl in PBS. Mice were anaesthetized by intraperitoneal (IP) injection of 2.25 mg ketamine and 0.45 mg xylazine, and the corneal epithelium was abraded using a 30-gauge needle. Through the abrasion was inserted a 33-gauge Hamilton syringe from which a 2 µl injection containing 1 x 105 conidia (Optimal inoculum size for induction of keratitis based on preliminary studies; data not shown) was released into the corneal stroma. Mice were examined daily under a stereomicroscope for corneal opacification, ulceration, and perforation. At set time points, animals were euthanized by CO2 asphyxiation, and were either placed in

10% formalin and embedded in paraffin and sectioned at 5 µm intervals, or excised and placed in 1 ml of sterile saline and homogenized for quantitative culture. For cyclophosphamide immunosuppression, mice were given 180 mg/kg cyclophosphamide

(Sigma-Aldrich) via I.P. injection at days 3 and 1 prior to infection (28). All animals were bred under specific pathogen-free conditions and maintained according to

76 institutional guidelines.

Depletion of c-fms expressing cells using Mafia mice

MAcrophage Fas-Induced Apoptosis (Mafia) mice are C57BL/6J mice harboring an eGFP and suicide-protein expressing transgene downstream of the macrophage and dendritic cell lineage- specific c-fms promoter(163, 170). As such all MACs/DCs in these mice, constitutively express eGFP and a membrane-bound suicide protein. To deplete c-fms expressing cells, Mafia mice received five daily ip injections (10 mg/kg) of the covalently-linked dimer (AP20187; Ariad Pharmaceuticals) followed by two days rest before the experiment. AP20187 has affinity for the FK506 binding protein region of the suicide protein; and homodimerization of the suicide proteins activates the intramolecular cytoplasmic Fas domains, with the subsequent induction of caspase-

8 mediated apoptosis in all MACs/DCs. Our recent study showed that this protocol results in loss of viable eGFP + cells in the cornea (163, 170).

Imaging Corneal Opacity and Fungal RFP expression.

Mice were sacrificed by CO2 asphyxiation and positioned in a thee-point stereotactic mouse restrainer. Corneal opacity (Brightfield), fungal proliferation (RFP; 580nm) and cellular infiltration (eGFP; 488nm), were visualized in the intact cornea using a high- resolution stereo fluorescence

77 MZFLIII microscope (Leica Microsystems) and Spot RT Slider KE camera

(Diagnostics Instruments). In some experiments, corneas were dissected and examined using an inverted Leica DMI 6000B microscope. All images were captured using

SpotCam software (RT Slider KE; Diagnostics Instruments).

Quantification of Corneal Opacification, Fungal dsRed and c-fms+ cell eGFP expression.

To quantify corneal opacity objectively, brightfield images of mouse corneas were analyzed using Metamorph software (Molecular Devices). Briefly, a constant circular region encompassing the cornea was defined, and the pixel intensity within this region summed to yield a numerical value, called the pixel intensity corresponding to the total amount of light reflected from the cornea (i.e. opacity). Similarly, fungal dsRed RFP and c-fms+ cell eGFP expression were quantified via Metamorph image analysis. All images were obtained with the same Spot RT Slider KE camera using the same Spot

Advanced Software under the same magnification, exposure (BF=0.4s; RFP=10s; eGFP=2s), gain (BF=1; RFP/eGFP=16), and gamma (BF/RFP/eGFP=1.85) parameters.

Quantification of Aspergillus Colony Forming Units (CFUs).

For assessment of fungal viability, whole eyes were homogenized under sterile conditions in 1 ml PBS, using the Mixer Mill MM300 (Retsch, Qiagen, Valencia, CA) at 33 Hz for 4 min. Subsequently, serial log dilutions were performed and plated onto bacteriologic-grade Sabouraud dextrose agar plates (Becton Dickenson). Following incubation for 24h at 37°C, the number of CFUs was determined by direct counting.

78 Identification of Fungi and Inflammatory Cell Recruitment.

Eyes were enucleated and fixed in 10% formalin in PBS (Fisher) for 24h. Tissues were then dehydrated in graded ethanol concentrations at room temperature (65% 1x, 80%

2x, 95% 1x, 100% 3x; 1 h for each change of solution), followed by three 1h changes of xylene, and 4 changes of paraffin at 60˚C under 15mm Hg vacuum to remove air bubbles. Five µm sections from the center of the cornea (as determined by noncontiguous iris morphology) were cut and stained with Periodic-Acid Schiff

(PASH) for identification of fungi and inflammatory cell recruitment.

Detection of β-glucan expression and neutrophil infiltration during live corneal infection.

To detect ß-glucan expression during live corneal infection, 5-µm paraffin sections were deparaffinized. Slides were blocked with 1.5% normal rabbit serum in PBS for 1h, then incubated with primary mouse anti-fungal ß-glucan IgM (BF-Div; Biothera/

Brown Univ.) diluted to 24 µg/ml with 1% BSA (Fisher) for 1h at 37°C. The slides were then washed 3x in PBS plus 0.05% Tween 20 (PBS-T; Sigma) and incubated with alexafluor-488 rabbit-anti-mouse IgM (Invitrogen) diluted to 1µg/ml in PBS for 1h at

37°C. The slides were washed 3x in PBS-T and imaged by fluorescence microscopy

(magnification, 400x). A similar process was performed to stain 5µm corneal sections for neutrophils using monoclonal rat-anti-mouse neutrophil IgG (NIMP-R14, AbCam,

Cambridge, MA), and alexafluor-488 tagged rabbit-anti-rat IgG (Invitrogen).

79 Detection and Quantification of -glucan Surface Expression by A. fumigatus grown in vitro

A.fumigatus strains were cultured for 3 days in VMM + 4% agar. Pure conidial suspensions were prepared from the 3-day culture, and 5 million conidia were added to

50 ml SDA broth in vented-cap 250 ml tissue culture flasks. The conidia were grown at

37C/ 5% CO2 for 0, 6, and 10h. At indicated timepoints, A.fumigatus was fixed with

4% paraformaldehyde for 30 min, washed 3x with PBS and spun onto Superfrost microscope slides (Fisher) using a Cytospin centrifuge. Subsequently, slides were blocked with 1.5% normal rabbit serum in PBS for 1h, then incubated with primary mouse anti-fungal ß-glucan IgM (BF-Div; Biothera/ Brown Univ.), diluted to 24 µg/ml with 1% BSA (Fisher) for 1 h at 37°C. The slides were then washed 3x in PBS-T and incubated with alexafluor-488 rabbit-anti-mouse IgM (Invitrogen) diluted to 1µg/ml in

PBS for 1h at 37°C. The slides were washed again 3x in PBS-T and imaged by fluorescence microscopy (magnification, 400x). Subsequently, images were analyzed using Metamorph software and the numerical output of average 488nm pixel intensity/area of fungal cells was used to quantify fungal -glucan surface expression.

To minimize the confounding variable of enhanced fluorescence due to cell clumping, only 488nm fluorescence emanating from isolated fungal cells was used for quantitation.

Detection of pSyk, IB, and Dectin-1 via Western Blot Analysis.

Cornea protein extracts were prepared by homogenization in cell lysis buffer (Cell

Signaling) plus 1mM phenylmethylsulphonyl-fluoride (PMSF) using a Tissue-Lyser

80 (described above). Similarly, cell culture protein extracts were prepared via lysis in cell-lysis buffer + PMSF. Total protein was quantified via Bicinchoninic Acid Assay

(Pierce), denatured with 2× Laemmli buffer (Sigma) and heated to 95 °C for 5 min. 20

µg total protein was loaded onto each well of a 10% polyacrylamide gel (BioRad), separated via electrophoresis, and transferred to nitrocellulose. Blots were stained accordingly with anti-Dectin-1 (RandD; MAB1756), anti-phosphoSyk (Cell Signaling;

2710), anti-pIB (Cell Signaling; 2859), and anti-ß-actin (Cell Signaling; 4967). HP- tagged secondary antibodies were purchased from Santa Cruz Biotechnologies. All blots were developed with GE Healthcare ECL Western Blotting Detection Reagent

(Amersham) or Supersignal West Femto Maximum Sensitivity Substrate (Pierce).

Intracellular NF-κB localization and cell-associated conidia in bone marrow derived macrophages (BMM)

Mice underwent euthanasia by CO2 asphyxiation, and femurs and tibias from 129SvEv and Dectin-1-/- mice were removed, cleaned, and centrifuged at 5000 x g for 45 s at 4˚C.

Any contaminating red blood cells were lysed in 5 ml RBC Lysis Buffer (eBioscience), and remaining bone marrow cells were cultured in bacteriologic grade petri dishes in 6 ml Macrophage Growth Medium (MGM:DMEM w/ L- Glutamine, Na-Pyruvate,

HEPES,10% FBS, P/S, 30% L929 cell conditioned medium). On day 5, and every 2 days thereafter, the cell supernatant was aspirated, and fresh MGM media was added.

Adherent cells were harvested between 7-14 days of culture, and counted. 2 x 104 cells were cultured onto sterile 18mm2 coverslips (Corning) in a 6 well-plate, and treated with LPS (100 ng/ml; positive control) or 6h A.fumigatus strain Af-BP swollen conidia

81 (MOI=100) for 15, 30 and 60 min. Following activation, BMM were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized using 0.1% Triton X-

100 in PBS for 1 min at RT, and incubated with rabbit anti-mouse p65 (1:100; eBioscience Ltd) in PBS containing 10% goat serum for 1h at RT. Coverslips were washed 2X with PBS and cells were incubated with Alexa Fluor 488-labeled goat anti- rabbit IgG antibody (Molecular Probes Inc.) in PBS at RT for 1h and washed 2x with

PBS. The cells were mounted on glass slides using Vectashield mounting medium with

DAPI (Vector Laboratories, UK), and examined by fluorescence microscopy, and average pixel intensity (green fluorescence) per nucleus (DAPI) was calculated using

Metamorph.

For cell association studies, bone marrow macrophages incubated with A.fumigatus strain Af-BP swollen conidia were washed and fixed in 4% paraformaldehyde as described above, then incubated 5 min with Calcufluor white (Sigma) at a 1:1 ratio with

10% KOH. After washing 2x with PBS, cells were examined by DIC and fluorescence microscopy, and associated conidia per 100 cells, and the percent cells with associated conidia was determined after direct examination of at least 300 cells per coverslip. Two coverslips were examined, and the mean and SD were calculated.

Results

Development of a murine model of Aspergillus keratitis

To visualize A.fumigatus during infection of the transparent mammalian cornea, we generated a monomeric dsRED RFP-expressing A. fumigatus strain. Figure S1 illustrates the structure of the plasmid, and shows expression of RFP at each lifecycle

82 stage of Aspergillus. To determine the role of the host response to these organisms in the cornea, C57BL/6 mice were either treated systemically with cyclophosphamide

(cyc), or were left untreated prior to injecting 1 x 105 Af293.1RFP conidia into the corneal stroma (Preliminary studies revealed that 1 x 105 conidia was the smallest inoculum in which organisms could be recovered from the cornea after 24h). Corneal opacification, fungal growth, survival, and cellular infiltration were assessed at each time point. Figure 1A shows that at 24h post-infection, immunocompetent C57BL/6 mice developed significant corneal opacity, which peaked at 48h, and persisted up to

72h post-infection. However, cyclophosphamide-treated mice had significantly lower corneal opacification scores than immunocompetent mice at 48 and 72h post-infection.

To characterize the host response in Aspergillus keratitis, 5µm corneal sections were stained with PASH and examined by brightfield microscopy. As shown in Figure

1B, there was a pronounced cellular infiltration in the corneas of immunocompetent mice between 24 and 72h, with neutrophils also present in the anterior chamber (for comparison, a PASH stained section of a normal mouse cornea is shown in

Supplementary Figure 2A). In contrast, there was less cellular infiltration in corneas of cyclophosphamide-treated mice at each time point. There was also enhanced fungal penetration though Descemet’s membrane and into the anterior chamber at 48 and 72h post-infection, and increased epithelial debridement in cyclophosphamide treated mice compared with immunocompetent mice. Figure 1C shows that

cells in the C57BL/6 cornea are NIMP-R14+ neutrophils, and that the numbers increased over time. Neutrophils were not detected in corneas of cyclophosphamide- treated mice at any time post-infection, and in contrast to immunocompetent mice,

83 longer-term infection of treated mice results in corneal perforation (data not shown).

To quantify corneal opacification and hypertrophic and filamentous fungal growth during corneal infection, we used image analysis software that provides numerical output for corneal opacification and for fungal dsRed expression for all animals in the experiment rather than representative mice. Figure 1D shows a representative image analysis derivation, which shows color (pixel) intensity. Using this method, we found that opacification was significantly lower in cyclophosphamide treated animals (Figure 1E). Conversely, fungal dsRed expression was significantly elevated in corneas of cyclophosphamide-treated mice compared with immunocompetent mice at each time point (Figure 1F). There was no detectable red fluorescence in uninfected mice (Figure S2). To examine fungal viability, eyes were homogenized, and the number of colony forming units (CFU) was determined by standard methods. Figure 1G depicts a 1 log-fold reduction in fungal CFU in immunocompetent mice between 48h and 72h post-infection, whereas CFU were not reduced in cyclophosphamide-treated C57BL/6 corneas over this time period.

Together, these findings identify a resistant phenotype in immunocompetent animals, where there is a pronounced neutrophil infiltration to the corneal stroma, resulting in controlled fungal growth. These findings also show a susceptible phenotype illustrated by cyclophosphamide treated mice, where neutrophil recruitment to the corneal stroma is impaired, fungal growth continues unabated, and the cornea eventually perforates.

84 Resident corneal c-fms+ macrophages and dendritic cells mediate cellular recruitment during Aspergillus keratitis

The predominant cells in the corneal stroma are keratocytes, which produce collagen and proteoglycans that comprise the extracellular matrix; however there is also a population of resident macrophages and dendritic cells at this site (29, 171). To determine if resident macrophages and dendritic cells mediate the initial recognition of

A. fumigatus in the cornea, we utilized Macrophage Fas Induced Apoptosis (Mafia) mice, which express eGFP and a membrane bound suicide protein under control of the myeloid-lineage specific c-fms promoter (163, 170). In these mice, all macrophages and dendritic cells express eGFP constitutively, and undergo cell-lineage specific apoptosis after cross-linking the FK506 binding domain of the membrane-bound suicide protein using the FK506 dimerizer AP20187 (163).

We showed previously that eGFP+ macrophages/dendritic cells are readily identified in the corneas of naïve untreated Mafia mice, and that eGFP expressing cells are depleted in AP20187-treated Mafia mice (170). To ascertain the role of macrophages and dendritic cells in Aspergillus keratitis, Mafia mice were treated with

AP20187, and infected with 1 x 105 Af293.1RFP conidia.

85 Figure 3.1 Aspergillus fumigatus keratitis in cyclophosphamide treated mice

A mouse model of Aspergillus keratitis was developed by injecting 1 x 105 Af293.1RFP conidia into the corneal stroma, and tracking disease progression over time. A. Slit- lamp microscopy of immunocompetent and cyclophosphamide-immunosuppressed (Cyc) C57BL/6J corneas infected for 24, 48, and 72h. Fluorescence microscopy of emitted 580nm light reveals fungal growth in the corresponding cornea. B. 5µm paraffin section of the central cornea were stained with Periodic Acid Schiff and Hematoxylin (PASH) to visualize cellular infiltration, fungal growth, and corneal pathology at 24, 48, and 72 h post-infection. C. 5µm paraffin sections were immunostained with anti-neutrophil NIMPR-14 to visualize neutrophil infiltration at 24, 48, and 72 h post-infection. D. Bright-light reflected from the cornea, and 580nm light emitted from the cornea were independently captured as TIFF images and the pixel intensity of each image was analyzed within a constant defined region encompassing the cornea to quantify corneal opacity or fungal RFP expression (corresponding to fungal biomass), respectively. E. Corneal opacity in immunocompetent and cyc-immunosuppressed C57BL/6 mice at 24-72h post-infection F. Fungal RFP expression in immunocompetent and cyc-immunosuppressed C57BL/6 mice at 24 and 72h post-infection. G. At each time point, eyes were enucleated, homogenized, and fungal viability assessed via serial dilutions on Sabouraud Dextrose agar. Data are representative of two independent experiments, with five mice per time point. Reprint permission obtained from publisher.

86 Corneas of untreated Mafia mice developed opacification at 24 and 48h after infection, consistent with increased eGFP+ cellular infiltration to the corneal stroma and dsRed expressing Aspergillus hyphae (Figure 2A). In contrast, corneas of AP20187-treated

Mafia mice had decreased opacification, and significantly increased dsRed expressing

Aspergillus hyphae (Figures 2A). Cellular infiltration to the corneal stroma was absent in infected AP20187-treated Mafia mice as shown by the absence of eGFP+ cells

(Figure 2A) and in histological sections (Figure 2B).

To determine if the impaired cellular infiltration is related to pro-inflammatory and chemotactic cytokine production, corneas were dissected from untreated and

AP20187-treated Mafia mice at 10h after intrastromal injection of Af293.1RFP conidia or PBS (trauma control), and prior to detectable cellular infiltration. Corneas were homogenized, and CXCL1/KC and IL-1ß production were examined by ELISA. Figure

2C shows that both CXCL1/KC and IL-1 were elevated in untreated, but not

AP20187-treated corneas from Mafia mice.

Figures 2D-F show image analysis based quantification of corneal opacification, eGFP+ cell infiltration, and RFP expression for individual corneas in the experiment and reveal significantly lower corneal opacification (Figure 2D) and eGFP+ cell infiltration (Figure 2E) in AP20187-treated Mafia mice. Conversely, dsRed hyphae (Figure 2E) and CFU (Figure 2F) were significantly higher in corneas of

AP20187-treated Mafia mice compared with untreated Mafia mice, indicating that cfms+ cells regulate fungal growth and survival (Figure 2G).

87 Taken together, these data show that resident c-fms+ macrophages and dendritic cells in the naïve cornea produce IL-1ß and CXCL1/KC, which likely mediate neutrophil recruitment into the cornea, and subsequently limit Aspergillus growth and survival.

Spleen tyrosine kinase (Syk) is phosphorylated during Aspergillus keratitis

As resident cfms+ macrophages and dendritic cells are essential for early cytokine production in the cornea after Aspergillus infection, they are likely the first cells to respond to germinating conidia. We therefore examined the pathogen recognition molecules initiating this response, including Dectin-1, TLR2 and TLR4.

Dectin-1 is a C-type lectin expressed by myeloid-derived cells that recognizes ß-glucan when it is exposed on the cell wall, and ß-glucan expression in Aspergillus occurs in germinating, but not dormant conidia, and in hyphal stages (60, 61, 125). We examined

ß-glucan and Dectin-1 expression and activation in the cornea after Aspergillus infection. As shown in Figure 3A, PASH stained corneas revealed swollen conidia (6h) and hyphae (24h); further, ß-glucan expression was apparent in both forms, especially at 24h when hyphal forms predominated, indicating that Aspergillus expresses the ligand for Dectin-1 during corneal infection.

To determine the effect of Aspergillus on Dectin-1 protein levels in the cornea, we dissected corneas 10h after infection or after injection with PBS (trauma controls), and processed the corneas for western blot analysis. Dectin-1 was not detected in naïve and PBS-injected corneas; however, as early as 10h after Aspergillus infection, Dectin-

1 was clearly expressed in the cornea (Figure 3B) Further, phosphorylated Spleen

88 Tyrosine Kinase (Syk) was elevated in infected corneas, compared with naïve and trauma controls (Figure 3C). Given that Syk phosphorylation is indicative of Dectin-1 activation (172), these findings indicate that not only is Dectin-1 expressed in the cornea, but that it is also activated during Aspergillus infection.

Dectin-1 mediates cytokine production and cellular infiltration after infection with

Af293.1RFP

As we found -glucan expression in vivo, and increased Dectin-1 / pSyk activation in infected corneas, we next examined the role of Dectin-1 during

Aspergillus keratitis. Dectin-1-/- and control 129SvEv mice were injected intrastromally with Af293.1RFP conidia, and corneal opacification, cellular infiltration, and fungal survival were measured as before. As shown in Figure 4A, corneal opacification was evident in 129SvEv corneas by 24h post-infection, increased at 48h and decreased at

72h, whereas corneal opacification was significantly lower in Dectin-1-/- mice at 24h and 48h. However, there were no significant differences between Dectin-1-/- and control, 129SvEv corneas in fungal dsRed expression (Figure 4A). To determine the role of Dectin-1 in cytokine production and cellular infiltration to the cornea, Dectin-1-/- and 129SvEv mice were infected as before, eyes were processed for histology, and 5µm sections were stained with PASH. As shown in Figure 4B, cellular infiltration in

129SvEv mice was similar to infected C57BL/6 mice, with pronounced cellular infiltration at each time point. In contrast, Dectin-1-/- corneas had impaired cellular infiltration at each time point. To examine if the decreased cellular infiltration in

Dectin-1-/- mice was associated with pro-inflammatory and chemotactic cytokine

89 Figure 3.2 The role of macrophages in A. fumigatus keratitis

Mafia mice were used to identify the resident corneal cell-types responsible for recognizing A.fumigatus and initiating cellular recruitment into the cornea A. Brightfield microscopy of Mafia mice +/- 5 day AP20187- dimerizer treatment at 24 and 48h post-infection. 488 nm fluorescence images indicate monocytic recruitment into the cornea and 580nm fluorescence indicates fungal RFP expression. B. 5µm sections of the central cornea at 24 and 48h post-infection were PASH stained and used to visualize cellular infiltration, fungal growth, and corneal pathology in treated vs. untreated Mafia mice. C. ELISA analysis at 10h post-infection in treated vs. untreated Mafia mice for CXCL1/KC and IL-1β. D. Corneal opacity in treated vs. untreated Mafia mice was objectively quantified using Metamorph software. E. c-fms+ cell eGFP expression and F. Fungal RFP expression were quantified at 24 and 48h post-infection. G. Eyes were enucleated, homogenized and subjected to serial plating and CFU quantification. Data are representative of two independent experiments, with five mice per time point. Reprint permission obtained from publisher.

90 production, corneas were dissected and homogenized at 10h post-infection (prior to detectable cellular infiltration), and CXCL1/KC and IL-1ß were measured by ELISA.

Consistent with impaired cellular infiltration, infected Dectin-1-/- corneas had significantly less CXCL1/KC and IL-1 compared with 129SvEv mice (Figure 4C).

Quantification by image analysis shows elevated corneal opacification in 129SvEv compared with Dectin-1-/- mice 24h and 48h after infection (Figures 4D), but no difference in fungal RFP expression (Figures 4E). Consistent with the latter observation, there were no differences in fungal CFUs between Dectin-1-/- and 129SvEv mice at any time point examined (Figure 4F).

These findings demonstrate that Dectin-1 regulates cytokine production in the cornea, cellular infiltration to the corneal stroma and development of corneal opacification; however, Dectin-1 expression was not required to regulate growth and survival of strain Af293.1RFP.

91 Figure 3.3 ß-glucan expression and Dectin-1 signaling in A. fumigatus keratitis

Fungal ß-glucan expression and Dectin-1 protein activation in the cornea during Aspergillus infection were assessed by IHC and western blot analysis, respectively. A. 5µm central cornea sections at 6 and 24h post-infection were PASH stained to visualize swollen conidia (6h) and hyphae (24h), while ß-glucan expression was detected using a mouse anti-fungal ß-glucan antibody. B. Western blot analysis using anti-Dectin-1 antibody or C. anti-pSyk antibody was performed on cornea protein lysates from naïve, 10h PBS injected (trauma control), and 10hr A. fumigatus infected corneas. Data are representative of two independent experiments, with three corneas per time point. [Endo- endothelium Epi- epithelium, Neuts- casein-elicited peritoneal neutrophils. MACs- thioglycolate-elicited peritoneal, macrophages. Reprint permission obtained from publisher.

92 Figure 3.4 Role of Dectin-1 in keratitis caused by A. fumigatus Af293.1RFP

To determine the role of Dectin-1 in recognizing A.fumigatus and initiating cellular recruitment into the cornea, we injected 1 x 105 Af293.1RFP conidia into the stroma of 129SvEv and Dectin-1-/- mice. A. Brightfield and fluorescence microscopy of corneas from 129SvEv and Dectin-1-/- mice at 24, 48, and 72h post-infection. B. 5µm PASH stained central cornea sections of infected 129SvEv and Dectin-1-/- mice at 24, 48, 72h reveal the extent of cellular infiltration, fungal growth, and corneal pathology in the absence of Dectin-1. C. ELISA analysis at 10h post-infection in 129SvEv vs. Dectin-1-/- mice for CXCL1/KC and IL-1β. D. Corneal opacity, E. Fungal RFP expression, and F. Fungal viability at set time-points post-infection. Data is representative of 5 independent experiments, 5 mice per time point. Reprint permission obtained from publisher.

93 Dectin-1 regulates growth and survival of high -glucan expressing A.fumigatus clinical isolates

Failure to detect increased survival of Af293.1RFP in Dectin-1-/- mice, despite decreased cellular infiltration at all stages post-infection, led us to hypothesize that fungal survival in Dectin-1-/- mice is dependent on the virulence of the infecting

A.fumigatus strain. We therefore infected Dectin-1-/- and 129SvEv mice with a clinical isolate from a patient with fungal keratitis. Injection of Strain Af-BP into the 129SvEv cornea induced corneal opacification at 24h post-infection, which increased after 48 and

72h (Figure 5A). In contrast to strain Af293.1RFP, the Af-BP isolate also caused engorgement of limbal blood vessels after 48h, and hemorrhage after 72h post-infection

(Figure 5A), and may be reflective of the characteristic angio-invasiveness of clinical

A.fumigatus isolates (173). Dectin-1-/- mice exhibited impaired cellular infiltration

(Figure 5B), and had significantly lower corneal opacification at 24 and 48h post- infection with Strain Af-BP (Figure 5C), which was similar to infection with strain

Af293.1RFP. However, in contrast to infection with Af293.1RFP, fungal CFUs recovered from infected corneas were significantly elevated in Dectin-1-/- compared with 129SvEv mice at 48 and 72h post-infection (Figure 5D). There were no apparent structural differences between naïve Dectin-1-/- and wild type corneas (data not shown).

To determine if Dectin-1 regulation of Af-BP but not Af293.1RFP survival is related to

-glucan surface expression, we cultured strains Af293.1RFP, Af-BP, and the lung isolate B-5233 in SDA media for 6h or 10h, and examined ß-glucan expression as described above. ß-glucan was not detected on resting conidia of Af293.1RFP or the clinical isolates (data not shown). However, at 6 h (swollen and germinating conidia),

94 there was significantly higher surface ß-glucan expression in the clinical isolates Af-BP and B-5233 compared with laboratory strain Af293.1RFP (Figure 5E, F). ß-glucan surface expression on hyphae after 10h growth was also quantified; as shown in Figure

5G,H, there were no significant differences among these strains at this time point.

These findings indicate that strains with high surface ß-glucan expression on germinating conidia are more likely to be detected by Dectin-1 and to induce a more pronounced cellular infiltrate, resulting in increased fungal killing.

Dectin-1 regulates A.fumigatus activation of bone marrow macrophages

Since macrophage depleted Mafia mice and Dectin-1-/- mice exhibited impaired cellular recruitment to the cornea during Aspergillus infection, we next examined the role of Dectin-1 in activation of bone marrow derived macrophages (BMMs) by

A.fumigatus. Af-BP swollen conidia were isolated after 6h incubation with Sabouraud dextrose media, fixed, and incubated with 129SvEv or Dectin-1-/- BMMs at an

MOI=100. After 15, 30, and 60 min, cells were lysed, and Syk and Ib phosphorylation were detected by western blot analysis, and NFB nuclear translocation was examined after incubation with anti-p65 antibody. Figure 6A shows Syk phosphorylation in

129SvEv BMMs after 15 min incubation, which was sustained for 30 and 60 min. In contrast, p-Syk was not detected in Dectin-1-/- BMMs until 60 min post-exposure. P-

Syk was not detected in naïve BMM or after LPS stimulation. Similarly, IB

95 Figure 3.5 Role of Dectin-1 in keratitis caused by a clinical isolate of A. fumigatus

To determine the role of Dectin-1 in Aspergillus keratitis during infection with a clinical isolate we injected 1 x 105 conidia from Strain Af-BP into the corneal stroma and tracked disease progression over time. A. Brightfield microscopy of corneas from 129SvEv and Dectin-1-/- mice at 24, 48, and 72h post-infection B. 5µm PASH stained central cornea sections of infected 129SvEv and Dectin-1-/- mice C. Corneal opacity , and D. Fungal viability at set time-points post-infection. Data is representative of 2 independent experiments, 10 mice per time point. E,G. Mouse anti-fungal ß-glucan IgM was used to detect ß-glucan surface expression in A.fumigatus swollen conidia/germlings (Strains Af293.1RFP, Af-BP-KO, B-5233) grown for 6h (E), or 10h (G) in SDA media. F,H. Quantification of ß-glucan surface expression in Strains Af293.1RFP, Af-BP-KO, and B-5233 grown for 6h (F) and 10h (H) in SDA media. ß-

96 glucan expression was determined using Metamorph software. Data are representative of two independent experiments. Reprint permission obtained from publisher. MOI=100. After 15, 30, and 60 min, cells were lysed, and Syk and Ib phosphorylation were detected by western blot analysis, and NFB nuclear translocation was examined after incubation with anti-p65 antibody.

97 phosphorylation was elevated in 129SvEv BMMs after 15 min incubation with swollen conidia compared with naïve BMM; however, elevated P-Syk was not detected in

Dectin-1-/- BMMs until 60 min incubation (Figure 6A). Additionally, A.fumigatus- induced CXCL1/KC production by BMMs from 129SvEv mice was significantly higher than BMMs from Dectin-1-/- mice (Figure 6B). As shown in Figure 6C, D, NF-

κB p65 nuclear translocation was apparent in 129SvEv BMM after 30 min and 60 min, but not in Dectin-1-/- BMMs within this time period. As cell-associated conidia were detected in the p65 translocation experiments, and Dectin-1 – mediated phagocytosis has been shown to inhibit cell signaling (174), we examined the effect of Dectin-1 on cell associated conidia. BMM were incubated with swollen conidia at 100 MOI as described above, then incubated with Calcofluor white to identify conidia (175). Cell association was examined by DIC and fluorescence microscopy, and quantified by direct counting. As shown in Figures 6E-G, Dectin-1-/- BMM had significantly less conidia associated with each macrophage than 129SvEv BMMs (Figure 6F). Similarly, the percent of total Dectin-1-/- BMM macrophages with associated conidia was also significantly lower than 129SvEv BMMs (Figure 6G).

Together, these data indicate that A .fumigatus conidia bind Dectin-1 on resident corneal macrophages, which then stimulates Syk and NFκB–dependent production of

CXCL1/KC that is important for neutrophil recruitment to the cornea.

A.fumigatus killing in the cornea is dependent on TLR4, but not TLR2 or MD-2 To determine the role of TLR2 and TLR4 in Aspergillus keratitis, C57BL/6, TLR2-/- and TLR4-/- mice were injected intrastromally with Af293.1RFP conidia, and corneal opacification, cellular infiltration, fungal growth and fungal survival were measured as

98 described above. As depicted in Figure 7A-C, there were no significant differences between TLR2-/- and C57BL/6 mice in any of these parameters, indicating that TLR2 has no role in corneal infection with these organisms. Similar results were obtained with clinical isolate Af-BP (data not shown).

Figure 7D, E illustrate that as with TLR2-/- mice, there were no significant differences in corneal opacification, cellular infiltration or fungal RFP expression between TLR4-/- and C57BL/6 corneas; however, in marked contrast to TLR2-/- mice, significantly more CFU were recovered from TLR4-/- mice after 48h compared with

C57BL/6 mice (Figure 7F), indicating an impaired ability of TLR4-/- mice to clear the infection, and suggesting a role for TLR4 in fungal killing. Increased CFU was also detected in TLR4-/- corneas infected with the Af-BP clinical isolate at 48h and 72h after infection (Table 1), indicating that the role for TLR4 is consistent among Af strains.

Despite the difference in CFU, there was no difference in cellular infiltration between

TLR4-/- and C57BL/6 and TLR2-/- corneas (Figure 7G, H), nor any differences in fungal RFP expression (Figure 7I, J).

Given the role for TLR4 in fungal viability, we also examined the role of TLR4 co-receptor MD-2, which binds of Gram negative bacteria. Corneas of

C57BL/6, TLR4-/-, and MD-2-/- corneas were infected with Af293.1RFP, and CFU were quantified after 24, 48, and 72h. As before, CFU recovered from corneas of TLR4-/- mice were significantly higher than C57BL/6 at 72h post-infection Figure 7K.

However, despite the documented role for MD-2 in responding to LPS, there was no difference in fungal survival in MD-2-/- compared with C57BL/6J mice, indicating that the role of TLR4 fungal killing is MD-2 independent.

99 Figure 3.6 Role of Dectin-1 in activation of bone marrow-derived macrophages.

Bone marrow-derived macrophages (BMM) from 129SvEv and Dectin-1-/- mice were incubated with A.fumigatus swollen conidia Strain Af-BP (MOI=100). A. Protein lysates from 0, 15, 30, and 60 min stimulated 129SvEv and Dectin-1-/- BMMs were examined via western blot analysis, using anti-pSyk and anti-pIB antibodies. B. 129SvEv and Dectin-1-/- BMM culture supernatants were harvested at 60 min post conidia exposure and CXCL1/KC levels assessed via ELISA. C. 129SvEv and Dectin- 1-/- BMMs were seeded onto coverslips, and exposed to conidia for 0, 15, 30, and 60 min. Subsequently, BMMs were fixed, permeabilized, stained with anti p65 primary antibody, and Alexafluor-488 tagged anti-rabbit secondary antibody, and visualized via fluorescence microscopy (40X). D. Image analysis using Metamorph Software was subsequently used to quantify p65 translocation to the nucleus of stimulated 129SvEv and Dectin-1-/- BMMs. E. Representative fields of 129SvEv and Dectin-1-/- BMMs 1h after incubation with conidia. Coverslips were incubated with Calcufluor white to detect conidia, and examined by DIC and fluorescence microscopy (conidia are visualized as blue). F. Number of conidia per 100 BMM; G. Percent BMM with associated conidia. At least 300 cells were examined in each group. Data represent mean +/- SEM of two BMM cultures, and similar results were found in two repeat experiments. Reprint permission obtained from publisher.

100 Cellular infiltration and fungal killing is dependent on MyD88, but not TIRAP or

TRIF,

Given that fungal survival is dependent on TLR4, and TLR4 signaling involves the adaptor molecules MyD88, MAL/TIRAP and TRIF (MyD88: myeloid differentiation primary- response gene 88; TIRAP: toll-interleukin 1 receptor domain containing adaptor protein, which is also called MAL:MyD88-adaptor-like protein;

TRIF: TIR-domain-containing adaptor protein inducing IFNβ (176)), we next examined the role of these adaptor molecules in cellular infiltration and fungal killing. C57BL/6,

MyD88-/-, TIRAP -/- and TRIF-/- mice were injected intrastromally with Af293.1RFP conidia, and markers of infection were examined as before.

Figure 8A shows that MyD88-/- mice had less corneal opacification scores at

24h compared with C57BL/6 mice, but not 48h after infection; conversely, MyD88-/- corneas also had increased fungal RFP (Figure 8A). We found impaired cellular infiltration in the corneal stroma of MyD88-/- mice compared with C57BL/6 mice at 24h

(Figure 8B); however, at 48h, there was an intense cellular infiltration into the MyD88-

/- corneas. Image analysis shows significantly lower corneal opacification (Figure 8C), but higher fungal RFP expression (Figure 8D) in MyD88-/- mice versus C57BL/6 mice.

Consistent with the latter observation, Figure 8F shows significantly increased fungal

CFU at 48h post-infection in MyD88-/-mice. These data demonstrate that MyD88 regulates early cellular infiltration and fungal survival in Aspergillus keratitis. Further, even though cellular infiltration is detected in MyD88-/-mice after 48h, there was no difference in CFU, indicating a role for MyD88 on the ability of neutrophils and infiltrating macrophages to kill Aspergillus. To determine if MyD88 is due to TLR4

101 signaling, we infected mice deficient in TIRAP, which is essential for MyD88 signaling by TLR4. We also infected mice deficient in TRIF, which mediates the TLR4 (and

TLR3), MyD88-independent signaling pathway (48). Figure 9A shows that TRIF-/- and

TIRAP-/- mice develop corneal opacification that is not different from C57BL/6 mice.

Similarly, the presence of dsRed Aspergillus in the corneas of TRIF-/- and TIRAP-/- mice was similar to C57BL/6 mice. Image analysis also showed no difference in opacity or dsRed expression, respectively, between C57BL/6, TRIF-/-, and TIRAP-/- mice (Figure 9B, C). In addition, there was no difference in fungal CFU among the three strains at either 24h or 48h (Figure 9D) or in cellular infiltration among these strains (Figure 9E). These findings indicate that although MyD88 has a critical role in

Aspergillus keratitis, there is no apparent involvement of TIRAP or TRIF.

IL-1R1 mediates cellular infiltration to the corneal stroma in Aspergillus keratitis

The role of MyD88, but not TIRAP in Aspergillus keratitis led us to conjecture that the

IL-1 receptor (IL-1R1), which signals through MyD88 independently of TIRAP, could mediate cellular recruitment during Aspergillus keratitis. To test this hypothesis, we injected 1 x 105 A. fumigatus conidia into the corneas of C57BL/6 and IL-1R1-/- mice.

Figure 10A shows that IL-1R1-/- mice had significantly lower corneal opacification and increased fungal RFP expression and survival at 24 and 48h post-infection. Conversely, there was significantly less cellular infiltration into the corneal stroma of IL-1R1-/-mice compared with C57BL/6 mice (Figure 10B). Image analysis revealed decreased corneal opacity at 24h (Figure 10C) and increased fungal dsRed expression at 48h

102 Figure 3.7 The role of TLR2, TLR4, and MD-2 in A. fumigatus keratitis

Corneas of TLR2-/-, TLR4-/-, MD-2-/-, and C57BL/6 mice were infected with A.fumigatus conidia as described above. A. Brightfield and fluorescence microscopy of corneas from TLR2-/- and C57BL/6 mice infected for 24 and 48h with strain Af293.1RFP. B. Corneal opacity quantification and C. Fungal viability post-infection in TLR2-/- and C57BL/6 mice. D. Brightfield and fluorescence microscopy of corneas from TLR4-/- and C57BL/6 mice infected with Af293.1RFP. E. Corneal opacity quantification and F. Fungal CFU post-infection of TLR4-/- and C57BL/6 mice. Note increased fungal survival at 48 hr post-infection in TLR4-/- mice. G. 5µm PASH stained central cornea sections of infected TLR2-/- and H. infected TLR4-/- and C57BL/6 corneas at 24 and 48 h post-infection. I. Fungal RFP expression in TLR2-/- and C57BL/6 mice post-infection. J. Fungal RFP expression in TLR4-/- and C57BL/6 mice post-infection. K. Fungal viability at 4h and 72h post-infection of MD2-/-, TLR4-/-, and C57BL/6 mice infected with A.fumigatus Strain Af-BP. Data are representative of six independent experiments, with five mice per time point. Reprint permission obtained from publisher.

103 Table 3.1 Fungal infection of TLR4-/- mice

C57BL/6 and TLR4-/- mice were infected with 1 x 105 A.fumigatus conidia (Strain Af293.1RFP or Af-BP). At set time-points post-infection, eyes were enucleated and plated on SDA media for CFU analysis. Data from three experiments are shown indicating the time post-infection (Time) in which a difference between C57BL/6 and TLR4 -/- mice was observed, colony forming units (CFU), standard error of the mean (SEM), and population size (n) per experiment. Reprint permission obtained from publisher.

104 Figure 3.8 The role of MyD88 in A. fumigatus keratitis

C57BL/6 and MyD88-/- mice were infected with Af293.1RFP as before, and examined at 24 and 48h post-infection A. Representative images from brightfield and fluorescence microscopy of MyD88-/- and C57BL/6 corneas. B. PASH stained 5 µm central corneal sections of infected MyD88-/- and C57BL/6 mice 24h and 48h post- infection. C. Corneal opacity quantification, D. Fungal RFP expression, and E. Fungal viability. Data are representative of two independent experiments, with five mice per time point. Reprint permission obtained from publisher.

105

post-infection in IL-1R1-/- mice compared to C57BL/6 mice (Figure 10D).

Correspondingly, Figure 10E shows increased fungal CFU 48h post-infection in IL-

1R1-/- mice compared to C57BL/6 mice. Taken together, these observations indicate that IL-1R1 mediates early cellular infiltration and fungal survival in Aspergillus keratitis, which is a similar phenotype to MyD88-/- mice.

Discussion:

Aspergillus is a major cause of visual impairment and blindness worldwide; however, the nature of the host response to these organisms in the cornea is not well understood. Our findings show that critical components of the innate immune response in the cornea include c-fms+ macrophages and dendritic cells in addition to Dectin-1,

TLR4, MyD88, and IL-1R1. We also showed that there was no role for MD-2, TLR2,

TIRAP or TRIF. Taken together, these observations are consistent with a sequence of events that is initiated by expression of β-glucan on germinating conidia in the corneal stroma, and recognition by Dectin-1 expressed on resident corneal macrophages and dendritic cells. Dectin-1 mediated activation of p-Syk, p-IB, and translocation of

NFB to the nucleus of these cells results in production of CXCL1/KC and IL-1β within 10h of infection, and recruitment of neutrophils to the corneal stroma.

Neutrophils kill Aspergillus by attaching to hyphae and releasing cytotoxic proteases, antimicrobial peptides, and reactive oxygen species (92). Although this inflammatory response causes corneal opacification, the corneas eventually heal, leaving scarified

106 tissue. However, blockade of this response at any of these stages allows the organisms to grow unimpaired, resulting in corneal perforation.

Germinating Aspergillus conidia encounter a dense, highly organized matrix in the corneal stroma, with anti-parallel layers of collagen separated by keratan sulfate proteoglycans that are essential for corneal transparency; however, the growing hyphae migrate through the stromal matrix and penetrate the basement (Descemet’s) membrane of the corneal endothelium, which forms the barrier to the anterior chamber. Unless killed by neutrophils entering the anterior chamber from iris vessels (seen as a hypopyon in infected individuals), hyphae can penetrate the posterior eye and cause endophthalmitis, at which point enucleation of the infected eye is often indicated.

Although the cornea was long considered to be an immune privileged tissue, it has been well established that macrophages and dendritic cells are resident in the corneal stroma and epithelium (41-46). Recently, a role for macrophages in mediating

Fusarium and Candida keratitis was identified (116). Similarly, in the present study we

107 Figure 3.9 The role of TIRAP and TRIF in A. fumigatus keratitis

Corneas of TIRAP -/-, TRIF-/-, and C57BL/6 mice were abraded and infected with Af293.1RFP. A. Representative brightfield and fluorescence microscopy of corneas at 24 and 48h post-infection. B. Corneal opacity quantification, C. Fungal RFP expression, and D. Fungal viability post-infection. E. 5µm PASH stained central cornea sections of infected Mal/TIRAP -/-, TRIF-/-, and C57BL/6 mice post-infection. Data are representative of three independent experiments, with five mice per time point. Reprint permission obtained from publisher.

108 Figure 3.10 The role of IL-1R1 in A. fumigatus keratitis

Corneas of IL-1R1-/- and C57BL/6 mice were infected with Af293.1RFP as before, and examined after 24h and 48h. A. Representative brightfield and fluorescence microscopy of corneas from IL-1R1-/- and C57BL/6 mice post-infection. B. 5 µm PASH stained central cornea sections of infected IL-1R1-/- and C57BL/6 mice at 24h and 48h post- infection. C. quantification of corneal opacity, D. Fungal RFP expression, and E. Fungal viability. Data are combined from two repeat experiments. Reprint permission obtained from publisher.

109 Figure 3.S1 Construction of RFP-Aspergillus fumigatus.

An improved monomeric dsRED RFP expressing A. fumigatus strain was developed to visualize A.fumigatus during live tissue infection. A. Primers used in construction of pRG3AMA1-RFP are listed. GTAC= Kpn1 recognition cut site; Italicized = DNA complementary to rfp on RgpdR and DNA complementary to gpdA on GrfpR B. The plasmid pRG3AMA1-RFP harbors rfp downstream of the constitutive Glyceraldehyde 3 phosphate dehydrogenase promotor (gpdA), allowing constant visualization of A.fumigatus under fluorescence microscopy C. Af293.1RFP shows similar morphological developmental progression from conidiaswollen conidia germ tube hyphae mycelial mass as the parental strain Af293.1 Insets shows micrographs of the individual morphological growth stages of Aspergillus fumigatus. Reprint permission obtained from publisher.

110 Figure 3.S2 Normal mouse cornea histology and eye

A. Normal mouse cornea showing epithelium (epi), corneal stroma, corneal endothelium (endo) and anterior chamber. B. 580 nm fluorescence image of naïve mouse cornea showing no background emission in the RFP spectrum. Reprint permission obtained from publisher.

111 identified c-fms+ resident macrophages and dendritic cells as essential mediators of cellular recruitment and fungal survival into the cornea during A.fumigatus infection, implicating bone marrow derived cells rather than corneal epithelial cells or fibroblasts as the cellular mediators of innate immune recognition (116).

In the current study, we found that ß-glucan is not only expressed on Aspergillus germinating conidia and hyphae in vitro as shown previously (60, 61, 125), but is also expressed during corneal infection. Furthermore, Syk phosphorylation was detected in infected corneas, and Syk and IκB were phosphorylated in bone marrow macrophages in a Dectin-1 dependent manner. Correspondingly, both NFκB translocation to the nucleus, and CXCL1/KC and IL-1β production were significantly lower in Dectin-1 deficient compared with control macrophages. Similarly, Dectin-1-/- corneas had impaired pro-inflammatory cytokine production and cellular infiltration compared with control corneas. These findings indicate that expression of Dectin-1 on resident corneal macrophages is essential for the initial innate immune recognition of A.fumigatus, for the subsequent pro-inflammatory cytokine response, and ultimately for cellular recruitment into the cornea.

We also found that the physical association of swollen conidia with bone marrow macrophages, which includes attachment and phagocytosis, is dependent on

Dectin-1 expression. This observation is consistent with reports showing Dectin-1 dependent phagocytosis of ß-glucan coated particles (174, 177), and that phagocytosis inhibits Dectin-1 signaling (174), indicating that macrophage interactions with hyphae that are too big to be ingested may have enhanced signaling compared with conidial forms. Our findings also indicate a possible role for Dectin 1 in early killing of swollen

112 conidia and germ tubes rather than hyphae, where we found no differences in ß-glucan staining among the A. fumigatus strains. Taken together, it is likely that during corneal infection, macrophages and neutrophils utilize Dectin-1 to bind conidia and germ tube developmental stages of A.fumigatus, which can be phagocytosed and presumably killed.

Results from the current study are consistent with reports on pulmonary aspergillosis in which germinating Aspergillus conidia in the lungs express ß-glucan, and Dectin-1 mediates cellular infiltration and fungal killing (60, 125). Our data are also in agreement with a recent study showing that Dectin-1-/- mice have lower cytokine and chemokine production in the lungs after intratracheal infection, resulting in impaired neutrophil recruitment and increased susceptibility to Aspergillus (129). The role of

Dectin-1 in responding to Aspergillus therefore appears to be conserved in the cornea and lungs.

Although studies with fungal cell wall components indicate that Dectin-1 and

TLR2 collaborate in recognizing β-glucan (133, 134, 178), we found no role for TLR2 in Aspergillus keratitis, indicating that Dectin-1 mediates cellular recruitment independently of TLR2. Additionally, TLR2-independent receptor collaboration occurs in a sequential manner during non-opsonic phagocytosis of C. albicans by macrophages, where Dectin-1, CR3, and mannose receptors collaborate in recognition and phagocytosis (179). In the current study, phagocytosis of swollen A. fumigatus conidia was impaired in Dectin-1-/- bone marrow macrophages, raising the possibility that a similar collaboration occurs during macrophage phagocytosis of this pathogen. In support of this notion, the mannose receptor was one of the few genes found to be

113 upregulated in a microarray analysis of Aspergillus –infected corneas (180). Future studies will determine if a similar mechanism occurs for macrophage phagocytosis of

Aspergillus conidia.

In pulmonary aspergillosis, TLR2 and TLR4 have no role in otherwise immunocompetent animals, although Aspergillus CFU were elevated in vinblastin- or cyclophosphamide-treated TLR2-/- and TLR4-/- mice (62, 136). In contrast, we showed increased Aspergillus CFU in TLR4-/-, but not TLR2-/- mice. Although this is consistent with our earlier observation that TLR4-/- mice have impaired clearance of Fusarium in the cornea (40), the mechanism of TLR4 regulation of Aspergillus survival has yet to be determined. Given that TLR4-/- mice exhibit defects in fungal killing but not cellular recruitment, we predict that the primary role for TLR4 is on infiltrating neutrophils and macrophages that mediate fungal killing rather than on resident macrophages, which regulate cell recruitment to the cornea. Further, as fungal clearance is unimpaired in

MD-2-/- corneas, and MD-2 is the binding site for the lipid A moeity of LPS (181, 182),

TLR4 likely recognizes Aspergillus hyphae at an MD-2 independent site of the receptor. TLR4 recognizes C. albicans o-linked mannosyl residues (133) and

Cryptococcus neoformans glucuronoxylomannan (183), although the ligand on

Aspergillus hyphae has yet to be identified. In addition to being the receptor for LPS,

MD-2 mediates TLR4 dimerization and cell signaling(176); therefore, it is possible that

TLR4 recognition of the fungal cell wall does not induce signaling. In support of this notion, we found that the absence of TIRAP or TRIF did not affect cellular infiltration or fungal survival. We also noted that although TLR4-/- mice have elevated Aspergillus

CFU, there was no difference in RFP expression between TLR4-/- and C57BL/6 mice.

114 RFP quantification measures total fungal load, but not viability, and CFU and RFP measurements correlated well in most studies; however, in TLR4-/- mice, more RFP+ hyphae appear to be viable, possibly due to production of fungistatic rather than fungicidal mediators in the tissue, including lactoferrin and lipocalin (77).

In the current study, MyD88-/- mice had delayed cellular infiltration and unimpaired fungal growth, which is similar to the role of MyD88 in pulmonary aspergillosis (136)

(184), and indicates an essential role for this adaptor molecule in Aspergillus keratitis.

However, although TLR4 signaling through MyD88 requires the accessory adaptor molecule TIRAP (176), we found no detectable effect on the progression of Aspergillus keratitis in the absence of TIRAP. We therefore examined the role of IL-1R1, which signals through MyD88 in the absence of TIRAP (176), and showed that IL-1R1-/- mice have a similar phenotype as MyD88-/- mice. As IL-1β is produced in the cornea early after infection, it seems reasonable to assume that the MyD88 dependence is due to IL-

1R1 signaling. In the corneal stroma, IL-1R1 is expressed not only by macrophages and dendritic cells, but also by resident keratocytes in the corneal stroma, which can differentiate into fibroblasts and produce pro-inflammatory and chemotactic cytokines, including CXCL1/KC (185-187). IL-1R1 also mediates the host response in pulmonary candidiasis (93), and we identified a similar role for IL-1R1 and MyD88 in trauma- induced and biofilm-associated Fusarium keratitis (40, 188), indicating that the cornea employs similar responses to regulate infection by filamentous fungi.

In conclusion, results of the current studies using a murine model of fungal disease demonstrate essential, though distinct roles for Dectin-1 and TLR4. There also appears to be a role for these receptors in human fungal disease as specific

115 polymorphisms in Dectin-1 and TLR4 genes are associated with susceptibility. A

Dectin-1 polymorphism resulting in an early stop codon was associated with reduced ß- glucan binding and increased susceptibility to Candida albicans infections (126, 189), whereas TLR4 polymorphisms correlate with susceptibility to aspergillosis in recipients of stem cell transplants (128). Taken together, results from human genetics studies and animal model studies combine to demonstrate that these receptors have an essential role in fungal infection, and are therefore potential targets for immunotherapy that are common to multiple fungal pathogens. Future studies will examine the potential of targeting receptors in prevention and treatment of fungal keratitis.

116 Figure 3.11 Working model of cornea inflammation

Dectin-1 on resident macrophages recognizes fungal β-glucan and mediates NFκB (p65) translocation into the nucleus and production of IL-1β and CXCL1/KC. The IL- 1β signals back through IL-1R1 on the same cell and neighboring cells resulting in further pro-inflammatory cytokine production. TLR4 but not MD-2expression on infiltrating cells is required to control fungal growth

117

Chapter 4.

Thioredoxin and superoxide dismutase enhance survival of hyphae

against CD18-dependent neutrophil NADPH oxidase activity

118 Summary

Filamentous fungi are an important cause of blindness and visual impairment worldwide. Using gene knockout mice in a model of fungal keratitis and a novel in vitro neutrophil-hyphae killing assay, we found that neutrophil NADPH oxidase is essential to control the growth of Aspergillus and Fusarium in the cornea, with no role for iNOS or myeloperoxidase, and that neutrophil oxidase and anti-fungal activity is dependent on CD18, but not Dectin-1. We used mutant A.fumigatus strains to show that the reactive oxygen species sensing transcription factor, Yap1, superoxide dismutases, and the Yap1-regulated thioredoxin antioxidant pathway are required for optimal hyphae survival in vivo and against oxidation by neutrophils, whereas we found no role for catalases, or LaeA-regulated 2° metabolites including gliotoxin. We also found that inhibition of thioredoxin using an anti-cancer drug (PX-12) increased the sensitivity of fungal hyphae to H2O2 and neutrophil mediated killing in vitro, and topical application significantly enhanced fungal killing in infected mouse corneas. Together, results of these studies identify critical host oxidative and fungal anti-oxidative mediators that regulate hyphae survival during infection. These findings also indicate that targeting fungal anti-oxidative defenses, via PX-12, may represent an overlooked and novel approach to treat fungal infections.

119 Introduction

Pathogenic fungi, such as Aspergillus and Fusarium species, can cause lethal pulmonary and systemic disease in immune suppressed individuals, including those with HIV infection (15, 190). These organisms are also a major cause of infectious blindness and corneal ulcers in immunocompetent individuals, and in contrast to systemic and pulmonary fungal infections, there is no indication that fungal keratitis patients are other than fully immunocompetent (12, 113). In the hot and humid southeastern United States, fungal infections of the cornea account for up to 35% of all corneal ulcers (16, 17). Globally, fungal infections of the cornea account for up to 65% of corneal ulcers with estimates of 80,000 total cases and 10,000 cornea transplants per year due to fungal infections in India alone (18, 115, 130, 145-147). Other risk factors for disease in the USA, Britain and Europe include contact lens wear, as illustrated by a

2005-6 fungal keratitis outbreak associated with a lens care product (111). A.flavus, A. fumigatus, F.solani and F.oxysporum are the main etiologic agents of fungal keratitis

(9). These organisms are prevalent in vegetative matter and suspended in air, and are inoculated into the corneal stroma via traumatic injury associated with agricultural work (9). Current treatment with topical anti-mycotics is often ineffective, with up to

60% of cases requiring corneal transplantation (9, 12, 25)

Neutrophils are the predominant cell type infiltrating fungal infected lungs and corneas, and contribute to tissue destruction by release of proteolytic and reactive oxygen and nitrogen species(85, 92). Our recent studies characterizing fungal- infected human corneas in India showed that neutrophils constitute >90% of cellular infiltrates in corneal ulcers in patients infected for < 7 days and >70% total infiltrate at

120 later stages of infection (30). Similarly, neutrophils are the first cells recruited to the corneal stroma in murine models of Aspergillus and Fusarium keratitis (30, 40), indicating that neutrophils are the main effector cells required for killing fungal hyphae.

A role for neutrophils in control of fungal infection is also suggested by the increased incidence of systemic and pulmonary fungal infections in patients with neutropenia

(15).

Neutrophils produce NADPH oxidase (NOX), which catalyzes the conversion of molecular O2 to superoxide anion with the release of reactive oxygen species (ROS) and protons into the extracellular space (92, 97). Individuals with inherited defects in

NOX such as in chronic granulomatous disease exhibit an increased incidence of bacterial and fungal infections, supporting the concept that the specific expression of

NOX by neutrophils is required to kill fungi (191). However, even though it is the hyphal stage of these organisms that is invasive, most studies on CGD patients and transgenic mice with mutations in NOX genes have focused only on the role of NOX in killing conidia (89, 90, 192, 193). Infected human corneas and lungs exhibit primarily hyphal stages of fungal growth and conidia are rarely detected. Given that hyphae are significantly larger in size, and are not readily phagocytosed, they are likely killed through distinct mechanisms not required for anti-conidial defenses and a recent study suggests that NOX is not required to control the growth of all filamentous fungi (194).

In the current study, we examined the role of ROS in killing Aspergillus and

Fusarium hyphae by human neutrophils and in a murine model of fungal keratitis. We show that hyphae activate neutrophil NADPH oxidase through CD18, and that NOX activation is essential for killing hyphae. In addition, utilizing mutant A.fumigatus

121 strains, we show that the ROS-sensing transcription factor Yap1, the ROS detoxifying- enzyme superoxide dismutase, and the Yap1-regulated thioredoxin antioxidant pathway, but not catalases or fungal 2° metabolites such as gliotoxin are required for resistance to oxidation by neutrophils. Lastly, using pharmacologic inhibitors of thioredoxin, we provide proof-of-concept that targeting fungal anti-oxidative stress responses can enhance fungal clearance from infected tissues and may represent a new avenue for treatment of fungal infections.

Materials and Methods

Source of mice.

All animals were treated in accordance with the guidelines provided in the

Association for Research in Vision and Ophthalmology ARVO statement for the Use of

Animals in Ophthalmic and Vision Research, and were approved by Case Western

Reserve University IACUC . C57BL/6 mice (6–12 wk old) were purchased from The

Jackson Laboratory (Bar Harbor, ME). LysM-eGFP mice were provided by Dr. Alex

Huang (CWRU), CybB-/- mice were provided by Dr. Brian Cobb (CWRU), and iNOS-/- mice were provided by Timothy Kern (CWRU Cleveland, OH). CD18-/- mice were kindly provided by Dr. Claire Doerschuk (UNC, NC), and CXCR2 -/- mice were kindly provided by Dr. Richard Ransohoff (Cleveland Clinic, OH). Dectin-1-/- and TLR4 -/- mice were kindly provided by Yoichiro Iwakura (Tokyo University) and Dr. Shizuo

Akira (Osaka University), respectively. All mice used in this study are on a C57BL/6 background.

122 Fungal strains, media, and growth conditions.

Table 1 list the genotype and phenotype of all strains utilized in this study.

Aspergillus fumigatus and A.flavus strains used in this study were cultured on Vogel’s

Minimal Media (VMM) w/wo 2% agar unless otherwise stated. Fusarium oxysporum, and F.solani, strains were cultured on sabouraud dextrose media. To visualize fungi in the transparent murine cornea, fluorescent strains of Aspergilli or Fusarium species expressing either RFP or GFP were utilized in this study. The A. fumigatus strain Af-

RFP constitutively expresses enhanced monomeric dsRed protein (gpdA promotor driven; pyrG1selection marker). The strain A. flavus 70-GFP constitutively expresses

GFP (gpdA promotor driven; niaD selection marker; kindly provided by Dr. Rajah

Rajasekaran at the USDA, New Orleans, LA (195). The strains FoxL-RFP & FoxL-

GFP are F.oxysporum lycopersici strains constitutively expressing either RFP or GFP which were kindly provided by Dr. Seogchan Kang (Penn State, University Park, PA).

The A. fumigatus Strain Af-BP was isolated from a keratitis patient at Bascom Palmer

Eye Institute (Miami, FL) provided by Darlene Miller. The A. flavus strain Aflav-

TN302 was isolated from a keratitis patient at Aravind Eye Hospital, Tamil Nadu, India and kindly provided by Dr. Lalitha Prajna. The Fusarium oxysporum strain 8996 was isolated from a keratitis patient at the Cole Eye Institute, Cleveland Clinic, OH. For our studies on gliotoxin, Dr. June Kwon-Chung (NIAID) kindly provided A.fumigatus strains: B-5233(WT), ∆gliP, and gliP-R. Additionally, Dr. Nancy Keller (U.W.

Madison) kindly provided the A.fumigatus strains: Af293 (WT), ∆gliZ, gliZ-R, ∆laeA, laeAR as well as the Aspergillus flavus strains: A.flavus NRRL3357 (WT), ∆laeA, laeAR. For our reactive oxygen species (ROS) scavenging studies, Dr. Jean Paul Latge

123 kindly provided A.fumigatus strains: G10 (WT), ∆catA, and ∆cat1/2, and AfKu80

(WT), ∆sod1/2/3, and Dal (WT), ∆yap1.

Construction of an A.fumigatus ∆trxA mutant

We performed a BLAST analysis on the A.fumigatus strain Af293 genome

(aspergillus genome.org) for proteins similar in sequence to the characterized

A.nidulans thioredoxin protein encoded by the gene trxA (196).Utilizing this approach, we identified 5 putative thioredoxin proteins in the A.fumigatus genome:

Afu5g11320/aspf29 (Score=348); Afu6g10300/aspf28 (251); Afu3g14970 (243);

Afu8g01090 (239); and Afu4g09090 (213). The top two hits, aspf29 and aspf28 are both functionally uncharacterized, however, they are both known human allergens suggesting high expression during infection (197). Table 2 list the primers utilized in this study. All PCRs and fusion PCRs were conducted using Clontech Advantage HD

DNA polymerase as previously described (205). Briefly, two separate fragments of the trxA gene were amplified: the first fragment was approximately 1-1.5 Kb upstream of the trxA gene (A. fumigatus template) using primers P1 and P3 and the second fragment was approximately 1-1.5 Kb downstream of the trxA gene (A. fumigatus template) using primers P4 and P6. A third fragment was amplified from the pyrG gene of A. nidulans using primers anpyrGF and anpyrgR. Primers P3 and P4 includes 20 bp of A. nidulans pyrG gene to facilitate fusion PCR. The DNA fragments were run on a 0.8% agarose gel; the bands were excised and cleaned with the Qiagen QIAquick gel

124 Table 4.1 Fungal strains utilized in this study

Reprint permission obtained from publisher.

125 Table 4.2 Primers utilized in this study

Name Sequence P1- upstream sense TTATGTAGGTGCTGGGAGTGG trxA P2- upstream sense AGCAGCTTCTTTGGCTTTCC trxA P3- antisense pyrG atctggtagacaagcactgaGTAGGTGAA trxA AACCGAG P4- sense pyrG trxA cttcgacaggtatcgaattcGCCCATTTTTG ACGAGG P6- downstream GAGCACTCTAGAACCCCAAGG antisense trxA anpyrGF tcagtgcttgtctaccaga anpyrGR gaattcgatacctgtcgaaag trxA del ck F use P1 trxA del ck R use anpyrGR Reprint permission obtained from publisher.

126 extraction kit (Maryland). Equal amounts of each fragment were used in the fusion PCR reaction (primer P2 and P6). The fusion PCR product was run on a 0.8% agarose gel to ensure that a single band was obtained. The band was then excised and gel purified with the Qiagen QIAquick gel extraction kit (Maryland). The resulting DNA was used to transform A. fumigatus AfΔku80 (CEA17) strain by protoplasting. The transformants were checked by PCR. Colonies that were positive were checked by Southern to ensure that only a single copy of the cassette was incorporated homologously.

Mouse model of Aspergillus and Fusarium keratitis

Aspergillus strains were cultured for 2-3 days on VMM in 25 cm2 tissue culture flasks. Fresh conidia were disrupted with a bacterial L-loop, harvested in 5 ml PBS, and filtered through sterile PBS-soaked cotton gauze in a 10 ml syringe to obtain pure conidial suspensions. Conidia were quantified using a haemacytometer and adjusted in

PBS to a final stock solution: A.fumigatus= 20,000 conidia/µl; A.flavus= 20,000 conidia/µl; F.oxysporum= 25,000 conidia/µl. Subsequently, mice were anaesthetized with 1.25% 2,2,2-tri-bromoethanol. The corneal epithelium of anaesthetized mice was abraded using a 30-gauge needle, through which a 2 µl injection containing conidia was released into the corneal stroma using a 33-gauge Hamilton syringe (Total -

A.fumigatus= 40,000 conidia; A.flavus= 40,000; F.oxysporum= 50,000.) The optimal inoculum size for induction of keratitis was based on preliminary studies (Data not shown.) Mice were examined daily under a stereomicroscope for corneal opacification, ulceration, perforation, and fungal fluorescent protein expression. At set time points, animals were euthanized by CO2 asphyxiation, and eyes were either placed in 10%

127 formalin and embedded in paraffin and sectioned at 5 µm intervals, or excised and placed in 1 ml of sterile saline and homogenized for quantitative culture. For depletion of neutrophils in mice, 400µg of anti-mouse neutrophil antibody (NIMP-R14) was injected at day 1 prior to infection. To detect ROS production in the mouse cornea during fungal infection, 2µl of 12.5 ng/ml carboxyfluorescein diacetate (CFDA;

Invitrogen) was injected into the stroma of anaesthetized mice and eyes images under standard GFP filters after 10min incubation at 25C. Subsequently, mice were euthanized and eyes processed accordingly. To inhibit reactive nitrogen species (RNS) production during corneal infection, 500µg of the irreversible iNOS inhibitor 1400W was injected IP into C57BL/6 mice at 6h, 24h, 48h post-infection (Cobb et al.) All animals were bred under specific pathogen-free conditions and maintained according to institutional guidelines.

Neutrophil-specific adoptive transfer mouse model used to study fungal killing

CXCR2-/- or CD18-/- mice are anaesthetized as described above and conidia from a fluorescent fungal are injected into the corneal stroma strain (Ex. 30,000 conidia from the RFP-expressing A.fumigatus strain Af-dsRed). Subsequently, naive bone-marrow derived neutrophils are isolated from the femur and tibia of WT and knockout mice

(described below). At 2h post-infection, one set of mice are left untreated, one set receive an I.V. injection of 4 million WT C57BL/6 neutrophils, and subsequent sets of mice receive 4 million neutrophils isolated from knockout mice. At 24h post-infection, infected mice are euthanized and fungal growth in the cornea is imaged under a fluorescent steroescope. The level of fluorescence emitted from infecting fungi is

128 quantified using image analysis software (Metamorph;described below) and used as a measure of fungal growth during infection.

Imaging and quantification of light reflected or emitted from infected mouse corneas.

Mice were sacrificed by CO2 asphyxiation and positioned in a three-point stereotactic mouse restrainer. Corneal opacity (Brightfield), fungal proliferation

(RFP/GFP), cellular infiltration (GFP) and CFDA dye oxidation (GFP spectra) were visualized in the intact cornea using a high-resolution stereo fluorescence MZFLIII microscope (Leica Microsystems) and Spot RT Slider KE camera (Diagnostics

Instruments). Importantly, all images were obtained with the same Spot RT Slider KE camera using the same Spot Advanced Software under the same magnification, exposure (BF=0.4s; RFP=10s; eGFP=2s), gain (BF=1; RFP/eGFP=4/16), and gamma

(BF/RFP/eGFP=1.85) parameters.

Metamorph imaging software (Molecular Devices, Downington PA) was used to quantify the Percent Area of Opacity and the Integrated Corneal Opacity. Firstly, a circular region of the same constant area was centered on the image of each cornea to standardize the area. Secondly, photos of corneas were converted into pseudo-color images thereby allowing a color visualization of corneal opacity using a scale ranging from purple (no opacity) to red (maximum opacity) that corresponds to increasing pixel intensity level. Either images of naïve corneas that were taken at the same time, or corneas within the data set with distinct regions of no disease were analyzed first in order to obtain a minimum pixel intensity value corresponding to no opacity (i.e

129 transparency). The mean value obtained from at least four naïve corneas or no disease regions was considered the minimum opacity threshold level and all pixels with an intensity level above this threshold were considered disease and included in the subsequent analysis. In addition the areas of glare (Pixel Intensity= 249-255) were demarcated and then set to zero, thereby eliminating glare from the subsequent analysis.

Percent Corneal Opacity = Percent of Cornea that is Opaque = (Area over the threshold

/ (total area of circle - area of glare)*100). Total Cornea Opacity = Sum of the intensity of all pixels within the circle above the minimum opacity threshold level and below the pixel intensity of glare as calculated by Metamorph Software. Metamorph was also used to quantify emission of fluorescent light from the cornea. Similarly a constant circular region encompassing the cornea was defined, and the sum of pixel intensity within this region summed to yield a numerical value, called the pixel intensity. For

CFDA dye emission the pixel intensity value was plotted, however, for quantification of fungal RFP emission or GFP+ cell infiltrates the pixel intensity for each eye was divided by the mean pixel intensity of uninfected eyes to obtain a ratio of infected/uninfected pixel intensity.

Quantification of Aspergillus colony forming units (CFUs).

For assessment of fungal viability, whole eyes were homogenized under sterile conditions in 1 ml PBS, using the Mixer Mill MM300 (Retsch) at 33 Hz for 4 min.

Subsequently, serial log dilutions were performed and plated onto bacteriologic-grade

Sabouraud dextrose agar plates (Becton Dickenson). Following incubation for 24h at

37°C (Aspergillus) or at 30°C (Fusarium), the number of CFUs was determined by

130 direct counting.

Identification of fungal growth patterns and neutrophil recruitment into the cornea.

Eyes were enucleated and fixed in 10% formalin in PBS (Fisher) for 24h. Five µm sections from the center of the cornea (as determined by noncontiguous iris morphology) were cut and stained with Periodic-Acid Schiff (PASH) for identification of fungi and inflammatory cell recruitment by standard methods. To detect infiltrating neutrophils, 5µm corneal sections were subjected to immuno-histochemical analysis using monoclonal rat-anti-mouse neutrophil IgG (NIMP-R14, AbCam, Cambridge,

MA), and alexafluor-488 tagged rabbit-anti-rat IgG (Invitrogen).

Isolation of human neutrophils from peripheral blood

Human neutrophils were isolated from normal donor blood using Ficoll density centrifugation. This procedure was reviewed and approved by the Institutional Review

Board at Case Western Reserve University/University Hospitals of Cleveland. After informed consent was obtained in accordance with the Declaration of Helsinki, 20ml peripheral blood was obtained from normal healthy donors. Red blood cells were separated from whole blood via incubation at 1*g for 20 min with 3% Dextran in PBS

(Sigma). The top clear layer containing leukocytes was transferred to a fresh 50ml conical tube to which was underlayed 10 ml Ficoll-Paque Plus (GE HealthCare). The cell suspension was centrifuged at 500*g for 20 min to separate mononuclear cells from neutrophils and the remaining RBCs. The overlying plasma and PBMC layers were

131 aspirated and the neutrophil/RBC pellet was resuspended in RBC lysis buffer (8.3g

NH4Cl, 1g KHCO3, 0.09g EDTA/ 1L ddH2O), incubated at 37C for 10min to lyse remaining RBCs, and spun at 300*g for 5 min at 4C. The lysis procedure was repeated as needed to obtain sufficient RBC lysis in cell preparations. Subsequently, cells were washed 2x in PBS and ultimately resuspended in RPMI + L-glutamine w/o Phenol Red

(Hyclone). The purified neutrophil cell suspension was counted via a haemacytometer and the purity of each cell preparation confirmed at >97% via Cytospin/Diff-quick staining followed by light microscopy.

Isolation of peritoneal and bone-marrow derived murine neutrophils

Murine thioglycolate-elicited peritoneal neutrophils and bone-marrow derived murine neutrophils were utilized in this study. To isolate peritoneal neutrophils, mice were injected with 1ml 4% thioglycolate (Aged >2weeks) at 16h and 3h prior to peritoneal lavage with cold 1X PBS. Isolated cells were transferred to a fresh 50ml conical tube in a total volume of 30ml to which was underlayed 10 ml Ficoll-Paque Plus (GE

HealthCare). Cells were then spun at 1200*g for 20 min at 4ºC . The overlying monocytic cell layer was aspirated and the underlying neutrophil layer was washed 3x with 50ml PBS and ultimately resuspended in RPMI + L-glutamine w/o Phenol Red

(Hyclone). The purified neutrophil cell suspension was counted via a haemacytometer and the purity of each cell preparation confirmed at >95% via Cytospin/Diff-quick staining followed by light microscopy.

Bone-marrow derived neutrophils were obtained from mice for particular assays in order to work with a naive, unstimulated cell population. Briefly, mice were

132 euthanized by CO2 asphyxiation, and femurs and tibias were removed, cleaned, and centrifuged at 5000×g for 45 s at 4°C. Any contaminating red blood cells were lysed in

5 ml RBC Lysis Buffer (eBioscience), and remaining bone marrow cells were pipetted onto a discontinous percoll gradient of 52%, 69%, and 78% . Cells were spun for 30 min at 1500*g at 25C. Following centrifugation, the neutrophils suspended in percoll at the 69%/78% interface and below were harvested, washed 2x in PBS in 50ml conical tubes, ultimately resupended in RPMI media + L-glutamine w/o Phenol Red (Hyclone), and counted on a haemacytometer. Neutrophil purity of >98% was confirmed upon each isolation, utilizing Cytospin and Diff-quick staining.

Detection of fungal growth using a neutrophil-hyphae co-incubation assay

An in vitro assay was developed to study the ability of mouse and human neutrophils to inhibit the growth of fungal hyphae from A.fumigatus, A.flavus, F.oxysporum, and F. solani species. Purified conidia (12,500/well for Aspergillus species and 100,000/well for Fusarium species) from fluorescent or non-fluorescent fungal strains were cultured in 200µl SDA media in black-walled 96 well plates with an optically clear bottom

(CoStar 3720) for several hours till early germ tubes were observed (A.fumigatus- 6h,

A.flavus- 4h, F.oxysporum- 6h, and F. solani - 6h). Wells were washed 2x with sterile ddH2O and incubated for a further 16h with either RPMI media w/o Phenol Red (+

5 Control for Fungal Growth), ddH2O (- Control), or 10 murine neutrophils purified from WT or KO mice or 2*105 human neutrophils purified from healthy donors +/- pharmacological inhibitors. Preliminary experiments identified the optimal fungi/neutrophil ratios per well required to resolve differences in fungal killing by

133 neutrophils (Data not shown.) For assays in which the desired phenotype was survival of the WT strain, sub-lethal doses of human neutrophils (50-100,000/ well) were added to fungi so that fungal survival defects in mutant fungi would be readily observable. At

16h post-exposure, wells were washed 3x with ddH2O to remove exhausted media and lyse residual neutrophils. Fungal growth was subsequently analyzed by microscopy and spectrophotometry as described below.

In assays utilizing fluorescent strains, fungal growth was visualized directly using standard GFP and RFP filters in an upright microscope (Zeiss). In order to visualize non-fluorescent fungal strain growth, 50µl calcofluor white stain (Binds chitin; Fluka 18909) was added to each well for 5 min in the dark. Subsequently, plates were washed 3x with ddH2O to remove non-specifically bound calcofluor white.

Calcofluor-stained fungi were subsequently visualized and imaged as stated above using standard DAPI filters. Following microscopic image acquisition, fungal growth was quantified using a 96-well fluorometer with the following excitation/ emission filters: RFP- 530/620 nm, GFP- 485/520 nm, Calcofluor White- 360/440nm (Synergy

HT; Biotek).

In order to inhibit neutrophil NADPH oxidase we performed preliminary experiments with diphenyliodonium (DPI) and apocynin (Apo) and identified 200µM and 30mM as the optimal concentrations for inhibition of ROS production upon stimulation with fungal hyphae. In order to inhibit iNOS in human neutrophils, we utilized the pharmacologic inhibitors: aminoguanidine HCl (Agd), and 2-Methyl-2- thiopseudoureasulfate (SMT). In addition, we utilized the pharmacologic MPO

134 inhibitors: Indomethacin (Indo) and 4-aminobenzoic hydrazide (4-AH) to examine the involvement of myeloperoxidase (MPO) and hypohalous acids in killing fungal hyphae.

Detection of extracellular ROS and NO release by neutrophils upon exposure to fungal hyphae

In this study, the extracellular release of reactive oxygen species (ROS) and nitric oxide

(NO) by neutrophils upon exposure to fungal hyphae were assayed using a dye that fluoresces upon oxidation with ROS (Carboxyfluorescein diacetate;CFDA) or a dye that changes spectrophotometric properties upon reaction with NO (Griess Reagent).

For these assays, purified conidia were cultured in sabouraud dextrose broth in black- walled, clear-bottom 96 well plates as described above. Subsequently, plates were washed twice with PBS and either 200µl RPMI+ L-glutamine w/o phenol red, PBS, or neutrophils in RPMI media were added to corresponding wells. Neutrophils and fungal hyphae were co-incubated for 2h at 37C/5% CO2. After 2h incubation, 50 µl of supernatant was transferred to wells containing either: 50 µl CFDA 25ng/ml

(Invitrogen; ROS) or 250µl Griess reagent (Invitrogen). Plates were incubated in the dark for 10 min (CFDA/DAF-FM) or 30min (Griess) at 37C and read using a fluorescent spectrophotometer at excitation/emission 485/520nm γ (CFDA/DAF-FM) or absorbance at 548nm γ (Griess).

Detection of neutrophil intracellular ROS levels upon exposure to fungal hyphae

Fungal conidia were plated at 12,500/well and cultured for 4-6h in SDB media at 37C/

5%CO2. Meanwhile, naive bone-marrow derived neutrophils were isolated as described

135 above and pre- loaded with 10µM CFDA at 37C for 10 min. Subsequently, plates were washed and 200,000 CFDA-pre-loaded neutrophils/well were added to hyphae and plates were spun at 300*g for 1min to enhance cell contact. Neutrophils were incubated at 37C for 1h excitation/emission of 485/520nm γand oxidized CFDA dye fluorescence analyzed via flow cytometry (excitation/emission of 485/520nm γ).

Determination of fungal sensitivity to oxidative stress

To study whether thioredoxin inhibition enhances susceptibility of fungal strains to

H2O2 , fungi were grown at 37°C in 200µl SDB in the presence of PX-12 or 1Cl(2,4) dinitrobenzene (1µM) for 2h. Subsequently, H2O2 was added at both lethal and sub- lethal concentrations (1µM-10mM). At 16h post-incubation fungal growth was analyzed by phase-contrast microscopy, stained with calcofluor white, and quantified by fluorometry as described above.

Detection of extracellular lactate dehydrogenase (LDH) in cell culture supernatants

To test the cellular of drugs used in this study, we exposed a panel of human cell lines (HEK293, RAW264.7 (Macrophage-like), HL-60 (Neutrophil-like),

10.1PRSVT (Human cornea epithelial cell) , HCTEPI (Human cornea epithelial cell) ,

MK/T-1(Human cornea fibroblast)) to 10µM and 100µM drug concentrations for 16h.

Subsequently, 20µl of cell supernatant were added to 100 µl LDH reaction mix (LDH- cytotoxicity assay; ABCAM), and incubated for 30 min at RT as per manufacturer's instructions. The absorbance was subsequently read at 450nm.

136 Inhibition of thioredoxin during fungal corneal infection

PX-12 (Sigma) was dissolved at 3mM in proprietary eye drop formulation kindly provided by Alcon laboratories. C57BL/6 mice were infected with A.fumigatus Af dsRed conidia as described above. at 0h and 6h post-infection 8µl eye drops were applied to infected corneas of anaesthetized mice and fungal dsRed expression imaged and quantified at 24h post-infection.

Statistical analysis.

Statistical analysis was performed for each experiment using an unpaired t test or

Tukey's one-way anova analysis (Prism, GraphPad Software). A p value < 0.05 was considered significant.

Results

Neutrophils have an essential role in regulating fungal growth in the cornea

To examine the role of neutrophils in fungal keratitis, we used two complementary approaches: systemic depletion of neutrophils from immune competent

C57BL/6 mice, and adoptive transfer of neutrophils into CXCR2-/- and CD18-/- mice. In the first approach, neutrophils were depleted from transgenic C57BL/6 mice expressing eGFP on the neutrophil-specific promoter LysM (LysM-eGFP mice (206)) by intraperitoneal injection of a neutrophil-specific monoclonal antibody (NIMPR-

14)while control mice were given rat isotype Ab. After 24h, corneas were infected with

A.fumigatus Af-dsRed conidia (40,000 in 2 µl), and neutrophil infiltration and fungal

137 growth were assessed in live corneas. We also examined the effect of neutrophils on corneal opacity.

Figure 1, A and B shows significantly increased eGFP+ neutrophils at 24h and

48h post-infection in control, isotype-treated mice, but not in neutrophil depleted

(NIMP) mice. Conversely, Figure 1A and C shows significantly decreased dsRed expressing fungal hyphae at 24h and 48h in isotype controls compared with NIMP treated mice, which is consistent with increased CFU in NIMP-treated mice (Figure

1D). We also found significantly lower corneal opacity in neutrophil-depleted mice as measured by both area of opacity and total corneal opacity (image analysis methods described in Fig S1) (Figure 1E and F). Figure 1A also shows a representative PASH- stained cornea section of a 48h infected LysM eGFP mouse with infiltrating cells in the stroma and anterior chamber, and few intact fungal hyphae. In contrast, the corneas of neutrophil-depleted mice exhibited reduced cellular infiltrates and prominent fungal hyphae in both the corneal stroma and the anterior chamber.

As a second approach, we utilized two mouse strains with known defects in neutrophil infiltration during infection, and adoptively transferred WT naive bone- marrow-derived neutrophils into these mice to study the specific role of neutrophils in killing fungi during corneal infection. CXCR2-/- neutrophils are unable to recognize and respond to ELR+ CXC chemokines, whereas CD18-/- neutrophils are unable to bind to

ICAM-1 on limbal vessel vascular endothelial cells (207, 208).

C57BL/6, CXCR2+/- and CXCR2-/- corneas were infected with A.fumigatus conidia as described above. Figure 1G shows fungal dsRed expression and corneal opacity in C57BL/6 and CXCR2+/- heterozygous mice, which both increase at 48h

138 post-infection. In contrast, infected CXCR2 -/- mice had significantly increased fungal dsRed expression (Figure 1H) and lower corneal opacity scores (Figure 1 I and J) at

24h and 48h compared to C57BL/6 mice. However, CXCR2-/- mice given C57BL/6 syngeneic neutrophils intravenously (iv) had significantly lower fungal dsRed values

(Figure 1H) and higher corneal opacity (Figure 1 I and J) compared to CXCR2-/- mice not receiving neutrophils, indicating that neutrophils contribute to both fungal killing and corneal opacity. Corneal sections from these mice show intense cellular infiltration, neutrophil recruitment, and minimal intact fungal hyphae at 48h post-infection in

C57BL/6 mice and CXCR2+/- mice, whereas CXCR2-/- corneas exhibited minimal cellular infiltrates, but abundant hyphae in the stroma and anterior chamber (Figure

1G). Following adoptive transfer of C57BL/6 neutrophils into CXCR2-/- mice, neutrophils were detected in the corneal stroma and fungal growth in the cornea was lower than in the absence of neutrophils (Figure 1G).

Similarly C57BL/6 neutrophils transferred to CD18-/- mice, prior to infection conferred a protective response with increased cell migration to the cornea and lower fungal load. Figure 1K and L shows that fungal dsRed expression at 48h was significantly increased in CD18-/- mice compared to C57BL/6 mice. However, CD18-/- mice given syngeneic bone-marrow derived neutrophils from transgenic C57BL/6

LysM-eGFP mice exhibited significantly decreased fungal dsRed expression (Figure

1L) along with significantly increased eGFP+ neutrophil infiltration (Figure 1M), and increased corneal opacity (Figure 1 N and O).

139 Together, these findings demonstrate that both CXCR2 and CD18 regulate neutrophil recruitment to fungal-infected corneas, and that neutrophils have an essential role in controlling fungal growth at this site.

Neutrophil NADPH oxidase activity is required for control of fungal growth during corneal infection

NADPH oxidase is an enzyme complex required for reduction of molecular

- oxygen (O2) to the superoxide anion (O2 ) (96). Superoxide is the limiting reagent in subsequent reactions leading to the transient synthesis of ROS with greater oxidative and fungal killing potential (97), and individuals with chronic granulomatous disease due to impaired NADPH oxidase function are unable to control microbial infections, and often succumb to filamentous fungal infections in the lung (15, 209).

CybB-/- mice lack the gene encoding the NADPH oxidase (NOX) subunit, gp91phox, and thus do not express a functional NOX complex. These mice are also more susceptible to Aspergillus lung infections (90). To determine the role of NOX in fungal keratitis, we infected CybB-/- mice with the RFP expressing strain of A. fumigatus (dsRed), and ROS levels in the corneas were measured at 48h post-infection after intrastromal injection of carboxyfluorescein diacetate (CFDA), which emits green fluorescence upon oxidation. Fungal dsRed expression and corneal opacification were measured by image analysis as described above. Figure 2, Aand B show CFDA activity in C57BL/6 and CybB-/- corneas at 48h post-infection; however,

140 Figure 4.1 Neutrophils are required for control of fungal growth during corneal infection

A. Transgenic C57BL/6 mice with neutrophil-specific eGFP expression downstream of the lysozyme promotor (LysM) were depleted of neutrophils utilizing neutrophil- specific NIMPR-14 antibody (I.P.) and infected with 40,000 A.fumigatus strain Af- dsRed conidia. Eyes were imaged at 24h and 48h post-infection for neutrophil infiltration (eGFP), fungal growth (dsRed), and corneal opacity (BF). In addition, Periodic Acid Schiff and Hematoxylin (PASH) stains were performed on 5µm sections of corneas at 48h post-infection. B. Metamorph software was utilized to quantify neutrophil infiltration (eGFP emission) and C. fungal dsRed expression. D. At 4h and 48h post-infection eyes were homogenized and plated on sabouraud dextrose agar (SDA) plates and colony forming units (CFU) quantified by direct counts. E. Metamorph software was utilized to quantify corneal opacity area and F. total cornea opacity. G. C57BL/6, CXCR2+/- , and CXCR2-/- mice were infected with 30,000 A.fumigatus strain Af-dsRed conidia. At 24h post-infection one group of fungal- infected CXCR2-/- mice were given 4 million adoptively transferred bone-marrow

141 derived neutrophils from a CXCR2+/+ C57BL/6 mice and at 48h eyes were imaged for fungal growth and cornea opacity. At 48h post-infection, corneas were excised and PASH stains were performed on 5µm sections. In addition, NIMP antibody and fluorescently tagged secondary antibody were utilized to identify neutrophils in 5µm sections. H. Metamorph software was used to quantify fungal dsRed expression, I. corneal opacity area, and J. total cornea opacity in infected corneas. K. Similar to CXCR2-/- mice, C57BL/6 mice and CD18-/- mice were infected with Af-dsRed conidia and at 24h post-infection one group of infected CD18-/- mice were given 4 million adoptively transferred bone-marrow derived neutrophils isolated from a LysM-eGFP mouse (eGFP+ neutrophils) and eyes were imaged at 48h. L. Fungal dsRed expression, M. eGFP+ neutrophil infiltration, N. cornea opacity area, and O. total cornea opacity were quantified using Metamorph software.

142 total fluorescence was significantly lower in CybB-/- corneas (there was no fluorescence in naïve corneas injected with CFDA, data not shown). Conversely, fungal dsRed expression and CFU (Figure 2, C and D) were elevated in CybB-/- compared with

C57BL/6 corneas, indicating impaired fungal clearance in CybB-/- corneas.

Interestingly, CybB-/- corneas had more severe disease than C57BL/6 mice, with significantly higher total cornea opacity scores (Figure 2, E and F), which correlated with increased neutrophil infiltration and formation of microabscesses and the presence of intact fungal hyphae (Figure 2G). As there is no defect in the ability of CybB-/- neutrophils to migrate to the cornea, it is likely that these findings represent ‘frustrated’ neutrophils that are unable to kill the hyphae, but can still recruit neutrophils to this site.

Very similar results were found when mice were infected with other pathogenic

Aspergillus and Fusarium species that cause keratitis (Figure S2 - S4), indicating that

ROS has a more general role in inhibiting growth of filamentous fungi.

To ascertain directly if the impaired fungal killing in CybB-/- mice is due to

NOX that is specifically produced by neutrophils, CD18-/- mice were infected with Af- dsRed as described above, and bone-marrow-derived neutrophils from C57BL/6 or

CybB-/- mice were injected intravenously 2h post-infection. Corneas were imaged 24h post-infection. Figure 2H and I show that fungal dsRed expression is significantly reduced following adoptive transfer of C57BL/6 neutrophils, whereas mice given

CybB-/- neutrophils have the same fungal dsRed expression as mice not receiving neutrophils. These data clearly demonstrate that NOX- dependent ROS production by neutrophils is essential for inhibiting fungal growth in the cornea.

143 Figure 4.2 Neutrophil NADPH oxidase is required for control of A.fumigatus fungal growth during corneal infection

A. C57BL/6 mice and CybB-/- mice were infected with 40,000 A.fumigatus strain Af- dsRed conidia. Eyes were imaged at 48h post-infection for ROS-mediated CFDA dye oxidation, fungal dsRed expression, and corneal opacity. B. CFDA dye oxidation, C. fungal dsRed expression, D. CFU, E. cornea opacity area, and F. total cornea opacity were quantified post-infection. G. 5µm sections of 48h infected fungal corneas were stained with PASH or neutrophil-specific NIMP antibody. H. CD18-/- mice were infected with A.fumigatus strain Af-dsRed conidia. At 2h post-infection, one set of mice were left untreated, the 2nd set received an i.v. injection of 4 million bone-marrow derived neutrophils (BMNs) isolated from C57BL/6 mice and a 3rd set received the same number of neutrophils isolated from CybB-/- mice. At 24h post-infection, eyes were imaged and I. fungal dsRed expression quantified using Metamorph software.

144 iNOS and reactive nitrogen species are not essential for control of fungal growth

The superoxide produced by NADPH oxidase can be converted to ROS or alternatively can react with nitric oxide (NO) produced by the enzymatic cleavage of arginine by inducible Nitric Oxide Synthase (iNOS), and can form the highly reactive nitrogen species, peroxynitrite (ONOO-) (97). If iNOS activity is high and significant amounts of NO are produced, the end products of NADPH oxidase activity will shift from production of ROS to production of reactive nitrogen species (RNS). To test the hypothesis that reactive nitrogen species are required for control of fungal growth during fungal keratitis, C57BL/6 mice were injected systemically with 1400W, which is an irreversible pharmacologic inhibitor of iNOS. In addition, the role of iNOS was examined in gene knockout mice.

Figure 3 shows that there were no significant differences in corneal opacification or fungal CFU between iNOS-/- and C57BL/6 mice, or between untreated and 1400W treated C57BL/6 mice. These findings indicate that in contrast to NADPH oxidase and reactive oxygen species, neither iNOS nor reactive nitrogen species have an essential role in control of fungal growth during corneal infection.

Human and murine neutrophil- mediated killing of A.fumigatus, A. flavus and

F.oxysporum hyphae is dependent on NADPH oxidase, but not iNOS or myeloperoxidase (MPO)

As neutrophil NADPH oxidase is required for control of fungal growth in vivo, but both conidia and hyphae are present during corneal infection, we developed a neutrophil-hyphae co-

145 Figure 4.3 iNOS is not required for control of fungal growth during corneal infection

A. C57BL/6, iNOS-/-, and 1400W-treated C57BL/6 mice were infected with A.fumigatus strain Af-BP conidia and eyes were imaged at 24h and 48h post-infection. B. Corneal opacity area, C. total cornea opacity, and D. CFU were quantified in infected corneas post-infection.

146 incubation fungal growth inhibition assay to determine which neutrophil mediators are required to limit the growth of fungal hyphae. A.fumigatus dsRed, A.flavus eGFP and

F.oxysporum FoxL GFP conidia were incubated in 96 well plates for 6h to allow time for germination and hyphal growth. For human neutrophil studies, neutrophils were isolated from the peripheral blood of normal volunteers, and added to each well at a

16:1 ratio in the presence of pharmacological inhibitors of either NADPH oxidase, iNOS or MPO. (NADPH oxidase: Diphenyliodonium (DPI), Apocynin (Apo); iNOS:

Aminoguanidine HCl (Agd), 2-Methyl-2-thiopseudoureasulfate (SMT)); MPO

(Indomethacin (Indo), 4-aminobenzoic hydrazide (4-AH)). For mouse neutrophil studies, neutrophils were isolated from the peritoneal cavities of C57BL/6, CybB-/-, and iNOS-/- mice and incubated with fungal hyphae as described above. Fungal growth was observed by fluorescence microscopy and measured by fluorescence spectroscopy.

Figure 4A shows hyphal growth of all three organisms after 16h incubation in

RPMI, but not PBS. However, in the presence of normal human neutrophils, fungal growth was clearly reduced, indicating that normal human neutrophils inhibit hyphal growth. However, addition of the NOX inhibitor DPI resulted in hyphal growth levels similar to RPMI alone, which is consistent with NOX dependent killing. Figures 4B-D show quantification of growth of A.fumigatus, A.flavus, and F.oxysporum, respectively.

For all three species, neutrophil mediated killing was inhibited in the presence of the

NADPH oxidase inhibitors DPI or Apo. In contrast, co-incubation with neutrophils and iNOS or MPO inhibitors resulted in fungal killing similar to that observed with neutrophils alone. Taken together, these data indicate that inhibition of hyphal growth by human neutrophils is dependent on NADPH oxidase but not iNOS or MPO.

147 As a complementary approach, we examined the role of NADPH oxidase and iNOS in CybB-/-, iNOS-/-, and C57BL/6 mouse neutrophils. As shown in Figure 4E,

A.fumigatus RFP expression in the presence of C57BL/6 neutrophils was lower than in

RPMI alone, consistent with impaired fungal growth. In contrast, RFP expression after incubation with CybB-/- neutrophils was significantly higher than after incubation with

C57BL/6 neutrophils and similar to incubation with no neutrophils, and is consistent with impaired ROS activity and fungal killing. Similar results were obtained for

A.flavus and F.oxysporum (Figure 4, F and G). However, fungal growth was not significantly different between iNOS-/- and C57BL/6 neutrophils (Figure 4H). Similar results were obtained for A.flavus and F.oxysporum (Figure 4, I and J). ROS production measured by CFDA assay correlates with the fungal killing assay, as ROS is detected after A. fumigatus incubation with C57BL/6, but not CybB-/- neutrophils, indicating that most of the ROS was dependent on NADPH oxidase. In contrast, there was no difference in NO production between CybB-/- and C57BL/6 neutrophils, and

ROS production by iNOS-/- neutrophils was not significantly different from C57BL/6 neutrophils (Figure S5).

Taken together, these data clearly demonstrate that NADPH oxidase and ROS production, but not iNOS or RNS production by murine neutrophils are essential for inhibiting the growth of A.fumigatus, A.flavus, and F. oxysporum hyphae.

ROS production and fungal killing is dependent on CD18, but not Dectin-1

Activation of NADPH oxidase is regulated by the physical separation of membrane and cytoplasmic components of the complex. However, following cellular

148 Figure 4.4 NOX but not iNOS or MPO is required for human and mouse neutrophils to control hyphae growth

Fungal conidia were cultured in SDB media for 6h in 96 well-plates. Subsequently, neutrophils were added to each well containing hyphae and the cells were co-incubated

149 for an additional 16h. In certain experiments, the NOX inhibitors (DPI and Apo), iNOS inhibitors (SMT, AgD), or MPO inhibitors (Indo, 4-AH) were added to the wells. A. A.fumigatus strain Af-dsRed was cultured either alone in PBS or RPMI or coincubated with 2x105 human neutrophils in RPMI or the same number of neutrophils in RPMI + the NOX inhibitor DPI. Fungal dsRed expression was imaged at 16h post incubation. Similar experiments were performed using eGFP expressing A.flavus (70-GFP), and GFP-expressing F.oxysporum (FoxL-GFP). B. A.fumigatus Af dsRed was co-incubated with neutrophils as described above for 16h, plates were washed, stained with calcofluor white and fungal chitin content determined via fluorometry. C. Similarly A.flavus (70-GFP) and D. F.oxysporum (FoxL-GFP) growth was quantified. E. To examine the role of NOX on mouse neutrophil-mediated killing of fungal hyphae we grew A.fumigatus dsRed conidia as described above for 6h and co-incubated them with thioglycolate-elicited peritoneal neutrophils from WT C57BL/6 and CybB-/- mice. At 16h post-infection, endogenous fungal fluorescent protein expression was quantified using fluorometry. Similar experiments were performed with F. A.flavus (70-GFP), and G. F.oxysporum (Fox-8996). H. Similarly, A.fumigatus dsRed, I. A.flavus (70- GFP), and J. F.oxysporum (FoxL-8996) growth was quantified following co- incubation with WT C57BL/6 and iNOS-/- mice. Abbreviations: Diphenyliodonium (DPI-100µM), apocynin (Apo-3mM), aminoguanidine HCl (Agd-1mM), 2-Methyl-2- thiopseudoureasulfate (SMT-100µM), Indomethacin (Indo-200µM), 4-aminobenzoic hydrazide (4-AH-100µM)

150 activation by pathogen recognition or cytokine receptors, p47phox in the cytoplasm is phosphorylated and translocated along with other cytoplasmic NOX components to the membrane, thereby forming the functional enzyme (88). Dectin-1 and CD18 recognize fungal cell-wall β-glucan and can activate NOX (95, 125), and Dectin-1-/- and CD18-/- mice exhibit impaired fungal clearance in cornea infection models (39, 95), suggesting a possible role for these receptors in neutrophil-mediated fungal killing. To examine if these cell surface receptors mediate NOX activation and ROS production by neutrophils following exposure to fungal hyphae, we loaded naive bone-marrow derived neutrophils with the ROS-sensitive dye CFDA, incubated them with

A.fumigatus hyphae and measured intracellular ROS-mediated CFDA dye oxidation using flow cytometry. Figure 5A shows that 91.5% of C57BL/6 neutrophils and 94.2% of Dectin-1-/- neutrophils exhibit high levels of ROS, compared with 31.4% of CD18-/- neutrophils, indicating that ROS production is dependent on CD18.

We also examined the role of neutrophil expressed CD18 and Dectin-1 in mediating killing of fungal hyphae by co-incubating A.fumigatus hyphae with purified thioglycolate elicited peritoneal neutrophils from C57BL/6, CD18-/-, or Dectin1-/- mice.

Figure 5B shows significant hyphal growth at 16h post-infection when hyphae were incubated in RPMI alone but not in PBS. These parameters were used as positive and negative controls. Figure 5B also shows that hyphae coincubated with either C57BL/6 or Dectin1-/- neutrophils show limited growth, whereas hyphae coincubated with CD18-

/- neutrophils show significant hyphal growth similar to that observed in RPMI alone.

In Figure 5C, the chitin content in the above experiment was quantified as described above. Taken together, these findings identify a major role for CD18, but not Dectin-1

151 Figure 4.5 CD18-dependent neutrophil NADPH oxidase activity is required to kill A.fumigatus hyphae

A. C57BL/6, CD18-/-, and Dectin1-/- BMNs were isolated, pre- loaded with the ROS- sensitive dye- CFDA, exposed to A.fumigatus hyphae for 1.5h and CFDA-dye oxidation analyzed by flow cytometry. B. Thioglycolate-elicited C57BL/6, CD18-/-, and Dectin1-/- peritoneal neutrophils were purified and exposed to A.fumigatus hyphae for 16h, stained with the chitin-binding dye calcofluor white, imaged using fluorescence microscopy, and C. fungal chitin content quantified via fluorometry.

152 in neutrophil NOX activation, ROS production, and fungal killing of A.fumigatus hyphae.

A.fumigatus antioxidant resistance to human neutrophils is dependent on superoxide dismutase and Yap1, but not catalases, gliotoxin, or LaeA-regulated secondary metabolites

As neutrophil NOX is essential for control of fungal growth in the cornea, and

NOX is required for both human and mouse neutrophil- mediated control of hyphal growth, we hypothesize that anti-oxidative mediators produced by Aspergillus and

Fusarium will scavenge ROS or inhibit NOX, and thereby impair neutrophil killing.

The transcription factor Yap1 is activated in A.fumigatus exposed to oxidative conditions, and regulates production of intracellular antioxidants and enzymes that convert ROS into less reactive products (101). For example, A.fumigatus superoxide dismutases (SOD1/2/3) convert superoxide to the less reactive hydrogen peroxide

(104), whereas A.fumigatus catalases (CatA/1/2) convert hydrogen peroxide to H2O

(103). In addition to ROS-catabolizing enzymes, filamentous fungi including

A.fumigatus produce secondary metabolites such as gliotoxin and fumagillin, which inhibit NADPH oxidase and are controlled by the master transcriptional regulator protein LaeA (107-109). We therefore examined the susceptibility of A.fumigatus strains with mutations in the Yap1 transcription factor (∆yap1), superoxide dismutases

(∆sod/1/2/3), catalases (∆catA, ∆cat1/2), or NOX-inhibiting 2° metabolites (∆gliP,

∆gliZ, ∆laeA) to human neutrophils.

153 To determine if Yap1 is required for hyphae survival in the presence of human neutrophils, we incubated ∆yap1 A.fumigatus hyphae with 1 x 105 human neutrophils, which are not sufficient to kill wild type Aspergillus (2x105 neutrophils were used above), and measured fungal biomass by calcofluor white binding as before. Figure 6A shows that RFU of WT-Dal hyphae grown in the presence of 1 x 105 human neutrophils was not significantly different from those grown in RPMI alone, indicating that WT-

Dal hyphae were not killed by 1 x 105 human neutrophils. In contrast, RFU of ∆yap1 mutants (derived from WT-Dal) incubated with the same number of neutrophils was significantly lower than WT-Dal, which is consistent with increased susceptibility of

∆yap1 mutants to killing by neutrophils. Similarly, RFU of the ∆sod1/2/3 mutant was significantly less than that of the WT-Ku80 strain (Figure 6B). However, there were no significant differences in RFU/fungal growth between ∆catA or ∆cat1/ ∆cat2 double mutants compared to the WT-G10 (Figure 6C), or ∆gliP and WT-B-5233 A.fumigatus strains (Figure 6D), or between ∆gliZ mutants or ∆laeA mutants and WT-Af293

(Figure 6E).

Taken together, these data show that the anti-oxidative pathways regulated by the transcription factor Yap1 and superoxide dismutases 1/2/3 are essential for optimal fungal growth in the presence of human neutrophils, whereas catalase A, catalase 1, catalase 2, gliotoxin and LaeA-regulated secondary metabolites have either a redundant or no role in blocking neutrophil- mediated killing.

154 Figure 4.6 Yap1, SOD 1/2/3 but not catalases or 2°metabolites mediate hyphae growth upon exposure to purified human neutrophils

A. The ∆yap1 and WT Dal1 A.fumigatus strains (12,500/well) were cultured for 6h to obtain hyphae and subsequently co-incubated with a sub-lethal MOI of human neutrophils (1x105) for 16h, at which time fungal growth was quantified by calcofluor- white staining and flourometry. B. Similarly, the fungal growth of the ∆sod1/2/3, WT- Ku80 , C. ∆catA, ∆cat1/2, WT-G10, D. ∆gliP, gliP-R, WT-B-5233, and E. ∆gliZ, gliZ-R, ∆laeA, laeA-R, WT-Af293 A.fumigatus strains were quantified 16h post- exposure to human neutrophils.

155

Yap1 and Superoxide dismutases 1/2/3 are required for fungal survival in vivo

Since the A.fumigatus transcription factor Yap1 and superoxide dismutases

1/2/3 regulate the survival of hyphae in the presence of human neutrophils, we next examined the role of these mediators in corneal infection. WT or mutant conidia were injected into the corneal stroma of C57BL/6 mice and corneal disease and fungal survival was examined as described above. We found that there was significantly less

CFU in corneas infected with ∆yap1 mutants compared to those infected with WT-Dal

(Figure 7A). These mutants also induced significantly less corneal opacity than WT-

Dal (Figure 7B-D). Similarly, ∆sod1/2/3 mutant CFU was significantly lower than

WT-Ku80 (Figure7E), although there was no detectable difference in corneal opacity

(Figure7F-H).

In contrast to ∆yap1 and ∆sod1/2/3 mutants, and consistent with the human neutrophil studies, mice infected with ∆catA or ∆cat1/2 showed no significant differences in CFU compared with WT-G10 (Figure 7I), and there were no significant differences in CFU between corneas infected with ∆gliZ, ∆gliP, or ∆laeA compared with either WT or reconstituted strains (Figure 7J, Figure S6). Taken together, these data reveal a critical role for Yap1- regulated antioxidant pathways and superoxide dismutases 1/2/3 in fungal keratitis, but either a redundant or no role for catalases, gliotoxin, or LaeA-regulated secondary metabolites.

156 Figure 4.7 Yap1, SOD 1/2/3 but not catalases or 2° metabolites mediate fungal growth during corneal infection

A. C57BL/6 mice were infected with 40,000 A.fumigatus conidia isolated from either ∆yap1 or the WT Dal1 strain, and fungal CFU were quantified at 4h and 48h post- infection. B. Eyes were imaged at 24h and 48h post-infection. C. Corneal opacity area and D. total cornea opacity were quantified. E. C57BL/6 mice were infected as described above with either ∆sod1/2/3 or the WT Ku80 strains, and fungal CFU were quantified at 4h and 48h post-infection. F. Eyes were imaged at 24h and 48h post- infection. G. Corneal opacity area and H. total cornea opacity were quantified. I. C57BL/6 mice were infected as described with either ∆catA, ∆cat1/2, or the WT G10 strain, and CFU were quantified in infected corneas post-infection. J. C57BL /6 mice were infected as described with either ∆gliZ, gliZ-R, ∆laeA, laeA-R, WT-Af293 or ∆gliP, gliP-R, WT-B-5233, and CFU were quantified in infected corneas post-infection.

157 Fungal thioredoxin mediates resistance to human neutrophils and oxidative stress and is required for fungal survival during corneal infection

Exposure of A.fumigatus to oxidative stress activates the transcription factor

Yap1, which up-regulates genes involved in anti-oxidative processes (101, 210). During oxidative stress, the Yap1- dependent proteins Allergen AspF3 and peroxiredoxin Prx1 are highly upregulated. These are putative thioredoxin peroxidases (peroxiredoxins) in the thioredoxin antioxidant pathway (101), and their peroxidatic residues reduce H2O2 to H2O, and are then oxidized and non-functional (211, 212).

Subsequently, thioredoxin protein reduces peroxiredoxins to their functional state, allowing further detoxification of H2O2 (212). Thioredoxin is then further reduced by the enzyme thioredoxin reductase using H+ equivalents from NADPH+ (212).

To examine the role of the thioredoxin pathway in mediating fungal anti- oxidative responses, we constructed an A.fumigatus strain, CEA-∆trxA which lacks aspf29, the gene with the highest homology to A. nidulans trxA (83). There are a total of five total putative thioredoxins encoded in the A.fumigatus genome (described in methods). Similar to the A.nidulans ∆trxA mutant (83), CEA-∆trxA exhibited a decreased growth rate and an increased susceptibility to H2O2 (data not shown).

Consistent with these observations, Figure 8A shows that fungal growth by the ∆trxA mutant after incubation with human neutrophils is significantly lower than the parent strain, indicating that the mutants are less resistant to killing by human neutrophils than the WT CEA10.

We also took a pharmacological approach to inhibit the function of total thioredoxin protein, utilizing the anti-cancer drug PX-12, which binds to the active site

158 of thioredoxin, and inhibits its ability to mediate redox reactions with target proteins and ultimately quench reactive oxygen species (122, 213, 214). Figure 8B shows that

A.fumigatus hyphae survive incubation with sub-lethal doses of human neutrophils, however upon inhibition of total thioredoxin protein with 10µM or 100µM PX-12 fungal growth is decreased, indicating that as with the single aspf29 knockout described above, thioredoxin is required for hyphae survival. To determine if PX-12 sensitizes hyphae to ROS, conidia were grown for 6h in SDB and incubated with hydrogen peroxide and PX-12 (1µM). Figure 8C shows that 5mM and 10mM H2O2 killed hyphae in the presence or absence of PX-12, whereas 1mM H2O2 alone showed no effect. However, in the presence of 1µM PX-12, this dose of H2O2 had anti-fungal activity. These findings demonstrate that Aspergillus thioredoxin mediates resistance to neutrophil and H2O2 oxidative stress.

Pharmacokinetic studies during a recent PX-12 phase 1 clinical trial noted that

PX-12 given at 400mg/m2/day for 72h is safe to administer to patients (122). However, to determine if PX-12 exhibits cytotoxicity to immune cells and cornea-specific cell types, several human myeloid and cornea cell lines were incubated for 16h with a dose- curve of PX-12 at which point the release of lactate dehydrogenase (LDH) into the surrounding media was assayed. In these assays, LDH release in the presence of PX-12 was not significantly different from media alone, indicating there was no cytotoxic effect of PX-12 (data not shown).

To determine if thioredoxin also impairs neutrophil mediated killing in vivo,

C57BL/6 mice were infected with dsRed expressing A.fumigatus, and given 8µl eye- drops of 3mM PX-12 or vehicle at 0 and 6h post-infection. Mice were sacrificed at 24h

159 Figure 4.8 Thioredoxin is required for hyphae survival during neutrophil exposure, oxidative stress, and corneal infection

A. To ascertain the role of thioredoxin in hyphae survival upon exposure to human neutrophils the single knockout A.fumigatus strain ∆trxA and WT CEA10 were co- incubated with a sub-lethal MOI of human neutrophils (1x105) for 16h, and fungal growth quantified as described above. B. To ascertain the role of the 5 putative thioredoxin proteins encoded in the A.fumigatus genome, hyphae were co-incubated with a sublethal MOI of human neutrophils in RPMI or neutrophils plus varying doses of the thioredoxin inhibitor PX-12. C. To examine the effect of thioredoxin inhibition on fungal growth during oxidative stress, A.fumigatus strain Af-dsRed was co- incubated with PX-12 and lethal and sublethal doses of H2O2 . D. To test the role of thioredoxin in mediating fungal survival during corneal infection, C57BL/6 mice were infected with A.fumigatus strain Af-dsRed. At 0 and 6h post-infection, PX-12 suspended at 3mM in a proprietary eye-drop formulation (Alcon laboratories) or vehicle was applied topically to the infected mouse corneas. At 24h post-infection, corneas were imaged, and E. fungal dsRed expression and F. CFU were quantified post-infection. G. C57BL/6 mice were infected with ∆trxA and WT CEA10 conidia and fungal CFU determined at 4h and 48h post-infection.

160 post-infection and imaged. Figure 8D shows no difference in corneal opacity between vehicle and PX-12 treatment, however, a significant reduction in fungal dsRed and

CFU (Figure 8, E and F) were observed in PX-12 treated mice. However, in vivo, there was no difference between ∆trxA and CEA10 in CFU (Figure 8G) or in corneal opacity (Figure S7). It is therefore likely that redundant expression of the five thioredoxin proteins encoded in the A.fumigatus genome mask the phenotype of the single gene knockout ∆trxA.

Taken together with in vitro findings with human neutrophils and H2O2, these data clearly demonstrate that A. fumigatus thioredoxin protein functions as an anti- oxidant during infection, impairs neutrophil mediated fungal killing, and is required for fungal growth during infection of the cornea.

161 Figure 4.S1 Quantification of Corneal opacification

Metamorph Imaging software (Molecular Devices, Downington PA) was used to determine the area of opacity and integrated corneal opacity. A circular region of constant area was centered on the images of corneas (upper panels). In order to standardize analysis of images within a given data set, 4 images of corneas with distinct regions of corneal opacification and no disease within the same eye were chosen. These standard eye images were converted into pseudo-color images to aid in opacity visualization (See panel above), Subsequently, the minimum threshold pixel intensity corresponding to opaque regions of the cornea were determined for each eye and averaged. All subsequent images within the data set were analyzed using the average minimum threshold pixel intensity value obtained during standardization. The threshold areas are clearly seen in the pseudocolor images (lower panels) with a scale shown of increasing pixel number from purple to red that correspond to areas of corneal opacity. Areas of glare (red arrows) were also demarcated and then set to zero, thereby eliminating glare from the subsequent analysis. To determine the area of opacity, the total area above the threshold within the circle was calculated and converted to percentage of total area of the circle minus areas of glares (i.e. Percent Corneal Opacity = (Threshold Area/ (Circle Area- Glare Area)*100). A second measure of corneal opacity was determined from the integrated intensity values, which record the total pixel numbers above the threshold. The more opaque corneas displayed a greater integrated intensity value (C57BL/6; CXCR2+/-) compared with less opaque corneas (CXCR2-/- ),and are represented in the data as Integrated Corneal Opacity. Upper panels: Naïve mice, C57BL/6 corneas , CXCR2+/-, and CXCR2-/- corneas infected with Af dsRed as shown in Figure 2. Lower panels: pseudocolor images generated by Metamorph.

162 Figure 4.S2 NOX is required to control A.fumigatus in vivo

A. C57BL/6 mice and CybB-/- mice were infected with 105 conidia from the A.fumigatus keratitis clinical isolate strain Af-BP. B. Corneal opacity area, C. total cornea opacity, and D. CFU were quantified in infected corneas post-infection.

163 Figure 4.S3 NOX is required to control A.flavus in vivo

A. C57BL/6 mice and CybB-/- mice were infected with 40,000 A.flavus 70-GFP conidia. Eyes were imaged at 24h and 48h post-infection for corneal opacity and fungal eGFP expression. B. Corneal opacity area, C. total cornea opacity, D. fungal eGFP expression, and E. CFU were quantified in infected corneas post-infection. F. C57BL/6 mice and CybB-/- mice were infected with 40,000 conidia from the A.flavus clinical isolate strain TN-302 and eyes were imaged at 24h and 48h post-infection for corneal opacity. G. Corneal opacity area, H. total cornea opacity, and I. CFU were quantified in infected corneas.

164 Figure 4.S4 NOX is required to control F.oxysporum in vivo

A. C57BL/6 mice and CybB-/- mice were infected with 20,000 F.oxysporum lysopersicum-RFP conidia. Eyes were imaged at 24h and 48h post-infection for corneal opacity and fungal dsRed expression. B. Corneal opacity area, C. total cornea opacity, D. fungal dsRed expression, and E. CFU were quantified in infected corneas post- infection. F. C57BL/6 mice and CybB-/- mice were infected with 50,000 conidia from the F.oxysporum clinical isolate strain 8996 and eyes were imaged at 24h and 48h post- infection for corneal opacity. G. Corneal opacity area, H. total cornea opacity, and I. CFU were quantified in infected corneas.

165 Figure 4.S5 Neutrophil NOX is required for ROS production

A.fumigatus strain Af-dsRed conidia (12,500/well) were cultured for 6h in black- walled, clear-bottom 96 well-plates before addition of purified thioglycolate-elicited peritoneal neutrophils from C57BL/6, CybB-/- iNOS-/- . At 2h post-infection, A. ROS and B. NO release by C57BL/6 and CybB-/- neutrophils into neutrophil-fungal coincubation supernatants were measured via fluorometry (CFDA dye oxidation) and spectrophotometry (Nitrosylation of Griess reagent), respectively. Similarly, At 2h post-infection C. ROS and D. NO release by C57BL/6 and iNOS-/- neutrophils into neutrophil-fungal coincubation supernatants

166 Figure 4.S6 Gliotoxin is not required for fungal infection

A. C57BL /6 mice were infected with 50,000 A.fumigatus conidia isolated from either ∆gliZ, gliZ-R, ∆laeA, laeA-R, WT-Af293 or ∆gliP, gliP-R, WT-B-5233, and eyes were imaged at 24h and 48h post-infection. In addition, at 48h post-infection CFDA dye was injected into 48h ∆gliP, gliP-R, or WT-B-5233- infected corneas and eyes imaged (GFP filter) to detect ROS- mediated oxidation of CFDA in infected corneas. B. Corneal opacity area, C. total cornea opacity, and D. ROS-mediated CFDA dye oxidation were quantified in infected corneas post-infection. E. C57BL/6 mice were infected with 50,000 conidia isolated from the A.flavus strains ∆laeA, laeA-R, WT- NRRL and eyes were imaged at 24h and 48h post-infection. F. Corneal opacity area, G. total cornea opacity, and H. CFU were quantified in infected corneas post-infection.

167 Figure 4.S7 TrxA is not required for fungal infection

A. C57BL/6 mice were infected with ∆trxA and WT CEA10 conidia and eyes were imaged at 24h and 48h. B. Cornea opacity area and C. total cornea opacity were quantified using Metamorph software.

168 Discussion:

In this study, we utilized a novel neutrophil adoptive transfer model and neutrophil depletion using NIMP-R14 antibody, which in contrast to GR1, is specific for murine neutrophils (215), to demonstrate that neutrophils are absolutely required to kill fungal hyphae during infection. These approaches are also more specific than shown in earlier studies (216-218) using chemical immunosuppression (219).

Subsequently, we identified the molecular interactions between neutrophils and fungal hyphae that results in fungal death. Conclusions from these studies are illustrated in

Figure 5.2 in the context of neutrophil oxidase production and anti-oxidants produced by hyphae.

Both Dectin-1 and CR3 (CD11b/CD18) recognize fungal β-glucan (220,

221), however, we identified an essential role for CD18, but not Dectin-1 in ROS production and killing of A.fumigatus hyphae. These findings are in agreement with observations that CR3 (CD11b/CD18) and not Dectin-1 is the major receptor on human neutrophils for β-glucan (94, 95), and more recently that CD18 mediates ROS production to A.fumigatus hyphae (222). In contrast, neutrophil ROS production in response to A.fumigatus swollen conidia is Dectin-1-dependent (129, 223). Although β- glucan is expressed on the surface of hyphae and swollen conidia (30, 125), it is unclear why CD18 but not Dectin-1 is required for NOX activation and killing of hyphae.

Potential explanations include increased cell surface expression of CD18 compared with Dectin-1 (224), or preferential Dectin-1 recognition of conidia compared to hyphae (60). However, it is also possible that differences in receptor-mediated 2° and

3° granule exocytosis result in different levels of NOX localization to the plasma

169 . membrane, production of extracellular O2 , and killing of fungal hyphae. This hypothesis will be addressed in future studies.

Neutrophils spread onto fungal hyphae allowing CD18/CD11b to recognize cell wall β-glucan, initiating an intracellular signaling cascade, which results in p47phox phosphorylation, translocation of cytoplasmic NOX components to the plasma membrane, and formation of a functional NOX enzyme (88). NOX subsequently

. . produces short-lived O2 within nanometer proximity to the fungal cell wall(88). O2 can oxidize cell wall components directly, be converted to the more stable H2O2, or enter the fungal cytoplasm through porins and anion channels on the fungal plasma membrane (97, 225). H2O2 can also be converted extracellularly by MPO into hypohalous acids which are likely too short-lived to enter the fungal cytoplasm or react with iNOS-derived NO forming peroxynitrite (ONOO-) (97). Our finding that the fungal cytoplasmic antioxidant thioredoxin and superoxide dismutases, but not catalases, are essential for hyphae survival (see below) suggests that during infection,

. neutrophil-derived O2 enters the fungal cytoplasm, and along with other ROS- derivatives (H2O2, etc.) mediates oxidation of essential cytoplasmic proteins and lipids leading to death of fungal hyphae.

Using NOX deficient CybB-/- mice and adoptive transfer of CybB-/- neutrophils, we are the first to demonstrate that neutrophil-specific expression of NOX

. and O2 production is required to control the growth of A.fumigatus in vivo. In addition, we demonstrated using specific inhibitors and gene knockout neutrophils that NOX is required for human and murine neutrophils to kill A. fumigatus, A. flavus, and F. oxysporum hyphae. In contrast to NOX, we found no role for either iNOS or MPO in

170 killing Aspergillus or Fusarium hyphae. Similar findings were obtained in experimental A.fumigatus lung infections in which NOX (90) but not MPO (226) or iNOS (192)are required to control fungal growth. Our findings are also in agreement with the enhanced susceptibility of chronic granulomatous disease (CGD) patients (15) but not MPO deficient patients to filamentous fungal infections (209). However, our findings differ from a study showing no role for NOX in killing A.nidulans in vitro

(194). Given that iNOS is required for optimal killing of readily phagocytosable

Candida (227)and Cryptococcus (228), and MPO is required for optimal responses to

Candida (226), it is possible that both enzymes are most effective intracellularly as opposed to extracellularly and thus not effective against hyphae.

Oxidation by neutrophils induces nuclear translocation of Yap1, and up- regulation of the H2O2 catabolizing thioredoxin-dependent peroxiredoxases, aspf29 and prx , and secreted catalases 1/2 (101). The current study identified the Yap1 transcription factor as essential for hyphae survival during corneal infection and upon exposure to neutrophils. These findings are in agreement with a report on C.albicans in which the Yap1 homologue, Cap1p, is required for antioxidative defenses (229).

However, our results differ from A.fumigatus lung infection studies which showed no role for Yap1 during infection of immunocompromised mice or upon exposure of neutrophils to swollen conidia (101, 210). These differences may be explained by the immune status of the infected mice and a morphotype-specific role for Yap1.

In this study, we did not detect a role for Yap1-regulated and secreted catalases 1/2 or for cytoplasmic CatA in hyphae survival. These findings are consistent with studies on experimental Aspergillus lung infection, and are likely due to the lack

171 of CatA expression in hyphae as opposed to conidia, and the unavailability of secreted

Cat1/2 for cytoplasmic antioxidant defense by hyphae (103). Further, Yap1 induces very high levels of intracellular peroxiredoxins following oxidative stress (210) and similar to catalases, peroxiredoxins reduce H2O2 to H2O, while being oxidized in the process (212). Subsequently, peroxiredoxins are cyclically reduced to their functional state by thioredoxin protein, which is itself reduced by thioredoxin reductase (212). The importance of thioredoxin protein in fungal antioxidant defense is evidenced by upregulated A.fumigatus thioredoxin reductase expression upon exposure to normal versus CGD neutrophils (230), and the essential role of thioredoxin for redox regulation in A. nidulans (196). In this study, our bioinformatic analysis, revealed five putative thioredoxins in the A.fumigatus genome, including the human allergens aspf29 and aspf28, which are highly expressed during human infection (197). In addition, utilizing the pharmacological thioredoxin inhibitor, PX-12 (214), we show that thioredoxin is required for hyphae survival in the presence of neutrophils in vitro, and also during corneal infection. We found that topical application of PX-12 was sufficient to restrict fungal growth, and taken together with similar findings with thioredoxin inhibitors in dermatophyte skin infection models (231), and our in vitro findings showing directly that PX-12 enhances H2O2 mediated killing (Figure 8) indicates that this or similar compounds that target the thioredoxin pathway can block fungal thioredoxins and thereby inhibit the predominant anti-oxidant defense utilized by these pathogens.

A.fumigatus also expresses three superoxide dismutases (SODs) which catalyze the conversion of superoxide to H2O2 (103, 104). SOD1 and SOD3 are both cytoplasmic, whereas SOD2 is restricted to the mitochondrial membrane (104). In this

172 study, we identified a role for the SODs in mediating hyphae survival during oxidation by neutrophils and during infection. Our findings are consistent with increased cytoplasmic SOD1 and SOD3 but not mitochondrial SOD2 expression by A.fumigatus hyphae in the presence of normal versus CGD neutrophils (230) and the increased susceptibility of A.fumigatus ∆sod1/2/3 mutants to killing by alveolar macrophages

(104). However, ∆sod 1/2/3 mutants showed no difference in survival in an immunocompromised lung infection model (104). Given that SOD3 is the most highly expressed SOD in hyphae under oxidative conditions, it is likely that SOD3 is functionally dominant in A.fumigatus hyphae. Future studies will address this hypothesis.

Lastly despite the fact that numerous 2° metabolites up-regulated by the transcription factor LaeA are reported to have immunosuppressive effects including gliotoxin, fumagillin, fumagatin, and helvolic acid (105, 107-109) we did not detect a role for these toxins in hyphae survival upon exposure to neutrophils or during infection. Given that 2° metabolite production by A.fumigatus in culture peaks at 48h post-infection, (232), it is possible that 2° metabolites may mediate fungal survival in patients, but not mice, due to the more chronic nature of human disease, typically weeks to months, compared to our acute murine model of fungal keratitis.

Successful control of filamentous fungal infections requires that hyphae are killed and eliminated from infected tissues. The current study was performed to enhance our understanding of the mechanism by which host cells kill hyphae and how hyphae resist attack. In this effort, we have identified superoxide production by neutrophil NADPH oxidase as the optimal effector, whose fungicidal activity if

173 therapeutically exploited, could be therapeutically useful. In addition, we identify both

CXCR2 and CD18 as possible targets to inhibit excessive neutrophil recruitment into fungal-infected tissues. Both CXCR2 and CXCR1 antagonists are currently utilized clinically (195). In addition, we have identified the fungal transcription factor Yap1, superoxide dismutases, and thioredoxin as potential drug targets to inhibit fungal anti- oxidative stress responses and enhance fungal killing. Lastly, we show proof-of-concept that targeting fungal anti-oxidative stress defenses using the thioredoxin inhibitor, PX-

12, limits fungal growth during infection. As PX-12 has successfully completed two

Phase 1 safety clinical trials for cancer therapy (122, 213), it is possible that PX-12 may safely be utilized clinically to increase fungal sensitivity to oxidative stress and decrease the threshold required for neutrophils to kill fungal hyphae during infection.

174

Chapter 5

Discussion and Future Directions

175 Data summary-Abstract

In collaboration with Aravind Eye Hospital in Tamil Nadu, India, we first characterized the human immune response during fungal keratitis using ulcerative material from infected corneas as well as infected whole corneas from patients undergoing corneal transplantation. Analysis of these tissues identified neutrophils as the major cellular infiltrate with smaller populations of macrophages and T cells. In addition, utilizing qPCR and RNA extracted from fungal-infected human corneas we identified elevated expression of: 1. fungal-sensing cell-surface receptors (Dectin-1,

TLR2, and TLR4), 2. neutrophil-recruiting cytokines (IL-8, IL-1β, IL-17) , 3. proteins involved in IL-1 β maturation (NALP3, ASC), and 4. cytokines associated with adaptive immune responses (IFN- ,IL-17, but not IL-4) .

Having characterized the human immune response during fungal keratitis, we next developed and utilized a novel mouse model of trauma-induced fungal keratitis and knockout mice to identify that ß-glucan on A.fumigatus germinating conidia activates Dectin-1 on resident macrophages to produce IL-1ß, and CXCL1/KC, which together with IL-1R1/ MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4 but not MD-2 dependent fungal killing. In the next series of studies we show that hyphae activate neutrophil NADPH oxidase via complement receptor 3 (CD11b/CD18) and utilizing a novel neutrophil adoptive transfer model that neutrophil NADPH oxidase is essential for killing hyphae in vivo.

In addition, utilizing mutant A.fumigatus strains, our mouse model , and a novel in vitro neutrophil-hyphae killing assay we show that the ROS-sensing transcription factor

Yap1, the ROS detoxifying-enzyme superoxide dismutase, and the Yap1-regulated

176 thioredoxin antioxidant pathway, but not fungal 2° metabolites such as gliotoxin are required for resistance to oxidation by neutrophils.

In conclusion, this work has significantly advanced our knowledge of the immune and microbial mediators of disease outcome during fungal keratitis. In this effort we identified: Dectin-1, IL-1R1, CXCR2, CD18, Syk, and MyD88 as potential anti-inflammatory targets to minimize excessive neutrophil recruitment during fungal keratitis. Lastly, we identify fungal superoxide dismutase and thioredoxin as potential anti-fungal targets to enhance fungal sensitivity to oxidative stress and neutrophils.

Working Model - Immune recognition of fungi during corneal infection

As shown in Figure 5.1, fungal conidia are inoculated into the corneal stroma through an epithelial defect. Conidia subsequently germinate, shed surface hydrophobins, and expose underlying conserved molecular patterns which are recognized by germ-line encoded pattern recognition receptors on resident tissue macrophages. In the context of

A.fumigatus infection, β-glucan is recognized by Dectin-1 expressed on macrophages leading to the phosphorylation of spleen tyrosine kinase (Syk) and the nuclear translocation of NFκB. Subsequently, both IL-1β and CXCL1/KC (Human- IL-8) transcripts are upregulated. Immature IL-1β protein is subsequently processed to mature

IL-1β by the NLRP3 caspase-1inflammasome and both IL-1β and CXCL1/KC are secreted. IL-1β binds to IL-1R1on the same cell in an autocrine fashion and to neighboring fibroblasts, macrophages, and epithelial cells leading to further production of CXCL1/KC and other pro-inflammatory cytokines. Neutrophils extravasate into the cornea via CD18/CD11a interaction with endothelial-expressed ICAM-1 and migrate

177 Figure 5.1 Working Model- Immune response to fungi during corneal infection

See text for details

178 within the cornea via CXCL1/KC interaction with CXCR1 and CXCR2. Upon encountering hyphae, CD18 on neutrophils mediates NOX activation and extracellular killing of fungal hyphae, whereas Dectin-1 on infiltrating monocytes mediates the phagocytosis of conidia. In addition, MD2-independent TLR4 activity mediates fungal killing in vivo through an unknown mechanism possibly involving activation by

DAMPs released by injured cells. In human fungal keratitis neutrophils in the cornea produce IL-17, which presumably stimulates CXCL1/KC production by macrophages, fibroblasts, and epithelial cells and recruits more neutrophils. Lastly, T cells infiltrating the cornea produce both IL-17 and IFNγ which recruit neutrophils and enhance monocytic phagocytic responses, respectively.

Working Model- Oxidative stress at the neutrophil-hyphae interface

As shown in Figure 5.2, neutrophil extravasation into the cytokine-laden cornea primes neutrophils for further activation by triggering 2° and 3° granule exocytosis and localization of gp91 and p22phox on the neutrophil cell membrane (88). At the infectious site, primed neutrophils wrap themselves around hyphae and engage β- glucan on the fungal cell wall via complement receptor 3 (CD11b/CD18) not Dectin-1 resulting in p47phox phosphorylation, functional NOX enzyme assembly, and production of extracellular superoxide. Neutrophil NOX but not MPO or iNOS activity is required for killing hyphae indicating that superoxide and hydrogen peroxide but not hypohalous acids or peroxynitrite are required to kill hyphae. Extracellular ROS enter the fungal cytoplasm through membrane diffusion, porins, or anion channels where they oxidize essential biomolecules. A.fumigatus hyphae sense oxidation of cytoplasmic oxidants via the transcription factor Yap1, which translocates to the nucleus and up-

179

Figure 5.2 Working Model-oxidative stress at the neutrophil-hyphae interface

See text for details

180 regulates the expression of thioredoxin-regulated peroxiredoxins that detoxify cytoplasmic hydrogen peroxide. In addition, A.fumigatus hyphae express two cytoplasmic superoxide dismutases (SOD1/3) and one mitochondrial SOD (SOD2), which detoxify intracellular superoxide. Yap1, the thioredoxin antioxidant pathways and superoxide dimutases are essential for hyphae survival during infection and upon oxidation by neutrophils. In contrast, we show no role for hydrogen-peroxide detoxifying catalases or NOX-inhibiting LaeA transcription factor-regulated 2° metabolites in mediating hyphae survival in vivo or in vitro. the cornea, and prime and activate neutrophil NOX. These hypotheses should be addressed in future studies.

Future focus- Inflammatory responses to fungi during infection

CR3 or LFA-1 mediated ICAM-1 interaction and extravasation

In this study, we show that CD18 is required to recruit neutrophils into the cornea. However, CD18 is found in heterodimeric association with one of four alpha integrins: CD11a (αL β2-LFA-1), CD11b (αM β2-CR3/Mac-1), CD11c (αXβ2,

ITAX,CR4), or CD11d (αDβ2, ITAD) and both CR3 and LFA-1 can bind to endothelial-expressed ICAM-1and mediate neutrophil arrest and transmigration into inflamed tissues(67). Therefore, it is possible that either LFA-1 or CR3 or both are required for neutrophil arrest and extravasation into the cornea. To answer this question it is recommended that bone-marrow derived neutrophils from C57BL/6, CD18-/-,

CD11a-/-, or CD11b-/- mice be adoptively transferred into fungal-infected CD18-/- mice and total neutrophils in the cornea at 24h post-infection be quantified by flow cytometry. If LFA-1 but not CR3 is required for neutrophil extravasation during fungal

181 infection then it is possible that the commercially available monoclonal antibody

Efalizumab (Raptiva) (67)can be utilized to inihibit LFA-1 mediated leukocyte recruitment w/o simultaneously abrogating CR3-dependent NOX activation and fungal killing.

CR3 or LFA-1 mediated migration through the cornea

Within the cornea, neutrophils encounter high concentrations of two immobilized keratan sulfate proteoglycans, keratocan and lumican, which maintain cornea hydration, transparency, and bind to ELR+ chemokines forming chemokine gradients(233-235). It is likely that CR3 binds to these proteoglycans via its I-domain leading to cellular activation and migration through the cornea(67). In addition, it was recently shown that neutrophil

CD18 is required for interaction with ICAM-1 expressed on the surface of keratocytes in the cornea possibly through either LFA-1 or CR3(236). ECM-stimulated β2 integrin activation is likely to enhance neutrophil migration through the cornea, and prime and activate neutrophil NOX. These hypotheses should be addressed in future studies.

Although β-glucan- mediated CR3 activation does not result in significant cytokine production(67), it is likely that CR3-dependent migration of resident macrophages through the cornea (ICAM-1/proteoglycans) enhances interaction with fungi and indirectly increases total cytokine production in the cornea. This hypothesis can be addressed by examining pro-inflammatory cytokine production in LPS-injected corneas of C57BL/6, CD18-/- , and CD11b-/- mice prior to cellular infiltration (6h). Any inflammatory stimulus that doesn’t activate CR3 can be utilized. In addition, in vivo

182 confocal microscopy early during infection of αCD18, αCD11b, or isotype antibody- treated MAFIA mice (GFP+ macs; subconjunctival injection) with RFP-expressing fungi can be utilized to visualize CR3-dependent resident macrophage migration during infection. ICAM-1 and keratocan knockout mice can additionally be studied to identify the cornea ECM ligands mediating CR3-dependent resident macrophage migration to the infectious foci.

CXCR1/2-mediated β2-integrin activation

In addition, we show a role for CXCR2 in neutrophil recruitment during fungal infection. However, this defect could be due to a lack of CXCR2-mediated inside-out signaling to the β2-integrins LFA-1 or CR3(67) or to the lack of CXCR2-dependent chemotaxis up the ELR+ chemokine gradient(207). To decipher the role of CXCR2 in mediating β2-integrin activation on inflamed endothelia C57BL/6 and CXCR2-/- mice should be infected and β2-integrin mediated neutrophil arrest on limbal post-capillary venules examined in vivo using confocal microscopy. If CXCR2 signaling is required for β2-integrin actvation, neutrophils will roll but not adhere to the inflamed endothelia.

In contrast, if CXCR2 is not required for β2-integrin activation, then CXCR2-/- neutrophils will extravasate into the cornea but remain in the peripheral cornea as they are unable to sense the ELR+ chemokine gradient.

ELR+ chemokine mediated CXCR1/2 activation

In addition, although we show significant CXCL1/KC production during fungal infection of mouse corneas, CXCR2 can also bind to CXCL2/MIP-2, CXCL5/LIX and

CXCL15/lungkine (207). Similar to a study by Lin et.al (83), the relative contribution

183 of each chemokine to cellular recruitment during fungal keratitis can be deciphered via in vivo antibody-mediated neutralization. However, unlike mice, humans express two receptors, CXCR1 and CXCR2 that bind to 7 ELR+ chemokines and the collagen breakdown product P-G-P(207). Although we detected high expression of CXCL8/ IL-

8 during human fungal keratitis we did not examine expression levels of CXCL1-7 or

CXCR1-2 and future studies should attempt to do so. A better understanding of the chemokines and receptors mediating neutrophil recruitment during human fungal keratitis can help guide appropriate therapies and translate findings in animal models to patients. CXCR1 and CXCR2 antagonists are currently available clinically(195).

Pattern recognition receptor-mediated fungal recognition

In this study, we show that Dectin-1 mediates pro-inflammatory cytokine production and neutrophil recruitment during A.fumigatus infection. However, it is unclear whether

Dectin-1 mediates recognition of Fusarium and other species that cause human fungal keratitis. Numerous toll-like receptors and C-type lectin receptors recognize fungal components(49). It is possiblethat other receptors or multiple receptors including

Dectin-2 or MINCLE mediate resident macrophage activation in response to A.flavus and Fusarium species. A thorough in vitro screen using macrophage-like RAW cells and siRNA technology is recommended to simultaneously knock-down multiple receptors and decipher recognition pathways in vitro. Subsequently, infection of mice lacking these receptors should be utilized to confirm an in vivo phenotype.

184 Distinctions between Dectin-1 and CR3-mediated signaling

Both Dectin-1 and CR3 bind to β-glucan and are expressed on the surface of resident macrophages(68, 125). At first glance, one would assume that during infection

β-glucan would be detected by both receptors and both Dectin-1 and CR3 would contribute equally to pro-inflammatory cytokine production and cellular recruitment.

However, two independent factors help to explain why Dectin-1 but not CR3 mediates cellular recruitment during cornea infection. 1. CR3 is likely to exist on the surface of resting cells in a folded conformation with its ligand-binding domains unexposed(67).

Therefore, unlike Dectin-1, CR3 is unable to bind to β-glucan without prior cellular activation. 2. As the infection progresses, it is likely that PRR activation on the same cell, cytokines released fom neighboring cells, or IL-1α/DAMPs released from tissue trauma activate resident macrophages and expose the ligand binding domains allowing

CR3 to bind to β-glucan or ECM components and mediate intracellular signaling.

However, despite mediating potent cellular responses, CR3 only activates the AP-1 transcription factor and not NFAT or NFκB(67). Thus unlike Dectin-1 which activates all three transcription factors(237), CR3 activation leads to relatively little cytokine production. It is interesting that although both Dectin-1 and CR3 signaling lead to Syk activation and a rise in intracellular Ca2+, Syk/CARD9/Bcl-10/MALT1-mediated

NFκB activation and Ca2+/calcineurin-mediated NFAT activation occurs with Dectin-1 signaling but not CR3 signaling (67, 237). Unlike Dectin-1, Syk recruitment during

CR3-signaling is mediated by the adaptor protein SLP76 and the ITAM-containing adaptor proteins FcRγ and DAP12(67). It is possible that the mechanism by which Syk is activated determines subsequent substrate specificity. It is also possible that upon

185 activation, CR3 but not Dectin-1signaling simultaneously activates inhibitors that block

Syk-mediated NFκB or calcineurin-mediated NFAT activation. Future experiments in

HEK cells using CR3 and Dectin-1 encoding plasmids, chemical inhibitors, and siRNA knockdown of potential signaling pathways should be utilized to examine these hypotheses.

CR3 and complement-mediated neutrophil recruitment

In addition to pattern recognition receptors, complement activation and binding of soluble C3a and C5a to the cell surface receptors C3aR and C5aR on macrophages can mediate neutrophil and monocyte recruitment into infected tissues (238). Although fungi can activate complement (239), few studies have addressed whether complement can mediate inflammation during fungal infection. Identification of complement deposition in the cornea is as simple as immunohistochemical staining of infected human and mouse tissues with an anti-C3d antibody. If complement deposition is observed, then knockout mice should be utilized to dissect its role during fungal keratitis. If complement mediates inflammation, then clinically available complement inhibitors may yield therapeutic potential (240).

IL-1 family-mediated neutrophil recruitment

In this study, we also show that IL1R1 is essential for inflammation during fungal keratitis. However, IL-1R1 has two potential ligands and future studies should examine whether this phenotype is due to IL1α, or IL-1β (241). During cornea infection, we show Dectin-1 dependent IL-1β production. Future studies should

186 investigate whether Dectin-1 upregulates IL-1β transcript, or activates inflammasomes which cleave this protein into its mature form or both? Once macrophages produce and secrete IL-1 family cytokines, these molecules can presumably feedback to the same macrophage or neighboring cells through IL-1R1(241). This potential autocrine feedback loop is likely essential for sufficient macrophage activation and cell recruitment during fungal infection. As mentioned previously, even though Dectin-1 provides the 1st signal of a fungal infection to resident tissue macrophages, the signal soon ceases upon internalization of the receptor. It is likely that Dectin-1 stimulated IL-

1β functions as a 2nd signal establishing an autocrine and paracrine feedback loop and further cell activation.

Inflammasome-mediated IL-1β and IL-18 processing

There is evidence to suggest that macrophages exposed to A.fumigatus process

IL-1β via the NLRP3 inflammasome(242). It is not known whether this occurs in vivo and no studies to date have examined the IPAF/NLRC4, NLRP1, or AIM2 inflammasomes(243). Infection of caspase-1, ASC, and specific inflammasome knockout mice should help to answer these questions. Although inflammasomes are likely important to the maturation of IL-1β and IL-18 during fungal infection, the mechanism by which fungi activate inflammasomes is currently unknown.

Phagocytosis of conidia often leads to fungal death, however, some phagocytosed conidia continue to grow forming germ tubes/hyphae which burst out of phagolysosomes (244). Recently, it was shown that leakage of lysosomal components into the cytosol can activatethe NLRP3 inflammasome(245). Given this phenomenon, it

187 is possible that in vivo fungal-mediated rupture of phagolysosomes mediates inflammasome activation. In addition, fungal-mediated rupture of phagolysosomes exposes fungal components to the cytosol. It was recently shown that NAIPS (NLR family, apoptosis inhibitory proteins) bind to bacterial flagellin in the cytosol mediating

IPAF/NLRC4 inflammasome activation (246). It is therefore possible that NAIPs or similar cytoplasmic proteins recognize fungal components in the cytosol and subsequently activate inflammasomes. Future experiments should address these hypotheses.

Inflammasome-independent IL-1β and IL-18 processing

During human cornea infection we identified RNA expression of NLRP3 and its signaling adaptor molecule ASC. Interestingly, we detected this in a population almost entirely composed of neutrophils suggesting that human neutrophils produce and process mature IL-1β during human infection. However, neutrophils also express a series of serine proteases within 1° granules including neutrophil elastase, cathepsin G, and proteinase 3 which cleave IL-1β into its mature form(85). If secreted these proteases could mediate cleavage of extracellular immature IL-1β released from lysed epithelial cells, stromal cells, macrophages, and neutrophils (85, 243). However, it is also possible that upon fusion of 1°granules with conidia-laden phagosomes that subsequently lyse, immature IL-1β along with other cytoplasmic proteins are cleaved resulting in mature IL-1β production and likely protease-mediated cell death. Whether this occurs in vivo is not clear, however, a recent study from our laboratory suggests

188 that IL-1β production during experimental P.aeruginosa infection is largely inflammasome- independent (Karmakar et.al).

Role of T-cell and neutrophil-derived IL-17-in fungal infection

In the current study, we identified CD4+ T cells in infected human corneas and in situ expression of both IFNγ and IL-17, but not IL-4 suggesting the presence of both

Th1 and Th17 cells. Interestingly, in corneal ulcer material which consisted of >95% neutrophils and 5% epithelial cells we identified high expression of IL-17 transcript, suggesting that IL-17 producing neutrophils are present during human fungal keratitis.

Mouse models in our laboratory have since confirmed that upon immunization Th1,

Th17, and IL-17+ neutrophils infiltrate the cornea. The exact role of IL-17 during fungal keratitis has yet to be determined. However, it is possible that IL-17 in the cornea can prolong neutrophil survival, enhance neutrophil recruitment, or enhance neutrophil fungicidal activity(247).

Role of IFNγ in early and late stage fungal infection

Apart from T cells, many cells can produce IFNγ including NK-T cells(248).

During experimental P.aeruginosa keratitis NK-T cells were shown to produce IFNγ early during infection(249). If the same is true for fungal keratitis, then early IFNγ production would increase resident macrophage-mediated phagocytosis of fungal conidia, thereby decreasing the chance of conidial germination into hyphae and the risk of fulminant infection with hyphae. In this capacity, early IFNγ production in the cornea could act as a gatekeeper regulating the potential for spores in the cornea stroma

189 to germinate into hyphae and cause intractable disease. In addition, IFNγ production during late stage disease would likely enhance the ability of phagocytic cells to clear conidia and dead hyphae from the cornea (250). If true, the result of this action would be removal of these pro-inflammatory stimuli from the cornea and decreased inflammation. In support of this hypothesis, preliminary studies in immunized mice show infiltration of IFNγ-expressing Th1 cells in the cornea during late stage-fungal keratitis, though their exact role has not been elucidated.

Future experiments should attempt to decipher these current knowledge gaps in our understanding of inflammatory responses during fungal infection of the cornea.

Future focus- Activation of neutrophil fungicidal mechanisms

Priming neutrophil NOX during fungal infection

In this study, we show that both CD18 and CXCR2 are required to recruit neutrophils into the cornea, however, both CD18-mediated adhesion and CXCR2 activation are implicated in “priming” neutrophil NOX (88). In addition, at low levels a plethora of other cytokines including TNFα and IL-1β have been shown to prime neutrophils in vitro(88). It is implied that “priming” neutrophil NOX during migration to the infectious foci enhances the ability of neutrophils to subsequently produce ROS upon engaging microbes. Given the level of pro-inflammatory stimuli and microbial ligands at the infectious foci it is likely that NOX will be sufficiently activated upon engagement of fungi and although “priming” is likely to occur in vivo its functional significance is likely redundant. To ascertain the in vivo kinetics of NOX “priming”, the status of S345 p47phox phosphorylation should be examined in protein extracts from

190 C57BL/6 neutrophils isolated from the blood prior to infection, upon extrvasating into the cornea (peripheral cornea), and upon engaging fungi within the cornea (central cornea). To ascertain whether NOX “priming” is functionally important in vivo a mouse strain with a single point mutation on S345 of p47phox should be constructed and infected. If important increased fungal growth and CFU should be observed in S345 mutant mice compared to WT mice.

CR3-mediated signaling and NOX activation

At the molecular level, CR3-mediated NOX activation requires Syk,

ADAP/PRAM, SLP76, vav 1/2/3, and PLCγ(67). The guanine nucleotide exchange factors vav1/2/3 mediate activation of the Rho GTPase Rac2 which is essential for

NOX activation (67, 92). However, since triple knockout strains were used in this study

(222), it is unclear whether vav1, vav2, or vav3 are required. NOX activation also requires serine/threonine kinase-mediated phosphorylation of serine residues in the carboxy terminus of p47phox(88). To date PKC, PAK, ERK1/2, and p38 have all been shown to mediate p47phox phosphorylation in other receptor signaling pathways(88).

During CR3-mediated NOX activation, both PKC and ERK2 are activated and thus are likely candidates for p47phox phosphorylation (67). To decipher their role experimentally, ROS activity should be measured in BM-neutrophils from WT, CD18-/-

, and CD11b-/- exposed to hyphae in the presence or absence of PKC and ERK2 inhbitors. To more thoroughly examine CR3-mediated NOX activation, similar experiments should be performed utilizing more serine/threonine kinase inhibitors

(JNK, p38, and PAK) and signaling molecule inhibitors (Syk, PI3K, etc.). A similar

191 approach was recently used to show that PI3K β and PI3Kδ mediates NOX activation in response to A.fumigatus hyphae(222). For greater relevance these studies should also be performed in human neutrophils utilizing blocking antibodies to inhibit CR3 activation.

β2-integrin or CR3-mediated neutrophil NOX activation and fungal killing

In this study, we showed that mouse neutrophils require CD18 but not Dectin-1 to activate NOX and kill fungal hyphae in vitro. However, CD18 can form heterodimers with 4 α integrins forming 4 distinct proteins on the neutrophil surface:

CD11a (LFA-1), CD11b (CR3), CD11c (CR4), or CD11d (ITAD)(67). As described above, CR3 uniquely contains a lectin-like binding site allowing β-glucan recognition and has been shown to mediate neutrophil recognition of β-glucan coated particles(68,

95). Though unlikely, our data using CD18-/- mice does not exclude the possibility that the observed phenotype is due to other CD18-dependent heterodimeric β2-integrins. To definitively show that CR3 not other β2 integrins is required for NOX activation and fungal killing ROS activity should be measured in C57BL/6 and CD11b-/- bone-marrow derived neutrophils upon exposure to hyphae. If CR3 is required then CD11b-/- neutrophils like CD18-/- neutrophils will exhibit minimal ROS activity. Blocking antibodies to CD18 and CD11b should be utilized to to explore the role of human neutrophil CR3 in killing fungal hyphae.

An important caveat to the interpretation of in vitro data from cells lacking CR3 is that CR3 mediates adherence to plastic(251). In our assays, C57BL/6 neutrophils plated onto plastic 96-well plates exhibited minimal ROS production in the absence of fungal stimulation, suggesting that CR3-dependent adherence to plastic does not

192 mediate NOX activation. However, this does not exclude the possibility that CR3- dependent adherence to plastic enhances neutrophil migration towards hyphae and secretory vesicle/3° granule exocytosis in vitro. Both these factors could contribute to the observed fungal killing deficiency in CD18-/- neutrophils. Future studies on neutrophil CR3-dependent fungal killing should examine these confounding variables prior to data interpretation.

CR3-mediated neutrophil NOX activation and fungal killing during infection

The same CR3 molecule can bind to both soluble fibrinogen via its I-domain and soluble β-glucan via its lectin-like domain(67). However, it is not clear if steric hindrance limits the ability of a single CR3 molecule to bind to immobilized ICAM-1 or ECM proteoglycans via its I-domain and also simultaneously bind to particulate β- glucan particles such as conidia or hyphae. It is more plausible that within a population of CR3 molecules some will bind I-like domain ligands and others will bind β-glucan, although this hypothesis has not yet been tested. In the cornea, CR3 interaction with

ECM proteins raises concerns on whether the in vitro data showing a role for CR3 in killing β-glucan-expressing hyphae can be extrapolated to live infection. If ECM proteins bind with higher affinity to CR3 than β-glucan (Km= 5 x 10-8)(252), they may non-competitively inhibit the binding of β-glucan to CR3 and block CR3-dependent fungal killing. In addition, soluble ECM proteins, such as MMP8-cleaved keratocan

(83, 235), would induce rapid receptor internalization resulting in less cell-surface CR3 for engagement of fungal β-glucan. However, soluble β-glucan is also present in the

ECM during fungal infection (253). In contrast to ECM proteins, soluble β-glucan

193 binding to CR3 is likely to non-competitively inhibit ECM protein binding, enhance

NOX priming at low concentrations, and mediate NOX activation at high concentrations.

The complexities of neutrophil CR3 interaction with multiple ligands in vivo and the impact of these interactions on β-glucan recognition and fungal killing necessitate investigation into the fungicidal role of CR3 in vivo. Since neutrophils require CD18 to infiltrate infected tissues we are unable to utilize CD18-/- mice or theadoptive transfer model to study the role of CR3 in vivo. However, CD11b-/- mice do not exhibit migration defects and can be utilized to study the role of CR3 in fungal killing in vivo (254). In addition, we can inject the corneas of C57BL/6 mice with either anti-CD11b antibody or isotype control, followed by injection of fungal conidia, and measurement of both fungal growth (RFP) and fungal CFU at 48h post-infection. This method allows neutrophil extravasation into the cornea and blocks neutrophil

CR3activation only within the cornea. For this experiment, it is essential to control for a possible role of CR3 in neutrophil migration through the cornea. Therefore, proper data interpretation from this experiment requires that the quantification of neutrophils in the periphery and central cornea are identical amongst experimental groups.

CR3 interaction with complement fragment iC3b during infection

Neutrophil CR3 can also bind to the inactive C3b derivative iC3b via its I- domain(67). In vivo, although there is likely to be C3b deposition on the surface of hyphae, iC3b deposition is likely negligible given that inactivation of C3b to iC3b requires soluble Factor I, CD46 and CR1 which are host membrane bound

194 proteins(238). Thus in vivo, it is likely that β-glucan is the only ligand for CR3 on the surface of hyphae. In contrast, CR1 does bind to C3b and can mediate leukocyte adherence and phagocytosis to opsonized microbes(238). It is therefore possible that

CR1 enhances leukocyte responses to opsonized fungi in vivo. Infection of C3-/- and

CR1-/- mice could be used to address this hypothesis. If a phenotype is observed in C3-/- mice further investigation of the complement pathway during fungal keratitis is warranted.

CR3 and Dectin-1 dependent NOX activation and phagocytosis of conidia

In contrast, neutrophil ROS production in response to A.fumigatus swollen conidia is Dectin-1-dependent (129, 223) and we and others have shown that Dectin-1 is essential for phagocytosis of conidia by macrophages (39, 125). In neutrophils, CR3 is the main receptor for β-glucan particles (95), suggesting that neutrophil-mediated phagocytosis of A.fumigatus conidia is CR3-dependent. Phagocytosis assays with knockout neutrophils and fungal conidia should be performed to examine this hypothesis. However, if neutrophils do preferentially utilize CR3 to phagocytose

A.fumigatus conidia, while macrophages preferentially use Dectin-1it is possible that the relative surface levels of CR3 and Dectin-1 differ between the 2 cell types. Resting neutrophils express CR3 on their surface, however, upon further activation secretory vesicles, and 3° and 2° granules fuse with the plasma membrane placing progressively more CD11b on the neutrophil surface(71). High CD11b surface expression likely results in high formation of CD11b/CD18 heterodimers (CR3) on the neutrophil surface. In contrast, macrophages do not contain rapidly mobilized CD11b+ granules

195 and likely rely more heavily on Dectin-1 for conidia phagocytosis. Flow cytometric analysis of Dectin-1, CD11b, and CD18 surface expression on resting and activated macrophages and neutrophils should be performed to test these hypotheses.

CR3 and Dectin-1 dependent recognition of fungal β-glucan

In addition, it is possible that Dectin-1 recognizes a specific type of β-glucan structure expressed on the conidial surface, whereas CR3 recognizes a β-glucan structure expressed on the surface of hyphae. β-glucans can vary in size (large or small), complexity (highly branched or linear), and structure (β (1,3) or β (1,6) )(252).

Biochemical analysis of the conidial vs. hyphal cell wall would have to be performed to determine the types of β-glucan expressed on A.fumigatus morphotypes. Currently available antibodies can distinguish between β (1,3) or β (1,6) glycosidic linkages, however, they yield no information on the size and complexity of different β-glucans which may be critical for receptor activation. Therefore, fungi which stain positive for

β-glucan may not necessarily mediate activation of Dectin-1 or CR3. A more accurate readout on whether a particular β-glucan+ fungus will activate Dectin-1 or CR3 can be achieved by staining cells with Dectin-1-Fc or CR3-Fc fusion proteins (60, 255). An in vitro screen of Dectin-Fc and CR3-Fc fusion protein binding to fungi isolated from patients with fungal keratitis can provide valuable predictive information on whether these fungi are recognized in vivo by these receptors. For example, if a particular fungus isolated from a patient with fungal keratitis binds strongly to Dectin-1Fc, then it is likely that Dectin-1 signaling inhibitors will be successful in inhibiting excessive

196 inflammatory responses in this patient. The turnaround time for such clinically useful information would only be approximately 1.5h and likely less upon optimization.

TLR4-mediated fungal killing during infection and DAMPs

In this study we identified a role for TLR4 in killing fungi during infection. In addition TLR4-dependent killing appeared to be MD-2 independent. However, in vitro we did not detect a role for either TLR4 or MD-2 in fungal killing. TLR4–mediated cellular activation is decreased upon exposure to C.albicans mutants lacking o- mannosylation suggesting though not proving that o-mannose is a TLR4 ligand (133).

However, TLR4 responses are also activated by molecules, termed danger associated molecular patterns (DAMPs- HSP60, HMGB1, etc.), that are released from necrotic cells (256). DAMPs are likelyabundant in the cornea during live infection but not in- vitro during neutrophil-hyhae killng assays and a recent report suggests that necrotic cells stimulate TLR4- activity independently of MD-2(257). Given that DAMPs are constitutively expressed in host cells and only activate receptors when located extracellularly(256), typical assays such as ELISAs or qPCR yield no relevant data with respect to the location and therefore function of DAMPs. Therefore DAMPs must be identified in situ using immunohistochemistry wih anti-DAMP antibodies. Given that sectioning of slides cuts through cells, it is imperative that DAMPs be localized in the

ECM and not within cells to conclude that they are indeed released during infection.

Alternatively, infected corneas of GFP+ mice can be excised, fixed, stained with a non-

GFP fluorescently tagged anti-DAMP-antibody, mounted on a slide and visualized by confocal Z-sectioning. This method has the added bonus of intact cells so that

197 extracellular DAMPs can be readily identified. NIMP antibody mediated depletion followed by wholemount cornea staining could be used to identify whether neutrophils themselves are the main source of DAMPs in vivo. This is highly likely given that neutrophils are the most abundant cell during corneal infection and they characteristically spew their contents into the ECM during normal function and cell death(71). It is likely that since most DAMPs are essential for cellular function few knockout mice will be available for this study. However, once DAMP release is identified specific anti-DAMP antibodies can be utilized to study the role of DAMPs in vivo.

In addition the hypothesis that DAMPs activate TLR4-mediated anti-fungal responses can be examined by exogenously adding DAMPs to neutrophil:hyphae killing assays using human neutrophils and anti-TLR4 antibody as well as neutrophils from C57BL6 and TLR4-/- mice. Addition of exogenous DAMPs to these cultures should enhance fungal killing by human and mouse neutrophils but not when TLR4 is blocked or absent. Similar studies can be performed with MD-2-/- mice and mice lacking other signaling adaptors in the TLR4 pathway (CD14, MAL, TRIF, MyD88,

IRAKs). Lastly, future studies should examine the extent to which DAMPs stimulate

TLR4-dependent NOX activation and degranulation in neutrophils. Since DAMPs denote tissue trauma but not necessarily infection it would be interesting to examine whether TLR4 stimulation through DAMPs as opposed to LPS, stimulates the release of relatively less cytotoxic 1° granules and NOX-derived ROS. Interestingly, fungi characteristically express toxins such as gliotoxin which can kill host cells(157). It is therefore possible that during infection fungal-derived toxins enhance DAMP release

198 from host cells and activate TLR4-dependent fungicidal activity. These hypotheses warrant further experimental investigation.

Neutrophil degranulation during infection

The anti-microbial arsenal of neutrophils is contained within secretory vesicles

(SV) and 1°, 2°, and 3° granules(70). The kinetics by which neutrophils degranulate during fungal infection has not been elucidated. Neutrophil exocytosis can be assessed by flow cytometry due to specific translocation of granule proteins to the neutrophil surface during exocytosis. Using this method, surface expression of CD35/CR1,

CD66b, and CD63/LAMP3 can be used to identify sv, 2° granule, and 1° granule exocytosis, respectively(70, 91). Unfortunately, no specific membrane-bound marker for 3° granules has been identified and exocytosis of 3° granules is detected experimentally by measuring extracellular MMP9/gelatinase secretion (91). To ascertain the kinetics of neutrophil degranulation in vivo, flow cytometry using the markers described above should be performed on neutrophils isolated from mouse blood pre and post infection, as well as from the peripheral, peri-central, and central corneas of infected mice. The functional significance of neutrophil degranulation during fungal infection should also be addressed in future studies. Rab27a is required for the plasma membrane translocation of sv and all 3 neutrophil granules. Rab27a knockout mice are available and infection of these mice and invitro characterization of neutrophils derived from these mice could help establish the role of neutrophil degranulation during fungal infection.

Subsequently, the role of specific granule subsets should be examined.

199 The neutrophil fungicidal arsenal- one granule at a time

The fungicidal potential of granule contents also warrants further investigation.

Two recent proteomic analyses have identified far more proteins than previously thought within human neutrophil granules and secretory vesicles (69, 72). This valuable data set should be analyzed thoroughly to uncover and examine potential novel proteins with fungicidal activity released during neutrophil degranulation. Neutrophil granule proteins with known or potential fungicidal activity will be discussed here. Infection of mice deficient in specific granule proteins, and in vitro fungal killing assays with knockout neutrophils, neutralizing antibodies, or purified granule proteins alone or in combination should be utilized to examine the role of specific granule proteins in killing fungal hyphae.

Tertiary granules- tissue migration and revving the engines

Tertiary granule exocytosis results in the release of fungicidal lysozyme

(unknown mechanism) and arginase-1 (arginine catabolism), and surface expression of

β2 microglobulin(70). β2-microglobulin is required for surface expression of the MHC- like HFE protein, which in turn stabilizes transferrin receptor 1(76). Fe-bound transferrin subsequently activates transferrin receptor 1 leading to upregulation of the hormone hepcidin(76). Local production of hepcidin likely mediates ferroportin degradation inhibiting the ability of local cells to release intracellular Fe. In addition, it is possible that IL-6 and IL-23 produced at the local site travel to the liver and induce hepatocyte production of hepcidin leading to systemic iron depletion(258, 259). These hypotheses warrant further experimental investigation.

200

Secondary granules- the give and take of iron and oxidation

Secondary granule exocytosis also results in enhanced surface expression of β2- microglobulin and continues the trend of iron sequestration by characteristically releasing the iron chelator lactoferrin, the bacterial siderophore-binding protein

NGAL/lipocalin 24p3, and the hemoglobin binding protein haptoglobin which prevents microbial acquisition of hemoglobin bound heme(71). In addition, the acute phase response lipocalin-family protein which binds neutral/basic drugs α1-acid glycoprotein/orosomucoid is released during 2° granule exocytosis(71). It is possible that like lipocalin 1/tear lipocalin (34) which sequesters fungal hydroxymate-type siderophores and Lcn24p3/NGAL which binds bacterial siderophores(78), orosomucoid can chelate microbial siderophores although this hypothesis has not been tested. It is likely that in vivo the chelation of extracellular iron and enhanced intracellular iron storage inhibits microbial growth but also enhances microbial sensitivity to oxidation as

Fe is essential for most redox reactions within a cell(76). It is likely no coincidence that

2° granules harbor the highest concentration of granule proteins affecting iron acquisition and also simultaneously mobilize the greatest intracellular stores of gp91/p22phox to the neutrophil surface(71). Likely neutrophils have evolved to fine- tune their killing efficiency by tapping into the synergistic antimicrobial effects of simultaneous oxidation and iron chelation. It is imperative that future experiments examining the microbicidal role of iron-related granule proteins be performed in the presence or absence of oxidative stress. Lastly, 2° granule fusion also releases the fungal opsonizer pentraxin 3(80)and the fungicidal protein and peptides: hCAP18-LL-

201 37(81) and α-defensins (HNP1-4)(82). Importantly, mouse neutrophils do not express

α-defensins (260, 261).

Primary granules- tissue destruction and the last stand

Finally, primary granule fusion results in the release of myeloperoxidase(71), which is required for optimal killing of C.albicans (226, 262)but not Aspergillus or

Fusarium hyphae (this study). 1° granule exocytosis also releases the serine proteases: elastase, cathepsin G and proteinase 3(85). Surprisingly, elastase is required for lung defense against A.fumigatus in immuncompetent mice although the mechanism is unclear(84). Whether elastase, cathepsin G, and PR3 have direct antimicrobial activity against fungal hyphae warrants further investigation, 1° granule exocytosis also releases high quantitites of fungicidal α-defensins HNP1-4 and the antimicrobial lipid A binding protein azurocidin/CAP37. Lastly, 1° granule exocytosis places the enzyme sialidase on the surface of neutrophils, which cleaves sialic acid on membrane proteins and enhances adhesion to the ECM(86) . It is possible that sialidase also enhances neutrophil adhesion to hyphae as well. These hypotheses warrant further investigation.

Future focus- Fungal defenses against neutrophil fungicidal mechanisms

Genetic dissection of fungal thioredoxins

In the current study, we performed a bioinformatic analysis which identified 5 putative thioredoxin proteins in the A.fumigatus strain Af293 genome. We subsequently mutated one gene, aspf29, which exhibited the highest homology to A.nidulans trxA

(196), encoding a functionally characterized thioredoxin. However, the single mutant did not exhibit decreased survival in vivo. We attribute this to functional redundancy

202 mediated by the other 4 putative thioredoxins. The construction of thioredoxin mutant strains with multiple gene deletions will likely be required to genetically assess the role of thioredoxin in fungal virulence.In this study, we utilized the thioredoxin inhibitor

PX-12 to simultaneously inhibit the function of all 5 putative fungal thioredoxins(214,

263). Using this approach, we showed that thioredoxin is essential for fungal survival in vivo and during exposure to human neutrophils.

The role of fungal peroxiredoxins in defense against oxidation

We hypothesize that thioredoxin mediates anti-oxidative defenses by regulating the redox state of hydrogen peroxide-catabolizing peroxiredoxins(264). Interestingly, the two genes most highly upregulated by Af-Yap1 (Aspf3 and Prx) are both thioredoxin –regulated peroxiredoxins suggesting an intricate role for them in anti- oxidative defense (101, 210). Construction of Aspf3 and Prx mutant strains would be required to further examine their role in mediating hyphae survival to neutrophils and during corneal infection. Interestingly, a covalent inhibitor of peroxiredoxins (conoidin

A) was recently shown to inhibit the ability of Toxoplasma gondii to cause infection in mice (265). It is possible that conoidin A or similar peroxiredoxin inhibitors, like PX-

12, can be utilized to inhibit fungal antioxidative stress defenses and enhance fungal clearance in vivo.

203 Sensitivity of fungal mitochondria to oxidative stress

During infection, significant energy is required by fungi to resists host defenses(266). To keep up with the demand, mitochondria must continuously produce

ATP at high rates resulting in high levels of mitochondrial-derived ROS (267). To minimize ROS-mediated damage, mitochondria exhibit their own anti-oxidative defenses including mitochondrial thioredoxins, superoxide dismutases, and glutathione

(268, 269). These defenses are crucial because ROS-mediated mitochondrial damage results in a lack of ATP and a halt of all biochemical pathways resulting in cell death(267). Given the importance of mitochondria to organism survival it is possible that neutrophil-derived oxidants kill hyphae via inducing mitochondrial damage. It is also possible that PX-12 increases fungal sensitivity to oxidative stress by inhibiting mitochondrial thioredoxin and making mitochondria more susceptible to neutrophil and self-derived oxidative stress. Future studies should address these hypotheses.

Genetic dissection of SODs and therapeutic potential of SOD inhibitors

In addition, this study identified a role for superoxide dismutases in mediating fungal survival. A.fumigatus expresses three superoxide dismutases(104). However,

SOD3 is the most highly expressed SOD in hyphae under oxidative conditions and it is likely that SOD3 mediates the phenotype observed with the SOD1/2/3 triple mutant(104). The SOD3 single mutant has been constructed and future studies should examine the role of SOD3 in mediating hyphae survival during infection of the cornea.

Interestingly, SOD inhibitors have been identified(269), although their ability to enhance fungal sensitivity to oxidative stress has not been examined.

204 Role of fungal toxins during chronic fungal keratitis

Although, we did not detect a role for gliotoxin, or LaeA-regulated 2° metabolites during infection or upon exposure to human neutrophils, these data should not be interpreted to suggest that fungal toxins do not mediate human fungal keratitis.

A.fumigatus 2° metabolite production in culture peaks at 48h(232). If the same toxin- production kinetics exists during infection, then our mouse model in which mice are sacrificed at 48h is not useful for the study of fungal toxins. In contrast, human disease typically last weeks to months allowing ample time for fungal toxin production and accumulation(20). In order to examine the role of fungal toxins during corneal infection, a chronic model of fungal keratitis should be established which more closely reflects human disease. Perhaps a rabbit model of fungal keratitis should be utilized given that rabbit corneas more accurately approximate the shape and size of the human cornea(270).

Role of fungal glutathione and HOG-MAPK in anti-oxidative stress defenses

In this study, we did not examine the glutathione pathway. It is likely that glutathione works in parallel with thioredoxin to maintain cytoplasmic redox homeostasis(271). However, to date the only components of the A.fumigatus glutathione pathway that have been functionally characterized are three glutathione transferases (272) . Likewise, we have not yet characterized the high osmolarity glycerol mitogen-activated protein kinase pathway (HOG-MAPK) pathway. Like Yap-

1, the HOG-MAPK pathway is activated by oxidative stress and results in the upregulation of genes involved in anti-oxidative stress(271). Both these pathways

205 await complete functional characterization and their roles in virulence have yet to be determined.

Therapeutic implications

Targeting inflammatory mediators during fungal infection

The human and mouse immune systems are remarkably similar, however, significant differences have been identified (260). It is therefore imperative to understand the differences between these two systems before attempting to translate experimental findings in mice to humans. Given this caveat, Figure 5.3 illustrates the potential drug targets that our experimental murine studies suggest would inhibit excessive cellular recruitment during fungal infection of the cornea.

Inhibiting Dectin-1mediated neutrophil recruitment

Dectin-1-mediated recognition of fungal β-glucan can be inhibited experimentally by soluble β-glucan analogues, such as laminarin, which bind to Dectin-1 but do not mediate receptor clustering and thus competitively inhibit receptor activation by particulate β-glucan(223, 252). The soluble β-glucan PGG-glucan (poly-[1-6]-β-

Dglucopyranosyl-[1-3]-β-D-gluco-pyranose) has been shown in mice to stimulate CR3- dependent killing of iC3b coated tumor cells without pro-inflammatory cytokine production(221). PGG-glucan has been proven safe in clinical trials and is now being tested for adjunct therapy in the treatment of various cancers (273). The fact that PGG-

206 Figure 5.3 Anti-inflammatory targets during fungal keratitis

See text for details

207 glucan does not induce pro-inflammatory cytokine production , likely means that it either does not bind Dectin-1 or like laminarin binds to Dectin-1 w/o receptor activation. Therefore during fungal infection, PGG-glucan treatment would likely result in the inhibition of excessive Dectin-1mediated cellular recruitment and enhancement of CR3-dependent fungicidal activity.

Inhibiting Syk-mediated neutrophil recruitment

The activation of numerous immune receptors including the B-cell receptor (BCR),

Dectin-1, CR3, and LFA-1 recruit and activate spleen tyrosine kinase (Syk) which mediates a plethora of cellular responses including antibody production, pro- inflammatory cytokine production, and leukocyte migration into inflamed/infected tissues(274). Syk inhibitors such as 3-(1- Methyl-1H-indol-3-yl-methylene)-2-oxo-2,3- dihydro-1H-indole-5-sulfonamide, have been used experimentally for decades and optimized propietary Syk inhibitors are now utilized to treat inflammatory diseases such as rheumatoid arthritis, allergies, and B-cell malignancies(274). Syk inhibitors therefore hold therapeutic potential for the inhibition of inflammation during fungal keratitis by targeting Dectin-1-mediated pro-inflammatory cytokine production and

CR3/LFA-1-mediated leukocyte extravasation and migration through the cornea.

Inhibiting IL-1R1 and CXCR1/2 mediated neutrophil recruitment

The Dectin-1/Syk pathway results in significant production of IL-1β which binds to IL-1R1 leading to further pro-inflammatory cytokine production.

Currently, there are 3 FDA approved IL-1 pathway antagonist (275). Anakinra is a

208 recombinant IL-1R antagonist that competitively inhibits IL-1α and IL-1β binding to

IL-1R1 inhibiting cellular activation (275). Rilonacept is a recombinant anti-IL1R antibody which blocks the ligand binding site of IL-1R1, and Canakinumab is an anti-

IL-1β antibody which blocks IL-1β binding to IL-1R1(276) . In addition, the Dectin-

1/Syk pathway leads to significant production of ELR+ chemokines in mice and

CXCL8/IL-8 production by human macrophages. IL-8 recruits neutrophils into the inflammatory sites by binding to the cell surface receptors: CXCR1 and CXCR2.

Currently, three CXCR1 and CXCR2 antagonists are in clinical trials for COPD, reperfusion injury, and cystic fibrosis(277). The potential therapeutic benefit of IL-1 pathway and CXCR2 antagonists in treating fungal keratitis should be examined in future studies.

Inhibiting β2-integrin and ICAM-1 mediated neutrophil recruitment

In addition to CXCR2, our study shows that neutrophil- expressed LFA-1

(CD18/CD11a) and/or CR3 (CD18/CD11b) interaction with endothelial-expressed

ICAM-1 is essential for neutrophil extravasation into the cornea. There are several inhibitors experimentally shown to inhibit LFA1/CR3-ICAM-1 interactions(67).

SAR1118 recently showed efficacy in a Phase 2 clinical trial for inflammatory dry eye disease(278). However for the treatment of fungal infections, the ideal inhibitor would block LFA-1 but not CR3, resulting in inhibition of excessive neutrophil recruitment into the cornea with preservation of CR3-mediated fungicidal activity.

209 Inhibiting IL-17 mediated neutrophil recruitment

Lastly, in human fungal keratitis we identified high IL-17 expression. IL-17 mediates neutrophil recruitment into infected tissues(247) and it is possible that blocking IL-17 can restrict excessive inflammation during cornea infection. The humanized anti-IL-17 antibody AIN457 recently showed significant efficacy in the treatment of psoriasis, rheumatoid arthritis, and non-infectious uveitis and it may also prove efficacious in fungal keratitis(279).

Future studies are warranted.

Targeting cell wall β-glucan and phagocyte fungicidal activity

β-glucan is a major cell wall constitutent of germinated fungi and can potentially be targeted therapeutically (Figure 5.4). Caspofungin is a relatively new antifungal agent which inhibits the enzyme β-glucan synthase(280). Decreased β-glucan synthesis inhibits further growth of the fungal cell wall. However, it also causes structural changes to the fungal cell wall leading to increased exposure of β-glucan

(281). Increased β-glucan exposure will likely lead to increased fungal recognition by phagocytic cells through Dectin-1 and CR3 resulting in enhanced fungicidal activity. In agreement with this hypothesis, three recent studies have shown that fungi treated with sublethal concentrations of echinocandins are more easily killed by human neutrophils(281-283). We propose that echinocandins hold therapeutic potential for the treatment of fungal keratitis via direct microbicidal activity and enhancement of β- glucan mediated activation of neutrophil fungicidal activity. Echinocandin-mediated enhancement of surface β-

210 Figure 5.4 Targeting the fungal cell wall

See text for details

211 glucan exposure could prove especially helpful for infections with organisms, such as

F.oxysporum and C.albicans whose hyphae exhibit little surface β-glucan to activate immune cells.

In addition, since β-glucan is a major constituent of the fungal cell wall, it is possible to target this molecule and other fungal-specific cell wall molecules, such as chitin, with antibodies or antibody-like molecule that would enhance complement activation and FcγR mediated opsonization. A fusion molecule consisting of the extracellular domain of Dectin-1 and Fc portion of an IgG antibody has been used to successfully decrease fungal burden in experimental lung infections with P jiroveci and

A.fumigatus(255, 284). The Dectin-1 domain binds to surface β-glucan whereas as the

Fc domain is recognized by FcRγ expressed on phagocytic cells enhancing cell-cell interaction and phagocytosis of opsonized fungi (255, 284). Similarly, antibodies specific to β-glucan and chitin are available or can be optimized to opsonize fungi during infection. These approaches hold therapeutic potential and should be examined further in future studies.

Targeting fungal anti-oxidative stress responses

The persistence of fungal hyphae in infected corneas causes the persistent release of pro-inflammatory stimuli, cellular recruitment, and both host and microbial-mediated tissue damage. Current antifungal treatment regimens (topical natamycin and voriconazole) are often unsuccessful, with up to 60% of patients requiring corneal transplantation (12, 25). Alternative treatment strategies are thereby warranted.

Currently available anti-mycotic agents target the fungal cell wall via pore formation or inhibition of the of molecules essential for cell wall formation (β-glucan,

212 ergosterol)(285). Though at times efficacious, these drugs do not exhibit activity against all fungi and many fungi exhibit intrinsic or acquired resistance limiting their use clinically(26). In addition, these molecules tend to be large (MW: caspofungin-

1,093 g/mole) limiting their ability to penetrate through epithelial barriers and penetrate deep into avascular tissues such as the cornea (286).

In this study, we hypothesized that targeting microbial anti-oxidative stress responses would exploit microbial weaknesses and maximize host defenses. In this effort, we utilized the thioredoxin inhibitor, PX-12, and showed that hyphae treated with PX-12 exhibited increased sensitivity to hydrogen peroxide and were killed more easily by human neutrophils. In addition, PX-12 treatment exhibited minimal toxicity to myeloid and cornea cell lines and enhanced fungal clearance in infected mouse corneas. As PX-

12 has successfully completed two Phase 1 safety clinical trials for cancer therapy (213,

287), it is possible that PX-12 may be utilized to treat human fungal infections.

Likewise, it is possible that the thioredoxin-regulated H2O2-degrading peroxiredoxins

(265) and superoxide dismutases (269) can be targeted therapeutically to enhance fungal sensitivity to oxidation. Future studies should examine these hypotheses.

213 Concluding remarks

In conclusion, studies described herein highlight the key mediators of inflammatory responses to fungi, the mechanism by which hyphae are killed in vivo, and how they resist being killed. Likewise, the ideas for future studies proposed within this text warrant further investigation and are likely to shed light on the complex dynamics of host-fungal interactions. Ultimately, it is my hope that the information obtained and ideas proposed in this cumulative work translate into practical therapeutic advancements to help save the vision and lives of patients infected with filamentous fungi.

214 References

1. Romani, L. 2011. Immunity to fungal infections. Nat Rev Immunol 11:275-288. 2. Bahn, Y.S., Xue, C., Idnurm, A., Rutherford, J.C., Heitman, J., and Cardenas, M.E. 2007. Sensing the environment: lessons from fungi. Nat Rev Microbiol 5:57-69. 3. Kabir G. Peay, P.G.K., Thomas D. Burns. 2008. Fungal Community Ecology: A hybrid beast with a molecular master. BioScience 58:799-810. 4. Leenders, A.C., van Belkum, A., Behrendt, M., Luijendijk, A., and Verbrugh, H.A. 1999. Density and molecular epidemiology of Aspergillus in air and relationship to outbreaks of Aspergillus infection. J Clin Microbiol 37:1752-1757. 5. Oliveira, M., Ribeiro, H., Delgado, J.L., and Abreu, I. 2009. The effects of meteorological factors on airborne fungal spore concentration in two areas differing in urbanisation level. Int J Biometeorol 53:61-73. 6. Aimanianda, V., Bayry, J., Bozza, S., Kniemeyer, O., Perruccio, K., Elluru, S.R., Clavaud, C., Paris, S., Brakhage, A.A., Kaveri, S.V., et al. 2009. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature 460:1117- 1121. 7. Organization, W.H. October 2011. Visual Impairment and Blindness- Key Facts. W.M. Centre, editor. 8. Whitcher, J.P., Srinivasan, M., and Upadhyay, M.P. 2001. Corneal blindness: a global perspective. Bull World Health Organ 79:214-221. 9. Srinivasan, M. 2004. Fungal keratitis. Curr Opin Ophthalmol 15:321-327. 10. 2011. How Common are fungal diseases? . In The Fungal Research Trust 20th Anniversary meeting;June 18th, 2011. London Fungal Research Trust. 11. Ibrahim, M.M., de Angelis, R., Lima, A.S., Viana de Carvalho, G.D., Ibrahim, F.M., Malki, L.T., de Paula Bichuete, M., de Paula Martins, W., and Rocha, E.M. 2012. A new method to predict the epidemiology of fungal keratitis by monitoring the sales distribution of antifungal eye drops in Brazil. PLoS One 7:e33775. 12. Thomas, P.A. 2003. Fungal infections of the cornea. Eye 17:852-862. 13. Tao, X., Shao, D., and Xue, W. 2011. Prevention of HIV-1 infection with antiretroviral therapy. N Engl J Med 365:1934-1935; author reply 1935. 14. Enoch, D.A., Ludlam, H.A., and Brown, N.M. 2006. Invasive fungal infections: a review of epidemiology and management options. J Med Microbiol 55:809-818. 15. Antachopoulos, C. 2010. Invasive fungal infections in congenital immunodeficiencies. Clin Microbiol Infect 16:1335-1342. 16. Liesegang, T.J., and Forster, R.K. 1980. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol 90:38-47. 17. Rosa, R.H., Jr., Miller, D., and Alfonso, E.C. 1994. The changing spectrum of fungal keratitis in south Florida. Ophthalmology 101:1005-1013. 18. Bharathi, M.J., Ramakrishnan, R., Meenakshi, R., Padmavathy, S., Shivakumar, C., and Srinivasan, M. 2007. Microbial keratitis in South India: influence of risk factors, climate, and geographical variation. Ophthalmic Epidemiol 14:61-69. 19. Crawford, F., Harris, R., and Williams, H.C. 2008. Are placebo-controlled trials of creams for athlete's foot still justified? Br J Dermatol 159:773-779. 20. Thomas, P.A. 2003. Current perspectives on ophthalmic mycoses. Clin Microbiol Rev 16:730-797.

215 21. Hohl, T.M., and Feldmesser, M. 2007. Aspergillus fumigatus: principles of pathogenesis and host defense. Eukaryot Cell 6:1953-1963. 22. Michielse, C.B., and Rep, M. 2009. Pathogen profile update: Fusarium oxysporum. Mol Plant Pathol 10:311-324. 23. Amaike, S., and Keller, N.P. 2011. Aspergillus flavus. Annu Rev Phytopathol 49:107-133. 24. Schwessinger, B., and Ronald, P.C. 2012. Plant Innate Immunity: Perception of Conserved Microbial Signatures. Annu Rev Plant Biol. 25. Prajna, N.V., Mascarenhas, J., Krishnan, T., Reddy, P.R., Prajna, L., Srinivasan, M., Vaitilingam, C.M., Hong, K.C., Lee, S.M., McLeod, S.D., et al. 2010. Comparison of natamycin and voriconazole for the treatment of fungal keratitis. Arch Ophthalmol 128:672-678. 26. Lalitha, P., Shapiro, B.L., Srinivasan, M., Prajna, N.V., Acharya, N.R., Fothergill, A.W., Ruiz, J., Chidambaram, J.D., Maxey, K.J., Hong, K.C., et al. 2007. Antimicrobial susceptibility of Fusarium, Aspergillus, and other filamentous fungi isolated from keratitis. Arch Ophthalmol 125:789-793. 27. Szczotka-Flynn, L.B., Pearlman, E., and Ghannoum, M. 2010. Microbial contamination of contact lenses, lens care solutions, and their accessories: a literature review. Eye Contact Lens 36:116-129. 28. Imamura, Y., Chandra, J., Mukherjee, P.K., Lattif, A.A., Szczotka-Flynn, L.B., Pearlman, E., Lass, J.H., O'Donnell, K., and Ghannoum, M.A. 2008. Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type, and susceptibility to lens care solutions. Antimicrob Agents Chemother 52:171- 182. 29. Knop, E., and Knop, N. 2007. Anatomy and immunology of the ocular surface. Chem Immunol Allergy 92:36-49. 30. Karthikeyan, R.S., Leal, S.M., Jr., Prajna, N.V., Dharmalingam, K., Geiser, D.M., Pearlman, E., and Lalitha, P. 2011. Expression of innate and adaptive immune mediators in human corneal tissue infected with Aspergillus or fusarium. J Infect Dis 204:942-950. 31. McDermott, A.M. 2009. The role of antimicrobial peptides at the ocular surface. Ophthalmic Res 41:60-75. 32. Zhou, L., Beuerman, R.W., Foo, Y., Liu, S., Ang, L.P., and Tan, D.T. 2006. Characterisation of human tear proteins using high-resolution mass spectrometry. Ann Acad Med Singapore 35:400-407. 33. Flanagan, J.L., and Willcox, M.D. 2009. Role of lactoferrin in the tear film. Biochimie 91:35-43. 34. Fluckinger, M., Haas, H., Merschak, P., Glasgow, B.J., and Redl, B. 2004. Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores. Antimicrob Agents Chemother 48:3367-3372. 35. Ueta, M., and Kinoshita, S. 2010. Innate immunity of the ocular surface. Brain Res Bull 81:219-228. 36. Sun, Y., Chandra, J., Mukherjee, P., Szczotka-Flynn, L., Ghannoum, M.A., and Pearlman, E. 2010. A murine model of contact lens-associated fusarium keratitis. Invest Ophthalmol Vis Sci 51:1511-1516.

216 37. Alarcon, I., Tam, C., Mun, J.J., LeDue, J., Evans, D.J., and Fleiszig, S.M. 2011. Factors impacting corneal epithelial barrier function against Pseudomonas aeruginosa traversal. Invest Ophthalmol Vis Sci 52:1368-1377. 38. Hassell, J.R., and Birk, D.E. 2010. The molecular basis of corneal transparency. Exp Eye Res 91:326-335. 39. Leal, S.M., Jr., Cowden, S., Hsia, Y.C., Ghannoum, M.A., Momany, M., and Pearlman, E. 2010. Distinct roles for Dectin-1 and TLR4 in the pathogenesis of Aspergillus fumigatus keratitis. PLoS Pathog 6:e1000976. 40. Tarabishy, A.B., Aldabagh, B., Sun, Y., Imamura, Y., Mukherjee, P.K., Lass, J.H., Ghannoum, M.A., and Pearlman, E. 2008. MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2. J Immunol 181:593-600. 41. Brissette-Storkus, C.S., Reynolds, S.M., Lepisto, A.J., and Hendricks, R.L. 2002. Identification of a novel macrophage population in the normal mouse corneal stroma. Invest Ophthalmol Vis Sci 43:2264-2271. 42. Chinnery, H.R., Humphries, T., Clare, A., Dixon, A.E., Howes, K., Moran, C.B., Scott, D., Zakrzewski, M., Pearlman, E., and McMenamin, P.G. 2008. Turnover of bone marrow-derived cells in the irradiated mouse cornea. Immunology. 43. Hamrah, P., and Dana, M.R. 2007. Corneal antigen-presenting cells. Chem Immunol Allergy 92:58-70. 44. Hamrah, P., Huq, S.O., Liu, Y., Zhang, Q., and Dana, M.R. 2003. Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 74:172-178. 45. Hamrah, P., Liu, Y., Zhang, Q., and Dana, M.R. 2003. The corneal stroma is endowed with a significant number of resident dendritic cells. Invest Ophthalmol Vis Sci 44:581-589. 46. Chinnery, H.R., Pearlman, E., and McMenamin, P.G. 2008. Cutting edge: Membrane nanotubes in vivo: a feature of MHC class II+ cells in the mouse cornea. J Immunol 180:5779-5783. 47. Brown, G.D. 2011. Innate antifungal immunity: the key role of phagocytes. Annu Rev Immunol 29:1-21. 48. Kawai, T., and Akira, S. 2007. TLR signaling. Semin Immunol 19:24-32. 49. Netea, M.G., Brown, G.D., Kullberg, B.J., and Gow, N.A. 2008. An integrated model of the recognition of Candida albicans by the innate . Nat Rev Microbiol 6:67-78. 50. Jouault, T., Ibata-Ombetta, S., Takeuchi, O., Trinel, P.A., Sacchetti, P., Lefebvre, P., Akira, S., and Poulain, D. 2003. Candida albicans phospholipomannan is sensed through toll-like receptors. J Infect Dis 188:165-172. 51. Tada, H., Nemoto, E., Shimauchi, H., Watanabe, T., Mikami, T., Matsumoto, T., Ohno, N., Tamura, H., Shibata, K., Akashi, S., et al. 2002. Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner. Microbiol Immunol 46:503-512. 52. Wagner, H. 2001. Toll meets bacterial CpG-DNA. Immunity 14:499-502. 53. Ariizumi, K., Shen, G.L., Shikano, S., Xu, S., Ritter, R., 3rd, Kumamoto, T., Edelbaum, D., Morita, A., Bergstresser, P.R., and Takashima, A. 2000. Identification of

217 a novel, dendritic cell-associated molecule, dectin-1, by subtractive cDNA cloning. J Biol Chem 275:20157-20167. 54. McGreal, E.P., Rosas, M., Brown, G.D., Zamze, S., Wong, S.Y., Gordon, S., Martinez-Pomares, L., and Taylor, P.R. 2006. The carbohydrate-recognition domain of Dectin-2 is a C-type lectin with specificity for high mannose. Glycobiology 16:422-430. 55. Jouault, T., El Abed-El Behi, M., Martinez-Esparza, M., Breuilh, L., Trinel, P.A., Chamaillard, M., Trottein, F., and Poulain, D. 2006. Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling. J Immunol 177:4679-4687. 56. Forsyth, C.B., Plow, E.F., and Zhang, L. 1998. Interaction of the fungal pathogen Candida albicans with integrin CD11b/CD18: recognition by the I domain is modulated by the lectin-like domain and the CD18 subunit. J Immunol 161:6198-6205. 57. Wells, C.A., Salvage-Jones, J.A., Li, X., Hitchens, K., Butcher, S., Murray, R.Z., Beckhouse, A.G., Lo, Y.L., Manzanero, S., Cobbold, C., et al. 2008. The macrophage-inducible C-type lectin, mincle, is an essential component of the innate immune response to Candida albicans. J Immunol 180:7404-7413. 58. Saijo, S., Fujikado, N., Furuta, T., Chung, S.H., Kotaki, H., Seki, K., Sudo, K., Akira, S., Adachi, Y., Ohno, N., et al. 2007. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol 8:39-46. 59. Dennehy, K.M., and Brown, G.D. 2007. The role of the beta-glucan receptor Dectin-1 in control of fungal infection. J Leukoc Biol 82:253-258. 60. Steele, C., Rapaka, R.R., Metz, A., Pop, S.M., Williams, D.L., Gordon, S., Kolls, J.K., and Brown, G.D. 2005. The beta-glucan receptor dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog 1:e42. 61. Gersuk, G.M., Underhill, D.M., Zhu, L., and Marr, K.A. 2006. Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states. J Immunol 176:3717-3724. 62. Balloy, V., Si-Tahar, M., Takeuchi, O., Philippe, B., Nahori, M.A., Tanguy, M., Huerre, M., Akira, S., Latge, J.P., and Chignard, M. 2005. Involvement of toll-like receptor 2 in experimental invasive pulmonary aspergillosis. Infect Immun 73:5420- 5425. 63. Wang, J.E., Warris, A., Ellingsen, E.A., Jorgensen, P.F., Flo, T.H., Espevik, T., Solberg, R., Verweij, P.E., and Aasen, A.O. 2001. Involvement of CD14 and toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae. Infect Immun 69:2402-2406. 64. Meier, A., Kirschning, C.J., Nikolaus, T., Wagner, H., Heesemann, J., and Ebel, F. 2003. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell Microbiol 5:561-570. 65. Netea, M.G., Warris, A., Van der Meer, J.W., Fenton, M.J., Verver-Janssen, T.J., Jacobs, L.E., Andresen, T., Verweij, P.E., and Kullberg, B.J. 2003. Aspergillus fumigatus evades immune recognition during germination through loss of toll-like receptor-4-mediated signal transduction. J Infect Dis 188:320-326. 66. Takeuchi, O., and Akira, S. 2010. Pattern recognition receptors and inflammation. Cell 140:805-820. 67. Abram, C.L., and Lowell, C.A. 2009. The ins and outs of leukocyte integrin signaling. Annu Rev Immunol 27:339-362.

218 68. O'Brien, X.M., Heflin, K.E., Lavigne, L.M., Yu, K., Kim, M., Salomon, A.R., and Reichner, J.S. 2012. Lectin site ligation of CR3 induces conformational changes and signaling. J Biol Chem 287:3337-3348. 69. Lominadze, G., Powell, D.W., Luerman, G.C., Link, A.J., Ward, R.A., and McLeish, K.R. 2005. Proteomic analysis of human neutrophil granules. Mol Cell Proteomics 4:1503-1521. 70. Borregaard, N., Sorensen, O.E., and Theilgaard-Monch, K. 2007. Neutrophil granules: a library of innate immunity proteins. Trends Immunol 28:340-345. 71. Amulic, B., Cazalet, C., Hayes, G.L., Metzler, K.D., and Zychlinsky, A. 2012. Neutrophil function: from mechanisms to disease. Annu Rev Immunol 30:459-489. 72. Uriarte, S.M., Powell, D.W., Luerman, G.C., Merchant, M.L., Cummins, T.D., Jog, N.R., Ward, R.A., and McLeish, K.R. 2008. Comparison of proteins expressed on secretory vesicle membranes and plasma membranes of human neutrophils. J Immunol 180:5575-5581. 73. Manicone, A.M., and McGuire, J.K. 2008. Matrix metalloproteinases as modulators of inflammation. Semin Cell Dev Biol 19:34-41. 74. Munder, M., Mollinedo, F., Calafat, J., Canchado, J., Gil-Lamaignere, C., Fuentes, J.M., Luckner, C., Doschko, G., Soler, G., Eichmann, K., et al. 2005. Arginase I is constitutively expressed in human and participates in fungicidal activity. Blood 105:2549-2556. 75. Tobgi, R.S., Samaranayake, L.P., and MacFarlane, T.W. 1988. In vitro susceptibility of Candida species to lysozyme. Oral Microbiol Immunol 3:35-39. 76. Hentze, M.W., Muckenthaler, M.U., Galy, B., and Camaschella, C. 2010. Two to tango: regulation of Mammalian iron metabolism. Cell 142:24-38. 77. Zarember, K.A., Sugui, J.A., Chang, Y.C., Kwon-Chung, K.J., and Gallin, J.I. 2007. Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion. J Immunol 178:6367-6373. 78. Devireddy, L.R., Hart, D.O., Goetz, D.H., and Green, M.R. 2010. A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell 141:1006-1017. 79. Levy, A.P., Asleh, R., Blum, S., Levy, N.S., Miller-Lotan, R., Kalet-Litman, S., Anbinder, Y., Lache, O., Nakhoul, F.M., Asaf, R., et al. 2010. Haptoglobin: basic and clinical aspects. Antioxid Redox Signal 12:293-304. 80. Garlanda, C., Hirsch, E., Bozza, S., Salustri, A., De Acetis, M., Nota, R., Maccagno, A., Riva, F., Bottazzi, B., Peri, G., et al. 2002. Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response. Nature 420:182-186. 81. Wong, J.H., Ng, T.B., Legowska, A., Rolka, K., Hui, M., and Cho, C.H. 2011. Antifungal action of human cathelicidin fragment (LL13-37) on Candida albicans. Peptides 32:1996-2002. 82. Hazlett, L., and Wu, M. 2011. Defensins in innate immunity. Cell Tissue Res 343:175-188. 83. Lin, M., Jackson, P., Tester, A.M., Diaconu, E., Overall, C.M., Blalock, J.E., and Pearlman, E. 2008. Matrix metalloproteinase-8 facilitates neutrophil migration through the corneal stromal matrix by collagen degradation and production of the chemotactic peptide Pro-Gly-Pro. Am J Pathol 173:144-153.

219 84. Tkalcevic, J., Novelli, M., Phylactides, M., Iredale, J.P., Segal, A.W., and Roes, J. 2000. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 12:201-210. 85. Pham, C.T. 2006. Neutrophil serine proteases: specific regulators of inflammation. Nat Rev Immunol 6:541-550. 86. Cross, A.S., and Wright, D.G. 1991. Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells. J Clin Invest 88:2067-2076. 87. Scapini, P., Lapinet-Vera, J.A., Gasperini, S., Calzetti, F., Bazzoni, F., and Cassatella, M.A. 2000. The neutrophil as a cellular source of chemokines. Immunol Rev 177:195-203. 88. El-Benna, J., Dang, P.M., and Gougerot-Pocidalo, M.A. 2008. Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Semin Immunopathol 30:279-289. 89. Pollock, J.D., Williams, D.A., Gifford, M.A., Li, L.L., Du, X., Fisherman, J., Orkin, S.H., Doerschuk, C.M., and Dinauer, M.C. 1995. Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9:202-209. 90. Morgenstern, D.E., Gifford, M.A., Li, L.L., Doerschuk, C.M., and Dinauer, M.C. 1997. Absence of respiratory burst in X-linked chronic granulomatous disease mice leads to abnormalities in both host defense and inflammatory response to Aspergillus fumigatus. J Exp Med 185:207-218. 91. Uriarte, S.M., Rane, M.J., Luerman, G.C., Barati, M.T., Ward, R.A., Nauseef, W.M., and McLeish, K.R. 2011. Granule exocytosis contributes to priming and activation of the human neutrophil respiratory burst. J Immunol 187:391-400. 92. Nauseef, W.M. 2007. How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219:88-102. 93. Bellocchio, S., Moretti, S., Perruccio, K., Fallarino, F., Bozza, S., Montagnoli, C., Mosci, P., Lipford, G.B., Pitzurra, L., and Romani, L. 2004. TLRs govern neutrophil activity in aspergillosis. J Immunol 173:7406-7415. 94. Lavigne, L.M., Albina, J.E., and Reichner, J.S. 2006. Beta-glucan is a fungal determinant for adhesion-dependent human neutrophil functions. J Immunol 177:8667- 8675. 95. van Bruggen, R., Drewniak, A., Jansen, M., van Houdt, M., Roos, D., Chapel, H., Verhoeven, A.J., and Kuijpers, T.W. 2009. Complement receptor 3, not Dectin-1, is the major receptor on human neutrophils for beta-glucan-bearing particles. Mol Immunol 47:575-581. 96. Lambeth, J.D. 2004. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 4:181-189. 97. Winterbourn, C.C. 2008. Reconciling the chemistry and biology of reactive oxygen species. Nat Chem Biol 4:278-286. 98. Reeves, E.P., Lu, H., Jacobs, H.L., Messina, C.G., Bolsover, S., Gabella, G., Potma, E.O., Warley, A., Roes, J., and Segal, A.W. 2002. Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416:291-297.

220 99. Remijsen, Q., Kuijpers, T.W., Wirawan, E., Lippens, S., Vandenabeele, P., and Vanden Berghe, T. 2011. Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ 18:581-588. 100. Bruns, S., Kniemeyer, O., Hasenberg, M., Aimanianda, V., Nietzsche, S., Thywissen, A., Jeron, A., Latge, J.P., Brakhage, A.A., and Gunzer, M. 2010. Production of extracellular traps against Aspergillus fumigatus in vitro and in infected lung tissue is dependent on invading neutrophils and influenced by hydrophobin RodA. PLoS Pathog 6:e1000873. 101. Lessing, F., Kniemeyer, O., Wozniok, I., Loeffler, J., Kurzai, O., Haertl, A., and Brakhage, A.A. 2007. The Aspergillus fumigatus transcriptional regulator AfYap1 represents the major regulator for defense against reactive oxygen intermediates but is dispensable for pathogenicity in an intranasal mouse infection model. Eukaryot Cell 6:2290-2302. 102. Belozerskaia, T.A., and Gessler, N.N. 2007. [Reactive oxygen species and the strategy of the antioxidant defense in fungi: a review]. Prikl Biokhim Mikrobiol 43:565- 575. 103. Paris, S., Wysong, D., Debeaupuis, J.P., Shibuya, K., Philippe, B., Diamond, R.D., and Latge, J.P. 2003. Catalases of Aspergillus fumigatus. Infect Immun 71:3551- 3562. 104. Lambou, K., Lamarre, C., Beau, R., Dufour, N., and Latge, J.P. 2010. Functional analysis of the superoxide dismutase family in Aspergillus fumigatus. Mol Microbiol 75:910-923. 105. Spikes, S., Xu, R., Nguyen, C.K., Chamilos, G., Kontoyiannis, D.P., Jacobson, R.H., Ejzykowicz, D.E., Chiang, L.Y., Filler, S.G., and May, G.S. 2008. Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence. J Infect Dis 197:479-486. 106. Sugui, J.A., Pardo, J., Chang, Y.C., Mullbacher, A., Zarember, K.A., Galvez, E.M., Brinster, L., Zerfas, P., Gallin, J.I., Simon, M.M., et al. 2007. Role of laeA in the Regulation of alb1, gliP, Conidial Morphology, and Virulence in Aspergillus fumigatus. Eukaryot Cell 6:1552-1561. 107. Perrin, R.M., Fedorova, N.D., Bok, J.W., Cramer, R.A., Wortman, J.R., Kim, H.S., Nierman, W.C., and Keller, N.P. 2007. Transcriptional regulation of chemical diversity in Aspergillus fumigatus by LaeA. PLoS Pathog 3:e50. 108. Tsunawaki, S., Yoshida, L.S., Nishida, S., Kobayashi, T., and Shimoyama, T. 2004. Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase. Infect Immun 72:3373-3382. 109. Fallon, J.P., Reeves, E.P., and Kavanagh, K. 2010. Inhibition of neutrophil function following exposure to the Aspergillus fumigatus toxin fumagillin. J Med Microbiol 59:625-633. 110. Gaujoux, T., Chatel, M.A., Chaumeil, C., Laroche, L., and Borderie, V.M. 2008. Outbreak of contact lens-related Fusarium keratitis in France. Cornea 27:1018-1021. 111. Chang, D.C., Grant, G.B., O'Donnell, K., Wannemuehler, K.A., Noble-Wang, J., Rao, C.Y., Jacobson, L.M., Crowell, C.S., Sneed, R.S., Lewis, F.M., et al. 2006. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 296:953-963.

221 112. Khor, W.B., Aung, T., Saw, S.M., Wong, T.Y., Tambyah, P.A., Tan, A.L., Beuerman, R., Lim, L., Chan, W.K., Heng, W.J., et al. 2006. An outbreak of Fusarium keratitis associated with contact lens wear in Singapore. JAMA 295:2867-2873. 113. Gower, E.W., Keay, L.J., Oechsler, R.A., Iovieno, A., Alfonso, E.C., Jones, D.B., Colby, K., Tuli, S.S., Patel, S.R., Lee, S.M., et al. 2010. Trends in fungal keratitis in the United States, 2001 to 2007. Ophthalmology 117:2263-2267. 114. Bharathi, M.J., Ramakrishnan, R., Meenakshi, R., Shivakumar, C., and Raj, D.L. 2009. Analysis of the risk factors predisposing to fungal, bacterial & Acanthamoeba keratitis in south India. Indian J Med Res 130:749-757. 115. Xie, L., Zhong, W., Shi, W., and Sun, S. 2006. Spectrum of fungal keratitis in north China. Ophthalmology 113:1943-1948. 116. Hu, J., Wang, Y., and Xie, L. 2009. Potential role of macrophages in experimental keratomycosis. Invest Ophthalmol Vis Sci 50:2087-2094. 117. Siddiqui, S., Anderson, V.L., Hilligoss, D.M., Abinun, M., Kuijpers, T.W., Masur, H., Witebsky, F.G., Shea, Y.R., Gallin, J.I., Malech, H.L., et al. 2007. Fulminant mulch pneumonitis: an emergency presentation of chronic granulomatous disease. Clin Infect Dis 45:673-681. 118. Martire, B., Rondelli, R., Soresina, A., Pignata, C., Broccoletti, T., Finocchi, A., Rossi, P., Gattorno, M., Rabusin, M., Azzari, C., et al. 2008. Clinical features, long- term follow-up and outcome of a large cohort of patients with Chronic Granulomatous Disease: an Italian multicenter study. Clin Immunol 126:155-164. 119. Gallien, S., Fournier, S., Porcher, R., Bottero, J., Ribaud, P., Sulahian, A., Socie, G., and Molina, J.M. 2008. Therapeutic outcome and prognostic factors of invasive aspergillosis in an infectious disease department: a review of 34 cases. Infection 36:533-538. 120. Denning, D.W., Follansbee, S.E., Scolaro, M., Norris, S., Edelstein, H., and Stevens, D.A. 1991. Pulmonary aspergillosis in the acquired immunodeficiency syndrome. N Engl J Med 324:654-662. 121. Livak, K.J., and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408. 122. Ramanathan, R.K., Abbruzzese, J., Dragovich, T., Kirkpatrick, L., Guillen, J.M., Baker, A.F., Pestano, L.A., Green, S., and Von Hoff, D.D. 2011. A randomized phase II study of PX-12, an inhibitor of thioredoxin in patients with advanced cancer of the pancreas following progression after a gemcitabine-containing combination. Cancer Chemother Pharmacol 67:503-509. 123. O'Donnell, K., Sutton, D.A., Rinaldi, M.G., Gueidan, C., Crous, P.W., and Geiser, D.M. 2009. Novel multilocus sequence typing scheme reveals high genetic diversity of human pathogenic members of the Fusarium incarnatum-F. equiseti and F. chlamydosporum species complexes within the United States. J Clin Microbiol 47:3851-3861. 124. O'Donnell, K., Sutton, D.A., Rinaldi, M.G., Sarver, B.A., Balajee, S.A., Schroers, H.J., Summerbell, R.C., Robert, V.A., Crous, P.W., Zhang, N., et al. 2010. Internet-accessible DNA sequence database for identifying fusaria from human and animal infections. J Clin Microbiol 48:3708-3718.

222 125. Hohl, T.M., Van Epps, H.L., Rivera, A., Morgan, L.A., Chen, P.L., Feldmesser, M., and Pamer, E.G. 2005. Aspergillus fumigatus triggers inflammatory responses by stage-specific beta-glucan display. PLoS Pathog 1:e30. 126. Ferwerda, B., Ferwerda, G., Plantinga, T.S., Willment, J.A., van Spriel, A.B., Venselaar, H., Elbers, C.C., Johnson, M.D., Cambi, A., Huysamen, C., et al. 2009. Human dectin-1 deficiency and mucocutaneous fungal infections. N Engl J Med 361:1760-1767. 127. Glocker, E.O., Hennigs, A., Nabavi, M., Schaffer, A.A., Woellner, C., Salzer, U., Pfeifer, D., Veelken, H., Warnatz, K., Tahami, F., et al. 2009. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N Engl J Med 361:1727-1735. 128. Bochud, P.Y., Chien, J.W., Marr, K.A., Leisenring, W.M., Upton, A., Janer, M., Rodrigues, S.D., Li, S., Hansen, J.A., Zhao, L.P., et al. 2008. Toll-like receptor 4 polymorphisms and aspergillosis in stem-cell transplantation. N Engl J Med 359:1766- 1777. 129. Werner, J.L., Metz, A.E., Horn, D., Schoeb, T.R., Hewitt, M.M., Schwiebert, L.M., Faro-Trindade, I., Brown, G.D., and Steele, C. 2009. Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus. J Immunol 182:4938-4946. 130. Bhartiya, P., Daniell, M., Constantinou, M., Islam, F.M., and Taylor, H.R. 2007. Fungal keratitis in Melbourne. Clin Experiment Ophthalmol 35:124-130. 131. Robinson, M.J., Osorio, F., Rosas, M., Freitas, R.P., Schweighoffer, E., Gross, O., Verbeek, J.S., Ruland, J., Tybulewicz, V., Brown, G.D., et al. 2009. Dectin-2 is a Syk-coupled pattern recognition receptor crucial for Th17 responses to fungal infection. J Exp Med 206:2037-2051. 132. Saijo, S., Ikeda, S., Yamabe, K., Kakuta, S., Ishigame, H., Akitsu, A., Fujikado, N., Kusaka, T., Kubo, S., Chung, S.H., et al. 2010. Dectin-2 recognition of alpha- mannans and induction of Th17 cell differentiation is essential for host defense against Candida albicans. Immunity 32:681-691. 133. Netea, M.G., Gow, N.A., Munro, C.A., Bates, S., Collins, C., Ferwerda, G., Hobson, R.P., Bertram, G., Hughes, H.B., Jansen, T., et al. 2006. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and Toll-like receptors. J Clin Invest 116:1642-1650. 134. Hise, A.G., Tomalka, J., Ganesan, S., Patel, K., Hall, B.A., Brown, G.D., and Fitzgerald, K.A. 2009. An essential role for the NLRP3 inflammasome in host defense against the human fungal pathogen Candida albicans. Cell Host Microbe 5:487-497. 135. Joly, S., Ma, N., Sadler, J.J., Soll, D.R., Cassel, S.L., and Sutterwala, F.S. 2009. Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome. J Immunol 183:3578-3581. 136. Bellocchio, S., Montagnoli, C., Bozza, S., Gaziano, R., Rossi, G., Mambula, S.S., Vecchi, A., Mantovani, A., Levitz, S.M., and Romani, L. 2004. The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J Immunol 172:3059-3069. 137. Yuan, X., and Wilhelmus, K.R. 2010. Toll-like receptors involved in the pathogenesis of experimental Candida albicans keratitis. Invest Ophthalmol Vis Sci 51:2094-2100.

223 138. Ramaprakash, H., Ito, T., Standiford, T.J., Kunkel, S.L., and Hogaboam, C.M. 2009. Toll-like receptor 9 modulates immune responses to Aspergillus fumigatus conidia in immunodeficient and allergic mice. Infect Immun 77:108-119. 139. Kasperkovitz, P.V., Cardenas, M.L., and Vyas, J.M. 2010. TLR9 Is Actively Recruited to Aspergillus fumigatus Phagosomes and Requires the N-Terminal Proteolytic Cleavage Domain for Proper Intracellular Trafficking. J Immunol 185:7614- 7622. 140. Sun, Y., Karmakar, M., Roy, S., Ramadan, R.T., Williams, S.R., Howell, S., Shive, C.L., Han, Y., Stopford, C.M., Rietsch, A., et al. 2010. TLR4 and TLR5 on corneal macrophages regulate Pseudomonas aeruginosa keratitis by signaling through MyD88-dependent and -independent pathways. J Immunol 185:4272-4283. 141. Hohl, T.M., Rivera, A., Lipuma, L., Gallegos, A., Shi, C., Mack, M., and Pamer, E.G. 2009. Inflammatory monocytes facilitate adaptive CD4 T cell responses during respiratory fungal infection. Cell Host Microbe 6:470-481. 142. Hebart, H., Bollinger, C., Fisch, P., Sarfati, J., Meisner, C., Baur, M., Loeffler, J., Monod, M., Latge, J.P., and Einsele, H. 2002. Analysis of T-cell responses to Aspergillus fumigatus antigens in healthy individuals and patients with hematologic malignancies. Blood 100:4521-4528. 143. Taylor, A.W. 2002. Neuroimmunomodulation and immune privilege: the role of neuropeptides in ocular immunosuppression. Neuroimmunomodulation 10:189-198. 144. Chowdhary, A., and Singh, K. 2005. Spectrum of fungal keratitis in North India. Cornea 24:8-15. 145. Dunlop, A.A., Wright, E.D., Howlader, S.A., Nazrul, I., Husain, R., McClellan, K., and Billson, F.A. 1994. Suppurative corneal ulceration in Bangladesh. A study of 142 cases examining the microbiological diagnosis, clinical and epidemiological features of bacterial and fungal keratitis. Aust N Z J Ophthalmol 22:105-110. 146. Perez-Balbuena, A.L., Vanzzini-Rosano, V., Valadez-Virgen Jde, J., and Campos-Moller, X. 2009. Fusarium keratitis in Mexico. Cornea 28:626-630. 147. Saha, R., and Das, S. 2006. Mycological profile of infectious Keratitis from Delhi. Indian J Med Res 123:159-164. 148. Wang, L., Sun, S., Jing, Y., Han, L., Zhang, H., and Yue, J. 2009. Spectrum of fungal keratitis in central China. Clin Experiment Ophthalmol 37:763-771. 149. Manikandan, P., Varga, J., Kocsube, S., Samson, R.A., Anita, R., Revathi, R., Doczi, I., Nemeth, T.M., Narendran, V., Vagvolgyi, C., et al. 2009. Mycotic keratitis due to Aspergillus nomius. J Clin Microbiol 47:3382-3385. 150. Kredics, L., Varga, J., Kocsube, S., Doczi, I., Samson, R.A., Rajaraman, R., Narendran, V., Bhaskar, M., Vagvolgyi, C., and Manikandan, P. 2007. Case of keratitis caused by Aspergillus tamarii. J Clin Microbiol 45:3464-3467. 151. Kredics, L., Varga, J., Kocsube, S., Rajaraman, R., Raghavan, A., Doczi, I., Bhaskar, M., Nemeth, T.M., Antal, Z., Venkatapathy, N., et al. 2009. Infectious keratitis caused by Aspergillus tubingensis. Cornea 28:951-954. 152. Allart, S., Lule, J., Serres, B., Jones, T., Davignon, J.L., Malecaze, F., and Davrinche, C. 2003. Impaired killing of HCMV-infected retinal pigment epithelial cells by anti-pp65 CD8(+) cytotoxic T cells. Invest Ophthalmol Vis Sci 44:665-671.

224 153. Gangneux, J.P., Camus, C., and Philippe, B. 2008. [Epidemiology of and risk factors for invasive aspergillosis in nonneutropenic patients]. Rev Mal Respir 25:139- 153. 154. Mahieu, L.M., De Dooy, J.J., Van Laer, F.A., Jansens, H., and Ieven, M.M. 2000. A prospective study on factors influencing aspergillus spore load in the air during renovation works in a neonatal intensive care unit. J Hosp Infect 45:191-197. 155. Ando, N., and Takatori, K. 1982. Fungal flora of the conjunctival sac. Am J Ophthalmol 94:67-74. 156. Fahad, B., McKellar, M., Armstrong, M., Denning, D., and Tullo, A. 2004. Aspergillus keratitis following corneal foreign body. Br J Ophthalmol 88:847-848. 157. Kwon-Chung, K.J., and Sugui, J.A. 2008. What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus? Med Mycol:1-7. 158. Kupfahl, C., Michalka, A., Lass-Florl, C., Fischer, G., Haase, G., Ruppert, T., Geginat, G., and Hof, H. 2008. Gliotoxin production by clinical and environmental Aspergillus fumigatus strains. Int J Med Microbiol 298:319-327. 159. Schwienbacher, M., Weig, M., Thies, S., Regula, J.T., Heesemann, J., and Ebel, F. 2005. Analysis of the major proteins secreted by the human opportunistic pathogen Aspergillus fumigatus under in vitro conditions. Med Mycol 43:623-630. 160. Monod, M., Capoccia, S., Lechenne, B., Zaugg, C., Holdom, M., and Jousson, O. 2002. Secreted proteases from pathogenic fungi. Int J Med Microbiol 292:405-419. 161. Xie, L., Zhai, H., Shi, W., Zhao, J., Sun, S., and Zang, X. 2008. Hyphal growth patterns and recurrence of fungal keratitis after lamellar keratoplasty. Ophthalmology 115:983-987. 162. Day, S., Lalitha, P., Haug, S., Fothergill, A.W., Cevallos, V., Vijayakumar, R., Prajna, N.V., Acharya, N.R., McLeod, S.D., and Lietman, T.M. 2009. Activity of against Fusarium and Aspergillus. Br J Ophthalmol 93:116-119. 163. Burnett, S.H., Kershen, E.J., Zhang, J., Zeng, L., Straley, S.C., Kaplan, A.M., and Cohen, D.A. 2004. Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J Leukoc Biol 75:612-623. 164. Sugui, J.A., Pardo, J., Chang, Y.C., Zarember, K.A., Nardone, G., Galvez, E.M., Mullbacher, A., Gallin, J.I., Simon, M.M., and Kwon-Chung, K.J. 2007. Gliotoxin is a of Aspergillus fumigatus: gliP deletion attenuates virulence in mice immunosuppressed with hydrocortisone. Eukaryot Cell 6:1562-1569. 165. Xue, T., Nguyen, C.K., Romans, A., Kontoyiannis, D.P., and May, G.S. 2004. Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch Microbiol 182:346-353. 166. Rasmussen, J.P., Bowring, F.J., Yeadon, P.J., and Catcheside, D.E. 2002. Targeting vectors for gene diversification by meiotic recombination in Neurospora crassa. Plasmid 47:18-25. 167. Toews, M.W., Warmbold, J., Konzack, S., Rischitor, P., Veith, D., Vienken, K., Vinuesa, C., Wei, H., and Fischer, R. 2004. Establishment of mRFP1 as a fluorescent marker in Aspergillus nidulans and construction of expression vectors for high- throughput protein tagging using recombination in vitro (GATEWAY). Curr Genet 45:383-389.

225 168. Jach, G., Pesch, M., Richter, K., Frings, S., and Uhrig, J.F. 2006. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nat Methods 3:597- 600. 169. May, G.S. 1989. The highly divergent beta-tubulins of Aspergillus nidulans are functionally interchangeable. J Cell Biol 109:2267-2274. 170. Chinnery, H., Carlson, E.C., Sun, Y., Lin, M., Burnett, S.H., Perez, V.L., Mcmenamin, P., and Pearlman, E. 2009. Bone marrow chimeras and c-fms conditional ablation (Mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation. J Immunol 182:2738-2744. 171. Akpek, E.K., and Gottsch, J.D. 2003. Immune defense at the ocular surface. Eye 17:949-956. 172. Rogers, N.C., Slack, E.C., Edwards, A.D., Nolte, M.A., Schulz, O., Schweighoffer, E., Williams, D.L., Gordon, S., Tybulewicz, V.L., Brown, G.D., et al. 2005. Syk-dependent cytokine induction by Dectin-1 reveals a novel pattern recognition pathway for C type lectins. Immunity 22:507-517. 173. Segal, B.H. 2009. Aspergillosis. N Engl J Med 360:1870-1884. 174. Hernanz-Falcon, P., Joffre, O., Williams, D.L., and Reis e Sousa, C. 2009. Internalization of Dectin-1 terminates induction of inflammatory responses. Eur J Immunol 39:507-513. 175. Luther, K., Rohde, M., Heesemann, J., and Ebel, F. 2006. Quantification of phagocytosis of Aspergillus conidia by macrophages using a novel antibody- independent assay. J Microbiol Methods 66:170-173. 176. O'Neill, L.A., and Bowie, A.G. 2007. The family of five: TIR-domain- containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7:353-364. 177. Underhill, D.M., Rossnagle, E., Lowell, C.A., and Simmons, R.M. 2005. Dectin-1 activates Syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood 106:2543-2550. 178. Dennehy, K.M., Ferwerda, G., Faro-Trindade, I., Pyz, E., Willment, J.A., Taylor, P.R., Kerrigan, A., Tsoni, S.V., Gordon, S., Meyer-Wentrup, F., et al. 2008. Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors. Eur J Immunol 38:500-506. 179. Heinsbroek, S.E., Taylor, P.R., Martinez, F.O., Martinez-Pomares, L., Brown, G.D., and Gordon, S. 2008. Stage-specific sampling by pattern recognition receptors during Candida albicans phagocytosis. PLoS Pathog 4:e1000218. 180. Wang, Y., Liu, T., Gong, H., Zhou, Q., Sun, S., and Xie, L. 2007. Gene profiling in murine corneas challenged with Aspergillus fumigatus. Mol Vis 13:1226- 1233. 181. Ohto, U., Fukase, K., Miyake, K., and Satow, Y. 2007. Crystal structures of human MD-2 and its complex with antiendotoxic lipid IVa. Science 316:1632-1634. 182. Kim, H.M., Park, B.S., Kim, J.I., Kim, S.E., Lee, J., Oh, S.C., Enkhbayar, P., Matsushima, N., Lee, H., Yoo, O.J., et al. 2007. Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell 130:906-917. 183. Shoham, S., Huang, C., Chen, J.M., Golenbock, D.T., and Levitz, S.M. 2001. Toll-like receptor 4 mediates intracellular signaling without TNF-alpha release in response to Cryptococcus neoformans polysaccharide capsule. J Immunol 166:4620- 4626.

226 184. Bretz, C., Gersuk, G., Knoblaugh, S., Chaudhary, N., Randolph-Habecker, J., Hackman, R.C., Staab, J., and Marr, K.A. 2008. MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus. Infect Immun 76:952-958. 185. Cubitt, C.L., Lausch, R.N., and Oakes, J.E. 1997. Differential induction of GRO alpha gene expression in human corneal epithelial cells and keratocytes exposed to proinflammatory cytokines. Invest Ophthalmol Vis Sci 38:1149-1158. 186. Lin, M., Carlson, E., Diaconu, E., and Pearlman, E. 2007. CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis. J Leukoc Biol 81:786-792. 187. Mahajan, V.B., Wei, C., and McDonnell, P.J., 3rd. 2002. Microarray analysis of corneal fibroblast gene expression after interleukin-1 treatment. Invest Ophthalmol Vis Sci 43:2143-2151. 188. Sun, Y., Chandra, J., Mukherjee, P.K., Szczotka-Flynn, L., Ghannoum, M., and Pearlman, E. 2009. A murine model of contact lens associated Fusarium keratitis. Invest Ophthalmol Vis Sci. 189. Plantinga, T.S., van der Velden, W.J., Ferwerda, B., van Spriel, A.B., Adema, G., Feuth, T., Donnelly, J.P., Brown, G.D., Kullberg, B.J., Blijlevens, N.M., et al. 2009. Early stop polymorphism in human DECTIN-1 is associated with increased candida colonization in hematopoietic stem cell transplant recipients. Clin Infect Dis 49:724- 732. 190. Milner, J.D., Sandler, N.G., and Douek, D.C. 2010. Th17 cells, Job's syndrome and HIV: opportunities for bacterial and fungal infections. Curr Opin HIV AIDS 5:179- 183. 191. Segal, B.H., and Romani, L.R. 2009. Invasive aspergillosis in chronic granulomatous disease. Med Mycol 47 Suppl 1:S282-290. 192. Philippe, B., Ibrahim-Granet, O., Prevost, M.C., Gougerot-Pocidalo, M.A., Sanchez Perez, M., Van der Meeren, A., and Latge, J.P. 2003. Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates. Infect Immun 71:3034-3042. 193. Rex, J.H., Bennett, J.E., Gallin, J.I., Malech, H.L., and Melnick, D.A. 1990. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis 162:523-528. 194. Henriet, S.S., Hermans, P.W., Verweij, P.E., Simonetti, E., Holland, S.M., Sugui, J.A., Kwon-Chung, K.J., and Warris, A. 2011. Human leukocytes kill Aspergillus nidulans by reactive oxygen species-independent mechanisms. Infect Immun 79:767-773. 195. Chapman, R.W., Phillips, J.E., Hipkin, R.W., Curran, A.K., Lundell, D., and Fine, J.S. 2009. CXCR2 antagonists for the treatment of pulmonary disease. Pharmacol Ther 121:55-68. 196. Thon, M., Al-Abdallah, Q., Hortschansky, P., and Brakhage, A.A. 2007. The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response. J Biol Chem 282:27259-27269. 197. Glaser, A.G., Menz, G., Kirsch, A.I., Zeller, S., Crameri, R., and Rhyner, C. 2008. Auto- and cross-reactivity to thioredoxin allergens in allergic bronchopulmonary aspergillosis. Allergy 63:1617-1623.

227 198. Rajasekaran, K., Cary, J.W., Cotty, P.J., and Cleveland, T.E. 2008. Development of a GFP-expressing Aspergillus flavus strain to study fungal invasion, colonization, and resistance in cottonseed. Mycopathologia 165:89-97. 199. Monod, M., Paris, S., Sarfati, J., Jaton-Ogay, K., Ave, P., and Latge, J.P. 1993. Virulence of alkaline protease-deficient mutants of Aspergillus fumigatus. FEMS Microbiol Lett 106:39-46. 200. da Silva Ferreira, M.E., Kress, M.R., Savoldi, M., Goldman, M.H., Hartl, A., Heinekamp, T., Brakhage, A.A., and Goldman, G.H. 2006. The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5:207-211. 201. Nierman, W.C., Pain, A., Anderson, M.J., Wortman, J.R., Kim, H.S., Arroyo, J., Berriman, M., Abe, K., Archer, D.B., Bermejo, C., et al. 2005. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438:1151-1156. 202. Bok, J.W., Chung, D., Balajee, S.A., Marr, K.A., Andes, D., Nielsen, K.F., Frisvad, J.C., Kirby, K.A., and Keller, N.P. 2006. GliZ, a transcriptional regulator of gliotoxin biosynthesis, contributes to Aspergillus fumigatus virulence. Infect Immun 74:6761-6768. 203. Horowitz Brown, S., Zarnowski, R., Sharpee, W.C., and Keller, N.P. 2008. Morphological transitions governed by density dependence and lipoxygenase activity in Aspergillus flavus. Appl Environ Microbiol 74:5674-5685. 204. Kale, S.P., Milde, L., Trapp, M.K., Frisvad, J.C., Keller, N.P., and Bok, J.W. 2008. Requirement of LaeA for secondary metabolism and sclerotial production in Aspergillus flavus. Fungal Genet Biol 45:1422-1429. 205. Yang, L., Ukil, L., Osmani, A., Nahm, F., Davies, J., De Souza, C.P., Dou, X., Perez-Balaguer, A., and Osmani, S.A. 2004. Rapid production of gene replacement constructs and generation of a green fluorescent protein-tagged centromeric marker in Aspergillus nidulans. Eukaryot Cell 3:1359-1362. 206. Faust, N., Varas, F., Kelly, L.M., Heck, S., and Graf, T. 2000. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96:719-726. 207. Viola, A., and Luster, A.D. 2008. Chemokines and their receptors: drug targets in immunity and inflammation. Annu Rev Pharmacol Toxicol 48:171-197. 208. Wilson, R.W., Ballantyne, C.M., Smith, C.W., Montgomery, C., Bradley, A., O'Brien, W.E., and Beaudet, A.L. 1993. Gene targeting yields a CD18-mutant mouse for study of inflammation. J Immunol 151:1571-1578. 209. Andrews, T., and Sullivan, K.E. 2003. Infections in patients with inherited defects in phagocytic function. Clin Microbiol Rev 16:597-621. 210. Qiao, J., Kontoyiannis, D.P., Calderone, R., Li, D., Ma, Y., Wan, Z., Li, R., and Liu, W. 2008. Afyap1, encoding a bZip transcriptional factor of Aspergillus fumigatus, contributes to oxidative stress response but is not essential to the virulence of this pathogen in mice immunosuppressed by cyclophosphamide and triamcinolone. Med Mycol 46:773-782. 211. Wood, Z.A., Schroder, E., Robin Harris, J., and Poole, L.B. 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28:32-40.

228 212. Koharyova, M., and Kolarova, M. 2008. Oxidative stress and thioredoxin system. Gen Physiol Biophys 27:71-84. 213. Ramanathan, R.K., Kirkpatrick, D.L., Belani, C.P., Friedland, D., Green, S.B., Chow, H.H., Cordova, C.A., Stratton, S.P., Sharlow, E.R., Baker, A., et al. 2007. A Phase I pharmacokinetic and pharmacodynamic study of PX-12, a novel inhibitor of thioredoxin-1, in patients with advanced solid tumors. Clin Cancer Res 13:2109-2114. 214. Welsh, S.J., Williams, R.R., Birmingham, A., Newman, D.J., Kirkpatrick, D.L., and Powis, G. 2003. The thioredoxin redox inhibitors 1-methylpropyl 2-imidazolyl and pleurotin inhibit hypoxia-induced factor 1alpha and vascular endothelial growth factor formation. Mol Cancer Ther 2:235-243. 215. Daley, J.M., Thomay, A.A., Connolly, M.D., Reichner, J.S., and Albina, J.E. 2008. Use of Ly6G-specific monoclonal antibody to deplete neutrophils in mice. J Leukoc Biol 83:64-70. 216. Bonnett, C.R., Cornish, E.J., Harmsen, A.G., and Burritt, J.B. 2006. Early neutrophil recruitment and aggregation in the murine lung inhibit germination of Aspergillus fumigatus Conidia. Infect Immun 74:6528-6539. 217. Stephens-Romero, S.D., Mednick, A.J., and Feldmesser, M. 2005. The pathogenesis of fatal outcome in murine pulmonary aspergillosis depends on the neutrophil depletion strategy. Infect Immun 73:114-125. 218. Mircescu, M.M., Lipuma, L., van Rooijen, N., Pamer, E.G., and Hohl, T.M. 2009. Essential role for neutrophils but not alveolar macrophages at early time points following Aspergillus fumigatus infection. J Infect Dis 200:647-656. 219. Barnes, P.J. 1998. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci (Lond) 94:557-572. 220. Brown, G.D., Herre, J., Williams, D.L., Willment, J.A., Marshall, A.S., and Gordon, S. 2003. Dectin-1 mediates the biological effects of beta-glucans. J Exp Med 197:1119-1124. 221. Vetvicka, V., Thornton, B.P., and Ross, G.D. 1996. Soluble beta-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells. J Clin Invest 98:50-61. 222. Boyle, K.B., Gyori, D., Sindrilaru, A., Scharffetter-Kochanek, K., Taylor, P.R., Mocsai, A., Stephens, L.R., and Hawkins, P.T. 2011. Class IA phosphoinositide 3- kinase beta and delta regulate neutrophil oxidase activation in response to Aspergillus fumigatus hyphae. J Immunol 186:2978-2989. 223. Goodridge, H.S., Reyes, C.N., Becker, C.A., Katsumoto, T.R., Ma, J., Wolf, A.J., Bose, N., Chan, A.S., Magee, A.S., Danielson, M.E., et al. 2011. Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'. Nature 472:471-475. 224. Vedder, N.B., and Harlan, J.M. 1988. Increased surface expression of CD11b/CD18 (Mac-1) is not required for stimulated neutrophil adherence to cultured endothelium. J Clin Invest 81:676-682. 225. Dickinson, B.C., and Chang, C.J. 2011. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat Chem Biol 7:504-511. 226. Aratani, Y., Kura, F., Watanabe, H., Akagawa, H., Takano, Y., Suzuki, K., Dinauer, M.C., Maeda, N., and Koyama, H. 2002. Relative contributions of

229 myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus. Med Mycol 40:557-563. 227. Elahi, S., Pang, G., Ashman, R.B., and Clancy, R. 2001. Nitric oxide-enhanced resistance to oral candidiasis. Immunology 104:447-454. 228. de Jesus-Berrios, M., Liu, L., Nussbaum, J.C., Cox, G.M., Stamler, J.S., and Heitman, J. 2003. Enzymes that counteract nitrosative stress promote fungal virulence. Curr Biol 13:1963-1968. 229. Bahn, Y.S., and Sundstrom, P. 2001. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans. J Bacteriol 183:3211-3223. 230. Sugui, J.A., Kim, H.S., Zarember, K.A., Chang, Y.C., Gallin, J.I., Nierman, W.C., and Kwon-Chung, K.J. 2008. Genes differentially expressed in conidia and hyphae of Aspergillus fumigatus upon exposure to human neutrophils. PLoS One 3:e2655. 231. Berdicevsky, I., Kaufman, G., Newman, D.J., and Horwitz, B.A. 2008. Preliminary study of activity of the thioredoxin inhibitor pleurotin against Trichophyton mentagrophytes: a novel anti-dermatophyte possibility. Mycoses. 232. Belkacemi, L., Barton, R.C., Hopwood, V., and Evans, E.G. 1999. Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin detection. Med Mycol 37:227-233. 233. Carlson, E.C., Lin, M., Liu, C.Y., Kao, W.W., Perez, V.L., and Pearlman, E. 2007. Keratocan and lumican regulate neutrophil infiltration and corneal clarity in lipopolysaccharide-induced keratitis by direct interaction with CXCL1. J Biol Chem 282:35502-35509. 234. Kao, W.W., and Liu, C.Y. 2002. Roles of lumican and keratocan on corneal transparency. Glycoconj J 19:275-285. 235. Carlson, E.C., Sun, Y., Auletta, J., Kao, W.W., Liu, C.Y., Perez, V.L., and Pearlman, E. 2010. Regulation of corneal inflammation by neutrophil-dependent cleavage of keratan sulfate proteoglycans as a model for breakdown of the chemokine gradient. J Leukoc Biol 88:517-522. 236. Gagen, D., Laubinger, S., Li, Z., Petrescu, M.S., Brown, E.S., Smith, C.W., and Burns, A.R. 2010. ICAM-1 mediates surface contact between neutrophils and keratocytes following corneal epithelial abrasion in the mouse. Exp Eye Res 91:676- 684. 237. Drummond, R.A., and Brown, G.D. 2011. The role of Dectin-1 in the host defence against fungal infections. Curr Opin Microbiol 14:392-399. 238. Zipfel, P.F., and Skerka, C. 2009. Complement regulators and inhibitory proteins. Nat Rev Immunol 9:729-740. 239. Speth, C., Rambach, G., Wurzner, R., and Lass-Florl, C. 2008. Complement and fungal pathogens: an update. Mycoses 51:477-496. 240. Ricklin, D., and Lambris, J.D. 2008. Compstatin: a complement inhibitor on its way to clinical application. Adv Exp Med Biol 632:273-292. 241. Dinarello, C.A. 2009. Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 27:519-550.

230 242. Said-Sadier, N., Padilla, E., Langsley, G., and Ojcius, D.M. 2010. Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase. PLoS One 5:e10008. 243. van de Veerdonk, F.L., Netea, M.G., Dinarello, C.A., and Joosten, L.A. 2011. Inflammasome activation and IL-1beta and IL-18 processing during infection. Trends Immunol 32:110-116. 244. Wasylnka, J.A., and Moore, M.M. 2003. Aspergillus fumigatus conidia survive and germinate in acidic organelles of A549 epithelial cells. J Cell Sci 116:1579-1587. 245. Hornung, V., and Latz, E. 2010. Critical functions of priming and lysosomal damage for NLRP3 activation. Eur J Immunol 40:620-623. 246. Kofoed, E.M., and Vance, R.E. 2011. Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity. Nature 477:592-595. 247. Korn, T., Bettelli, E., Oukka, M., and Kuchroo, V.K. 2009. IL-17 and Th17 Cells. Annu Rev Immunol 27:485-517. 248. Schroder, K., Hertzog, P.J., Ravasi, T., and Hume, D.A. 2004. Interferon- gamma: an overview of signals, mechanisms and functions. J Leukoc Biol 75:163-189. 249. Roy, S., Sun, Y., and Pearlman, E. 2011. Interferon-gamma-induced MD-2 protein expression and lipopolysaccharide (LPS) responsiveness in corneal epithelial cells is mediated by Janus tyrosine kinase-2 activation and direct binding of STAT1 protein to the MD-2 promoter. J Biol Chem 286:23753-23762. 250. Soehnlein, O., and Lindbom, L. 2010. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol 10:427-439. 251. Draskovic-Pavlovic, B., Van Der Laan, L.J., Pejnovic, N., Dijkstra, C.D., and Colic, M. 1999. Differential effects of anti-rat CD11b monoclonal antibodies on adhesiveness. Immunology 96:83-89. 252. Goodridge, H.S., Wolf, A.J., and Underhill, D.M. 2009. Beta-glucan recognition by the innate immune system. Immunol Rev 230:38-50. 253. Mori, T., Ikemoto, H., Matsumura, M., Yoshida, M., Inada, K., Endo, S., Ito, A., Watanabe, S., Yamaguchi, H., Mitsuya, M., et al. 1997. Evaluation of plasma (1-->3)- beta-D-glucan measurement by the kinetic turbidimetric Limulus test, for the clinical diagnosis of mycotic infections. Eur J Clin Chem Clin Biochem 35:553-560. 254. Lu, H., Smith, C.W., Perrard, J., Bullard, D., Tang, L., Shappell, S.B., Entman, M.L., Beaudet, A.L., and Ballantyne, C.M. 1997. LFA-1 is sufficient in mediating neutrophil emigration in Mac-1-deficient mice. J Clin Invest 99:1340-1350. 255. Rapaka, R.R., Goetzman, E.S., Zheng, M., Vockley, J., McKinley, L., Kolls, J.K., and Steele, C. 2007. Enhanced defense against Pneumocystis carinii mediated by a novel dectin-1 receptor Fc fusion protein. J Immunol 178:3702-3712. 256. Chen, G.Y., and Nunez, G. 2010. Sterile inflammation: sensing and reacting to damage. Nat Rev Immunol 10:826-837. 257. Chun, K.H., and Seong, S.Y. 2010. CD14 but not MD2 transmit signals from DAMP. Int Immunopharmacol 10:98-106. 258. Ganz, T., and Nemeth, E. 2011. Hepcidin and disorders of iron metabolism. Annu Rev Med 62:347-360. 259. Armitage, A.E., Eddowes, L.A., Gileadi, U., Cole, S., Spottiswoode, N., Selvakumar, T.A., Ho, L.P., Townsend, A.R., and Drakesmith, H. 2011. Hepcidin regulation by innate immune and infectious stimuli. Blood 118:4129-4139.

231 260. Mestas, J., and Hughes, C.C. 2004. Of mice and not men: differences between mouse and human immunology. J Immunol 172:2731-2738. 261. Eisenhauer, P.B., and Lehrer, R.I. 1992. Mouse neutrophils lack defensins. Infect Immun 60:3446-3447. 262. Aratani, Y., Koyama, H., Nyui, S., Suzuki, K., Kura, F., and Maeda, N. 1999. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase. Infect Immun 67:1828-1836. 263. Baker, A.F., Dragovich, T., Tate, W.R., Ramanathan, R.K., Roe, D., Hsu, C.H., Kirkpatrick, D.L., and Powis, G. 2006. The antitumor thioredoxin-1 inhibitor PX-12 (1- methylpropyl 2-imidazolyl disulfide) decreases thioredoxin-1 and VEGF levels in cancer patient plasma. J Lab Clin Med 147:83-90. 264. Rhee, S.G., and Woo, H.A. 2011. Multiple functions of peroxiredoxins: peroxidases, sensors and regulators of the intracellular messenger HO, and protein chaperones. Antioxid Redox Signal 15:781-794. 265. Haraldsen, J.D., Liu, G., Botting, C.H., Walton, J.G., Storm, J., Phalen, T.J., Kwok, L.Y., Soldati-Favre, D., Heintz, N.H., Muller, S., et al. 2009. Identification of Conoidin a as a Covalent Inhibitor of Peroxiredoxin Ii. Org Biomol Chem 7:3040-3048. 266. Hube, B. 2009. Fungal adaptation to the host environment. Curr Opin Microbiol 12:347-349. 267. Murphy, M.P. 2009. How mitochondria produce reactive oxygen species. Biochem J 417:1-13. 268. Zhang, X., Zheng, Y., Fried, L.E., Du, Y., Montano, S.J., Sohn, A., Lefkove, B., Holmgren, L., Arbiser, J.L., Holmgren, A., et al. 2011. Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes. Free Radic Biol Med 50:811-820. 269. Fulda, S., Galluzzi, L., and Kroemer, G. 2010. Targeting mitochondria for cancer therapy. Nat Rev Drug Discov 9:447-464. 270. Ishibashi, Y., Kaufman, H.E., and Kagawa, S. 1986. Comparison of the pathogenicities of Fusarium solani and Candida albicans in the rabbit cornea. J Med Vet Mycol 24:369-376. 271. Chauhan, N., Latge, J.P., and Calderone, R. 2006. Signalling and oxidant adaptation in Candida albicans and Aspergillus fumigatus. Nat Rev Microbiol 4:435- 444. 272. Burns, C., Geraghty, R., Neville, C., Murphy, A., Kavanagh, K., and Doyle, S. 2005. Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus. Fungal Genet Biol 42:319-327. 273. Babineau, T.J., Marcello, P., Swails, W., Kenler, A., Bistrian, B., and Forse, R.A. 1994. Randomized phase I/II trial of a macrophage-specific immunomodulator (PGG-glucan) in high-risk surgical patients. Ann Surg 220:601-609. 274. Mocsai, A., Ruland, J., and Tybulewicz, V.L. 2010. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat Rev Immunol 10:387-402. 275. Gabay, C., Lamacchia, C., and Palmer, G. 2010. IL-1 pathways in inflammation and human diseases. Nat Rev Rheumatol 6:232-241. 276. Goldbach-Mansky, R. 2009. Blocking interleukin-1 in rheumatic diseases. Ann N Y Acad Sci 1182:111-123.

232 277. Horuk, R. 2009. Chemokine receptor antagonists: overcoming developmental hurdles. Nat Rev Drug Discov 8:23-33. 278. Semba, C.P., Torkildsen, G.L., Lonsdale, J.D., McLaurin, E.B., Geffin, J.A., Mundorf, T.K., Kennedy, K.S., and Ousler, G.W. 2012. A Phase 2 Randomized, Double-Masked, Placebo-Controlled Study of a Novel Integrin Antagonist (SAR 1118) for the Treatment of Dry Eye. Am J Ophthalmol. 279. Hueber, W., Patel, D.D., Dryja, T., Wright, A.M., Koroleva, I., Bruin, G., Antoni, C., Draelos, Z., Gold, M.H., Durez, P., et al. 2010. Effects of AIN457, a fully human antibody to interleukin-17A, on psoriasis, rheumatoid arthritis, and uveitis. Sci Transl Med 2:52ra72. 280. Letscher-Bru, V., and Herbrecht, R. 2003. Caspofungin: the first representative of a new antifungal class. J Antimicrob Chemother 51:513-521. 281. Lamaris, G.A., Lewis, R.E., Chamilos, G., May, G.S., Safdar, A., Walsh, T.J., Raad, II, and Kontoyiannis, D.P. 2008. Caspofungin-mediated beta-glucan unmasking and enhancement of human polymorphonuclear neutrophil activity against Aspergillus and non-Aspergillus hyphae. J Infect Dis 198:186-192. 282. Katragkou, A., Chatzimoschou, A., Simitsopoulou, M., Georgiadou, E., and Roilides, E. 2011. Additive antifungal activity of anidulafungin and human neutrophils against Candida parapsilosis biofilms. J Antimicrob Chemother 66:588-591. 283. Tullio, V., Mandras, N., Scalas, D., Allizond, V., Banche, G., Roana, J., Greco, D., Castagno, F., Cuffini, A.M., and Carlone, N.A. 2010. Synergy of caspofungin with human polymorphonuclear granulocytes for killing Candida albicans. Antimicrob Agents Chemother 54:3964-3966. 284. Mattila, P.E., Metz, A.E., Rapaka, R.R., Bauer, L.D., and Steele, C. 2008. Dectin-1 Fc targeting of aspergillus fumigatus beta-glucans augments innate defense against invasive pulmonary aspergillosis. Antimicrob Agents Chemother 52:1171-1172. 285. Sable, C.A., Strohmaier, K.M., and Chodakewitz, J.A. 2008. Advances in antifungal therapy. Annu Rev Med 59:361-379. 286. Gaudana, R., Jwala, J., Boddu, S.H., and Mitra, A.K. 2009. Recent perspectives in ocular drug delivery. Pharm Res 26:1197-1216. 287. Ramanathan, R.K., Stephenson, J.J., Weiss, G.J., Pestano, L.A., Lowe, A., Hiscox, A., Leos, R.A., Martin, J.C., Kirkpatrick, L., and Richards, D.A. 2011. A phase I trial of PX-12, a small-molecule inhibitor of thioredoxin-1, administered as a 72-hour infusion every 21 days in patients with advanced cancers refractory to standard therapy. Invest New Drugs.

233