Host-Pathogen Interactions in Aspergillus Fumigatus Infection

NEUTROPHILS, NUTRITIONAL IMMUNITY AND NETS: HOST-PATHOGEN INTERACTIONS IN ASPERGILLUS FUMIGATUS INFECTION

HEATHER L. CLARK

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Department of Pathology

CASE WESTERN RESERVE UNIVERSTIY

January 2017

Case Western Reserve University School of Graduate Studies

We hereby approve the thesis/dissertation of

Heather L. Clark

Candidate for the degree of Doctor of Philosophy in Pathology

Committee Chair:

George Dubyak

Committee Members:

Clive Hamlin Amy Hise Eric Pearlman Theresa Pizarro

Date of Defense:

August 22nd, 2016

We also certify that written permission has been obtained for any proprietary material contained therein

Table of Contents

List of Tables ...... 7

List of Figures ...... 8

Acknowledgements ...... 11

List of Abbreviations ...... 13

Abstract ...... 17

Chapter 1: Introduction ...... 19

Fungi and Human Disease ...... 20

Burden of Fungal Disease ...... 20

Fungal Keratitis ...... 22

Current Anti-Fungal Therapy ...... 24

Immune Responses to Aspergillus fumigatus infection: A Critical Role for Neutrophils ...... 27

Neutrophil Recruitment ...... 27

The Neutrophil Anti-microbial Arsenal ...... 29

Neutrophils and Nutritional Immunity ...... 31

S100 Proteins in Nutritional Immunity ...... 33

Neutrophil Extracellular Traps ...... 36

Neutrophil Recognition of Fungi ...... 38

Intracellular Signaling in NETosis ...... 42

NETs in Tissue Damage ...... 47

Other Immune Responses in Aspergillus Infection ...... 48

Aspergillus fumigatus Virulence Determinants ...... 49

The Fungal Cell Wall and Immune Evasion ...... 50

Antioxidant Mechanisms ...... 52

Nutrient Acquisition ...... 53

Hypothesis and Goals ...... 57

Chapter 2: Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection ...... 59

Abstract ...... 60

Introduction ...... 61

Materials and Methods ...... 63

Results ...... 70

Discussion ...... 79

Figures ...... 87

Chapter 3: Atovaquone impairs mitochondrial function and metal homeostasis, and differentially inhibits growth of Aspergillus and Fusarium clinical isolates ...... 95

Abstract ...... 96

Introduction ...... 97

Materials and Methods ...... 99

Results ...... 103

Discussion ...... 108

Figures ...... 115

Chapter 4: A Role for Neutrophil Extracellular Traps in Aspergillus fumigatus Infection ...... 124

Abstract ...... 125

Introduction ...... 127

Materials and Methods ...... 130

Results ...... 135

Discussion ...... 141

Figures ...... 148

Chapter 5: Data Summary and Discussion ...... 155

Calprotectin and nutritional immunity to A. fumigatus ...... 156

Atovaquone as a novel anti-fungal agent ...... 158

Neutrophil extracellular traps in A. fumigatus infection ...... 160

Chapter 6: Future Directions and Preliminary Data ...... 165

NET-independent Calprotectin Release from Neutrophils ...... 166

S100 Proteins in Fungal Infection...... 167

Novel Anti-microbial Peptides in Fungal Infection ...... 168

Copper Homeostasis in Aspergillus fumigatus virulence and Nutritional Immunity ...... 172

CR3 Signaling in Neutrophils...... 174

Dectin-2/Dectin-3 Signaling in NET formation ...... 175

Cytokine Regulation of Neutrophil Responses ...... 176

Antibodies, Pentraxins and Fc Receptors in Fungal Infection ...... 179

Modulating NET Formation: Implications for Therapy ...... 183

Concluding Remarks ...... 186

Preliminary Figures ...... 185

References ...... 190

List of Tables

Table 1.1 Human fungal pathogens 20

Table 1.2 Spectrum of fungal species in fungal keratitis 22

Table 2.1 Aspergillus fumigatus strains used in this study 64

Table 3.1 Keratitis isolate strains and inhibitory concentrations 102

List of Figures

Figure 1.1 Corneas of fungal keratitis patients 23

Figure 1.2 Anti-fungal drugs and their targets 25

Figure 1.3 Morphology of pathogenic fungi 26

Figure 1.4 Neutrophils in corneal ulcers of fungal keratitis patients 27

Figure 1.5 Microbicidal functions of neutrophils 30

Figure 1.6 Crystal structure of calprotectin 34

Figure 1.7 Neutrophil extracellular traps induced by IL-8 36

Figure 1.8 Pattern recognition receptors in fungal recognition 39

Figure 1.9 PAD4 mediates histone citrullination 45

Figure 1.10 Simplified view of NETosis 46

Figure 1.11 Cell wall components of pathogenic fungi 51

Figure 1.12 Model of A. fumigatus Zn transport 55

Figure 2.1 Effect of neutrophil calprotectin on A. fumigatus corneal infection 87

Figure 2.2 Effect of neutrophil calprotectin on A. fumigatus hyphal growth

in vitro 88

Figure 2.3 Zn and Mn binding contribute to calprotectin Anti-Aspergillus activity 89

Figure 2.4 ZafA mediated zinc uptake in A. fumigatus virulence and susceptibility to calprotectin 90

Figure 2.5 Conidia killing by neutrophils is independent of calprotectin 91

Figure 2.S1 Supplementary Figure 1 92

Figure 2.S2 Supplementary Figure 2 93

Figure 2.S3 Supplementary Figure 3 94

Figure 3.1 Atovaquone inhibits growth of filamentous fungal keratitis isolates 115

Figure 3.2 Atovaquone disrupts mitochondrial potential and ATP

production 116

Figure 3.3 Atovaquone reduces labile intracellular Zn 117

Figure 3.4 Atovaquone increases metal toxicity and disrupts vacuolar acidification 118

Figure 3.5 ABC transporter overexpression reduces sensitivity to

atovaquone and azoles 119

Figure 3.6 Summary of atovaquone activity on filamentous fungi 120

Figure 3.S1 Supplementary Figure 1 121

Figure 3.S2 Supplementary Figure 2 122

Figure 3.S3 Supplementary Figure 3 123

Figure 4.1 Aspergillus fumigatus induces NETs from human and mouse neutrophils 148

Figure 4.2 Curdlan induces NET formation through CR3, but not Dectin-1 149

Figure 4.3 NET formation requires PAD4 activation and NETs contain citrullinated histones (murine) 150

Figure 4.4 NET formation requires PAD4 activation and NETs contain citrullinated histones (human) 151

Figure 4.5 CR3 is required for inhibition of A. fumigatus growth in vitro 152

Figure 4.6 CR3 is required for calprotectin release in vitro 153

Figure 4.7 NETs are formed in vivo during A. fumigatus corneal infection 154

Figure 6.1 Aspergillus fumigatus copper uptake is required for growth in the presence of neutrophils and in vivo. 187

Figure 6.2 GM-CSF and IL-1β increase neutrophil inhibition of

A. fumigatus hyphae in vitro. 188

Figure 6.3 Serum opsonization increases neutrophil inhibition of

A. fumigatus hyphae in vitro. 189

Acknowledgements

This collection of work is the result of love, support, friendship and mentorship from many people. Firstly, I would like to thank my thesis advisor and mentor, Dr. Eric Pearlman, for the incredible opportunity to pursue this work in your laboratory. I cannot thank you enough for creating a positive and supportive atmosphere in the lab that fostered my academic success. Furthermore, you provided me with the freedom to pursue my interests and to develop into an independent scientist. I will owe my future successes to the strong foundation of creativity, perseverance and integrity that I have built in your lab.

In addition I would like to thank all the members of the Pearlman lab, including Yan Sun, Sixto Leal, Patricia Taylor, Sanhita Roy, Mausita Karmakar,

Steven de Jesus Carrion, Chairut Vareechon, Tristan de Jesus and many others, who have served as mentors, peers and friends and with whom I have made many fond memories inside and outside of the lab. I am also grateful for the wonderful support from the CWRU visual science research core, the administration for the CWRU Departments of Ophthalmology and Visual Science and Pathology, the CWRU Medical Scientist Training Program, as well as the UC

Irvine Institute for Immunology, which became my second home during the past year. I would like to thank my thesis committee members, Dr. George Dubyak,

Dr. Amy Hise, Dr. Theresa Pizzarro and Dr. Clive Hamlin for their valuable guidance throughout my PhD training.

Finally, I would like to thank my family and friends for their support, love and patience during the past four years. To my husband Micah Clark, thank you

11 for always supporting my academic pursuits, even when they take me across the country!

Abbreviations

ABC transporter – atp-binding cassette transporter

AmB – amphotericin B

AMP – antimicrobial peptide/protein

AspHE – Aspergillus hyphal extract

Atg5 - Autophagy protein 5

ATP – adenosine triphosphate

Bcl10 – B-cell lymphoma/leukemia 10 protein

CARD9 - Caspase recruitment domain-containing protein 9

CBPA – chronic bronchopulmonary aspergillosis

CGD – chronic granulomatous disease

CLEC – c-type lectin

CP – calprotectin

CR3 – complement receptor 3

Cu - copper

DAMP – damage associated molecular pattern

DED – dry eye disease

DPI – diphenyl iodonium

ELISA – enzyme linked immunosorbent assay

Erk – extracellular signal-related kinase

FcR – Fc receptor

FK – fungal keratitis fMLP - N-Formylmethionyl-leucyl-phenylalanine

GAG – galactosaminogalactan

GM-CSF – granulocyte macrophage colony stimulating factor

H2O2 – hydrogen peroxide

H3cit – citrullinated histone H3

HMGB-1 – high mobility group protein 1

HOCl – hypochlorous acid

ICAM1 - Intercellular Adhesion Molecule 1

Ig – immunoglobulin

IGPD - imidazoleglycerol-phosphate dehydratase

IL-1R – interleukin 1 receptor

IL-6 – interleukin 6

IL-1B – interleukin 1 beta

IRAK Interleukin-1 receptor-associated kinase

IRF - Interferon regulatory factor

ITAM - immunoreceptor tyrosine-based activation motif

ITIM - immunoreceptor tyrosine-based inhibition motif

LAD – leukocyte adhesion deficiency

LAIR-1 – leukocyte associated Ig-like receptor 1

LC3 - microtubule-associated protein 1 light chain 3

LPS – lipopolysaccharide

Malt1 - Mucosa-associated lymphoid tissue lymphoma translocation protein 1

Mek – mitogen activated protein kinase kinase

MMP – matrix metalloprotease

Mn - manganese

MPO – myeloperoxidase

MRSA – methicillin-resistance Staphylococcus aureus mTOR - mechanistic target of rapamycin

MyD88 - Myeloid differentiation primary response gene 88

NADPH - Nicotinamide adenine dinucleotide phosphate

NE – neutrophil elastase

NET – neutrophil extracellular trap

NFAT- Nuclear factor of activated T-cells

NFkB - nuclear factor kappa-light-chain-enhancer of activated B cells

NK cell – natural killer cell

NOX – nadph oxidase

PAD4 – peptidyl arginine deiminase 4

PAMP – pathogen associated molecular pattern

PBMC – peripheral blood mononuclear cell

PCR – polymerase chain reaction pDC – plasmacytoid dendritic cell

PI3K – phosphoinositide-3-kinase

PKC – protein kinase C

PTX3 – pentraxin 3

MA – phorbol 12-myristate 13-acetate

PRR- pattern recognition receptor

ROS – reactive oxygen species

Siglec-9 – sialic-acid binding immunoglobulin-type lectin 9

SIRL-1 – signal inhibitory receptor on leukocytes 1

SLE – systemic lupus erythematosus

SOD – superoxide dismutase

Syk – spleen tyrosin kinase

T helper cell type 17

TLR – toll-like receptor

TNF-α – tumor necrosis factor alpha

TRAF- TNF receptor associated factor

Zn - zinc

Neutrophils, Nutritional Immunity, and NETs: Host-Pathogen Interactions in Aspergillus fumigatus Infection

Abstract

HEATHER L. CLARK

The filamentous fungal pathogen Aspergillus fumigatus is a leading cause of corneal infection (keratitis). Neutrophils are the most abundant cell type in corneal ulcers of fungal keratitis patients and are critical to controlling fungal growth, through reactive oxygen species (ROS) production and release of antimicrobial proteins (AMPs). We investigated the role of the neutrophil AMP calprotectin (CP) and neutrophil extracellular trap (NET) release in A. fumigatus infection, as well as the role of zinc (Zn) acquisition as a virulence determinant of

A. fumigatus. Furthermore, we studied the effect of atovaquone as a novel anti- fungal agent targeting fungal Zn homeostasis.

We demonstrated that neutrophil CP is required for control of A. fumigatus corneal infection and hyphal growth through Zn and manganese chelation.

Furthermore, we found that the A. fumigatus transcription factor ZafA is required for zinc uptake and virulence in vivo or in the presence of neutrophils and CP.

Due to the importance of Zn acquisition in fungal virulence, we investigated the effect of atovaquone, a ubiquinone analog, that was shown to disrupt Zn homeostasis in yeast, on filamentous fungi. We found that atovaquone

17 significantly inhibited growth of Aspergillus clinical keratitis isolates in vitro through disruption of mitochondrial activity and intracellular zinc homeostasis.

Finally, NETs have been shown to contribute to CP release and immunity microbial infection. We found that the fungal polysaccharide β 1,3 glucan induced

NET release which was mediated in part by complement receptor 3 (CR3), ROS production and peptidyl arginine deiminase 4 (PAD4) activation. However, we found that CR3, but not PAD4, was required for neutrophil CP release and killing of hyphae in vitro. Finally, we identified NET formation in vivo in corneas of mice infected with A .fumigatus by citrullinated histone H3 (H3cit) antibody staining, which is a specific marker of NETs.

In conclusion, we found an essential role for neutrophil CP and CR3 signaling in controlling A. fumigatus infection. We also found that A. fumigatus Zn acquisition is required for virulence and that targeting Zn homeostasis of fungi with atovaquone may represent a new anti-fungal strategy.

Chapter 1: Introduction

Fungi and Human Disease

Burden of Fungal Disease. Fungi are a kingdom of eukaryotic organisms

that is comprised of yeasts, molds and mushrooms. Fungi are found throughout

the environment and are therefore encountered daily by humans. Many fungi,

however, have the ability to cause disease in both plants and animals, including

human hosts (Table 1.1). Superficial infections of the skin, mucosa and cornea

caused by dermatophytes, yeasts and molds, respectively, and including

Table 1.1: Human Fungal Pathogens

20 athlete’s foot, ringworm, onychomycosis, oral and vulvovaginal candidiasis, and corneal keratitis, are extremely common (1). Invasive infections occur primarily in immune compromised hosts and have a mortality rate of greater than 50%, causing 1.5 million deaths per year (1). In particular, HIV/AIDs-mediated immune suppression leads to millions of cases of invasive fungal disease, including cryptococcal meningitis and pneumocystis pneumonia, caused by Cryptococcus neoformans, Cryptococcus gattii and Pneumocystis jirovecii, respectively, which are inhaled as airborne particles found in the environment or from other human hosts (2). Immune compromise as a result of hematopoietic stem cell transplant, solid organ transplant, genetic deficiencies in immune function, or immune suppressive treatment also increase the risk of contracting an invasive fungal infections (3-5). Candida yeasts that cause superficial infections can also cause severe invasive infection upon contamination of implanted medical devices (6).

Filamentous molds, primarily Aspergillus and Fusarium species, can cause severe pulmonary and invasive disease in immune compromised patients, particularly following hematopoietic stem cell transplant, and also in those with previous lung pathology, including asthma, chronic obstructive pulmonary disease and tuberculosis infection (3, 7). The number of invasive, chronic pulmonary and corneal Aspergillus infections are estimated at 200,000, 3 million and 1 million per year, respectively (1). In some cases healthy individuals can contract pulmonary and systemic fungal infections caused by the dimorphic fungi

Histoplasma, Blastomyces, Coccidioides and Paracoccidioides, which are found in the environment of specific geographical areas (8). Finally, fungi that are

21 normally not pathogenic can be introduced to the body through surgery or trauma and cause severe infections that are extremely difficult to eradicate, as was the case with an outbreak of Exserohilum CNS infections associated with contaminated steroid injections (9). A lack of surveillance and reporting of fungal infections worldwide means that the estimates of fungal disease burden are likely low.

Table 1.2: Spectrum of fungal species in fungal keratitis (18). Reprint permission obtained from Wolters Kluwer Health, Inc.

Fungal Keratitis. Fungal keratitis is a painful infection of the cornea which occurs in immune competent individuals and can result in blindness. This infection came to public attention in the United States in 2005 when several hundred cases of FK were diagnosed in the U.S., Europe and Asia (10). This outbreak was caused by the filamentous fungus Fusarium and was associated with the use of a contact lens care solution that was unable to inhibit fungal growth, particularly in the setting of poor lens care and hygiene (11, 12).

Although this outbreak was widely reported, an estimated 1 million cases of FK occur annually worldwide, largely in developing countries with tropical climates that facilitate fungal growth in the environment (1, 13). Furthermore, although contact lens use is a risk factor for keratitis caused by the filamentous fungi

Fusarium and Aspergillus, and Candida albicans yeast, many cases of FK occur due to ocular trauma. Studies of FK in India and China found that FK was primarily associated with trauma and occurred during the harvest seasons when agricultural workers are more likely to suffer injuries that become contaminated with fungal spores from the environment (14-17). Filamentous fungi including

Aspergillus and Fusarium species are the most commonly identified fungi in these studies, as demonstrated by Gopinathan et al. in Table 1.2 (18, 19). FK results in a robust inflammatory response and recruitment of leukocytes into the corneal stroma and anterior chamber, which causes opacification of the cornea

Figure 1.1: Corneas of patients infected with Aspergillus flavus (A) or Fusarium solani (B) Arrow shows neutrophil infiltrate in the anterior chamber (21). Reprint permission obtained from Oxford University Press. and scarring that results in irreversible blindness (Figure 1.1) (20, 21). In the case of the 2005-2007 Fusarium outbreak 34% of patients required corneal transplants to restore vision (12). A murine model of Aspergillus fumigatus FK has revealed many important aspects of the role of the innate immune system in fungal recognition and clearance (22-26). Because FK occurs in immune

23 competent hosts, study of this model allows for an understanding of how the intact immune system responds to fungal infection which can inform new therapeutic approaches to treating these devastating infections, even in immune deficient patients.

Current Anti-fungal Therapy. Treatment for fungal infections is difficult and the arsenal of anti-fungal agents available is limited. The classes of anti- fungal agents include the azoles, which inhibit ergosterol synthesis resulting in accumulation of toxic sterol intermediates, echinocandins, which target β 1,3 glucan synthesis, pyrimidine analogs that inhibit nucleic acid synthesis, and polyenes which disrupt cellular membranes (Figure 1.2) (27). Resistance to azole agents is emerging in Aspergillus fumigatus and ranges from 0.6 -29.6%, due to point mutations in the CYP51A gene, and is thought to be in part due to agricultural use of azole anti-fungals (28). Long-term treatment of chronic aspergillosis patients with azoles also results in resistance in clinical isolates

(29). Echinocandin resistance has been identified in both Candida and

Aspergillus species (30). Additionally, emerging non-fumigatus species of

Aspergillus have demonstrated intrinsic resistance to amphotericin B (AmB) (7,

28). Many Fusarium isolates are also resistant to echinocandins, AmB and some azoles. In addition to mutations or upregulation of the drug target genes, resistance to anti-fungal agents is mediated by efflux transporters, such as ABC transporters, or sequestration of drugs in biofilms and intracellular vesicles (31).

In the case of AmB, reductions in cell membrane ergosterol as well as increased anti-oxidant capability may confer resistance (32). Furthermore, treatment of FK

presents unique challenges to drug delivery. Topical polyene anti-fungals are

first-line therapy but penetration into the corneal stroma is poor. Topical azoles or

oral or subconjunctival AmB or azoles can also be used depending on

susceptibility and severity of infection (33). Overall, treating fungal infections

remains a challenge and development of new anti-fungal agents is of great

importance.

Figure 1.2: Antifungal drugs and their targets (27). Reprint permission obtained from the American Society for Microbiology.

Figure 1.3: Morphology of pathogenic fungi (34). Reprint permission obtained from Macmillan Publishers Ltd. Nature Reviews Immunology 2004.

The remainder of this work will focus on the immune response to and virulence of Aspergillus fumigatus, a leading cause of both FK and invasive fungal infection in immune compromised hosts. This filamentous fungus is disseminated throughout the environment as 3 µM spores (conidia) which can germinate and grow into long septate filaments called hyphae under favorable conditions, shown in Figure 1.3 in comparison to other pathogenic fungi (34).

Due to a concentration of 1-100 conidia/m-3 in the air, it is estimated we inhale several hundred conidia per day, which are normally cleared by lung phagocytes in healthy individuals but can cause devastating infections when immune responses are delayed or impaired (35, 36). In FK, hyphae are formed before large numbers of immune cells are recruited to the normally avascular cornea to control infection. An understanding of how the immune system normally clears

26 fungal infection, and conversely how fungi undermine immune defenses, will allow for development of more effective therapies for fungal infections.

Immune Responses to Aspergillus fumigatus infection: A Critical Role for

Neutrophils

Neutrophil Recruitment. Neutrophils are the most abundant white blood cell in the body and are rapidly mobilized to respond to infection or other inflammatory stimuli. In corneal scrapings of Aspergillus and Fusarium keratitis patients, neutrophils comprise >95% of infiltrating cells (Figure 1.4) (21).

Antibody-mediated neutrophil depletion in a mouse model of keratitis results in massively increased fungal growth in the cornea (25). Furthermore, neutrophils

Figure 1.4: Scrapings of corneal ulcers from FK patients A) Fungal hyphae. B) cellular infiltrate. C) Quantification of neutrophils and mononuclear cells in corneal scrapings (21). Reprint permission obtained from Oxford University Press.

27 are rapidly recruited upon A. fumigatus pulmonary infection and are essential for preventing hyphal growth and invasion in the lung (37). Neutrophil recruitment requires chemokines which are produced rapidly upon infection by epithelial cells and resident immune cells. In A. fumigatus keratitis recognition of the fungal cell wall polysaccharides β glucan and α mannan by the C-type lectin (CLEC) receptors Dectin-1 an d Dectin-2 on resident macrophages is required for chemokine production, neutrophil recruitment, and faster clearance of infection

(22, 23). Similarly, Dectin-1 is required for chemokine production, neutrophil recruitment and survival in a mouse model of pulmonary aspergillosis (38). A separate study found that lung epithelium signaling through IL-1R/MyD88 is essential for early chemokine production while myeloid-specific CARD9 dependent chemokine production drives later neutrophil recruitment (39).

Polymorphisms in Dectin-1 and another CLEC DC-SIGN were found to be associated with increased risk of invasive pulmonary aspergillosis in immunocompromised patients (40). A role for toll-like receptors (TLRs) on both resident immune cells and epithelial cells has also been described. Bone marrow macrophages or mice lacking TLR1, 2, 4 or 6 were found to be deficient in cytokine and chemokine production in response to A. fumigatus conidia exposure or pulmonary infection (41). It was demonstrated that TLR9 is recruited to phagosomes upon ingestion of A. fumigatus conidia by murine macrophages in a

Dectin-1 dependent fashion, although a specific role for TLR9 in the immune response was not described (42, 43). Therefore, there is an indispensable role for epithelium and non-neutrophil myeloid cells in the response to fungal

28 infection; however, we will focus on the critical role of neutrophils and the neutrophil mechanisms responsible for control of Aspergillus infection.

The Neutrophil Anti-microbial Arsenal. Recent studies have revealed neutrophils as highly heterogeneous regulators of inflammation and immunity, however these cells are still most recognized as efficient anti-microbial effector cells. Neutrophils possess a unique and complex set of tools for combatting bacteria, fungi and parasites (Figure 1.5) (44). A key function of neutrophils in both inflammation and anti-microbial immunity is the production of reactive oxygen species (ROS), which occurs primarily through the NADPH oxidase complex NOX2 (45). NOX2 is a multi-protein complex that is assembled on the plasma or phagosome membranes following activation and granular fusion, which delivers NOX2 gp91phox subunit to the membrane. The active complex facilitates delivery of electrons from NADPH to oxygen to form superoxide radicals (46, 47). Superoxide can be converted to hydrogen peroxide by superoxide dismutase (SOD). Furthermore, neutrophil azurophilic granules contain myeloperoxidase (MPO), which can convert hydrogen peroxide to the radicals hypochlorous acid (HOCl) and hypothiocyanous acid. ROS, particularly

HOCl can directly kill many different types of pathogens through reactions with organic molecules, including oxidation and cleavage of proteins and nucleic acids which disrupts metabolism and membrane integrity (48, 49). A critical role for

NOX2 mediated ROS production in anti-fungal immunity is highlighted by the recurrent Aspergillus infections seen in chronic granulomatous disease (CGD) patients, who possess genetic defects in components of the NOX2 complex (5,

50). Similarly, NOX2-deficient knockout mice demonstrate impaired clearance of

infection in both corneal and pulmonary A. fumigatus infection models (25, 51).

MPO deficiency is the most common genetic defect of neutrophils. Despite a

reported role for MPO in in vitro killing of fungi and NET formation, these patients

are typically not more susceptible to fungal infections (50). In addition to direct

antimicrobial activity, ROS have a complex role in inter and intracellular

signaling. A key example of this is the requirement for ROS production in the

process of NET formation, which will be discussed later in this chapter (52).

Figure 1.5: Microbicidal functions of neutrophils (44). Reprint permission obtained from Macmillan Publishers Ltd. Nature Reviews Immunology 2006.

In addition to oxidative defenses, neutrophils possess a broad array of

proteins with anti-microbial activity, including enzymes, cationic peptides and

proteins that chelate essential nutrients. The majority of these proteins are

packaged into intracellular compartments called granules. Neutrophils possess

30 three distinct types of granules, azurophilic/primary, specific/secondary and gelatinase/tertiary granules which each contain a characteristic set of proteins(44). Azurophilic granules contain a large amount of MPO as well as the proteases neutrophil elastase (NE), cathepsin G and proteinase 3 and cationic anti-microbial peptides called defensins. Specific granules contain the iron- binding proteins lactoferrin and lipocalin and NOX2 subunits, while gelatinase granules contain large amounts of the matrix metalloprotease (MMP) gelatinase and some lactoferrin. These granules are released in a regulated manner in response to specific stimuli and calcium signaling. Neutrophils also contain intracellular secretory vesicles, which contain membrane-bound receptors and other proteins. Secretory vesicles are rapidly mobilized upon adhesion to endothelium, followed by tertiary, secondary and lastly azurophilic granule fusion

(53). Neutrophils also possess other proteins with anti-microbial activity outside of the granules, including the cytoplasmic protein calprotectin (CP) and the nuclear histone proteins, both of which have anti-microbial activity upon release from neutrophils (54, 55).

Neutrophils and Nutritional Immunity. Metals are essential nutrients required for pathogen growth and the immune system has evolved complex mechanisms of sequestering nutrients to limit infection, which is known as nutritional immunity (56). Neutrophils have a key role in nutritional immunity through the release of metal-binding anti-microbial proteins that sequester nutrients including iron, zinc, manganese and copper.

Iron is an essential metal for both mammalian hosts and pathogens, due to its role in oxidative respiration and other vital cellular functions. Access to iron is normally restricted in the body, by its incorporation in hemoglobin of red blood cells, storage intracellularly with the protein ferritin, and extracellular binding by transferrin protein in the circulation (56). Iron is even more tightly controlled during acute inflammation and infection, primarily by up-regulation of hepcidin in hepatocytes which drives intracellular iron storage in the liver (57, 58). Iron is also sequestered locally at the site of infection through lactoferrin and lipocalin proteins released from neutrophils and mucosal surfaces (57, 58). Neutrophil lactoferrin was shown to inhibit growth of A. fumigatus conidia in vitro (59). In a mouse model of A. fumigatus keratitis, both systemic and corneal levels of iron regulatory proteins, including transferrin, transferrin receptor, lipocalin, hepcidin and haptoglobin, were elevated and treatment of infected mice with topical lactoferrin or lipocalin-1 further reduced fungal growth (60).

Zinc is a second trace metal which is also extremely important to cellular function, including many transcription factors and the Cu/Zn-SOD, which combats oxidative stress (56). Zn storage is highly regulated and most Zn is either bound to albumin in the blood, or stored intracellularly in vacuoles or by binding enzymes, transcription factors or metallothionein proteins (61, 62). The serum concentration of Zn is approximately 15 μM, however free (labile) Zn is a mere 0.08 μM (63, 64). Interestingly, the cornea stroma has a very high total Zn content of 42-81 µg/g (65). Zn concentrations are modulated by expression of Zn influx (ZIP1-14) and efflux channels (ZnT1-10) and metallothioneins (62, 66). Zn,

32 like iron, is further restricted during acute phase inflammation by up-regulation of

Zn importer ZIP14 and metallothioneins in hepatocytes (66, 67). The Zn-binding

S100 proteins such as CP, which is abundant in neutrophils, contribute to local control of Zn during infection.

In addition to zinc and iron, manganese and copper are transition metals that require tight regulation for proper cellular homeostasis. Manganese is not well studied but is most known for its role in controlling oxidative stress through

Mn-dependent SOD while Cu is required for several key processes in fungal survival and pathogenesis including respiration, Cu/Zn-SOD activity, melanin production and iron acquisition (68-70). Cu is bound in the blood by albumin and ceruloplasmin but in contrast to iron and zinc, copper levels in serum rise during infection, although Cu content varies widely among tissues and is differentially modulated during infection, as demonstrated in fungal infections with C. albicans and C. gattii (71, 72). Little is known about Mn regulation in infection, however, calprotectin also binds Mn which was found to be essential for its anti-bacterial activity (73). A potential role for Cu in A. fumigatus infection will be discussed in more detail in Chapter 6.

S100 Proteins in Nutritional Immunity. S100 proteins are a family of small calcium binding proteins that have diverse expression patterns and functions in vertebrates (74). S100A8 and S100A9 have been of interest in infection and inflammation research, due to their high constitutive expression in neutrophils. S100A8 and S100A9 make up 40% of neutrophil cytoplasmic protein and related protein S100A12 (in human neutrophils only) accounts for 5% (75-

77). This abundance points to the importance of these proteins in intracellular signaling processes within neutrophils, including calcium signal transduction related to migration, phagocytosis and NOX2 activation (78-82). In the extracellular space, a shift in calcium concentration allows for Zn and Mn binding by S100A8/A9 heterodimers and heterotetramers, known as calprotectin (Figure

1.6) (73, 83, 84). CP has been shown to inhibit growth of A. fumigatus and A. nidulans in vitro and C. albicans in vivo and in vitro, an effect which is reversible by Zn supplementation (85-87). S100A12 levels are also elevated during inflammation and S100A12 homodimers exhibit calcium dependent binding to Zn which contributes to its anti-microbial activity against C. albicans and bacteria with deficient Zn uptake systems (88). S100A12 can also bind Cu although little is known about this phenomenon and its relevance in vivo.

S100A8 and S100A9 proteins are released in large amounts from activated neutrophils and other myeloid and non-myeloid cells despite the lack of a classical secretion signal for the Golgi mediated secretion pathway (89). In

Figure 1.6: Crystal structure of calprotectin (S100A8/S100A9), S1 and S2 indicate transition metal binding sites, Mn ion shown in purple, Ca ions shown in gray (84). Reprint permission obtained from the National Academy of Sciences, Copyright 2013.

34 activated monocytes, S100A8/A9 secretion was dependent on mitochondrial ATP production, protein kinase C (PKC) activation and tubulin polymerization and did not involve cell death (89). This pathway was also distinct from secretion of IL-1β, which follows a non-classical secretion pathway that is not well defined. In neutrophils, release of calprotectin has largely been attributed to NET formation.

Urban and colleagues demonstrated that extracellular CP was found only after

>40% of neutrophils had formed NETs in response to PMA and not when cells were stimulated with degranulation inducer fMLP (87). However, in this study only approximately 30% of the CP detected was associated with NETs. Similarly, another study found that CP release correlated with cell death of neutrophils in whole blood (90). However, a recent study found S100A8/A9 and S100A12 were secreted from neutrophils stimulated with monosodium urate crystals independently of NET release, necrosis, or microvesicle release. Interestingly, this study found that release depended on NADPH oxidase as well as potassium efflux, which shares similarities with IL-1β release (91). Therefore calprotectin release may occur through multiple distinct mechanisms and further studies are warranted.

In addition to neutrophils, epithelial cells can also produce S100A8 and

S100A9 and corneal epithelial production of these proteins was detected in a model of Pseudomonas aeruginosa keratitis (92, 93). Furthermore, many other members of the S100 family of proteins that are produced by different immune and non-immune cell types, can bind transition metals and may have a role in nutritional immunity, which will be discussed in more detail in Chapter 6.

Figure 1.7: Neutrophil extracellular traps induced by IL-8. A) Neutrophil elastase B) DNA C) Histone-DNA complex (55). Reprinted with permission of the American Association for the Advancement of Science.

Neutrophil Extracellular Traps. In 2004, Zychlinsky and colleagues described novel structures formed by activated neutrophils which were filaments of extracellular chromatin decorated with histones and granular proteins. These structures were named neutrophil extracellular traps (Figure 1.7) (55). This seminal study described NET formation, or NETosis, in response to gram- positive and gram-negative bacteria and found that NET formation alone could kill up to 30% of the bacterial burden by trapping the bacteria and neutralizing their virulence factors. NET formation was found to be a distinct cell death pathway, separate from apoptosis and necrosis. Neutrophils stimulated with PMA undergo morphological changes whereby nuclear and granular membranes disintegrate while the plasma membrane remains intact and annexin V negative.

The chromatin decondenses, in contrast to condensation and fragmentation seen in apoptosis, and eventually the plasma membrane ruptures releasing the NET

(94). Since 2004, numerous studies have described NET formation in response to bacterial, fungal, parasitic and viral infections. Furthermore, NET formation has

36 been described in other inflammatory conditions and is thought to contribute to tissue damage and pathology.

Several studies have described NET formation in response to the fungal pathogens C. albicans, A. fumigatus, C. neoformans and P. brasiliensis, although a critical role for NETs in killing of fungi is still unclear (87, 95-98). NET formation was demonstrated in vitro in response to Aspergillus nidulans and the effect of

NETs on fungal growth was reversible by addition of Zn or blocking antibodies to

CP (85, 86). Similarly, CP release into NETs was demonstrated in response to C. albicans (87). NETs were also identified in vivo by DNA staining in lungs of mice administered swollen A. fumigatus conidia and to a lesser extent resting conidia.

In that study, NET formation did not contribute to killing of conidia but did reduce the respiration rate of hyphae after 9-12 hours (99). A second study showed that administration of live A. fumigatus hyphae into the airway of mice produced NETs in alveoli and bronchi, identified by extracellular stretches of DNA, MPO and histones (51). The dimorphic fungus P. brasiliensis was also found to induce

NETs, although NETs were not required for killing (97). An acapsular strain of C. neoformans induced NET formation and NETs were fungicidal against acapsular and wild-type C. neoformans. Similarly, A. fumigatus hyphae were found to produce an extracellular polysaccharide galactosaminogalactan (GAG), which inhibited NET formation in vitro (100). These studies suggest a role for NETs in anti-fungal immunity but more studies are needed to establish a definitive role of this response during infection. Furthermore, no role for NETs has been described in fungal keratitis and very little is known about NET formation in the cornea. One

37 study found increased extracellular DNA at the ocular surface during experimental infection with P. aeruginosa (101). In order to define a role for NETs in fungal infection, an understanding of the receptors and pathways mediating

NETosis is imperative.

Neutrophil Recognition of Fungi. Numerous stimuli for NETosis have been described including PAMPs, DAMPs, cytokines and chemokines, crystals and chemical stimulants. Fungal PAMPs are primarily polysaccharide antigens found in the cell wall including chitin and chitosan, β 1,3 and β 1,6 glucans and α mannans(102). β 1,3 glucan is a major component of the cell wall of A. fumigatus and other fungi with strong immune activating properties and β-glucan particles were shown to induce NETosis through a Src kinase/Syk and ROS dependent mechanism (103). Furthermore, β-glucan in combination with the extracellular matrix protein fibronectin triggered rapid NETosis, however, in that study

NETosis was independent of ROS (104). Neutrophils possess numerous pattern recognition receptors (PRRs) for recognition of fungal PAMPs (Figure 1.8), but we will focus this discussion on the β 1,3 glucan receptors Dectin-1 and

Complement receptor 3, CR3 (105). Dectin-1 is a C-type lectin receptor (CLEC).

CLECs are a superfamily of carbohydrate binding receptors which includes

Dectin-1, Dectin-2, Dectin-3, Mincle and DC-SIGN, all of which have been associated with immune responses to fungal infection (106). Polymorphisms in

Dectin-1, 2 and 3 as well as CARD9 are associated with increased superficial and invasive fungal disease including candidiasis, aspergillosis and cryptococcosis (107). These CLECs share a common signaling pathway

Figure 1.8: Pattern recognition receptors involved in fungal recognition and downstream signaling (105). Reprint permission obtained from Macmillan Publishers Ltd. Nature Reviews Immunology 2015.

downstream of receptor clustering and phosphorylation of ITAM motifs on the receptor or associated adapter molecules, which involves activation of Syk kinase and protein kinase Cδ (PKCδ), followed by activation of the CARD9-

Bcl10-Malt1 complex and finally activation of transcription factors including NFκB

NFAT, IRF1 and IRF5 (108). CLEC activation results in numerous downstream effects including phagocytosis, inflammasome activation, cytokine production and

ROS production. Dectin-1 is constitutively expressed on neutrophils and Dectin-1 was also found to mediate ROS production and killing of A. fumigatus germinated

39 conidia by thioglycollate elicited neutrophils (38). One study found that Dectin-1 drove NET formation in response to P. brasiliensis exposure in vitro, however, a separate study demonstrated that Dectin-1 negatively regulated NETosis, therefore a role for Dectin-1 in NET formation is unclear.

The β2 integrin CR3/Mac-1, a heterodimeric receptor composed of CD11b and CD18, was found to be the main β 1,3 glucan receptor on human neutrophils and Dectin-1-/- mouse neutrophils did not exhibit impaired killing of A. fumigatus hyphae in vitro, while CD18-/- neutrophils did have impaired killing (25, 109). CR3 is expressed on leukocytes and binds ICAM-1 on endothelium, as well as iC3b at its canonical binding site, the I-domain (110). CR3 has a critical role in neutrophil adhesion and chemotaxis, which is evidenced by the immune disorder leukocyte adhesion deficiency (LAD), which can result from mutations in CD18 or kindlin-3 which is required for CR3 activation, and predisposes patients to bacterial and fungal infections (50). Canonical activation of CR3 on neutrophils by ICAM, fibrinogen, or many other stimuli results in ROS production and degranulation and requires Syk activation through the ITAM containing adaptors DAP12 and

FcRγ (111). A distinct lectin-like binding site was identified on the C-terminal region of the CD11b molecule of CR3 that can bind to β 1,3 glucan (112). Both

Dectin-1 and CR3 also activate PKCδ and promote ROS production, degranulation and intracellular killing of C. albicans yeast and PKCδ-/- mice were highly susceptible to systemic candidiasis and pulmonary aspergillosis (113).

CR3 was also found to mediate non-oxidative killing of C. albicans yeast through lactoferrin delivery from granules to the phagosome (114). CR3 has been

40 implicated in NET formation in several models. Hantavirus was found to use CR3 as an entry receptor and subsequently induce ROS production and NETosis in neutrophils, which contributed to tissue damage during infection. NETosis was blocked by Src kinase or NOX2 inhibitors (115). In addition to β-glucan, C. albicans secreted protein Pra1p was found to be a ligand for CR3. A. fumigatus protein Aspf2 is a homolog of Pra1p and therefore could potentially be recognized by CR3 as well. Another study found that blocking CR3 with antibody inhibited LPS-induced NET formation, so it is possible that there is role for CR3 in NET formation, independent of β glucan recognition (116). Overall, both

Dectin-1 and CR3 have an important role in the anti-fungal immune response and a more thorough understanding of their distinct roles in neutrophil function is needed.

As previously mentioned, the fungal cell wall contains numerous PAMPs in addition to β-glucans and therefore there is likely role for other PRRs including

CLECs and TLRs in neutrophil recognition of fungi and anti-fungal responses.

Dectin-2 expression was identified in activated neutrophils and was shown to increase ROS production and killing of A. fumigatus hyphae (117, 118). Dectin-2 can also form heterodimers with a second CLEC, Dectin-3. Both Dectin-2 and

Dectin-3 were identified on plasmacytoid dendritic cells (pDCs) and contributed to fungal killing of Aspergillus and Cryptococcus, respectively (119, 120).

Interestingly, Dectin-2 mediated extracellular trap formation and CP release by pDCs, although only 1% of pDCs formed these structures. Mincle is a CLEC that was shown to recognize lipid antigens in the dermatophyte fungus Malassezia

41 and in a separate study Mincle expression on human neutrophils positively correlated with neutrophil anti-Candida activity, but no role has been demonstrated in Aspergillus infection (121, 122). Furthermore, Mincle activation mediated phagocytosis and NET formation in a bacterial pneumonia model (123).

Neutrophils also express all TLRs except for TLR3, which use the adapter

MyD88 to signal through IRAK, TRAF6 and TAK1 to activate NFkB and regulate transcription of cytokines and other processes(124). It was found that TLR2,

TLR4 and TLR9 expression were increased in neutrophils exposed to A. fumigatus conidia and hyphae and that TLR2 and TLR4 knock-out mice had higher fungal burden in a pulmonary aspergillosis model (125). In A. fumigatus keratitis, TLR4-/- mice also demonstrated higher CFU in the cornea with equal cellular infiltration to wild-type, suggesting a defect of TLR4-/- neutrophils in killing

A. fumigatus. No role for TLRs in NET formation during fungal infection has been described, however, TLRs are implicated in NETosis and tissue damage in many models of autoimmunity and inflammation including ischemia-reperfusion injury, vasculitis, and viral infection (126-128). Damage-associated molecular patterns

(DAMPs), or molecules release from injured tissue, including HMGB1 were found to stimulate TLR-mediated NETosis which suggests that both PAMPs and

DAMPs may contribute to immune activation during infection. Finally, neutrophils express both activating and inhibitory Fc receptors which can modulate neutrophil activity and will be discussed further in Chapter 6.

Intracellular Signaling in NETosis. The pathways underlying NET formation downstream of receptor activation are incompletely understood.

Chemical stimulation with PMA or calcium ionophores has been used in many studies to define signaling pathways leading to NETosis, which may not fully recapitulate NET formation in an infectious setting. However, despite the need for further studies, a role for ROS production and NE and PAD4 activation have been most well established.

Many studies of NETosis have found a dependence on NOX2-mediated

ROS production and neutrophils of patients with CGD were unable to form NETs to Aspergillus and other pathogens, although this was recently challenged (85,

129). Furthermore, NOX2-deficient mice did not form NETs in response to pulmonary challenge with A. fumigatus hyphae and these mice developed increased neutrophilic inflammation along with elevated CFU (51). However, recently NOX-independent NETosis has been described in response to calcium ionophores, which induced mitochondrial ROS production and activation of small conductance potassium channel SK3 (130). Leishmania promastigotes were shown to induce both a NOX and PAD4-dependent NETosis as well as a rapid,

NOX and PAD4-independent NETosis (131). Finally, rapid, ROS-independent

NETosis was found in response to stimulation with β glucan in concert with fibronectin. Therefore, there are likely multiple pathways to NET formation depending on the stimulus, microenvironment and timing.

Zychlinsky and colleagues found that NE translocation out of the primary granules and into the nucleus, where it mediates histone degradation, is required for NETosis. This translocation requires ROS production and MPO activity, which allow transfer of activated NE from the primary granule, through the cytoplasm to

43 the nucleus where it mediates cleavage of histones (132). NE knock-out mice did not form NETs in a Klebsiella pneumonia model of infection (133). A role for NE is further supported by the finding that a SerpinB1 knock-out mice, which lacks an endogenous inhibitor of NE, has an increased NET response to numerous stimuli in vitro and a P. aeruginosa lung infection model (134).

In addition to NE-mediate histone degradation, histone citrullination is thought to be a critical step in chromatin decondensation leading to NET formation. Citrullination is mediated by the enzyme peptidyl arginine deiminase

IV (PAD4) (Figure 1.9)(135). The PAD enzymes are a family of five calcium- activated enzymes highly conserved among vertebrates and that catalyze citrullination, which is conversion of arginine to citrulline resulting in loss of positive charge (136). PAD4 contains a nuclear localization signal and is found constitutively in the nucleus of granulocytes (137). PAD4 was found to be essential for NET formation in vitro in response to LPS, PMA and hydrogen peroxide and for neutrophil killing of Shigella flexneri and group A Streptococcus pyogenes in vitro and in a mouse model of necrotizing fasciitis, although only in the case of a DNAase I deficient strain of bacteria (136, 138). Many early studies of PAD4 used the F-amidine or Cl-amidine inhibitors, which have non-specific effects. Recently, newer PAD4 inhibitors were developed that have a promising role in inhibiting NET formation for both investigative studies and therapeutic purposes (139). In support of calcium-mediated activation of PAD4, calcium influx was required for NET formation in response to IL-8 or calcium ionophore treatment and was upstream of NOX2-mediated ROS production (140). PAD4

44 expression in a non-granulocyte cell line was also sufficient to induce release of

NET-like structures in the presence of calcium elevation(141). Recently PAD4- independent mechanisms of NET release have been described, therefore adding to the complex and stimulus-dependent signaling that regulates NET formation.

Other signaling molecules have been implicated in NET formation in response to fungal pathogens and molecules. β glucan stimulated NET formation through a Src-kinase and Syk-dependent mechanism and β glucan plus

Figure 1.9: PAD4 mediates histone citrullination and chromatin decondensation (135). Adapted by permission from Macmillan Publishers Ltd. Nature Chemical Biology 2014.

Figure 1.10: Simplified view of NETosis: A) Receptor engagement B) adherence and intracellular signaling C) activation of PAD4 and granule components D) release of chromatin and other proteins into NET (144). Reprint permission obtained from Rockefeller University Press. fibronectin stimulated NETosis and was dependent on ERK phosphorylation

(103). PMA and calcium ionophores were demonstrated to activate different PKC isoforms, PKCα and PKCζ, respectively, to stimulate NET formation through separate pathways (142). As previously mentioned, PKCδ activation was necessary for neutrophil killing of C. albicans, although that study did not address

NET formation. A screen for signaling pathways downstream of PKC activation in

NETosis identified that c-Raf, MEK and ERK activation were required for NET formation in response to PMA, platelet-activated factor or the bacteria

Helicobacter pylori (143). That study showed that the c-Raf-MEK-ERK signaling was required for NADPH oxidase activation. A simplified view of signaling underlying NET formation is shown in Figure 1.10 (144). Several studies point to a role for autophagy signaling in NET formation. Autophagosomes were identified in PMA-stimulated neutrophils and engulfed ribosomes and granules and underwent lysosomal fusion. Blocking PI3K prevented chromatin decondensation and drove neutrophils towards apoptosis (145). Furthermore, inhibition of mTOR,

46 an inhibitor of autophagy, enhanced NETosis in response to fMLP (146). A recent study also demonstrated that C. albicans induced autophagy and that

PI3K inhibitors suppressed autophagy and NET formation. Interestingly, that study also found that NFκB inhibition prevented NETosis. It is unclear what role

NFkB would have in NET formation given that NETosis is thought to be independent of changes in gene expression (96, 147).

NETs in Tissue Damage. In addition to a protective role during infection,

NETs are implicated in pathology of many autoimmune diseases and inflammatory states. One study found a role for NETs in ischemic liver injury, whereby released DAMPs promoted NET formation and NETs contributed to further hepatocyte and Kupffer cell death (127). NETs also contributed to increased liver damage in a model of MRSA sepsis (148). NETs can induce direct epithelial and endothelial cytotoxicity through histone release, which may contribute to tissue pathology (149). However, NETs also contain numerous molecules, such as LL-37 and HMGB1, that promote inflammation by stimulating pro-inflammatory cytokine production from macrophages and dendritic cells, including IL-1β and IFN-α, which are thought to drive the adaptive immune responses and pathology of chronic autoimmune diseases such as systemic lupus erythematosus (150, 151). Due to the action of PAD4, NETs also contain citrullinated antigens which prime autoantibody production in rheumatoid arthritis and likely other autoimmune diseases (152). In support of NETs a mediator of inflammatory damage, mutations in PAD4 leading to overexpression are linked to rheumatoid arthritis and ulcerative colitis in some populations and PAD4 is

47 overexpressed in the brain of multiple sclerosis patients (136). NET formation in the cornea may also be detrimental to the corneal integrity and architecture.

Although there is little known about NET formation in the eye, two studies have found that NET formation is increased at the ocular surface in dry eye disease

(DED) and may be due to reduced nuclease activity and hyperosmolarity (153,

154). These studies suggest NET formation may drive chronic inflammation in

DED. In summary, NETs have a complex role in anti-microbial immunity and inflammation. A better understanding of the pathways underlying NET formation will allow for development of therapeutic interventions that either stimulate or prevent NET formation depending on the infectious or inflammatory insult.

Other Immune Responses in Aspergillus Infection

Neutrophils are critical mediators of the anti-fungal response, however, successful prevention and clearance of infection involves coordinated responses from epithelial surfaces, tissue resident myeloid cells and recruited innate and adaptive leukocytes. As previously described, macrophages and dendritic cells are critical for initial recognition of infection and production of chemokines that recruit neutrophils and other leukocytes and prime adaptive responses. Both inflammatory monocytes and pDCs are recruited to lungs of mice infected with A. fumigatus and contribute directly to fungal killing (119, 155). Natural killer (NK) cells were found to directly kill A. fumigatus hyphae (156). Constant exposure to fungal antigens in the environment also drives adaptive responses to fungal pathogens and one study of healthy humans found diverse CD4+ and CD8+ T- cell responses to a panel of A. fumigatus hyphal antigens (157). Aspergillus

48 specific T-cells were expanded in patients with invasive aspergillosis and can be isolated and further expanded ex vivo with potential for therapy in invasive fungal disease (158). Aspergillus reactive T-cells isolated in patients recovering from invasive aspergillosis were found to have a Th1 phenotype, indicating a protective role for type 1 responses, whereas Th2-type responses underlie chronic aspergillosis (159). In an A. fumigatus keratitis model, Th1 and Th17-type cells were recruited but only IL-17 production was required for protection (26).

Loss of CD4+ T-cells in HIV/AIDS predisposes patients to many opportunistic fungal infections and highlights a critical role for adaptive immunity. Ongoing vaccine design efforts have identified that protective responses can be induced by immunization with heat-killed S. cerevisiae yeast or whole glucan particles

(160, 161). Furthermore, in fungal keratitis, the ocular surface has a unique role in the immune response. The epithelium and tear film contain and produce an array of anti-microbial peptides, including alpha and beta defensins, cathelicidins, lipocalin and lactoferrin, which can contribute to anti-microbial defenses (162,

163). Additionally, ocular epithelium expresses a range of TLRs which can recognize pathogens, resulting in chemokine production and subsequent inflammatory responses (164). It is important to understand the actions of each cell and tissue to fully appreciate the immune response to fungal infection.

Furthermore neutrophil cross-talk with other immune cells is just beginning to be appreciated and adds to the complexity of the immune response (165).

Aspergillus fumigatus virulence determinants

Given the array of anti-microbial strategies the immune system employs to combat infection, pathogens must overcome these mechanisms in order to survive. A. fumigatus possesses a number of virulence factors and systems that allow it to survive in the face of immune insults including ROS, anti-microbial proteins and NETs.

The Fungal Cell Wall and Immune Evasion. The cell wall of Aspergillus fumigatus contains a core of β 1,3 glucans with β 1,6 branches, with covalently bound chitin, chitosan and galactomannan, along with an amorphous fraction composed of α 1,3 mannans (Figure 1.11) (102). As previously discussed, immune cells recognize many of these components, in particular β 1,3 glucans, therefore, both A. fumigatus conidia and hyphae use different strategies to evade immune recognition. Firstly, the conidial surface is coated by a protein, RodA, which forms a hydrophobic and immunologically inert layer. Removal of RodA protein from conidia allows activation of dendritic cells and alveolar macrophages

(166). Furthermore, RodA deficient A. fumigatus mutants displayed β glucan and

α mannan on the conidia surface and activated macrophages via Dectin-1 and

Dectin-2, resulting in increased phagocytosis and accelerated clearance of infection in vivo compared with the WT strain (22). RodA deficient conidia were also found to induce more robust NET formation by neutrophils in vitro, however, this finding may not be relevant in vivo during keratitis as neutrophils are typically recruited following germination of conidia when RodA is lost (99). Conidia also contain an outer layer of melanin. Removal of three genes involved in early melanin synthesis resulted in an abnormal conidia surface and allowed for

Figure 1.11: Cell wall components of pathogenic fungi (102). Reprint permission obtained from Macmillan Publishers Ltd. Nature Reviews Microbiology 2016. conidial activation of dendritic cells and PBMCs. Blockade of Dectin-1, TLR4 or mannose receptor reduced activation of PBMCs by the melanin mutant, supporting that melanin masks antigenic cell wall constituents (167). Additionally, melanin was shown to inhibit phagolysosomal acidification, a critical step in conidial killing, in both macrophages and neutrophils (168). Furthermore, melanin was found to inhibit LC3-dependent phagocytosis in macrophages, which depends on atg5 and NADPH oxidase activation and results in killing of conidia

(169). Copper metabolism is linked to melanin synthesis, therefore nutritional immunity may have an impact on Aspergillus immune evasion (170).

The hyphal cell wall structure is variable and plastic, depending on environmental conditions. A. fumigatus can form biofilms under some conditions, including in aspergillomas and CBPA, where hyphae are embedded in an

51 extracellular matrix of GAG, galactomannans, α 1,3 glucans, hydrophobins and melanins (171). During invasive infection a biofilm is not formed, however, hyphae still express GAG, a secreted polysaccharide composed of galactose and

N-acetyl-galactosamine that is synthesized by two UDP-glucose 4-epimerases,

Uge5 and Uge3. Presence of GAG was found to promote fungal survival during pulmonary infection and A. fumigatus deficient in Uge3 had reduced virulence in vivo (172, 173). GAG suppresses immune responses through multiple mechanisms; firstly, GAG induces expression of IL-1R antagonist by PBMCs, which suppresses protective T-cell responses (174). Secondly, GAG alters PMN expression of surface molecules that promote NK-cell interaction and Fas- dependent apoptosis (175). Thirdly, GAG was found to inhibit NET formation by neutrophils (100). Since GAG was shown to mask β glucans on the hyphal surface, this supports the hypothesis that β glucan recognition drives NET formation (173).

Antioxidant Mechanisms. Aspergillus fumigatus possesses several mechanisms to combat oxidative stress upon exposure to immune cells. Firstly,

A. fumigatus expresses four superoxide dismutases (SODs) which include three

Mn-dependent SODs (SOD2-4) and one Cu/Zn-dependent SOD (SOD1). While inactivation of SOD4 was lethal, a SOD1/2/3 deficient mutant showed increased susceptibility to killing by phagocytes in the lungs of infected mice and by neutrophils in vitro (176). While a SOD1/2/3 mutant strain demonstrated impaired virulence in a keratitis model, virulence was not impaired in an immunosuppressed pulmonary aspergillosis model (25, 176). The dependence

52 of SOD activity on transition metals means this antioxidant response is also tightly linked to nutrient metabolism. Depletion of high-affinity Cu transporters

CtrA2 and CtrC reduces expression of SOD1 (177). Furthermore, A. fumigatus possess a thioredoxin antioxidant system whereby the thioredoxin peroxidases neutralize H2O2 and are restored to a functional state by thioredoxin to allow further antioxidant activity. Production of these thioredoxin peroxidases depends on transcription factor Yap1, which is essential for virulence in vivo during keratitis but not in an immune suppressed pulmonary model (178, 179). Inhibiting this pathway with the thioredoxin inhibitor PX-12 reduced fungal burden in vivo during corneal infection (25). Fungal antioxidant machinery clearly has an important role in fungal homeostasis and survival and discrepancies in virulence studies may reflect differences in the immune environment between tissues or immune competent versus immune suppressed hosts.

Nutrient Acquisition. Aspergillus fumigatus also has nutrient acquisition systems that allow the fungus to compete with immune-mediated nutrient deprivation. A. fumigatus possesses a number of proteases and lipases to break down tissues and acquire nutrients (180). Iron is an essential metal that has been well studied in the context of infection. As previously discussed, iron sequestration is driven by hepatic hepcidin, in response to acute phase inflammatory mediators, and locally by anti-microbial proteins such as lactoferrin and lipocalin, which are found in neutrophil granules and at mucosal surfaces

(57, 181). A. fumigatus can acquire iron through a reductive assimilation pathway or by siderophore mediated acquisition (180). Siderophores are secreted iron

53 binding molecules that re-associate with specific receptors, including MirB in A

.fumigatus at the fungal membrane to deliver iron to the cell (182). The reductive pathway of ferrous iron uptake, which involves reduction, re-oxidation and transport of iron through a complex of FetC and FtrA, was found to be dispensable for virulence in vivo (183). The siderophore mediated ferric iron uptake, conversely, was found to be essential for fungal survival in both the pulmonary aspergillosis and keratitis models (60, 183). Zinc is another essential trace metal that has been less well studied in fungal infection. It has been demonstrated in vitro that many pathogenic fungi are exquisitely sensitive to Zn limitation, more so than other trace metals including iron (184). A. fumigatus does not have as robust of an acquisition system for zinc as it does for iron, which is perhaps why it is so sensitive to low Zn conditions. Three putative Zn transporters have been identified that are up-regulated in response to Zn starvation (Figure 1.12) (185). The ZrfA and ZrfB transporters are expressed in low Zn, acidic conditions, while ZrfC is expressed in low Zn, alkaline or neutral conditions (186, 187). ZrfA, B and C and another protein, AspF2, are up- regulated by activation of the Zn-sensitive transcription factor ZafA (187, 188).

The function of AspF2 is unclear, however its C. albicans ortholog Pra1 was shown to be a secreted Zn binding protein that interacts with C. albicans ZrfC ortholog Zrt1 (189). This study suggested that Pra1 could act in a similar manner to siderophores to deliver Zn to the fungal cell. ZafA and ZrfC-deficient A. fumigatus strains have impaired growth in a pulmonary aspergillosis model, but the role and function of AspF2 has not been studied (188, 190). Targeting

54 siderophore synthesis with HMG CoA-reductase inhibiting statins ameliorated disease in A. fumigatus keratitis, so Zn acquisition may represent another pathway that can be targeted therapeutically (60, 191) .

Figure 1.12: Model of A. fumigatus Zn transport in Zn- replete and Zn- limiting conditions (185). Reprint permission obtained from Springer.

Copper is also required for several key processes in fungal survival and pathogenesis including SOD function and iron homeostasis. Cu uptake by model organism S. cerevisiae requires reduction to Cu(I) and subsequent transport

55 through high affinity Cu transporters Ctr1 and Ctr3. Ctr2 transports Cu from storage vacuoles to the cytosol. Recently, two homologs of Ctr1, which contained two transmembrane domains and multiple Met-X-Met Cu binding motifs, were identified in A. fumigatus and named CtrA2 and CtrC (177). Interestingly these two genes were previously identified as up-regulated in A. fumigatus hyphae upon exposure to neutrophils (192). Deletion of these transporters resulted in increased sensitivity to low Cu or iron, and to oxidative stress imposed by H2O2 and mutants had reduced SOD1 and melanin-synthesizing laccase expression

(177). The CtrA2/CtrC double mutant strain was also more susceptible to killing by neutrophil-like HL-60 cells, although no difference in virulence was observed in an immune suppressed BALB/C mouse model of pulmonary infection. Recent work found that while serum copper rose during systemic candidiasis, tissue levels, particularly in the kidney actually declined over the course of infection. In response, C. albicans switched expression from Cu/Zn SOD1 to Mn SOD3 and also upregulated Cu transporter Ctr1 (72). Similarly, during Cryptococcus infection in the lung high Cu levels are encountered and loss of Ctr1 and Ctr4 Cu transporters does not affect virulence, however, during CNS infection Cu levels are extremely low and Ctr1/Ctr4 are required for virulence (71). Cu dependent laccases Abr1 and Abr2 and a Cu transporter CtpA were identified in A. fumigatus and necessary for melanin production, however, interestingly a CtpA mutant had increased virulence in a Galleria melonella infection model. The importance of Cu metabolism in A. fumigatus virulence will be discussed further in Chapter 6.

In addition to a role for metal uptake systems in A. fumigatus survival, a recent study found an essential role for the histidine biosynthesis pathway in metal homeostasis and virulence of A. fumigatus. Histidine often comprises the metal binding residues of proteins and it was found that disrupting histidine synthesis through deletion of imidazoleglycerol-phosphate dehydratase (IGPD) impaired A. fumigatus growth in low iron as well as in excess iron, Cu, Mn and Zn metal shock, and the IGPD mutant had impaired virulence in G. melonella (193).

Aspergillus fumigatus has many strategies for overcoming the immune response to survive in human hosts, however, many of these pathways represent druggable targets that will be the focus of future anti-fungal drug development.

Hypothesis and Goals.

The goal of this work is to increase our understanding of the role of neutrophils in anti-fungal immunity and to identify fungal virulence factors that promote disease in an effort to find new strategies for managing fungal infection. We will use our established mouse model of fungal keratitis, which exhibits a robust neutrophil response, and will also use purified primary neutrophils from mice and humans to address the following questions. Given the significance of calprotectin anti-microbial activity in other infectious disease models and the abundance of this protein in neutrophils, we hypothesized that calprotectin will have a critical role in controlling Aspergillus fumigatus growth during corneal infection.

Furthermore, we reasoned that A. fumigatus zinc uptake will be required for fungal growth during infection and that blocking zinc uptake represents a new therapeutic strategy. We have shown that blocking iron uptake by inhibiting A.

57 fumigatus siderophore synthesis effectively inhibits fungal growth and virulence, therefore our goal is to extend the concept of targeting fungal nutrient acquisition to zinc.

We also considered that zinc uptake can be targeted using atovaquone, an antimalarial agent shown to disrupt zinc homeostasis in yeast. Thirdly, we hypothesized that neutrophil extracellular traps will play a role in limiting fungal growth and infection, through release of calprotectin and other anti-microbial proteins. The presence of NET formation in many microbial infections suggests a critical role for this neutrophil pathway in controlling infectious diseases. We aim to address the significance of NETs in fungal infection using a PAD4-deficient mouse, which has a specific defect in NET formation, but not other neutrophil functions. Overall, these studies will further our understanding of neutrophil anti- fungal strategies that may be co-opted for new anti-fungal treatments.

Furthermore, we hope to identify A. fumigatus virulence factors that can be targeted with novel therapeutic agents.

Chapter 2: Zinc and Manganese Chelation by Neutrophil S100A8/A9 (Calprotectin) Limits Extracellular Aspergillus fumigatus Hyphal Growth and Corneal Infection Published in: Clark HL, Jhingran A, Sun Y, Vareechon C, de Jesus Carrion S, Skaar EP, Chazin WJ, Calera JA, Hohl TM, Pearlman E. Journal of Immunology. 196 [1] 336-44. January 2016. Copyright 2016. The American Association of Immunologists, Inc. Reprint permission obtained from publisher.

Abstract

Calprotectin, a heterodimer of S100A8 and S100A9, is an abundant neutrophil protein which possesses anti-microbial activity primarily due to its ability to chelate Zn and Mn. In the current study, we showed that neutrophils from calprotectin-deficient S100A9-/- mice have an impaired ability to inhibit

Aspergillus fumigatus hyphal growth in vitro, and in infected corneas in a murine model of fungal keratitis; however, the ability to inhibit hyphal growth was restored in S100A9-/- mice by injecting recombinant calprotectin. Further, using recombinant calprotectin with mutations in either the Zn and Mn binding sites or the Mn binding site alone, we show that both Zn and Mn binding are necessary for calprotectin’s anti-hyphal activity. In contrast to hyphae, we found no role for neutrophil calprotectin in uptake or killing of intracellular A. fumigatus conidia either in vitro, or in a murine model of pulmonary aspergillosis. We also found that an A. fumigatus ∆zafA mutant, which demonstrates deficient Zn transport, exhibits impaired growth in infected corneas and following incubation with neutrophils or calprotectin in vitro as compared to wild-type. Collectively, these studies demonstrate a novel stage - specific susceptibility of A. fumigatus to Zn and Mn chelation by neutrophil-derived calprotectin.

Introduction

Aspergillus fumigatus causes severe pulmonary and disseminated infections in patients with compromised immune systems due to solid organ and bone marrow transplant, HIV/AIDS and genetic immunodeficiencies (7, 194).

However, Aspergillus and Fusarium species are also major causes of corneal infections (keratitis), which occur in immune competent individuals and are a significant cause of blindness worldwide (13). Spores (conidia) enter the cornea through epithelial abrasions and germinate into filamentous hyphae. Neutrophils are the predominant cell type in early stage corneal ulcers and in murine models of fungal keratitis (21, 23, 195), where they play an essential role in regulating hyphal growth. Previously we reported that neutrophils combat hyphal growth in the cornea through reactive oxygen species (ROS)1 and iron limitation, and that

A. fumigatus possesses antioxidant and iron acquisition mechanisms to allow for survival (24, 25).

In addition to iron, zinc and manganese are also required for fungal growth, and an important immune defense strategy is based on sequestering these metals, termed nutritional immunity (196). Calprotectin (CP) is a heterodimer of S100A8 and S100A9, members of the S100 family of calcium binding proteins, which exhibits anti-microbial effects on bacteria, fungi and protozoa through sequestration of Zn and Mn at two binding sites formed at the dimer interface (54, 73, 197). CP comprises ~40% of total protein in the

61 neutrophil cytoplasm and is also produced and secreted by other myeloid and non-myeloid cells, including macrophages, epithelial cells and keratinocytes under inflammatory stimuli (75, 198, 199). Neutrophil CP is also reported to mediate intracellular activities, including NADPH oxidase activation and ROS production, and cytoskeletal rearrangement (81, 82).

In the current study, we identify an essential role for neutrophil CP in regulating growth of the hyphal stage of Aspergillus in vitro and in a murine model of fungal keratitis. We also show CP-dependent chelation of both Zn and

Mn is required to compete with the A. fumigatus ZafA-regulated Zn transporter system. In marked contrast, using fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability (200), we found no role for CP in neutrophil killing of the conidia stage of A. fumigatus either in vitro or in a murine pulmonary challenge model with conidia.

Together, these observations identify a stage - specific role for neutrophil CP in combatting A. fumigatus infections. Given the importance of this pathogen as a cause of severe pulmonary, systemic and corneal disease, these findings may lead to development of more targeted therapies for these infections.

Materials and Methods

Mouse Strains

All animals were used in accordance with the guidelines of the CWRU

Institutional Animal Care and Use Committee (IACUC) and the MSKCC IACUC.

S100A9-/- mice were provided by Paul Fidel (LSU, Baton Rouge, LA). CD18-/- mice were originally provided by Claire Doerschuk (UNC, Chapel Hill, NC). Age and sex matched C57BL/6 mice were purchased from Jackson Laboratories (Bar

Harbor, ME). For pulmonary challenge experiments, S100A9 and WT BM chimeric mice were generated by reconstituting lethally irradiated (9.5 Gy) recipients (C57BL/6.SJL mice) with 2-5 x 106 S100A9(-/-) or C57BL/6 BM cells and resting them for 6-8 weeks prior to use in experiments. Enrofloxacin treatment for 14 days in drinking water was given to prevent bacterial infections.

Animal studies were compliant with all applicable provisions established by the

Animal Welfare Act and the Public Health Services Policy on the Humane Care and Use of Laboratory Animals.

Fungal Strains and Growth Conditions

Strains used in this study are identified in Table I. Strains were grown on

Sabouraud Dextrose Agar (201) (dsRed strains) or Vogel’s minimal medium

(VMM) + 2% agar +/- 0.5 mM ZnSO4 (for Zn-deficient strains). Strains were

63 grown at 37 ºC for 3-5 days for sporulation and conidia were isolated by disruption in PBS and filtration through sterile cotton gauze.

Mouse Model of Fungal Keratitis

Mice were anesthetized and the corneal epithelium was penetrated with a

30G needle and 2 µl of conidial suspension (25,000 conidia/µl PBS) was injected into the corneal stroma with a 33G Hamilton syringe. Mice were imaged under a stereomicroscope at 24-48 hours pi. Whole eyes were homogenized in sterile

PBS in a Mixer Mill MM300 (Retsch). Serial dilutions were plated in SDA (dsRed strains) or VMM + 2% agar +/- 0.5 mM ZnSO4 (Zn-deficient strains) and incubated 24h at 37 ºC. CFU was counted after 24h. dsRed expressing A. fumigatus eyes were imaged at 24-48h post-infection and opacity (pixel intensity) and integrated intensity of fluorescence was quantified using Metamorph software as described (23). In some experiments, recombinant calprotectin or

PBS (10µl) was injected into the sub-conjunctival space at the time of infection.

Table 2.1: Aspergillus fumigatus strains used in this study

Murine Conidia Pulmonary Challenge Model

Intratracheal infections were performed as described earlier (200). Briefly, a blunt 20G needle was used to deliver conidia into the trachea of anesthetized mice. BAL and lung tissues were processed for flow cytometry as previously described (37). Briefly, single cell suspension of lungs and BAL cells were stained with the following Abs: anti-Ly6G (clone 1A8), anti-CD11b (clone M1/70), anti-CD45.1 (clone A20), anti-CD45.2 (clone 104), anti-Ly6B.2 (clone 7/4).

Neutrophils were identified as CD45+CD11b+Ly6CloLy6G+Ly6B.2+ cells. The data were collected on a BD LSR II flow cytometer and analyzed on FlowJo, version

9.7.6 (Treestar, Ashland, OR).

FLARE Conidia Preparation and In Vitro Assay

A. fumigatus strain 293 (AF293) was genetically modified to express dsRed and FLARE conidia were labeled as described previously (200). In all experiments, % uptake = (dsRed+AF633+ + dsRed-AF633+/total) and % viability =

(dsRed+AF633+/ dsRed+AF633+ + dsRed-AF633+). For in vitro experiments,

FLARE conidia were added at MOI 2 to 1 x 106 peritoneal neutrophils in RPMI +

5% FBS for 8h at 37 ºC. Cells were washed and re-suspended in FACS buffer.

Data were collected on a BD Accuri C6 flow cytometer and analyzed on Accuri

C6 and FlowJo software.

Isolation of Mouse Peritoneal Neutrophils

Mice were injected IP with 3% thioglycollate at 18h and 3h prior to euthanasia. The peritoneal cavity was lavaged with sterile PBS and neutrophils were purified by negative selection using an EasySep mouse neutrophil enrichment kit (StemCell, Vancouver, BC). Neutrophil purity of > 90% was verified by Wright-Geimsa staining. Neutrophils were re-suspended in RPMI

1640. To obtain neutrophil lysates, purified neutrophils at 2 x 106 cells/ml in RPMI

1640 were freeze-thawed at -80ºC for 3 cycles. Lysates were spun at 10,000xg for 10 min and supernatants were collected.

Isolation of Human Neutrophils

Whole blood was collected from healthy donors between age 18-65 in accordance with the Declaration of Helsinki guidelines and the Institutional

Review Board of the University of California Irvine. Red blood cells were separated in 3% Dextran (Sigma Aldrich, St. Louis, MO) PBS and neutrophils were purified from remaining cells by overlay on a Ficoll (GE Healthcare) density gradient and centrifugation at 500xg for 25 min. Remaining RBCs were lysed and neutrophils were re-suspended in RPMI 1640. Purity (>90%) was assessed by flow cytometry using anti-human CD16 and CD66b antibodies (eBioscience, San

Diego, CA). Live/Dead fixable violet (Thermo Fisher) was used to gate live cells and positive populations were identified using FMO controls.

In Vitro Hyphal Growth Assays

Conidia (3,000 conidia/well) were grown in VMM +/- ZnSO4 (Zn-deficient strains) for 6h to hyphal stage. Hyphae were washed 2X in PBS. Live neutrophils, lysates or recombinant CP in RPMI were incubated with hyphae for

16h at 37 ºC. For some experiments ZnSO4 (Sigma Aldrich 83265) or MnSO4

(Sigma Aldrich M7899, St. Louis, MO) was added to media. Hyphae were stained with 50 µl Caclofluor white stain (undiluted)/well (Sigma Aldrich 18909) and washed 3X in ddH20. Fluorescence at 360/440 nM was assessed using a

Synergy HT plate reader (Biotek, Winooski, VT). % Fungal mass is calculated as a as percent of total hyphae when grown in RPMI media alone

(experimental/control x 100).

Histology and Immunohistochemistry

Whole eyes were fixed in 10% phosphate buffered formalin, paraffin embedded and sectioned. PASH and GMS staining were performed by the

CWRU Visual Science Research Center histology core. For immunohistochemistry, sections were treated with proteinase K (Dako 2015-011,

Carpinteria, CA) and blocked in 1.5% serum. Mouse S100A9 polyclonal Ab (2

µg/ml, R&D Systems AF2065, Minneapolis, MN), Mouse S100A8 polyclonal Ab

(2 µg/ml R&D Systems AF3059), or Anti-mouse neutrophil antibody NIMP-R14

(20 µg/ml) were used, followed by staining with Alexafluor 488 Chicken anti-goat

IgG (1:2000, Life Technologies A2467, Grand Island, NY) or Alexafluor 488 Goat anti-rat IgG (1:250, Life Technologies A11006). Slides were imaged at 200-400X.

Neutrophil quantification from histological sections was obtained by measuring %

NIMP-R14 positive area/cornea using Metamorph software.

Flow Cytometry of Cornea Cell Suspensions.

Corneas were dissected at 24-48h pi and treated with 1X collagenase for

1-2 h. Cell suspensions were washed in 1X PBS + 5% FBS, incubated with anti- mouse CD16/CD32 (clone 93, eBioscience 16-0161-86, San Diego, CA)(to block

Fc receptors) and stained with anti-mouse neutrophil antibody NIMP-R14-PE

(abcam ab125259, Cambridge, MA) or isotype for 1h and live/dead fixable far read stain (Thermo Fisher). For intracellular staining, cells were fixed and permeabilized using an intracellular staining kit (eBioscience). Cells were incubated with Mouse S100A9 polyclonal Ab ( R&D Systems AF2065), Mouse

S100A8 polyclonal Ab ( R&D Systems AF3059), or isotype for 45 min, washed and incubated with Alexafluor 488 Chicken anti-goat IgG (1:2000, Life

Technologies A2467) for 30 min, washed, resuspended in FACS buffer and analyzed in a BD Accuri C6 (San Jose, CA). Analysis was performed using

Accuri C6 software. Cells were gated on FSC/SSC, followed by live cells.

Cytokine and calprotectin quantification by ELISA

Corneas were dissected at 6-48h post-infection and homogenized in 150

µl 1X PBS using a Mixer Mill MM300 (Retsch) and lysates were analyzed for mS100A8 (R&D DY3059), mS100A9(R&D DY2065), mCXCL1/KC(R&D DY453), mCXCL2/MIP-2(R&D DY452)

or mMPO(R&D DY3667), according the manufacturer instructions. For neutrophil lysates, purified C57BL/6 neutrophils at a concentration of 1 x 106/ml were lysed in 0.5% Triton X-100 in PBS and 10-fold serial dilutions were analyzed for mS100A8 and mS100A9.

Recombinant Calprotectin Methods

Recombinant human wild-type CP, and the CP∆Zn/Mn and CP∆Mn mutants, were expressed, purified and tested for activity as described previously

(73).

Statistical Analysis

A Mann-Whitney U test was used for unpaired comparison of two groups for all in vivo and biological replicates. In vitro experiments are shown as one representative experiment with a minimum of three technical replicates per experiment and groups compared using the Student’s t-test. Survival data was analyzed by long rank test. All statistical analyses were performed with

GraphPad Prism software, v6.0c (La Jolla, CA). A p value < 0.05 was considered significant. (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001)

Results

Neutrophil calprotectin regulates growth of A. fumigatus hyphae in the cornea.

To evaluate the role of CP in regulating A. fumigatus hyphal growth, corneas of C57BL/6 and CP-deficient S100A9-/- mice were infected with A. fumigatus dsRed conidia, and hyphal growth was monitored at 24 and 48h post- infection (pi), which we reported is the peak time of corneal inflammation and neutrophil infiltration (23). Fungal burden was measured by dsRed imaging and

CFU.

S100A9-/- mice had elevated dsRed fluorescence at 24 and 48h and significantly elevated CFU at 48h compared to C57BL/6 (Fig 2.1A-C). Since CP is reported to mediate neutrophil chemotaxis (202), we examined whether

S100A9-/- mice have impaired neutrophil recruitment during infection. Neutrophils from infected corneas were quantified by flow cytometry of corneal cell suspensions, IHC in corneal sections and myeloperoxidase (MPO) ELISA on corneal lysates. There was no significant difference in neutrophil numbers between C57BL/6 and S100A9-/- (Fig 2.1D and 2.S1C-D), indicating that the increased hyphal burden in S100A9-/- mice is not due to impaired neutrophil migration. We also measured corneal opacity as an indicator of inflammation, and found no significant difference between S100A9-/- and C57BL/6 mice (Fig

2.S1A-B). Furthermore, infected corneas of C57BL/6 and S100A9-/- mice had comparable levels of the neutrophil chemokines CXCL1 and CXCL2 (Fig 2.S1E-

F), which are essential for neutrophil chemotaxis in pulmonary and cornea infection models (23, 39).

To determine whether neutrophils are the source of CP during infection, adjacent corneal sections were immunostained with NIMP-R14, or with S100A8 and S100A9 antibodies. S100A8 and S100A9 staining increased from 24 to 48 h and coincided with NIMP-R14 staining (Fig 2.1E). Neutrophils, but not S100A8 or

S100A9 were detected in corneal sections or single cell suspension from infected

S100A9-/- mice (Fig 2.S1G, H), which is consistent with previous reports that

S100A8 is not expressed in the absence of S100A9 (203). Although S100A8 and S100A9 are also produced by epithelial cells (199), we did not detect

S100A8 or S100A9 in the corneal epithelium. Total S100A8 and S100A9 proteins in infected corneas were quantified by ELISA and found to be elevated at 6, 24 and 48h post-infection compared with naïve C57BL/6 mice (Fig 2.1F). As an additional indicator that infiltrating myeloid cells rather than resident epithelial cells are the source of S100A8 and S100A9 in infected corneas, we infected corneas of CD18-/- mice, which lack the β2 subunit of CD11/CD18 integrin required for neutrophil trans-endothelial migration to the cornea, and found significantly lower levels of both proteins (Figs 2.1F and 2.S1G) (25).

As an additional approach to determine if neutrophils are the primary source of S100A8 and S100A9, corneal cell suspensions were stained for intracellular S100A8 and S100A9 and cell surface NIMP-R14 and examined by flow cytometry. We found that >90% S100A8+ and S100A9+ cells were also

NIMP-R14+ (Fig 2.1G, 2.S1H).

Finally, to determine whether exogenous CP could impair hyphal growth during infection, S100A9-/- mouse corneas were infected with A. fumigatus- dsRed, and 1 µg total recombinant CP was administered into the subconjunctival space simultaneously where it will diffuse into the corneal stroma.

Fig 2.1H-I shows that mice administered CP had significantly less hyphal mass than those given vehicle only, as measured by dsRed fluorescence, indicating that CP has a direct protective effect during A. fumigatus corneal infection.

Collectively, these data indicate that neutrophil-derived CP has an essential role in regulating A. fumigatus infection in the cornea.

Neutrophil calprotectin regulates growth of A. fumigatus hyphae in vitro.

To determine if neutrophil CP directly limits A. fumigatus hyphal growth, peritoneal neutrophils from C57BL/6 and S100A9-/- mice were incubated with A. fumigatus hyphae. Fungal mass was measured following staining with calcofluor white, which binds cell wall chitin and can be quantified by fluorimetry (25). We found that 2 x 105 live neutrophils was the minimum number that significantly inhibited fungal growth in vitro (Figure 2.S1I), although lysates from 1 x 105 neutrophils also significantly inhibited fungal growth (Figure 2.S1J), the difference likely a consequence of only partial CP release from live neutrophils.

We also quantified S100A8 and S100A9 in neutrophil lysates by ELISA, and found that murine neutrophils contained ~0.076 pg/cell (Fig. 2.S1K).

Hyphae grown in medium alone exhibited normal branching morphology; however in the presence of C57BL/6 neutrophils, hyphal morphology was distinct, with short hyphal filaments and increased branching (Fig 2.2A). In contrast, hyphae incubated with S100A9-/- neutrophils resemble hyphae grown in

RPMI alone (Fig 2.2A), indicating a direct effect of CP on growing hyphae.

Calcofluor quantification revealed significantly higher fungal mass when incubated with S100A9-/- compared to C57BL/6 neutrophils (Fig 2.2B, Fig 2.S1I).

S100A8/A9 is reported to mediate production of reactive oxygen species (ROS) through NADPH oxidase, which is the dominant source of ROS and requires live cells (81),(25). Therefore, to determine if there is a contributing role for ROS in

CP – mediated hyphal killing, neutrophils were lysed and incubated with hyphae.

We found that similar to live neutrophils, there was significantly higher fungal mass when hyphae were incubated with S100A9-/- compared to C57BL/6 neutrophil lysates (Fig 2.2C), indicating that ROS is not required for neutrophil

CP activity.

Taken together with the in vivo studies, these data indicate that neutrophils have a non-redundant role in controlling A. fumigatus infection through CP-mediated inhibition of hyphal growth.

Calprotectin zinc and manganese chelation contribute to anti-fungal activity.

To examine the direct effect of CP on A. fumigatus hyphal growth in vitro and to assess the relative contribution of Zn and Mn chelation in CP activity, wild-

73 type A. fumigatus hyphae were incubated with recombinant CP in the presence of 1µM or 5µM MnSO4 and increasing concentrations of ZnSO4.

Hyphae incubated with 25µg/ml recombinant CP exhibited the short hyphal filaments and increased branching shown above in the presence of

C57BL/6 neutrophils (Figs 2.3A and 2.2A). To determine if hyphal growth can be rescued by Zn or Mn, we measured A. fumigatus hyphal growth following incubation with CP in the presence of ZnSO4 and MnSO4. We found that hyphal mass in the presence of 50 µg/ml (~1.4µM) CP was significantly lower than hyphae grown in RPMI alone (Fig 2.3B). As 1 mole of CP can bind either 2 moles of Zn or 1 mole of Zn plus 1 mole of Mn (73), rescue of A. fumigatus hyphal growth to 100% was found to require > 2 µM ZnSO4, whereas 2µM ZnSO4 was sufficient to rescue in the presence of 1µM or 5µM MnSO4. Conversely, an excess (5µM) of Mn alone only partially rescued growth (Fig 2.3B), which is likely due to Mn occupying only one of the two CP binding sites.

As a complementary approach to determine the relative contribution of Zn and Mn, we performed experiments with recombinant CP mutants with altered binding sites to either Mn alone or Zn and Mn (73): CP∆Mn, lacking high affinity binding of Mn but with completely normal binding of Zn, and CP∆Zn/Mn, lacking high affinity binding of both Zn and Mn. Fig 2.3C shows that whereas hyphal mass in the presence of wild-type CP was approximately 10% of growth in medium alone, hyphal mass in the presence of CP∆Mn was significantly higher, though still inhibitory, In contrast, hyphal growth was unimpaired in the presence

74 of the CP∆Zn/Mn mutant, indicating complete loss of anti-fungal activity (Fig

2.3D).

Finally, to demonstrate that neutrophil anti-fungal activity depends on metal chelation, 2 x 105 purified human neutrophils were incubated with wild-type

A. fumigatus hyphae in vitro +/- ZnSO4 or MnSO4 and fungal growth was measured. Neutrophils inhibited fungal growth more than 95%, which was reversed in the presence of ZnSO4 (5 µM), but not when MnSO4 (5 µM) was added (Fig 2.3E-F).

ZafA mediated zinc uptake contributes to A. fumigatus virulence during corneal infection and resistance to calprotectin.

A. fumigatus expresses the Zn sensitive transcription factor ZafA under Zn limiting conditions, which up-regulates the expression of the Zn transporters ZrfA,

ZrfB and ZrfC and the putative Zn binding protein Aspf2 (186-188). To determine whether ZafA mediated Zn uptake is necessary to compete with calprotectin, parent strain (WT) and ∆zafA mutant A. fumigatus hyphae were incubated with a

5 x 104 C57BL/6 or S100A9-/- neutrophils or with recombinant CP, and hyphal growth was measured following calcofluor staining.

This low number of C57BL/6 neutrophils did not inhibit growth of the WT

A. fumigatus hyphae; however, growth of ∆zafA hyphae was significantly lower

(Fig 2.4A), indicating increased sensitivity of this mutant. In contrast growth of

∆zafA mutants was not inhibited when incubated with the same number of

S100A9-/- neutrophils (Fig 2.4A). Consistent with these data, growth of the ∆zafA mutant was significantly less than WT when incubated with recombinant CP (Fig

2.4B), indicating that ZafA is required for optimal growth in the presence of CP.

To determine if ZafA is required for growth in the cornea, C57BL/6 mice were infected with WT or ∆zafA mutant conidia, and CFU/eye were assayed after

48h. Corneas infected with ∆zafA had less hyphae in the corneal stroma than those infected with WT, as detected by GMS staining (Fig 2.4C), and significantly lower CFU than WT (Fig 2.4D). There was no significant difference in the number of neutrophils in the corneas of mice infected with WT vs. ∆zafA A. fumigatus when quantified by NIMP-R14 reactivity of corneal sections and image analysis (Fig 2.4E-F). Growth of ∆zrfC, ∆zrfAB and ∆aspf2 A. fumigatus in the cornea was not significantly different from the WT (Fig 2.S2D-F). Further, although there was less CFU in a ∆zrfABC mutant, this strain also did not grow as well as the WT or ∆zafA in vitro (Fig 2.S2A-C).

Together, these data indicate that ZafA protein is essential for virulence during corneal infection and ZafA mediated Zn uptake competes with CP mediated Zn sequestration.

Calprotectin is not required for conidial phagocytosis and killing.

In addition to metal chelation, S100A8 and S100A9 have been shown to promote neutrophil phagocytosis and intracellular killing of bacteria by ROS

(204). Therefore to determine if CP contributes to neutrophil phagocytosis and

76 conidial killing in vitro, peritoneal neutrophils from C57BL/6 or S100A9-/- mice were incubated with fluorescent Aspergillus reporter (FLARE) conidia as described (200). FLARE conidia express dsRed protein, which acts as an indicator of live conidia and is extinguished upon loss of conidial viability. FLARE conidia also incorporate Alexa Fluor 633 extracellularly, which persists even after loss of conidial viability (200). Thus, FLARE conidia can be harnessed to monitor conidial uptake and viability during cellular encounters with phagocytes, since phagocytes that contain live (DsRed+ Alexa Fluor 633+) conidia can be distinguished from counterparts that contain killed (DsRed- Alexa Fluor 633+) conidia) (155, 200). Representative images of neutrophils containing live or dead

FLARE conidia, or bystander neutrophils, are shown in Fig 2.S3A.

Neutrophils were incubated with FLARE conidia for 8h, and the total and viable conidia were quantified by flow cytometry. There was no significant difference in either conidial uptake or viability between C57BL/6 and S100A9-/- neutrophils (Fig 2.5A-B), indicating that CP is not required for intracellular conidial killing in vitro.

To examine whether CP expression in hematopoietic cells is required for host defense against respiratory A. fumigatus pulmonary infection, we generated

CD45.2+ S100A9-/-  C57BL/6.SJL bone marrow chimeric mice, together with

CD45.2+ S100A9+/+  C57BL/6.SJL controls. Both groups of chimeric mice were infected with FLARE conidia by the intratracheal route, and at 12h pi, single cell suspensions from bronchoalveolar lavage fluid (BALF) and the lung were assessed for conidial uptake and killing. Conidial phagocytosis and viability was

77 indistinguishable between S100A9-/- neutrophils and control neutrophils (Fig

2.5C-D). In addition, total S100A9-/- and S100A9+/+ neutrophil counts were similar in both groups (Fig 2.5E), and mice that lack S100A9 in hematopoietic cells did not succumb to infection (Fig 2.S3B).

Thus, CP expression in radiosensitive hematopoietic cells, including neutrophils, is dispensable for host defense against A. fumigatus conidia. Collectively, these data demonstrate that CP acts in a fungal stage-specific manner to restrict extracellular A. fumigatus hyphal growth and is dispensable for intracellular conidial killing.

Discussion

Nutritional immunity is an emerging concept that addresses the importance of nutrient regulation by the immune system as a means of controlling pathogen growth and survival (56, 205). Essential nutrients required by pathogens include amino acids, lipids and transition metals such as iron, zinc, manganese and copper. For example, iron is an essential cofactor for many cellular processes(206), and we reported that neutrophils regulate A. fumigatus hyphal growth by sequestering iron, and that treating infected corneas with iron- binding protein lactoferrin or inhibiting A. fumigatus iron-binding siderophore synthesis with HMG-CoA reductase inhibitors (statins) reduced fungal burden

(24). In the current study we extend our understanding of nutritional immunity in

A. fumigatus infection to include a role for neutrophil calprotectin.

CP is an abundant cytosolic protein in neutrophils(75), and we found that neutrophils are the primary source of CP during infection of the cornea. Previous work has shown that neutrophils are essential for control of A. fumigatus keratitis in mice (25). We did not observe S100A8/A9 staining in corneal epithelium, despite a report of epithelial S100A8/A9 production in a Pseudomonas keratitis model (93). A study in the lung also showed that leucopenic mice had very low

CP expression, indicating that neutrophils are the primary source of CP in

Aspergillus infection (207). The high levels of S100A8 and S100A9 protein in infected corneas is consistent with reports that calprotectin can reach levels as high as 1 mg/ml in abscess fluid(197). The ELISA assay may in fact

79 underestimated the concentration of CP in the tissue and in neutrophils due to the ability of S100A8 and S100A9 to form higher-order oligomers, which likely still have Zn and Mn chelating activity (208). We also demonstrated that CP is essential for control of A. fumigatus growth in the cornea but not in a conidia pulmonary challenge model. Further, we compared the ability of CP-deficient

S100A9-/- neutrophils to inhibit hyphal growth with their ability to kill A. fumigatus conidia using fluorescent Aspergillus reporter (FLARE) conidia and found that whereas S100A9-/- neutrophils could not limit hyphal growth, there was no deficiency in their ability to kill intracellular conidia. This is in contrast to reports that S100A8/A9 may promote phagocytosis and ROS production in response to

E. coli and zymosan (82, 204). Our findings describe CP as a specific regulator of extracellular A. fumigatus hyphal growth. Previous work identified neutrophil

ROS and iron-chelating enzymes as essential in anti-fungal responses during keratitis (24, 25). Furthermore, neutrophils contain numerous anti-microbial peptides and enzymes including defensins, cathelicidin, proteases and chitinases, which potentially mediate killing and degradation of fungal pathogens

(209, 210). Therefore CP likely inhibits growth early in infection, which complements other anti-fungal mechanisms.

Because the corneal stroma is avascular, infiltrating cells are recruited from limbal vessels in the peripheral cornea and migrate through the dense corneal stroma to the site of infection. Thus neutrophils do not reach the site of infection until hyphae are formed. In contrast, during A. fumigatus pulmonary infection neutrophils are rapidly recruited to the highly vascularized lung, and

80 control the organisms through phagocytosis of conidia (37). Thus in the cornea, anti-hyphal responses are essential for control of infection.

These results also fit well with the recent finding that neutrophil extracellular traps (211) are generated specifically in response to large extracellular pathogens such as fungal hyphae (95). The anti-fungal activity of

NETs has been attributed to CP in Candida albicans dermal abscess and intranasal infection models and in vitro in response to Aspergillus nidulans (85,

87). NETs have been identified in lungs of A. fumigatus infected mice (51, 99); however, rather than using resting conidia as used in the current study and which are the form that is normally inhaled and phagocytosed, those reports instilled the lungs with conidia that had been previously incubated for 7h, or with hyphal fragments. Although we are investigating NET formation during fungal corneal infection, those studies and ours indicate that CP, and therefore CP-laden NETs, are important to regulate growth of extracellular hyphae rather than conidia.

However, Urban et al. demonstrate only ~30% of CP was contained within NETs while the remainder is found in the supernatant or associated with cellular remnants (87). This suggests the possibility of NET-independent secretion of CP.

In activated monocytes S100A8 and S100A9 proteins were shown to be released in a non-classical, energy-dependent manner by associating with microtubules

(89). Further studies on CP release by neutrophils and other cell types are warranted.

Several properties have been reported for CP, including direct chemotactic activity and chemoattractant response in neutrophils (78, 202).

However, we found no difference in the number of neutrophils recruited to infected corneas of S100A9-/- mice or in S100A9-/- neutrophil migration to lungs, and no significant difference in corneal opacity between C57BL/6 and S100A9-/- mice. This observation is consistent with a recent report demonstrating that although S100A8/A9 can induce neutrophil chemotaxis, its absence does not affect neutrophil recruitment in vivo in a murine model of vaginal candidiasis

(199). Further, calprotectin is reported to promote phagosomal ROS production in response to zymosan particles or E. coli (82, 204); however, we found no impairment of S100A9-/- neutrophils in the phagocytosis or cytotoxic activity against conidia. In contrast, lung neutrophils that are defective in NADPH oxidase activity show a defect in conidial killing when mice are infected intratracheally with the FLARE conidia (200). In the current study, regulation of hyphal growth by calprotectin does not require NADPH oxidase, as our findings with live neutrophils were replicated using neutrophil lysates. Therefore, CP appears to have no role in either neutrophil recruitment or in ROS production.

Instead, we show that CP anti-fungal activity depends on binding of Zn and Mn. Addition of Zn or Zn and Mn was found to reverse CP mediated

Aspergillus anti-fungal activity in vitro (207). The current study extends these findings using CP mutants that lack the ability to bind either Zn and Mn or just Mn to show the relative contribution of each of these transition metals. We found that a CP∆Zn/Mn mutant had no anti-fungal activity, whereas a CP∆Mn mutant had only partial loss of activity. Furthermore, Zn alone rescued fungal growth in the presence of CP or human neutrophils, whereas Mn alone did not. These data

82 indicate that Zn chelation is essential for anti-fungal activity, whereas Mn has only a minor contributing role. This is in contrast to bacteria, in which Mn binding has a significant role in the anti-microbial activity of CP (73, 212). CP-mediated

Mn chelation has been shown to inhibit superoxide dismutase activity and reduce the virulence of Staphylococcus aureus (213). Zn is required for the function of numerous enzymes and transcription factors in A. fumigatus, whereas Mn function is not well characterized, although it is required for superoxide dismutase function (176).

A. fumigatus growth requires a Zn uptake system comprising the Zn- sensitive transcription factor ZafA, which is required for expression of Zn transporters ZrfA, ZrfB and ZrfC, and the putative Zn binding protein Aspf2 (186-

188, 207). ZafA and ZrfC are required for virulence in a pulmonary aspergillosis model in which mice were immunosuppressed (188, 207). This Zn transporter system is also important in C. albicans and Cryptococcus gatti, where it is required for yeast survival in vivo (189, 214). In the current study, we show that

ZafA is also essential for virulence during corneal infection and that ZafA- deficient A. fumigatus strain has increased susceptibility to inhibition by neutrophils and CP. In contrast to pulmonary infection, we found no role for ZrfC in vivo during corneal infection. The difference between these and previous findings has yet to be determined; however, cornea has a higher Zn content that other tissues, including the lung and the blood (215); therefore ZrfA and ZrfB may compensate for ZrfC in this setting as ZrfA and ZrfB expression is elevated in the

∆zrfC mutant (207). A ZrfABC-deficient strain also demonstrated less virulence in

83 vivo as well as significantly impaired growth in vitro. The difference in growth of this mutant compared to ZafA-deficient A. fumigatus may be due to low level constitutive (ZafA-independent) expression of Zrf transporters on the ZafA mutant, which are completely absent in ZrfABC-deficient strain (185). Due to its importance in both pulmonary and corneal infections, ZafA may be a target for anti-fungal therapies (191).

Although the A. fumigatus Zn transport system has been characterized, specific Mn transporters have yet to be identified in A. fumigatus. However, a recent report showed that A. fumigatus siderophores that bind iron can also bind

Mn (216).

Although the Zn transport system is important in A. fumigatus survival in the lungs, our current findings using the fluorescent Aspergillus reporter (FLARE) viability assay showed that there was no significant difference in viable conidia in neutrophils from infected lungs of S100A9-/- bone marrow chimeric mice compared with C57BL/6 mice, which taken together with in vitro assays indicate that CP is not required for host survival in a pulmonary challenge model in which the immune system is otherwise intact. CP is likely also important in the immunosuppressed condition where hyphae are present as leucopenic mice have reduced CP expression in the lung and increased fungal burden (207), and patients who develop Aspergillus pulmonary infections are often either neutropenic or have neutrophil dysfunction (217).

Interestingly, there is also a CP – independent mechanism of Zn sequestration that targets intracellular pathogens. Macrophages can limit Zn

84 uptake by fungal pathogen Histoplasma capsulatum by sequestering metals from the phagosome using the transporters Slc30a4 and Slc30a7 and metallothioneins (218). It is unclear whether this mechanism is relevant in A. fumigatus conidial killing in the phagosome, however, since conidia are dormant, whereas the yeast form of H. capsulatum is replicating, zinc sequestration may not influence conidia survival.

We found that recombinant human CP inhibited Aspergillus growth at concentrations as low as 6.25 µg/ml. In vitro assays with murine neutrophils required greater than 1 x 105 C57BL/6 neutrophils to inhibit growth similarly.

Human neutrophils were found to contain an average of 25 pg CP per neutrophil(219), whereas we found that murine neutrophils contained only ~0.076 pg/cell. Importantly, although human and mouse S100A8 and S100A9 share

59% protein sequence identity, minimum inhibitory concentrations may differ.

The high level of sequence identity and the fact that all metal ligand residues are identical suggest the Zn and Mn affinities are the same; however, dissociation constants have only been measured for recombinant human CP (73).

Furthermore, release of CP from live neutrophils is likely less than 100% and indeed, a lysate from 1 x 105 neutrophils effectively inhibited Aspergillus growth

(87).

Treatment of Aspergillus infections is difficult due to a limited range of anti- fungal agents with variable efficacy and significant associated toxicity. We demonstrated that exogenous CP could control fungal growth in S100A9-/- mice.

Therefore, treatment with CP could represent a therapeutic strategy for

85 neutropenic patients with fungal infections. Overall, these studies suggest that inhibiting fungal Zn and Mn acquisition through chelators, such as calprotectin, or through new inhibitors of ZafA or other components Zn and Mn transport may represent a new therapeutic approach (191).

Chapter 3: Atovaquone impairs mitochondrial function and metal homeostasis, and differentially inhibits growth of Aspergillus and Fusarium clinical isolates

Abstract

Aspergillus and Fusarium are filamentous fungi that cause severe pulmonary and systemic infections in immune compromised patients, and also blinding corneal infections in immune competent individuals. As Zn acquisition is essential for fungal growth, we examined the effect of Atovaquone, a hydroxy-

1,4-naphthoquinone ubiquinone analog demonstrated to disrupt Zn homeostasis in yeast, on these pathogenic fungi. We found that atovaquone has significant anti-fungal activity against Aspergillus and Fusarium clinical keratitis isolates; however, Fusarium clinical isolates are significantly more susceptible (IC50:

0.001 – 0.032 µM) when compared with Aspergillus isolates (IC50: 0.29 – 0.62

µM). Further, we demonstrate that 1 µM atovaquone significantly inhibits labile intracellular Zn levels and increases sensitivity to metal shock in Aspergillus, whereas 100 µM atovaquone is required to see similar effects in Fusarium. Since vacuolar acidification and ABC transporters are required for storage of intracellular free Zn, we found that atovaquone reduced vacuolar acidification and was less effective against an ABC-transporter overexpressing strain of A. fumigatus compared to WT. Atovaquone also acted synergistically with voriconazole and itraconazole. Furthermore, mitochondrial potential and ATP production was reduced in both Aspergillus and Fusarium upon atovaquone treatment. Collectively, these studies demonstrate that atovaquone has anti- fungal activity against filamentous fungal pathogens by disrupting both metal homeostasis and mitochondrial function, and therefore exhibits potential as a novel anti-fungal agent.

Introduction

Aspergillus and Fusarium are ubiquitous in the environment, but can cause severe pulmonary, dermatologic and systemic infections in patients with genetic or induced immune deficiencies, which frequently result in death from uncontrolled fungal growth (4, 220-222). Fusarium species are also an important cause of contact lens related corneal ulcers in immune competent individuals in the industrialized world, and very commonly in developing countries following ocular injury (13, 223, 224). Aspergillus species are also a major cause of keratitis. These infections are painful, blinding and frequently require corneal transplantation. Current topical and systemic anti-fungal agents have limited efficacy, and there are several reports of resistance to commonly used drugs such as azoles (30, 225-227). Furthermore, many anti-fungal agents have severe side effects, including infusion reactions and nephrotoxicity (228, 229). Overall, there is an unmet need for new drugs that target filamentous fungi, and which are safe and effective (230, 231).

Atovaquone is a hydroxy-1,4-naphthoquinone, which is an analog of ubiquinone that binds to cytochrome b and interferes with the electron transport chain and respiration in Plasmodium species, including P. falciparum,

P. ovale and P. malariae (232-234). Atovaquone has therefore been used for prophylaxis and treatment for malaria, and for other parasitic protozoa including

Toxoplasma, Babesia, and Leishmania (235-237). Atovaquone has also been used to treat fungal infections, including Pneumocystis pneumonia (PCP) in HIV patients (238). Although not shown for P. pneumoniae, atovaquone was found to

97 disrupt Zn homeostasis in the yeasts Candida albicans and Saccharomyces cerevisiae (239). However, the effect of atovaquone on filamentous fungi has yet to be examined. We, and others demonstrated that Zn, Mn and iron limitation by neutrophils inhibits Aspergillus growth in vitro and in experimental pulmonary and corneal infections (24, 207, 240). Intracellular storage of transition metals is also required to limit toxicity of these molecules, and regulation of metal uptake and storage is essential for pathogen survival.

In the current study, we examined the effect of atovaquone on the growth of Aspergillus and Fusarium clinical isolates, and found that atovaquone functions as an effective anti-fungal agent by disrupting both mitochondrial function and ABC transporters. We also show a pronounced difference in sensitivity and drug targets between Fusarium and Aspergillus species.

Materials and Methods

Fungal Strains and Growth Conditions

Fungi were cultured on sabouraud dextrose agar at 300F for Fusarium, or

370F for Aspergillus for 48-72h until conidiation. Conidia (spores) were harvested by scraping the plates using an L-spreader, re-suspending conidia in sterile PBS and filtering through sterile gauze to remove hyphae. Conidia were then used or stored at 40F. Conidia were plated at 5 x 103/ml in 200 ul of SD broth in black 96- well plates with clear bottoms. Conidia were incubated until germination to hyphal stage (6 hrs Aspergillus, 18-24 hrs Fusarium) for dose responses. Hyphae were then incubated with pharmacological agents and incubated until media alone wells reached maximal growth (18 h for Aspergillus, 48 h for Fusarium).

Dose Response and IC50 calculation

For dose responses, hyphae were treated with 2-fold serial dilutions of atovaquone (or DMSO control) in RPMI 1640 for 18 hrs (Aspergillus) or 48 hrs

(12). Atovaquone was purchased from Sigma-Aldrich and stock solution (25 mM) was prepared in DMSO. Following incubation media was removed and hyphae were stained with Calcofluor white (Sigma-Aldrich) at 50 µl/well for 10 min at RT. Wells were washed 3X in ddH20 and fluorescence was quantified in a

Bio-tek Cytation 5 at 360/440 nM. Hyphae were imaged on Cytation 5 at 4X using DAPI filters and Brightfield.

Mitochondrial Potential and ATP measurement

Hyphae were treated with atovaquone, Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or DMSO in RPMI 1640 for 2 hrs. Following incubation media was removed and fresh RPMI 1640 (no phenol red) containing JC1 (2 µM) probe was added and incubated for 15-30 min and fluorescence was read at 514/529,

590, per manufacturer instructions (MitoProbe JC1 Assay Kit, ThermoFisher

Scientific). According to the manufacturer “JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio.” For ATP measurements, hyphae were treated with atovaquone or media alone in 200 µl

RPMI 1640 for 2 hrs. 175 µl media was removed and 25 µl of BacTiter-Glo

Reagent (Promega) was added to remaining 25 µl culture media and incubated for 10 min at RT. BacTiter-Glo Reagent generates a luminescent signal proportional to the amount of ATP in a sample using a proprietary luciferase.

Luminesence was measured by plate reader.

Cell Death Assay

Hyphae were treated with atovaquone or amphotericin B (Sigma-Aldrich) in RPMI plus 0.25 µM SYTOX green extracellular nucleic acid stain

(ThermoFisher Scientific). Only dead cells with permeable membranes will take up SYTOX dye and fluoresce. Fluorescence was recorded after 2 hrs

(Aspergillus) or 8 hrs for Fusarium at 504/523 nm.

Metal Shock and Zn measurements and imaging

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Hyphae were incubated with atovaquone, TPEN (Sigma-Aldrich) or media only for 2 hrs. Media was removed and PBS containing 2 µM Zinbo-5 (Santa

Cruz Biotechnology) or RPMI 1640 containing 25 µM Zinquin (Santa Cruz

Biotechnology) was added to wells and incubated for 15 min. PBS or RPMI was removed and fresh PBS or RPMI 1640 was added to wells. Fluorescence was read at 358/463 for Zinbo-5 or 368/490 for Zinquin. For metal shock experiments conidia were grown to germination in SDB. Plates were washed 2X in sterile PBS and RPMI +/- atovaquone +/- ZnSO4, MnSO4 or CuSO4 (Sigma-Aldrich) was added. Fungi were incubated for 18 hrs (Aspergillus) or 48 hrs Fusarium and stained with calcofluor as described.

Acidic Vacuole Quantification

Hyphae were treated with atovaquone or media only for 2 hrs. After incubation media was removed at 200 µl of RPMI 1640 with 1 µM Lysosensor

Green DND-189 (ThermoFisher Scientific) was added and incubated for 30 min.

Media was removed and fresh RPMI 1640 was added to well. Fluorescence was read at 443/505.

Azole Sensitivity

WT or HspA-Cdr1A A. fumigatus, generously provided by Scott Moye-

Rowley (U Iowa), were grown for 6 hrs in SDB, washed 2X in sterile PBS and

RPMI 1640 +/- atovaquone +/- voriconazole or itraconazole (Cayman Chemical) was added. Hyphae were incubated for 18 hrs and stained with calcofluor as described.

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Statistical analysis

All experiments were performed with 3 or more replicate wells for each condition. GraphPad Prism software was used for all statistical analysis.

Significance was determined using a student’s t-test. IC50 values were calculated using the log(inhibitor) vs. response equation, where 100% fungal mass indicates growth hyphal growth in media alone.

Table 3.1: Keratitis isolate strains and Inhibitory concentrations

Strain IC50 (µM) 95% CI Source

Aspergillus fumigatus 0.294 (0.247-0.351) Bascom Palmer Eye Institute,

BP Miami FL

Aspergillus fumigatus 0.627 (0.548-0.717) University Hospitals Cleveland,

UH Cleveland OH

Aspergillus flavus TN- 0.500 (0.399-0.628) Aravind Eye Hospital, Tamil Nadu,

302 India

Fusarium oxysporum 0.011 (0.0078-0.015) Cole Eye Institute, Cleveland

8996 Clinic, Cleveland, OH

Fusarium oxysporum 0.005 (0.003- 0.008) CDC

CB69081

Fusarium oxysporum 0.001 (0.0006-0.0024) CDC

CB 69361

Fusarium solani 0.046 (0.041-0.053) CDC

CB69147

Fusarium solani (0.023-0.044) CDC

CB69701 0.032

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Results

Atovaquone inhibits growth of Aspergillus and Fusarium clinical isolates

To determine whether atovaquone inhibits growth of filamentous fungi, fungal spores (conidia) were grown to the hyphal stage, incubated with atovaquone at different concentrations, and growth was quantified using

Calcofluor white, which binds chitin and can be quantified by fluorimetry (24, 25,

240). Inhibitory concentrations (IC50) of atovaquone against clinical isolates of fungal keratitis cases revealed that IC50 values (Table 3.1) for Fusarium species were significantly lower (10-100 fold) than Aspergillus species, indicating increased susceptibility of Fusarium compared with Aspergillus. Representative dose response curves are presented in Figure 3.1A, and representative images of A. fumigatus BP and F. oxysporum 8996 stained with calcofluor are shown in

Figure 3.S1. A. fumigatus hyphae incubated with atovaquone exhibit significantly impaired hyphal growth compared to growth in RPMI media alone and impaired fungal growth to a comparable degree with other currently used polyene and azole anti-fungal agents, including voriconazole and Amphotericin B (Fig 3.1B).

Atovaquone disrupts mitochondrial potential and ATP production

Atovaquone is a ubiquinone analog that inhibits electron transport chain

(ETC) activity in Plasmodium, which then regulates mitochondrial potential and

ATP production (232). We investigated the effect of atovaquone on ETC function and ATP production by filamentous fungi. Hyphae were incubated for 2 h with

100 µM atovaquone, which completely inhibited fungal growth, and mitochondrial

103 potential was measured using JC-1, a fluorescent indicator of mitochondrial potential. ATP was measured using the BacTiter-Glo luminescence assay.

We found that the mitochondrial potential of A. fumigatus and F. oxysporum was significantly reduced following incubation with 100 µM atovaquone (Fig 3.2A,B). The effect of atovaquone was similar to that of CCCP, a well - characterized inhibitor of mitochondrial potential. Similarly, A. fumigatus and F. oxysporum ATP production was significantly lower than untreated controls in the presence of atovaquone (Fig 3.2C,D) To differentiate between cell death and growth inhibition, hyphae were incubated with atovaquone or anti-fungal agent amphotericin B (AmB), and cell death was measured using the SYTOX green nucleic acid stain, which is impermeable to intact membranes, but intercalates with DNA in dead cells. We found significantly decreased fungal viability following atovaquone or AmB treatment compared with controls. (Fig

3.E,F, Fig 3.S2).

Together, these data show that atovaquone inhibits mitochondrial function and ATP production, resulting not only in inhibition of fungal growth, but also increased hyphal death.

Atovaquone reduces labile intracellular Zn in Aspergillus, but not Fusarium

As atovaquone was found to impair Zn uptake by S. cerevisiae and C. albicans, and we and others showed that Zn is required for hyphal growth (239,

240), we examined the effect of atovaquone on intracellular Zn. Free, or labile,

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Zn was measured using Zinbo-5 and Zinquin fluorescent probes, and the Zn chelator TPEN was used as a positive control.

Representative images revealed bright Zinbo-5 staining in untreated A. fumigatus hyphae, but not following incubation with atovaquone or TPEN (Fig

3.3A). Quantification of Zinbo-5 and Zinquin showed significantly less free Zn in

A. fumigatus and F. oxysporum following incubation with atovaquone; however, whereas 1 µM reduced A. fumigatus Zn levels, 100 µM atovaquone was required to see the same effect in F. oxysporum (Fig 3.3B-E). Taken together with the data in Figure 1, these findings indicate that the effect of atovaquone on A. fumigatus is at is least in part by disrupting cellular Zn levels, whereas the effect of atovaquone on Fusarium appears to be independent of Zn modulation.

Atovaquone inhibits vacuole acidification

To examine whether an exogenous source of Zn would rescue the effect of atovaquone on Aspergillus. Hyphae were incubated with 1 µM atovaquone

(which inhibited hyphal growth) together with an excess of ZnSO4, MnSO4 or

CuSO4. However, rather than restoring growth, the presence of these metals further inhibited A. fumigatus hyphal growth (Fig 3.4A). These concentrations of

MnSO4 in the absence of ATV inhibited A. fumigatus growth whereas excess

ZnSO4 or CuSO4 at these levels had a minimal effect on growth (Fig 3.S3A). In contrast, addition of these metals had no significant effect on growth of Fusarium alone or in the presence of ATV, except for MnSO4 which did reduce growth in the presence of ATV (Fig 3.4B, S3B).

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Despite the essential role for Zn in fungal metabolism, free Zn and other metals can be toxic to cells at high levels (56). Therefore, free metals are stored in specialized vacuoles, which require acidification by v-type ATPases to facilitate metal uptake (241). To examine the effect of atovaquone on vacuolar acidification, we measured total acidic vacuoles using the pH sensitive probe

Lysosensor green that has been used to measure Zn in vacuoles (242).

We found that Fusarium and Aspergillus hyphae treated with atovaquone for 2 h had significantly lower Lysosensor green fluorescence compared with

RPMI controls(Fig 3.4C,D), indicating that the existing vacuoles are less acidic or that there are fewer acidic compartments in treated organisms.

Overall, these data indicate that for Aspergillus, atovaquone disrupts metal storage in vacuoles by inhibiting vacuolar acidification, which may explain the increased toxic effects of Zn and other metals in the cell. In contrast, although atovaquone also inhibits vacuolar acidification in Fusarium, the anti-fungal activity appears to be largely independent of disrupted metal storage in vacuoles.

Aspergillus ABC transporters regulate atovaquone and azole sensitivity

ABC transporters in S. cerevisiae are essential for Zn storage in yeast as loss of ABC transporters results in increased sensitivity to metal toxicity (243).

Furthermore, atovaquone inhibits ABC transporter activity in mammalian HEK cells(244) and ABC transporters mediate resistance to azoles (245). To examine the role of ABC transporters on atovaquone and azole sensitivity of filamentous fungi, an A. fumigatus mutant that overexpresses the ABC transporter Crd1A

106 was incubated with atovaquone alone or together with voriconazole or itraconazole, and growth was measured compared to the wild-type, parent strain.

As shown in Figure 3.5A, Atovaquone plus voriconazole was more effective than either drug alone in inhibiting growth of the WT Aspergillus strain.

However, the Crd1 overexpressing mutant hasp-A/abcA strain showed increased resistance to atovaquone alone or in combination with voriconazole. Similar results were found in separate experiments where both strains were incubated with atovaquone alone or in combination with itraconazole (Fig 3.5B).

These data indicate that Aspergillus ABC transporters regulate sensitivity to atovaquone and azoles, and imply that atovaquone disrupts metal storage in

A. fumigatus by inhibiting ABC transporter activity.

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Discussion

Atovaquone is currently used to prevent and treat parasitic infections with

Plasmodium, Babesia, Toxoplasma and the opportunistic yeast Pneumocystis carinii (238, 246). In this study we describe that activity of atovaquone against the filamentous fungi Aspergillus fumigatus, A. flavus, Fusarium oxysporum and

F. solani.

Atovaquone is an analog of ubiquinone/Coenzyme Q, which transfers electrons from dehydrogenases to cytochromes in the electron transport chain

(247). Atovaquone competitively inhibits coenzyme Q activity by binding to the ubiquinol oxidation pocket and collapses the mitochondrial membrane potential in

Plasmodium (247, 248). While ATP levels were not reduced in atovaquone- treated Plasmodium, ATP levels in P. carinii were significantly decreased with atovaquone treatment (249, 250) indicating atovaquone may have multiple mechanisms of action that differ between pathogens. Atovaquone inhibited P. carinii growth at 0.3 – 3 µM, compared with 10-100 nM required for Plasmodium.

Here we demonstrate that atovaquone inhibits the growth of Aspergillus in a similar range as P. carinii (IC50 0.294-0.5 µM) and Fusarium at much lower levels similar to Plasmodium (IC50 0.001-0.046 µM). The effective range of atovaquone against filamentous fungi is also well below steady-state plasma concentrations measured in patients which can reach 15-30 µg/ml (40-82

µM)(251). Therefore it is possible to reach inhibitory concentrations in vivo.

Filamentous fungi, especially Aspergillus species cause opportunistic lung and systemic infections in HIV infected and other immune compromised individuals;

108 therefore atovaquone may be an effective prophylactic or therapeutic strategy for these patients.

Plasmodium, Leishmania and P. carinii resistance to atovaquone is due to mutations in the coenzyme Q binding site (252-254). Because filamentous fungi are opportunistic and human-to-human transmission does not occur, it seems unlikely that atovaquone resistance would develop; however spontaneous mutations have been described in P. jirovecii and T. gondii, which may be due to high spontaneous mutation rate in mitochondrial DNA (255). However, new derivatives of atovaquone are under development that may have improved efficacy and circumvent development of resistance (256-258).

Given that P. carinii and A. fumigatus cytochrome b shares 74% similarity, it is likely that the mode of action of atovaquone on Aspergillus is similarly to that of P. carinii. In addition to directly targeting oxidative phosphorylation through inhibition of electron transport, atovaquone disrupts Zn homeostasis in an S. cerevisiae reporter system and in the pathogenic yeast C. albicans(239). Zn is essential for the catalytic activity of some 300 enzymes, and therefore contributes to growth and survival of microbial pathogens (259). We, and others reported that blocking Zn uptake inhibits growth of Aspergillus and other fungal pathogens, and that Zn acquisition is essential for Aspergillus virulence in corneal and pulmonary infections (207, 240).

Although Zn is required for hyphal growth, divalent cations such as Zn are toxic to cells if not effectively stored within the cell (259). Yeast cells have a specific vacuole that is required for Zn homeostasis, and which mediates

109 transition metal uptake through Zrc1p and Cot1p proteins. Zn uptake occurs in exchange for H+ ions, and is mediated by V-type ATPases that also maintain the vacuole acidification required for non-toxic storage of Zn and other metals (259).

A. fumigatus ZrcA is the putative ortholog of Zrc1p (260), although the orthologues in Fusarium have not been reported. In the current study, we demonstrate using Zinbo-5 and Zinquin dyes that labile Zn is present in untreated

A. fumigatus hyphae, but is decreased following incubation with atovaquone.

Interestingly, labile Zn is not reduced in F. oxysporum hyphae. Further, addition of excess Zn does not rescue growth, but instead leads to increased fungal killing, suggesting that atovaquone increases the toxicity of Zn, and also Mn and

Cu, possibly by blocking storage and detoxification of free metals within the cell.

Mislocalization of metals to the cytosol or other compartments can be detrimental to cells in part by replacing iron in Fe-S centers of enzymes, and by interfering with mitochondrial activity. Overall, Fusarium was less sensitive to metal toxicity which may indicate the presence of additional methods of regulating metal homeostasis in this organism.

We found that not only did atovaquone reduce acidic compartments, likely vacuoles, but there was also lower ATP in treated hyphae, thereby supporting a role for atovaquone in inhibiting V-type ATPases. This effect could be due to reduced ATP levels, or to a direct effect of atovaquone on V-type ATPases. In support of this, V-type ATPase deficient S. cerevisiae yeast and Aspergillus nidulans exhibit defects in growth and vacuolar acidification, and are susceptible

110 to metal shock (261). V-type ATPases have therefore been proposed as a therapeutic target for anti-fungal drug development (261, 262).

In addition to V-type ATPases, the ATP Binding Cassette (ABC) G family transporters of S. cerevisiae Pdr18p, Pdr5p and Pdr15p were shown to interact with Zn transporters Zrtp and Zrc1p, which transport Zn across the plasma membrane and vacuolar membrane, respectively (243). Pdr15 and to a lesser extent pdr5 and Pdr18 mutant yeast strains exhibited heightened sensitivity to

Zn shock, indicating that ABC transporters are also essential for normal Zn homeostasis, including Zrc1p mediated Zn storage in vacuoles (243). A. fumigatus homologs of Zrt1p and Zrc1p, termed ZrfA and ZrcA, have been identified (186, 260). Recently it was found that atovaquone inhibits activity of the

ABC transporter BCRP/ABCG2 in HEK293 cells at 0.23 µM (244). It is unknown whether atovaquone affects fungal ABC transporter activity. Enhancing metal sensitivity of pathogens could be a valuable tool due to recent development of metal-based anti-microbial agents, such as QBP, a form of 8-hydroxyquinolone that concentrates Cu in the macrophage phagosome and enhances killing of fungal pathogen Cryptococcus neoformans (263).

ABC transporters in Aspergillus and Fusarium are also implicated in azole resistance, including the A. fumigatus ABC transporters AbcA and AbcB, which have high sequence similarity to S. cerevisiae Pdr5, (245). Also, Fusarium graminearum ABC transporters ABC1, 3 and 4 contribute to azole resistance and virulence in plant hosts(264). Given that atovaquone potentially inhibits ABC transporters, it could increase sensitivity to currently used azole drugs in addition

111 to disrupting metal storage and mitochondrial function. Consistent with this possibility, we show in the current study that fungi treated with atovaquone together with either itraconazole or voriconazole has a greater inhibitory effect on fungal growth than either atovaquone or azole alone. Further, the synergistic effect of atovaquone does not occur when an ABC transporter is overexpressed as in the hspA-abcA mutant, indicating that atovaquone also affects ABC transporter activity. These findings show that atovaquone increases the effectiveness of azoles, and indicates that combination therapy could be an effective treatment, particularly in the case of azole resistant fungi. Although humans also possess ABC transporters, atovaquone has a good safety profile and is widely used, but this activity could potentially affect drug-drug interactions, since ABC transporters are required for many cellular processes including transport of drugs, nutrients and metabolites, including metals.

Atovaquone is typically given orally and is effective for pulmonary and systemic infections. However, as Aspergillus and Fusarium also cause severe corneal infections, it is unclear whether oral drug delivery would produce high enough levels in this tissue. One indication that they can reach the cornea is that lipophilic drugs such as atovaquone, when taken orally, can accumulate in the basal corneal epithelium (265). Although this is associated with a corneal epithelium defect, symptoms are typically minor and resolve with cessation of treatment. New delivery methods are under investigation, including cyclodextrin compounds, which form binding pockets for lipophilic drugs to facilitate distribution and release, and have been shown to improve ocular delivery of

112 voriconazole (266). One study demonstrated that atovaquone complexed with cyclodextrin nanoparticles had increased oral bioavailability of atovaquone and resulted in higher and longer lasting plasma levels of the drug (267).

Furthermore, new technologies including contact lens release and ocular

“nanowafers” are also likely to improve ocular drug delivery and increase effectiveness (268, 269).

In summary, atovaquone kills and/or inhibits growth of filamentous fungi through at least two mechanisms (Figure 3.6). Firstly, atovaquone acts on the mitochondria to inhibit electron transport and reduce the mitochondrial potential, resulting in decreased ATP production and cell death. Secondly, atovaquone disrupts metal homeostasis either directly by inhibiting ABC transporters, which are required for non-toxic storage of metal, or indirectly through reducing ATP levels, resulting in impaired V-type ATPase mediated vacuolar acidification and sequestration of Zn. This results in the presence of increased Zn in the the cytosol where it disrupts enzyme function and induces oxidative stress (270).

Interestingly, although Fusarium hyphal growth was impaired by lower atovaquone concentrations than Aspergillus, the mechanism of action appears to be distinct. In contrast to Aspergillus, there was little effect on free Zn in response to atovaquone, whereas there was decreased mitochondrial potential. Based on this observation, it seems likely that the effect of atovaquone on Fusarium is primarily on the mitochondria.

Despite differences in the mode of action, Atovaquone is a potential new therapy for Aspergillus and Fusarium infections when given either alone or

113 together with azoles. Furthermore these studies indicate that fungal metal homeostasis, V-type ATPases and ABC transporters represent new targets for development of anti-fungal agents.

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Chapter 4: A Role for Neutrophil Extracellular Traps in Aspergillus fumigatus infection

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Abstract

Aspergillus fumigatus is a leading cause of corneal infections in healthy individuals. Neutrophils are the most abundant cell type in corneal ulcers of patients with fungal keratitis and neutrophils are critical to controlling fungal growth, through ROS production and release of antimicrobial proteins including calprotectin. Neutrophil extracellular traps are released in response to bacteria, fungi and parasites and contain CP, therefore we investigated a role for NETs in neutrophil responses to A .fumigatus. We found that A. fumigatus hyphal extracts and the fungal cell wall polysaccharide β glucan induced NET release, measured by SYTOX green fluorescence of extracellular DNA. Furthermore, using blocking antibodies or CR3-deficient CD18-/- mouse neutrophils, we found that the β glucan receptor CR3, but not Dectin-1, was required for NET formation. We also showed that DNA release was dependent on ROS production, which was mediated by CR3 signaling. Using PAD4-/- mouse bone marrow neutrophils and

PAD4 inhibitors in human peripheral blood neutrophils, we found that PAD4 was required for NET formation. In an in vitro A. fumigatus hyphal killing assay we found that blocking CR3 inhibited neutrophil killing of hyphae but PAD4 had little role in killing hyphae in vitro. Furthermore, CD18-/- neutrophils, but not PAD4-/- neutrophils demonstrated impaired release of CP in response to β glucan, compared to neutrophils from C57BL/6 mice. Finally, we identified NET formation in vivo in corneas of mice infected with A .fumigatus by citrullinated histone antibody staining, which is a specific marker of NETs. We conclude that fungal β glucan induces NET formation through a CR3-ROS-PAD4 dependent

125 mechanism. Furthermore, CR3 activation is required for killing of A. fumigatus by neutrophils and for CP release, however PAD4-mediated NETosis is not required for killing or CP release in vitro, although further studies of A. fumigatus cornea and lung infection in PAD4-/- mice are warranted.

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Introduction

Aspergillus fumigatus is a filamentous fungal pathogen that is responsible for severe pulmonary and invasive infections in immunocompromised patients, as well as being a leading cause of corneal infection (keratitis) in healthy individuals. Neutrophils are the most abundant responding cell during acute A. fumigatus keratitis in infected individuals and in murine models, where we showed they are essential for controlling fungal growth (21, 25). Studies in human patients and mouse models of A. fumigatus keratitis and pulmonary infection have identified a role for reactive oxygen species (ROS) in control of infection (25, 51). Furthermore, neutrophils contain numerous anti-microbial proteins that have diverse effects on microbial pathogens. Among these proteins, lactoferrin, lipocalin and calprotectin were shown to control fungal growth during

A. fumigatus keratitis (24, 240). These proteins chelate iron, Zn and Mn, which are essential for pathogen growth.

NETs are structures formed of DNA, histones and anti-microbial proteins extruded from either live or dying neutrophils that contribute to sequestration and killing of pathogens. NET formation occurs via an active process that involves protein kinase C, Raf, Mek and Erk activation, which leads to activation of

NADPH oxidase, neutrophil elastase and peptidyl arginine deiminase 4. NE and

PAD4 then mediate decondensation of nuclear chromatin, which is released from the cell, forming a NET (96, 133, 138, 142, 143, 145). NETs have a demonstrated protective role in numerous bacterial, fungal and parasitic infections (55, 87, 271). Pulmonary challenge with A. fumigatus hyphae or

127 swollen conidia was shown to induce NETosis in murine models (99).

Furthermore, NADPH oxidase-deficient mice, which cannot generate ROS, are unable to form NETs and succumb to pulmonary aspergillosis, although this has recently been challenged (51, 129). Additionally, calprotectin release was found to be, in part, dependent on NET formation in response to Aspergillus and C. albicans (86, 87). Therefore, NETs may have a critical role in controlling fungal infection, however several studies did not find a significant role for NETs in killing

A. fumigatus and other fungi (97, 99, 129). Furthermore, NET formation during corneal infection is not well studied. One study of Pseudomonas aeruginosa keratitis described extracellular DNA isolated from the corneal surface, suggesting NET formation occurs in the cornea (101). However, NET formation was also detected in the cornea in dry eye disease is hypothesized to promote chronic inflammation, therefore NETs may have either a protective, or damaging role during infection (153).

The receptors driving NET formation in response to fungal pathogens are not well defined, however fungal polysaccharide β 1,3 glucan -induced NETosis depended on Src-kinase and Syk (103). Interestingly, the A. fumigatus cell wall contains galactosaminogalactan (GAG), an extracellular polysaccharide that masks β glucans and can inhibit NETosis (100). In separate studies, the β glucan

C-type lectin receptor Dectin-1 was found to either drive or to inhibit NETosis induced by the fungi P. brasiliensis or C. albicans, respectively (95, 272). CR3

(αMβ2 integrin, CD11b/CD18, Mac-1) also binds β glucan and is a key β glucan receptor on neutrophils (109). However, only one report found that CR3 was

128 important in NETosis in response to β glucan, and in that case NET formation also depended on binding of extracellular matrix protein fibronectin (104).

Interestingly, that study found that NET formation was independent of NADPH oxidase, in contrast to other reports. Finally peptidyl arginine deiminase 4 (PAD4) is thought to be important in NET formation. PAD4 is an enzyme found constitutively in the neutrophil nucleus that can citrullinate histones, which converts arginine to citrulline resulting in a loss of positive charge, which in turn mediates chromatin decondensation and was shown to be required for NET formation in response in bacterial and viral infection and also sterile inflammation

(127, 138, 139). A role for PAD4 has not been studied in the context of fungal infection.

In the current study we investigated NET formation in response to A. fumigatus and identified a role for CR3, ROS and PAD4 in NETosis in response to β glucan. Furthermore, we demonstrated that CR3 signaling was essential for

ROS production, NET formation, hyphal killing and CP secretion from neutrophils. Finally, we showed that NETs are formed in vivo during A. fumigatus corneal infection.

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Materials and Methods

Mouse studies

All animals were used in accordance with the guidelines of the Case

Western Reserve University and University of California, Irvine Institutional

Animal Care and Use Committee (IACUC). CD18-/- mice were originally provided by Claire Doerschuk (University of North Carolina, Chapel Hill, NC). Age and sex matched C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor,

ME). PAD4-/- mice were provided by Dr. Kerri Mowen (Scripps Research

Institute). All animal experiments were conducted with sex- and age-matched mice and animal studies were compliant with all applicable provisions established by the Animal Welfare Act and the Public Health Services Policy on the Humane

Care and Use of Laboratory Animals.

Aspergillus fumigatus strains and culture

The strain used in these studies was a keratitis clinical isolate obtained from the Bascom-Palmer Eye Institute, A. fumigatus BP. Fungi were cultured on sabouraud dextrose agar at 37 ºC for 3-5 days for sporulation and conidia were isolated by disruption in PBS and filtration through sterile cotton gauze. For hyphal killing experiments conidia were grown in sabouraud dextrose broth

(SDB), at 3000 conidia/well of 96-well plate until germination (6 hrs), washed in

1X sterile PBS and co-incubated with neutrophils. For AspHE preparation, conidia were grown in 500 mL SDB at 37 ºC for 5 days. SDB was removed from the fungi by vacuum filtration and fungal mass was homogenized in a mortar and

130 pestle under liquid nitrogen. Fungal homogenate was re-suspended in RPMI

1640 without phenol red and filtered through a 70 µM cell strainer. Protein content was measured by BCA assay (ThermoFiser) and AspHE stocks were stored at -20 ºC.

Neutrophil isolation

Human neutrophils were isolated from the blood of healthy donors age 18-

50. All human studies were approved by the University of California, Irvine institutional review board. Whole blood was mixed 1:1 with 3% Dextran-PBS for

20 min to sediment red blood cells. The upper layer was overlaid on Ficoll

Hypaque (GE Healthcare) and centrifuged at 500xg for 25 min. The pellet containing granulocytes and RBCs was treated with 1X RBC Lysis Buffer

(eBioscience) and spun at 300xg for 5 min to obtain a granulocyte pellet. Cells were resuspended in RPMI 1640 and counted. Typical purity was > 95%. Total bone marrow was isolated from the tibias and femurs of mice and neutrophils were purified using the EasySep mouse neutrophil enrichment kit (Stemcell).

Typical purity was >90%.

SYTOX Green assay for NETosis

Neutrophils were re-suspended in RPMI 1640, no phenol red (Life

Technoloiges) plus 2% FBS. In some assays neutrophils were incubated at 37 ºC with cytokines, inhibitors or blocking antibodies for 30 min prior to stimulation.

Diphenyliodonium (DPI) was purchased from Sigma Aldrich and a 10 mM stock solution in DMSO was prepared. GSK484, 199 and 106 were purchased from

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Cayman chemical and 40 mM stock solutions were prepared in DMSO. Blocking antibody for CR3, LEAF purified anti-human/mouse CD11b, M1/70 was purchased from Biolegend. Anti-Dectin-1 22H8 blocking antibody was purchased from Invivogen. 2 x 105 cells/well for human or 1 x 105 cells/well for mice, were added to black 96-well plates with optical bottoms (Corning) +/- RPMI only, PMA

(25 nM), curdlan (100 µg/ml) or AspHE (100 µg/ml) and 1 µM SYTOX Green nucleic acid stain (Life Technologies). Plates were incubated at 37 ºC, 5% CO2 in a Biotek Cytation 5 imaging plate reader and fluorescence was measured every 30 min at 504/523 nm for up to 24 hours. Plates were imaged at 40-200X using brighfield or GFP filters.

ROS Quantification

Neutrophils were re-suspended in RPMI 1640, no phenol red (Life

Technologies) plus 2% FBS. In some assays neutrophils were incubated at 37 ºC with cytokines, inhibitors or blocking antibodies for 30 min prior to stimulation. 2 x

105 cells/well were added to black 96-well plates with optical bottoms (Corning)

+/- RPMI only or curdlan (100 µg/ml) plus 500 µM luminol (Sigma Aldrich) and luminescence was measured every 2 min for 2 h on a Biotek Cytation 5.

H3cit Immunofluorescence

Neutrophils were resuspended in RPMI 1640, no phenol red, 2% FBS at 2 x 106 cells/ml. In some assays neutrophils were incubated at 37 ºC with cytokines, inhibitors or blocking antibodies for 30 min prior to stimulation. Cells were plated on glass bottom 8-well chamber slides (ibidi) or poly-l-lysine coated

132 glass coverslips (Neuvitro) +/- RPMI only or curdlan (100 µg/ml) for up to 24 hrs.

Media was removed and cells were fixed for 30 min in 4% formaldehyde in PBS.

Cells were permeabilized in 0.1% Triton-X100 in PBS for 15 min and washed in

PBS. Cells were stained for H3cit (Abcam 5103) 1:100 and neutrophil elastase

(SCBT) 1:50 overnight at 4 ºC. Slides were washed in PBST and donkey anti- rabbit Alexafluor 488 or donkey anti-goat Alexafluor 568 (Life Technologies) were added for 1 hr at RT. Slides were washed in PBST and 100 µl of PBS-DAPI was added to slides. Slides were imaged at 200X on a Biotek Cytation 5 using GFP,

RFP and DAPI filters and on a Leica confocal at 600X.

S100A8 ELISA

Mouse neutrophils were incubated in RPMI 1640 + 10% FBS + 20 ng/ml

GM-CSF (R&D systems) +/- Curdlan (100 µg/ml) or AspHE (100 µg/ml) at 1 x 105 cells/well in 96-well plates. Plates were incubated at 37 ºC for 2 or 16 hrs, spun at 300xg for 3 min and cell-free supernatants were collected. mS100A8 was measured by ELISA (R&D systems) per manufacturer instructions.

Fungal Growth Inhibition Assay

Conidia were grown to hyphal stage in a 96-well plate as described above.

Purified neutrophils at 0.5-2 x 105/well in RPMI 1640 were added to hyphae for

18 hrs at 37 ºC. Following incubation media was removed and wells were stained with 50 µl Calcofluor white chitin stain (Sigma Aldrich) for 10 min at RT. Wells were washed 3X in ddH2O and fluorescence was read on a Biotek Cytation 5 at

360/440 nM.

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Corneal Infection Model and Immunohistochemistry

Mice were anesthetized and the corneal epithelium was penetrated with a

30G needle and 2 µl of conidial suspension (25,000 conidia/µl PBS) was injected into the corneal stroma with a 33G Hamilton syringe. At 48 hrs post-infection, mice were euthanized and whole eyes were fixed in 10% phosphate buffered formalin for 24 h, paraffin embedded and sectioned at 5 µm. Paraffin sections were deparaffinized using xylenes and ethanol. Antigen retrieval was performed by heating to 95 for 20 min in Tris-EDTA buffer (pH 9.0)v. Slides were blocked in

PBS + 2% donkey serum for 20 min at RT. Slides were stained for H3cit (Abcam

5103) 1:100 overnight at 4 ºC. Slides were washed in PBST and stained with donkey anti-rabbit Alexaflour 488 (life technologies) for 1 hr at RT. Slides were washed in PBST and coverslips were mounted with Vectashield hardset with

DAPI. Slides were imaged at 200X on a Biotek Cytation 5 using GFP, RFP and

DAPI filters.

Statistical Analysis

In vitro experiments are shown as one representative experiment with a minimum of three technical replicates and groups compared using the Student’s t-test. All statistical analyses were performed with GraphPad Prism software, v6.0c (La Jolla, CA). A p value < 0.05 was considered significant. (* P ≤ 0.05, **

P ≤ 0.01, *** P ≤ 0.001)

134

Results

Aspergillus fumigatus induces neutrophil extracellular traps from human and mouse neutrophils

We established a quantitative assay for NET formation by incubating neutrophils in the presence of Aspergillus fumigatus hyphal extract (AspHE), particulate β glucan (Curdlan) or positive-control PMA and measuring extracellular DNA release over time by SYTOX Green fluorescence, a cell- impermeable DNA stain. All three stimuli induced a significant increase in DNA release from human neutrophils over time compared to media alone, shown as relative fluorescence units (RFU); total SYTOX is shown as the area under the curve (AUC) (Fig 4.1A, B). NETs in live cell cultures have been described as

“diffuse and cloud-like structures (142).” Representative images of stimulated neutrophils are shown in Figure 4.1C, and NETs appear as diffuse patches of

SYTOX green fluorescence. Mouse bone marrow neutrophils also released DNA in response to AspHE and Curdlan (Fig 4.1D, E), although PMA did not stimulate mouse neutrophils (not shown). Both ROS-dependent and ROS independent methods of NET formation have been described (51, 104). To determine whether

ROS is essential for NET formation in response to A. fumigatus, human neutrophils were incubated with stimuli in the presence of the NADPH oxidase inhibitor diphenyl iodonium (DPI). DPI inhibited DNA release in response to all stimuli (Fig 4.1F, G), indicating a requirement for NADPH oxidase-generated

ROS in NET formation.

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CR3 mediates NET formation and ROS production in response to Curdlan

Neutrophils constitutively express both the C-type lectin Dectin-1 and the

β2-integrin CR3, which, in addition to integrin activity, has a domain that binds to the fungal polysaccharide β glucan (112). To determine the role of these receptors in NET formation, human neutrophils were incubated with curdlan in the presence of blocking antibodies to either CR3 or Dectin-1. We found that Ab to CR3 significantly reduced DNA release by neutrophils in response to curdlan

(Fig 4.2A, B). In contrast, Ab to Dectin-1 did not reduce NET formation; conversely, neutrophils exhibited increased DNA release in the presence of

Dectin-1 blocking antibody (Fig 4.2C,D). Since ROS has a role in DNA release, we also investigated whether CR3 signaling stimulated ROS production. We stimulated neutrophils with curdlan in the presence of blocking antibody to CR3 and measured total ROS production by luminol assay, shown as relative luminescent units (RLU) and as area under the curve (AUC). We found that blocking CR3 significantly reduced the oxidative burst in response to curdlan (Fig

4.2E, F). Finally, we stimulated bone marrow neutrophils from C57BL/6 or from

CD18-/- mice, which do not express the CD18 component of the CR3 heterodimer and therefore cannot signal through CR3. CD18-/- neutrophils had significantly reduced DNA release in response to curdlan compared with WT neutrophils (Fig

4.2G,H). RFU were normalized to fluorescence of unstimulated neutrophils due to baseline differences in WT and CD18-/- neutrophils.

NETs contain citrullinated histone H3, and PAD4 is required for NET formation

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PAD4 is found in the neutrophil nucleus and mediates histone citrullination, which is required for NET formation in infection and autoimmunity.

These studies found that PAD4-/- mouse neutrophils are unable to form NETs

(138, 273). Citrullinated histone H3 was also identified in lungs of mice infected with C. albicans (95). To determine whether PAD4 is required for NET formation, we measured extracellular DNA release from bone marrow neutrophils of

C57BL/6 and PAD4-/- mice in response to curdlan. Compared with WT, PAD4-/- neutrophils released significantly less DNA following curdlan stimulation (Fig

4.3A,B). RFU and AUC were normalized to neutrophils in RPMI only, due to different baseline fluorescence between WT and PAD4-/- neutrophils.

Neutrophils were incubated with curdlan, and assessed for citrullinated histone H3 (H3cit) staining by immunocytochemistry. We found that C57BL/6 neutrophils form characteristic NET structures in response to curdlan, which are composed of DNA strands that are positive for H3cit, whereas PAD4-/- neutrophils retain their lobular nuclei and are negative for H3cit (Fig 4.3C).

To investigate if there is a role for PAD4 in NET formation by human neutrophils, we used novel PAD4 inhibitors that were recently developed and shown to inhibit NET formation in vitro (139). We incubated human neutrophils with the PAD4 inhibitor GSK484 or with the negative control compound GSK106.

GSK484 significantly reduced DNA release in response to curdlan, whereas

GSK106 did not inhibit DNA release (Fig4.4A-D).

Neutrophils were stimulated in vitro and assessed for the presence of

H3cit by immunocytochemistry. Neutrophils incubated with curdlan exhibited

137 characteristic NET structures, and were positive for H3cit; conversely, unstimulated neutrophils showed minimal H3cit staining (Fig 4.4E). Collectively, these data clearly demonstrate an essential role for PAD4 in driving NET formation by human and mouse neutrophils.

CR3 is required for inhibition of A. fumigatus growth in vitro

Although NET formation has been described in fungal infection, it remains unclear whether NETs are essential for killing A. fumigatus hyphae and for controlling infection. To determine whether NETs regulate fungal growth in vitro, neutrophils were incubated with A. fumigatus hyphae in the presence of the anti-

CR3 blocking antibody, or with PAD4 inhibitors GSK484, GSK199 and the control compound GSK106.

Neutrophils effectively inhibit A. fumigatus growth over an 18 h time course, as measured by calcofluor chitin stain, which stains total fungal mass by binding cell wall chitin. In the presence of blocking antibody to CR3 fungal growth is restored to approximately 80% of growth in media alone (Fig 4.5A).

Neutrophils incubated with PAD4 inhibitor GSK484 also showed an impaired ability to control fungal growth; however GSK106 had no effect (Fig 4.5B).

GSK484 was cytotoxic at concentrations higher than 5 µM; therefore we cannot eliminate the possibility off-target effects of this inhibitor on neutrophil function or viability. We also tested the ability of mouse bone marrow neutrophils from WT,

CD18-/- or PAD4-/- to control A. fumigatus growth in vitro. Fungal growth was limited in the presence of 2 x 105 C57BL/6 neutrophils, whereas there was no significant difference in A. fumigatus incubated with neutrophils from CD18-/- mice

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(Fig 4.5C). There was no significant difference in fungal mass when hyphae were incubated with neutrophils from PAD4-/- mice compared with C57BL/6 neutrophils

(Fig 4.5D). Together, these data demonstrate that CR3 signaling is required for fungal growth inhibition and/or killing by neutrophils in vitro, whereas there is no apparent role for PAD4.

CR3 is required for calprotectin release in vitro

Calprotectin (CP), which is essential for neutrophils to control A. fumigatus growth in vitro and in vivo, is thought to be released by NET formation (87, 240).

To determine if there is a role for CR3 or PAD4 in calprotectin release, bone marrow neutrophils from C57BL/6, CD18-/- and PAD4-/- mice were incubated with curdlan or AspHE for 2 or 16 hrs, and the CP subunit S100A8 was measured in cell-free supernatants by ELISA.

Both WT and PAD4-/- neutrophils released comparable amounts of

S100A8 protein in response to curdlan or AspHE, which was detected within 2 hrs, which is prior to significant DNA release as measured by SYTOX fluorescence (Fig 4.6A-B). In contrast, CD18-/- neutrophils did not release

S100A8 in response to curdlan, but did release S100A8 in response to AspHE after 16 hrs incubation (Fig 4.6B).

These data indicate that CP is released from neutrophils in the absence of

PAD4-dependent NET formation, and that release in response to curdlan requires CR3 activation. Since PAD4-/- neutrophils can secrete CP, which is required to inhibit fungal growth, this could explain why PAD4-/- neutrophils do not

139 exhibit impaired anti-fungal activity in vitro (Fig 4.5D). NETs may still have a role in vivo in retaining anti-microbial proteins such as CP at the site of infection and in contact with pathogens, whereas this role would be irrelevant under vitro conditions.

NETs are formed in vivo during A. fumigatus corneal infection

To determine whether NETs are formed in vitro during A. fumigatus corneal infection, WT C57BL/6 mice were infected with 50,000 A. fumigatus conidia in the corneal stroma. After 48 hrs, eyes were fixed and sectioned.

Corneal sections were stained for the presence of NET marker H3cit and total nuclei (DAPI). Corneas of infected mice were positive for H3cit, which was localized to areas of dense cellular infiltrates (Fig 4.7A). These data demonstrate

NET formation during corneal infection. The role for NETs in infection will be assessed in future studies by measuring fungal burden and inflammation in corneas of WT or PAD4-/- infected mice. Due to the inability of CD18-/- neutrophils to cross the endothelium and enter the cornea during infection, these mice cannot be used to assess NET formation in infected corneas.

140

Discussion

Neutrophils have an essential role in the control of fungal infections. We previously identified a critical role for ROS and the anti-microbial proteins lactoferrin, lipocalin and calprotectin in controlling A. fumigatus keratitis in vivo and fungal growth in vitro (25, 240, 274). Neutrophil extracellular traps are structures formed of extruded DNA and anti-microbial proteins released from dying neutrophils during an inflammatory insult, but a clear role for NETs in the control of infection has not been established in part due to a lack of specific inhibitors or knock-out mouse models for NETs until recently. In this study we demonstrate that NETs are formed in response to A. fumigatus cell wall polysaccharide β glucan in a CR3 and ROS-dependent manner. Furthermore, using PAD4-/- mice and PAD4 inhibitors, which were shown to inhibit NET formation and have no other reported effects on neutrophil function, we show that

NET formation is dependent on PAD4 activation and histone citrullination. Finally we have demonstrated that NET formation occurs in a mouse model of A. fumigatus keratitis.

Studies of NET formation in infection and autoimmunity have identified many stimuli and receptors, however, the pathway mediating NET formation to A. fumigatus has not been fully elucidated. β glucan is a major cell wall polysaccharide of fungi recognized by both the C-type lectin Dectin-1 and CR3, which are constitutively expressed on neutrophils(109). We found that curdlan, a particulate form of β glucan induced robust extracellular DNA release from neutrophils, which is characteristic of NET formation. Furthermore, NETs were

141 positive for citrullinated histone H3, a specific marker of PAD4 activation and

NET formation. Previous work has found conflicting roles for Dectin-1 in NET formation. We found that blocking Dectin-1 enhanced NET formation, which agrees with a recent study describing Dectin-1 as a negative regulator of

NETosis (95). Conversely, we found that blocking CR3 reduced DNA release, indicating a role for CR3 in NETosis. Furthermore, we showed that CR3 was required for ROS production in response to curdlan and that ROS was essential for NET formation. Previously, CD18-/- mouse neutrophils, which do not express functional CR3, were found to have impaired ROS production and killing of A. fumigatus in vitro, but NET formation was not assessed (25). In this study, in addition to confirming that CD18-/- neutrophils show impaired anti-hyphal activity, we demonstrate that CD18-/- neutrophils also have impaired NET formation in response to curdlan, which suggests that CR3-mediated NET formation may contribute to anti-fungal activity of neutrophils. One previous study found that β glucan binding by CR3 induced NETosis but this also depended on presence of the extracellular matrix protein fibronectin and was ROS-independent, therefore the mechanism of NET formation is distinct from our findings (104). A recent study reported that blocking CR3 inhibited neutrophils ability to prevent A. fumigatus conidial germination and growth, but not rapid killing of hyphae and the activity of CR3 against conidia was attributed to release of iron-chelating lactoferrin from granules, rather than ROS production or NET formation (129). In contrast we found that CR3 was required for ROS production, which is essential for controlling A. fumigatus growth and infection. However, we also found that

142

CD18-/- neutrophils did not release the anti-microbial protein calprotectin in response to curdlan, therefore CR3 may also have a role in release of anti- microbial peptides, including CP and lactoferrin. However, since lactoferrin is found in granules and CP is cytosolic, the mechanisms of their release are likely different. Furthermore, β glucan was found to induce NETs in a Src-kinase and

Syk-dependent manner, but a receptor was not identified in that study (103).

Both CR3 and Dectin-1 can activate Syk, but given our findings we conclude that

CR3 is the major β glucan receptor mediating NET formation.

Many studies of NET formation have found a dependence on ROS production. A loss of NET formation was reported in both human neutrophils from chronic granulomatous disease (CGD) patients, who possess defects in NADPH oxidase, and in NADPH oxidase deficient mice (p47phox-/-) (51, 85). NET formation in response to C. albicans hyphae was also reduced in the presence of antioxidant N-acetylcysteine (96). However, recently ROS-independent NETosis has been described in response to C. albicans β glucan in concert with the extracellular matrix fibronectin, as well as in response to Leishmania parasites and immune complex stimulation (104, 131, 275). In the case of both β glucan/fibronectin and Leishmania stimulation, NET formation occurred rapidly, in less than 30 minutes, whereas in our studies significant DNA release is not observed until several hours after stimulation. Furthermore a recent study found that CGD neutrophils did in fact form NETs in response to C. albicans but not to

PMA (129). It is likely that there are multiple mechanisms for NET release depending on the stimulus and microenvironment, however due to the high

143 sensitivity of isolated neutrophils to stimulation, differences in in vitro conditions and time points observed could result in disparate findings. Indeed most NET studies observe neutrophils up to only 3-4 hrs following stimulation. In our

SYTOX Green assay, PMA induced NET formation occurred by 2-3 hrs however curdlan and AspHE did not produce significant difference in DNA release over control neutrophils until approximately 6 hours or later, which was reproducible across multiple human donors. Under these conditions we found that NET formation depended wholly on ROS and was inhibited by the NADPH oxidase inhibitor DPI, therefore we conclude that ROS production is required for NET formation.

PAD4 is a member of the peptidyl arginine deiminase family found constitutively in neutrophils (137). PAD4 was shown to mediate histone citrullination and allow chromatin decondensation which allows release of DNA into NETs (276). Although PAD4 dependent NETosis was demonstrated in mouse models of bacterial and parasitic disease as well as autoimmunity, a role for PAD4 in fungal infection has not been described (131, 138). We found a decrease in DNA release in response to PAD4 inhibitors or in PAD4-/- mice, suggesting a role for PAD4 in NETosis. However, inhibition of NETosis was partial compared with blocking CR3 or ROS, which completely abrogated DNA release. This may suggest PAD4 independent NET formation, which has been described in response to Leishmania parasites. However, in contrast to our findings, that study found PAD4-independent NETosis occurred within 10 minutes of stimulation (271). It is also possible that alternate cell death pathways

144 are activated in the absence of NETosis that could result in DNA release observed in the setting of PAD4 inactivation. In the case of chemical inhibitors, they may incompletely inhibit PAD4 activity, but findings were similar to PAD4-/- neutrophils which exhibit complete loss of PAD4. Recently, new PAD4 inhibitors were developed based on Cl-amidine that inhibit NET formation and could be compared with the compounds used in this study for inhibition of NETosis (277).

Despite reports of NET formation in A. fumigatus and C. albicans infection, a direct role of NETs in killing fungi is controversial. Several studies found evidence of killing of C. albicans yeast or A. fumigatus hyphae, while other studies found a minimal role of NETs in controlling A. fumigatus infection (96, 99,

129). A recent study found no role for NETs in A. fumigatus hyphal killing in vitro, however, this study only observed killing after 1 hr of incubation, which is not sufficient time for robust NET formation in our study (129). Furthermore, if NET anti-fungal activity depends on CP-mediated Zn sequestration, which has been suggested, then NETs may only contribute to inhibition of fungal growth over time, or fungistasis, rather than immediate cytotoxicity (85). While blocking CR3 abrogated the anti-fungal activity of neutrophils in our study, this could be due to several mechanisms other than NETosis, including direct ROS production and lactoferrin or CP release as previously discussed. In contrast, PAD4-/- neutrophils, which should only exhibit defects in NET formation, controlled fungal growth in vitro equally well to wild-type neutrophils. PAD4-/- neutrophils also released comparable levels of calprotectin in vitro even after only 2 hrs of stimulation with curdlan, suggesting CP may be released by mechanisms other

145 than NETosis. These findings do not preclude a role for NETs in fungal infection, however. In contrast to in vitro conditions where neutrophils and fungi are contained in a small volume such that anti-microbial effectors released by any mechanism are kept in contact with fungi, NETs may serve a role in vivo to retain anti-microbial proteins in close proximity to the infectious agent, thereby maximizing their activity and preventing diffusion of the AMPs or spread of the pathogen. Furthermore, NETs may have other roles in regulating inflammation and immune responses in vivo. We demonstrate that the NET-specific marker

H3cit is present in corneas of mice infected with A. fumigatus, therefore future studies should investigate A. fumigatus keratitis and pulmonary infection in WT and PAD4-/- mice and assess both fungal burden and inflammatory markers in vivo. These results can also be compared with effects in NE-/- mice which do not form NETs, however, this knockout mouse has demonstrated defects in neutrophil transmigration and phagocytosis, whereas PAD4 has not been implicated in any other critical neutrophil functions (278).

Despite the initial description of NETs as anti-microbial structures, it is now appreciated that NETs contribute significantly to tissue damage and amplification of inflammatory responses. A pathogenic role for NETs in propagating inflammation has been described in numerous diseases including rheumatoid arthritis, lupus, vasculitis, acute respiratory distress syndrome and sepsis. Furthermore, histones, which are abundant in NETs have direct cytotoxic activity (149). Although very little work has been done on NET formation in the eye, two studies of dry eye disease (DED) found increased extracellular DNA on

146 the ocular surface of DED patients and that hyperosmolarity seen in DED promotes NETosis(153, 154). During keratitis, the inflammatory response is a key driver of corneal damage leading to blindness, and thus future studies on

NET formation in A. fumigatus keratitis should assess corneal inflammation and opacity as a measure of tissue damage. In the presence of effective anti-fungal therapy or if NETs have a detrimental role in keratitis pathology, NET formation could be blocked using inhibitors of PAD4 described here. PAD4 inhibitors may be ideal for preventing damage while preserving anti-fungal activity of neutrophils because PAD4-/- mice still released CP, which was shown to be protective during infection. Conversely, if NET formation is protective in keratitis by controlling fungal growth, then promoting NETosis during infection could accelerate clearance. One strategy for accelerating NETosis would be to prevent A. fumigatus inhibition of NET formation. A. fumigatus hyphae express GAG, a polysaccharide that was shown to mask β glucan recognition and reduce NET formation in vitro (100). Inhibiting GAG synthesis or treating with exogenous hydrolase enzymes, which have been used to disrupt bacterial biofilms and could be tested for GAG digestion, could promote NET-mediated immunity (279).

Furthermore, treating with anti-fungal echinocandin drugs was shown to increase

β glucan expression in hyphae, which could also enhance neutrophil responses to fungi (280).

Overall, these studies describe a role for CR3, ROS and PAD4 in NET formation in response to β glucan and provide new insights into how NET formation can be modulated to fine-tune immune responses to fungal infection.

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Chapter 5: Data Summary and Discussion

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In these studies we have expanded upon our understanding of how neutrophils control corneal infection caused by the pathogen A. fumigatus. We have found an essential role for neutrophils in mediating nutritional immunity through metal sequestration and in turn identified how A. fumigatus competes with nutritional immunity to maintain virulence. We have also found a novel anti- fungal agent that targets metal homeostasis to limit fungal growth. Finally, we have gained an understanding of how neutrophil extracellular traps are formed in response to fungi and set the stage for future studies on NETs in fungal keratitis.

Calprotectin and Nutritional Immunity to A. fumigatus. Previous work by our lab and others has established a central role for neutrophils in anti-fungal immunity (21, 25, 118). In Chapter 2, we extend our understanding of neutrophil function to include a non-redundant role for CP in limiting fungal growth in the cornea in a mouse model of fungal keratitis. Furthermore, we demonstrated that

CP was important for limiting hyphal growth but not for intracellular killing of conidia, indicating a specific role of CP in control of extracellular pathogens.

Using recombinant CP with mutated metal binding residues, we found that Zn binding, and in part Mn binding, contributed to CP anti-fungal activity. Finally, we demonstrated that injection of recombinant CP could inhibit fungal growth in the cornea, which indicates the therapeutic potential of CP or other chelating agents in fungal infection. A caveat of this approach is that sub-conjunctival injection is not optimal for treatment in keratitis patients, therefore topical delivery of these proteins should be assessed for effectiveness. However, recent use of a related

Zn-binding S100 protein, S100A7 as a treatment for fungal infections in the skin

156 and lungs, supports a future therapeutic role for S100 proteins as anti-microbial agents (281). Furthermore, mice express lower levels of both S100A8/A9 mRNA and protein than humans; therefore there may be an even more significant role for CP in humans than demonstrated in the mouse model (219, 240).

In addition to direct anti-microbial activity, S100A8 and S100A9 have many potential roles in the immune response, including acting as DAMPs and stimulating immune responses through interaction with TLR4 and RAGE. These roles can also be considered in the setting of infection, where they may activate neutrophils or other immune and epithelial cells through autocrine and paracrine stimulation. Previously our lab has demonstrated that TLR4-defiicient mice exhibited impaired clearance of fungal keratitis. It would be interesting to investigate a role for S100A8/A9 in modulating immune responses to fungal infection through TLR4.

Given the importance of Zn in A. fumigatus growth in the presence of neutrophils or CP, we investigated the role of A. fumigatus Zn acquisition in virulence. We found that deletion of Zn-sensing transcription factor ZafA impaired virulence of A. fumigatus in corneal infection. Although Zn channel ZrfC is the main target gene of ZafA under neutral or alkaline pH conditions, a ZrfC deficient strain did not show impaired virulence, which could be due to ZrfA/ZrfB up- regulation in the absence of ZrfC. However, targeting ZafA could represent a new strategy for treating fungal infections, particularly because homologs of ZafA are also found in other pathogenic fungi including Candida and Cryptococcus spp.

(282). We did not address Mn transport as a virulence factor; however this will be

157 important for future studies of A. fumigatus virulence. Since fungi possess both

Cu/Zn and Mn-dependent SOD, which can neutralize superoxide and SOD are important for virulence in A. fumigatus keratitis, it would be interesting to investigate whether CP increases oxidative stress in fungi using ROS probes

(25). Aside from oxidative stress, the mechanism of S100A7 killing of fungi was found to be intracellular Zn chelation that resulted in apoptosis, so this mechanism could also be studied in the context of CP (281). It is unknown whether CP can cross the fungal cell wall and membrane or whether it acts only extracellularly. A better understanding of how transition metals specifically affect fungal metabolism would open new avenues for development of anti-fungal agents. In a broader sense these studies highlight the importance of nutritional immunity in controlling infection and as a strategy that can be exploited in development of new anti-fungal drugs.

Atovaquone as a novel anti-fungal agent. Because Zn acquisition was essential for A. fumigatus virulence, we sought to target Zn uptake as a potential therapeutic tool. We selected the anti-malarial agent atovaquone based on the finding that it reduced free Zn levels in S. cerevisiae and C. albicans yeasts, which have similarities to A. fumigatus in Zn uptake(239). In Chapter 3, we show that atovaquone can inhibit growth of Aspergillus and Fusarium clinical keratitis isolates at an effective dose easily obtainable by oral administration. Because the anti-malarial activity of atovaquone depends on disruption of mitochondrial respiration, we investigated mitochondrial function and found that atovaquone inhibited mitochondrial potential and ATP production in A. fumigatus.

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Furthermore, we found that similar to yeast, atovaquone reduced free Zn in A. fumigatus, using fluorescent probes for free, or labile, Zn. Interestingly, adding excess Zn to the medium did not rescue growth, which would be expected if the target of atovaquone was ZafA-Zrf-mediated Zn uptake.

In contrast, excess Zn resulted in greater inhibition of A. fumigatus growth.

Since most “free” Zn in cells is stored in specialized vacuoles to prevent toxicity, we hypothesized that atovaquone may disrupt the normal cellular distribution of

Zn. We found that acidic vacuoles, which store transition metals, were reduced upon atovaquone treatment. Furthermore, ABC transporters are thought to mediate Zn uptake into vacuoles and we found that an ABC-transporter overexpressing strain of A. fumigatus was more resistant to atovaquone than

WT. Finally, because resistance to azole anti-fungal drugs is mediated in part through ABC transporters, we found that atovaquone and the azoles voriconazole or itraconazole had synergistic activity against Aspergillus, which was reduced in the ABC transporter overexpressing strain. These data indicate that atovaquone targets ABC transporter activity, which could be an important tool to improve susceptibility of resistant strains to currently used anti-fungal drugs.

Both v-type ATPases and ABC transporters require ATP for activity, therefore the effect of atovaquone on these transporters could be direct or indirect due to reduced ATP levels. Furthermore, ABC transporters also act as efflux pumps for azoles and other compounds, so we cannot rule out the possibility that ABC transporter overexpression increases efflux of atovaquone

159 from the fungal cell, thereby reducing its activity. Another caveat to this study is the lack of testing in vivo in the keratitis model, due to difficulty solubilizing the lipophilic drug for topical delivery. However, atovaquone could also be tested against filamentous fungi in a pulmonary model of invasive aspergillosis, since it is currently used to treat Pneumocystis fungal pneumonia.

Strategies for effective ocular delivery of atovaquone were discussed in

Chapter 3. However, future studies could also evaluate effectiveness in a pulmonary aspergillosis model, where orally-delivered drug is more likely to reach the site of infection. It is unclear whether atovaquone would be effective in conjunction with chelating agents such as CP, since toxicity of atovaquone is enhanced in the presence of increased metals. However, neutropenic patients that may have very low CP levels at the site of infection could still benefit from this drug. Given the activity of atovaquone against parasites and now filamentous fungi, this should also be tested as a potential treatment in other models of fungal disease, alone and in conjunction with other established and novel anti-fungal drugs.

An interesting finding from this study was that although Fusarium species were more susceptible to atovaquone than Aspergillus, the mechanism of action did not appear to be through reducing free Zn in the vacuoles. A future study would therefore examine the differences in Zn uptake and storage in Fusarium, which is a major cause of fungal keratitis worldwide.

Neutrophil Extracellular Traps in A. fumigatus infection. Given the importance of CP in A. fumigatus infection, and that NETs are reported to contain

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CP, we investigated if there is a role for NETs in fungal infection and if CP release is associated with NETosis (87). In Chapter 4 we demonstrated that A. fumigatus extract, and specifically the polysaccharide β glucan could induce NET formation in human and mouse neutrophils. NET formation was impaired by blocking the β glucan receptor CR3, but not Dectin-1. As we only studied

Curdlan, which is particulate β 1,3 glucan, future studies could examine if other cell wall components, including β 1,6 glucans induce NET formation. We also found that blocking NADPH oxidase with DPI inhibited NET release, indicating an essential role for ROS production. A caveat to this approach is that DPI also inhibits flavoproteins, therefore more specific NOX inhibitors or NADPH-deficient mouse neutrophils should also be studied, although several studies have already reported a role for ROS in NET formation using these mice. We then used PAD4-

/- mice or PAD4 inhibitors to investigate a role for this enzyme in mediating

NETosis and found that loss/inhibition of PAD4 inhibited NET release. The observation that PAD4 inhibition only partially blocked NET formation indicate additional, PAD4-independent pathways of NET formation, which would be interesting to examine.

We also found that blocking CR3, but not PAD4, impaired neutrophil killing of A. fumigatus hyphae in vitro as well as release of CP in vitro. We observed mild inhibition of fungal killing with PAD4 inhibitor GSK484, but as these inhibitors are relatively unstudied, we cannot rule possible off-target effects, particularly since

GSK484 exhibited cytotoxicity at higher doses.

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These data also suggest that CR3 contributes to the anti-fungal response in addition to NET formation, which may include ROS production and anti-microbial protein release by mechanisms other than NETosis. CR3 signaling downstream of β glucan activation should be addressed to fully understand the effects of CR3 on anti-fungal responses and reveal more targets for immune modulatory therapy. Furthermore, these data demonstrate that NET formation may not be required for CP release as previously thought. Other possible mechanisms of CP release are discussed in Chapter 6. This is a potentially important finding if NET formation is found to cause excessive tissue damage in keratitis and blocking it could relieve damage without compromising the anti-fungal activity of CP or other neutrophil activities.

Lastly, we demonstrated that NETs are formed in vivo during A. fumigatus keratitis through detection of citrullinated histone H3 in infected corneas. An investigation of the role of NETs in fungal keratitis is warranted using WT and

PAD4-/- mice to assess fungal burden and corneal damage by opacification measurement. Our in vitro findings in PAD4-/- neutrophils do not preclude a role for PAD4 or NETs in vivo, since NETs could function to localize CP and other proteins in close contact with fungi in vivo, which would be irrelevant in the context of confined in vitro conditions. Furthermore, it is possible that the mouse model may not fully recapitulate human infection. Human neutrophils exhibit a more robust NET response than murine neutrophils. Human neutrophils produce much larger NET structures and a greater percentage of neutrophils undergo

NETs(283). Therefore, in mice the NET response may not play as large of a role

162 in immunity, whereas in humans it could play a larger role during infection.

However, some of the observations of NETs in vitro may also be attributed to differences in activation states of peripheral blood neutrophils used for human studies, versus bone marrow and peritoneal neutrophils used in mouse studies. It would be interesting to look for NET markers such as H3cit in corneas of fungal keratitis patients given transplants to compare the extent of NETosis with that seen in mouse corneas.

NET components can further stimulate immune responses through activation of other immune cells, therefore NETs may have a regulatory role in inflammation in vivo that is not reflected in a single cell-type in vitro assay. If NET formation proves beneficial to controlling infection in vivo, strategies to promote NETosis can be employed to enhance anti-fungal immunity, which are detailed in Chapter

Overall, these studies advance our understanding of the immune response to fungal infection and fungal virulence factors, both of which provide avenues for the investigation of novel treatments for fungal infection. The importance of nutrient acquisition for pathogen survival has been demonstrated for bacterial, fungal and parasitic diseases, therefore the development of nutrient chelating agents, potentially based on S100 proteins, could represent an effective route for treating many types of infection. Although not a metal chelating agent, atovaquone also shows potential as an anti-fungal agent and we have already demonstrated benefits in conjunction with currently used anti-fungal drugs.

Although it remains to be determined whether NETs are protective or damaging

163 in fungal keratitis, modulation of NET formation either by promoting or inhibiting it may also be of broad significance for infections and inflammatory diseases.

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Chapter 6: Future Directions and Preliminary Data

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NET-independent Calprotectin Release from Neutrophils

The calprotectin proteins S100A8/S100A9 do not possess Golgi secretion signals and therefore are secreted through a non-classical mechanism that is not well defined. Although NET formation was thought to be the primary mechanism by which CP is released, our data and a recent study find that CP can be released independently of NET formation (87, 91). We found that PAD4-/- mice secreted equal levels of CP into the supernatant compared with WT, despite impaired NET formation. Another study found CP was secreted independently of degranulation, NET formation, cell death or microvesicles, however, that study only assessed secretion in response to monosodium urate crystals, therefore, we cannot rule out different secretion mechanisms depending on the stimulus(91).

Other mechanisms of non-classical secretion have been described that could be involved in S100A8/A9 secretion including secretory lysosomes or secretory autophagosomes, both of which can fuse with the plasma membrane to release cargo extracellularly (284, 285). A. fumigatus conidia were found to recruit autophagy proteins to the phagosome, suggesting A. fumigatus can induce autophagy signaling, although this has not been shown in neutrophils (286). A recent study found that related protein S100A11 was secreted from mesothelioma cell lines by peroxisome-mediated secretion, a non-classical secretion system similar to lysosomal secretion (287). Localization of CP with different intracellular compartments including lysosomes, peroxisomes and autophagosomes could be assessed by electron or fluorescent microscopy before and after stimulation of cells for CP release. Furthermore, inhibitors of

166 these pathways or deletion of critical proteins, including Atg5 for autophagy, either through use of knock-out mouse primary neutrophils or knock-down in neutrophil cell lines, if shown to secrete CP, could be used to determine if these mechanisms are required for CP release.

S100 Proteins in Fungal Keratitis

25 S100 proteins have been identified in humans, all of which bind calcium and undergo conformational changes that are integral to their function in numerous physiological processes. In addition to S100A8/A9, S100A1, 2, 3, 5, 6,

7, 12, 15, 16, and S100B bind Zn. In addition S100A12, S100B and A13 also have the ability to bind Cu (288). It is therefore possible that S100 proteins other than CP may have a role in nutritional immunity during fungal infection.

S100A12 is abundant in neutrophils and can binding both Zn and Cu.

S100A12 was shown to deplete Zn from media and to inhibit Candida spp. growth in vitro, in a calcium dependent fashion (88). One recent study found that

S100A12 was produced by neutrophils to a comparable level with S100A8 during

H. pylori gastritis and that S100A12 could decrease bacterial growth in vitro

(289). Growth was restored by addition of Zn but not Cu. However, a role for Cu depletion cannot be excluded, particularly in the extracellular milieu which contains other Zn chelating proteins, thereby potentially increasing the likelihood that S100A12 would preferentially chelate Cu. Also, fungi may be more sensitive to Cu depletion than bacteria, since fungi contain a Cu/Zn-dependent SOD as a critical antioxidant mechanism. S100A12 was also shown to kill the filarial parasite Brugia malayi and this effect was not reversible by addition of Zn (290).

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Further studies of the anti-microbial activity of S100A12 are warranted but the lack of S100A12 in mice makes this a challenge. Recombinant S100A12 could be administered to mice during corneal infection, by subconjunctival injection, and fungal growth could be assessed by CFU and fluorescence. Additionally,

S100A12 could be knocked down in human neutrophil and macrophage cell lines, or alternatively overexpressed, and conidiocidal and anti-hyphal activity can be measured. This may need to be done in conjunction with knock-down of

CP due to the strong anti-microbial effect of CP. To determine the contribution of

Zn versus Cu binding, recombinant proteins with mutated binding residues for Zn or Cu could be generated, however since the Zn/Cu site is shared it is unclear whether mutations could be made to affect binding of only one metal.

Interestingly, S100A12 was recently described as a critical anti-mycobacterial protein of macrophages and showed a role in controlling growth of M. leprae

(291). Therefore, it would also be interesting to see whether S100A12 contributes to intracellular killing or inhibition of germination of A. fumigatus conidia in macrophages and/or neutrophils. Interestingly, Zn chelation by S100A12 was found to inhibit matrix metalloprotease (MMP) activity in atherosclerosis and

MMPs from neutrophils contribute to corneal damage in keratitis, so treatment with S100A12 could potentially have a dual role in limiting infection and preventing tissue destruction (292, 293).

The S100 family includes other Cu-binding proteins which may have a role in nutritional immunity. S100B was found to be expressed in corneal epithelial cell lines in response to A. fumigatus (294, 295). S100B expression could be

168 assessed by PCR or immunohistochemistry in corneas of mice infected with A. fumigatus. If detected the activity of recombinant S100B could be tested against fungi in vitro and in vivo. Knock-down of S100B in the cornea by shRNA injection could determine whether this protein has a role in infection.

S100A7, also known as psoriasin, and related protein S100A15 are produced mainly by keratinocytes and upregulated upon inflammation (296, 297).

Interestingly, S100A7 is also constitutively expressed by cornea, conjunctiva, lacrimal duct and corneal epithelial cell lines and was further increased by incubation of cell lines with pro-inflammatory cytokines and bacterial supernatants (298). Recently reduced S100A7 was shown to induce apoptosis in the fungi Tinea pedes and Aspergillus fumigatus, through intracellular Zn chelation (281). Furthermore, a recent study found that S100A7 and S100A5 were also expressed by circulating leukocytes, including neutrophils, in psoriasis

(299). S100A7/A15 expression should be evaluated in corneas of mice infected with A. fumigatus, for expression in both epithelium and myeloid cells.

Specifically the reduced form of S100A7 had fungicidal activity, so the presence of reduced vs. oxidized S100A7 would be important to differentiate. A. fumigatus infected mice could be treated with redS100A7 and fungal burden measured by

CFU or fluorescence.

S100A6 expression was identified in neutrophils in colorectal adenocarcinoma tissue by immunohistochemistry. S100A13 expression was found to be increased in neutrophil stimulated with LPS. Therefore, further

169 investigation of S100 protein expression in neutrophils in response to different types of infectious and inflammatory stimuli should be evaluated.

In addition to anti-microbial activity, many of the S100 proteins have reported roles in modulating inflammation, including neutrophil function. Many

S100 proteins have been found to activate TLR4 promote inflammatory responses including (198, 300, 301). Therefore, therapeutic use of S100 proteins in infection requires an understanding of how these proteins affect all aspects of the immune response. Blocking harmful pro-inflammatory effects of S100 proteins has been achieved using inhibitors such as quinolone-3-carboxamide which prevents S100A9 binding to TLR4 (302). Finally, as demonstrated with reduced vs. oxidized S100A7, S100 proteins can undergo various post- translational modifications including phosphorylation, oxidation/reduction and nitrosylation which can modulate their activity and will be an important consideration in understanding the biology of these proteins (303).

Novel Antimicrobial Peptides in Fungal Infection.

In addition to S100 proteins, numerous anti-microbial proteins (AMPs) have been identified but relatively unstudied in fungal infections. Recently an IL-

20 family cytokine, IL-26, was identified in Th17 cells and was found to have direct antimicrobial activity due to its unusually high charge of +18.1(304). While the authors did not demonstrate activity against C. albicans at high concentrations, this was using a less active recombinant IL-26 which was shown to be approximately 100-fold less potent than native IL-26 produced by T-cells

(304). The activity of natively produced IL-26 should be tested against a panel of

170 pathogenic fungi for activity in vitro. Expression of IL-26 in Th17 cells was found to depend on the transcription factor RORγT, therefore it would be interesting to investigate whether neutrophils can produce IL-26 in response to stimulation, because they have been shown to express IL-17 in a RORγT dependent fashion

(118). Th17 cells also have a role in later stages of A. fumigatus keratitis, therefore Th17-derived IL-26 may also contribute to anti-fungal immunity (26).

Another recently described AMP, AP-57/c10orf99, has a charge of +14 and demonstrated anti-microbial activity against Aspergillus niger, which suggests potential activity against other filamentous fungi (305). This protein was identified in the skin and mucosal tissues, and it would be interesting to investigate expression in infected corneas by PCR and immunohistochemistry.

Additionally, recombinant AP-57 could be produced and tested against other types of pathogenic fungi in vitro.

Interestingly, it is hypothesized that the GAG expressed by A. fumigatus may inhibit binding of some antimicrobial proteins to the fungal surface, therefore removing GAG as a therapeutic strategy could enhance innate defenses.

Similarly, C. albicans produces an extracellular mucin Msb2, which was shown to protect fungi against the AMPs LL-37, histatin-5, α-defensin 1 and β defensin

1(306). Preventing GAG production or removing GAG from the hyphal cell wall could allow for better activity of native or therapeutic AMPs. Essential components of the GAG synthetic pathway, including Uge3, Agd3 and the newly identified glycoside hydrolase Sph3 could be targeted with small-molecule inhibitors to block GAG production (307). Furthermore, exogenous glycoside

171 hydrolase enzymes that also degrade GAG could be used therapeutically, as a similar strategy has been employed to degrade exopolysaccharides in biofilm during bacterial infection (279). A. fumigatus strains deficient in Uge3, Agd3 or

Sph3 can be tested for in vitro susceptibility to different AMPs and therapeutic hydrolase preparations could be tested in vitro or in vivo during keratitis to determine whether they improve killing of fungi in the presence of neutrophils and

AMPs.

Copper Homeostasis in Aspergillus fumigatus virulence and Nutritional

Immunity

We, and others have described a critical role of iron and Zn in A. fumigatus infection. As discussed in Chapter 1, Cu is required for Cu/Zn-SOD activity, melanin production and iron acquisition in A. fumigatus. However, Cu is also toxic in high levels and the immune system targets many pathogens, particularly bacteria, with Cu excess as an antimicrobial strategy(68, 263, 308).

The importance of Cu excess versus scarcity is just beginning to be understood in the context of fungal infection. In systemic Candidiasis, for which the kidney is a major target organ, Cu levels in the kidney are elevated early in infection and later reduced. In turn, C. albicans switches its expression from Cu efflux and

Cu/Zn SOD to Cu importer and Mn SOD expression(308). Similarly, during infection with Cryptococcus, pulmonary Cu levels are high and the fungus down- regulates Cu transporters, whereas upon dissemination to the CNS, low Cu levels require Cu transporter expression for virulence (71). High affinity Cu transporters, CtrA2 and CtrC, were recently identified in A. fumigatus. Deletion of

172 these transporters was found to reduce SOD and laccase expression and increase susceptibility to oxidative stress, iron starvation and conidia killing by neutrophil-like HL-60 cells. No difference in survival was seen in mice infected intratracheally with WT or ∆ctrA2/C A. fumigatus. Due to tissue differences in Cu, we cannot rule out a role for CtrA2 and CtrC in corneal or invasive infections, particularly in the setting of intact immune responses, including ROS production and S100A12 secretion, which may contribute to enhanced killing of Cu-mutant

A. fumigatus.

We investigated the role for Cu transport in vitro and in a model of A. fumigatus keratitis. Although a ∆ctrA2/C strain of A. fumigatus exhibited impaired germination and growth in RPMI 1640 in the absence of added CuSO4, if conidia were grown in SDB + CuSO4 to hyphal stage and then media was replaced with

RPMI 1640, the mutant grew similarly to WT. We grew WT or ∆ctrA2/C conidia to hyphae and then neutrophils in RPMI +/- CuSO4 were added. Compared to WT,

∆ctrA2/C showed reduced growth in the presence of neutrophils (Figure 6.1A).

Addition of CuSO4 partly restored growth of ∆ctrA2/C although it was still reduced compared to WT. Due to the importance of iron and Zn in fungal growth we would not expect full rescue of growth with Cu alone and since Cu and iron uptake are linked, iron limitation by neutrophils may explain reduced growth in the mutant in the presence of sufficient Cu. Secondly, WT and ∆ctrA2/C conidia were injected intrastromally in WT C57BL/6 mice and CFU was measured 72 hrs after infection. ∆ctrA2/C infected mice exhibited lower CFU/cornea although not significant (Figure 6.1B). There is little data about the Cu content of the cornea

173 compared with other tissues, therefore it is possible that if Cu content is low the

∆ctrA2/C conidia may not germinate well in the cornea, thereby evading immune recognition. In this case un-germinated conidia would grow in vitro upon CFU calculation raising the observed CFU despite decreased virulence compared to

WT. Histology with GMS staining for fungi could elucidate whether un-germinated conidia or hyphae are present in infected eyes. In these preliminary studies we found decreased growth of ∆ctrA2/C A. fumigatus in the presence of neutrophils and during corneal infection, suggesting Cu uptake by pathogenic fungi may represent a new target. Further studies should evaluate the susceptibility of the the ∆ctrA2/C strain to specific neutrophil AMPs including lactoferrin, CP and

S100A12.

CR3 Signaling in Neutrophils

CR3 signaling is complex due to presence of multiple ligand binding sites and ligands as well as multiple activation states of the receptor. In the case of fungal infection we are interested in the CR3 signaling downstream of particulate

β glucan recognition. One comprehensive study of CR3 signaling in response to

β glucan shows increased phosphorylation in proteins involved in transcriptional control and mRNA processing and splicing, along with some changes in PRR signaling associated molecules including ERK2 and TRAF, however these studies were done in a leukemia cell line and used soluble rather than particulate

β glucan (309). Similarly, a soluble β glucan fragment was found to activate Syk and PI3K, but required dual ligation of both the canonical and lectin binding sites of CR3 (310). Unopsonized C. albicans yeast were shown to activate CR3

174 through Syk, PI3K and CARD9 and unopsonized A. fumigatus conidia similarly activate CR3 through PI3K, both which led to NADPH-oxidase independent inhibition of germination (114). Since we show evidence that CR3 activates the respiratory burst in neutrophils in response to curdlan and is required for NET formation and hyphal killing or growth inhibition, better understanding of the signaling pathways downstream of CR3 for non-oxidative fungal killing, oxidative burst and NET formation is imperative. A comprehensive analysis of CR3 signaling pathways can be done using a phosphoprotein array following stimulation with different fungal and β glucan preparations in the presence or absence of blocking antibodies. Furthermore, numerous stimuli including cytokines and chemokines, as well as extracellular matrix binding and transmigration can affect the expression and activation state of CR3, therefore binding and signaling of β glucan may be altered in an inflammatory environment that may not adequately be reflected in our in vitro environment (311). Peritoneal neutrophils vs. bone marrow neutrophils from mice may reflect neutrophils that have transmigrated in response to chemokines which may be more representative of physiologic responses. Signaling pathways identified by phosphoprotein array can be validated using specific inhibitors of signaling molecules and measuring NET formation, ROS production and fungal killing.

Dectin-2/Dectin-3 Signaling in NET formation

In addition to CR3 and Dectin-1, which were discussed in Chapter 1, neutrophils have the potential to express many other PRRs, including Dectin-2, under specific circumstances. Both IL-6/IL-23 and GM-CSF stimulation were

175 found to induce Dectin-2 expression on neutrophils and Dectin-2 signaling resulted in enhanced ROS production and A. fumigatus hyphal killing in vitro(117,

118). Dectin-3 was recently found to form heterodimers with Dectin-2 and enhance activation in response to fungal α mannans (312). Furthermore, Dectin-

2 and Dectin-3 expression on pDCs was found to enhance anti-fungal activity against A. fumigatus and C. neoformans, respectively, and Dectin-2 engagement induced formation of NET-like structures from pDCs, which contained calprotectin (119, 120). Dectin-3 expression on neutrophils has not been investigated. Dectin-2 and Dectin-3 expression should be assessed on neutrophils following stimulation with a panel of pro-inflammatory chemokines, cytokines, DAMPs and PAMPs to determine whether expression can be induced and under what conditions. A role for dectin-2 and dectin-3 in neutrophil function, including oxidative burst, phagocytosis, AMP release and NET formation can be assessed using the methods described in Chapters 2 and 4. Dectin-2/3 can be blocked with antibodies or knock-out mouse neutrophils can be compared to wild-type neutrophils. Furthermore, since CLECs can activate NFκB, a role for

Dectin-2/3 in cytokine production by neutrophils should also be assessed (313).

Cytokine Regulation of Neutrophil Anti-fungal Responses

In vitro studies using peripheral blood neutrophils may not fully recapitulate neutrophil activity in an inflammatory environment where numerous cytokines and chemokines are found in the milieu. For example a recent study found that neutrophils primed with chemoattractant gradients of fMLP or LTB4 were more effective at phagocytosis of A. fumigatus conidia and blocking hyphal

176 growth (314). Therefore, it is important to understand the effects of these molecules on neutrophil function in vivo.

GM-CSF is up-regulated during pulmonary infection with A. fumigatus. In a model of pulmonary challenge with A. fumigatus conidia, GM-CSF receptor- deficient mice had impaired fungal clearance and increased mortality. Despite normal neutrophil recruitment in these mice, neutrophils demonstrated impaired conidia uptake and killing as well as NADPH oxidase activation. Furthermore,

GM-CSF was shown to increase NET release by calcium crystals and fungal pathogen P. brasiliensis (272, 315). GM-CSF treatment was also found to reduce incidence of invasive fungal disease and infection-related mortality in hematopoietic stem cell transplant patients, compared with G-CSF treatment

(316).

Aspergillus as well as other fungal pathogens are known to activate the

NLRP3 and AIM2 inflammasomes which drives IL-1β and IL-18 production, both of which were protective in a pulmonary aspergillosis model (317-319).

Interestingly, it was recently reported that polymorphisms in IL-1β resulting in decreased cytokine production were associated with invasive fungal infections in solid-organ transplant recipients (320). IL-1β mRNA was more than 1000-fold up- regulated in corneas of patients with Aspergillus or Fusarium corneal infection and IL-1R-/- mice had increased fungal burden in a keratitis model, although this may be attributed to delayed inflammatory cell recruitment. However, these cytokines may also have a range of effects on neutrophil anti-fungal activity.

Indeed it was found that IL-1R-/- bone marrow neutrophils had impaired anti-

177 hyphal activity against A. fumigatus compared to WT neutrophils (321). That study also found an essential role for IL-1α in neutrophil recruitment during pulmonary aspergillosis, although a direct effect of IL-1α on neutrophil function was not investigated. Furthermore, another IL-1 family cytokine, IL-33 which is released from dying cells, was found to increase CR3 expression, phagocytosis,

ROS production and therefore killing of C. albicans yeast by neutrophils and IL-

33 was protective in an invasive candidiasis model (311).

The levels of GM-CSF, IL-1 family cytokines and other chemokines and cytokines should be measured in lysates of infected corneas at different time points using a cytokine array. Cytokines that are significantly elevated during infection can be used to stimulate neutrophils in vitro and assess responses including NET formation, oxidative burst, phagocytosis of conidia and hyphal killing. We isolated peripheral blood neutrophils by ficoll gradient and treated them with 20 ng/ml of either GM-CSF or IL-1β. We then incubated these neutrophils with A. fumigatus hyphae in vitro and measured fungal mass after 18 hrs using Calcofluor white chitin staining. We found that priming of neutrophils with either GM-CSF or IL-1 β increased neutrophil killing of hyphae (Figure 6.2A,

B). This effect was reproducible in three human donors, however in future studies using human neutrophils, expression of cytokine receptors should be evaluated for each donor because differences in expression may underlie variable responses. It would also be interesting to measure receptor expression by PCR and flow cytometry, including CR3, CLEC and TLR expression, following cytokine stimulation, since these receptors can be induced upon stimulation and

178 have significant effects on neutrophil activity. Furthermore, the necessity of these cytokines can be tested in vivo by using knock-out mouse strains or treating infected mice with recombinant cytokine and measuring fungal burden and corneal disease. An understanding of how inflammatory mediators affect specific neutrophil responses will allow for a fine-tuning of immune responses to maximize anti-microbial activity and minimizing tissue damage. In addition to testing the effects of IL-1 cytokines on neutrophils, it would also be interesting to investigate the necessity of neutrophil-specific inflammasome activation and IL-

1β production during fungal infection, since neutrophils were recently found to be an important source of IL-1β in other infections (322-324).

Antibodies, Pentraxins and Fc Receptors in Fungal Infection

Human and mouse neutrophils express an array of Fc receptors, which bind immunoglobulins. Neutrophils constitutively express the low-affinity FcγRIIA and FcγRIIIb (human) or FcγRIII and FcγIV (mouse), which bind immune complexes to activate neutrophils. Furthermore, activated neutrophils express the high-affinity FcRγI and the inhibitory FcγRIIb as well as FcαR and FcεR.

FcRs undergo phosphorylation at ITAM motifs which then activates Syk, PLCγ2 and PI3K, similar to CR3, leading to ROS production and Ca2+ influx(325).

Furthermore, there is cross-talk between FcRs and CR3 and it was shown that engaging FcγRIIA on monocytes could enhance CR3 mediated phagocytosis, while FcγRI inhibited it(326). Additionally, CR3, FcγRIII and FcγRII were found to cause synergistic activation of the respiratory burst in human neutrophils (327).

In the autoimmune diseases systemic lupus erythematosus (SLE) and

179 rheumatoid arthritis it was demonstrated that immune complexes could stimulate

NETosis through activation of the FcγRIII and CR3, which act as signaling partners. Blocking FcγRIII, CD18 and CD11b with antibodies inhibited NETosis.

This process also depended on ROS production and activation of Src, Syk, Akt,

Erk1/2, PI3K and p38, which shares similarities to findings from other NETs studies (See Chapter 1) (328). Similarly another study found that crosslinking

FcγRIIIb, but not FCγRIIa, induced NETosis and was dependent on Syk, PKC and MEK/ERK signaling and ROS production (329). In a mouse model of collagen induced arthritis, NETosis was dependent on FcγR signaling and Syk activation. In contrast, another study found that FcγRIIA mediated NETosis in response to soluble immune complexes. (330). Finally, Aleyd and colleagues showed that IgA opsonization of bacteria enhanced NETosis and depended on

FcαRI and ROS production (331). Interestingly in a model of S. pneumonia otitis media, it was demonstrated that B-cell deficient mice had reduced NET formation but also reduced bacterial burden, suggesting that inflammation and NET formation are potentially detrimental during infection (332). Overall, these studies indicate a significant role of antibodies and Fc-receptor activation on neutrophil function.

Although antibody responses have been detected in fungal infection, little work has been done to identify a role for FcRs and antibodies in filamentous fungal disease. In the setting of serum opsonization in vitro, FcγRs are required for killing both C. albicans conidia and A. fumigatus hyphae (114). The mechanism of killing was found to depend on ROS production in both studies.

180

We evaluated the ability of human serum opsonization to enhance killing of A. fumigatus hyphae in vitro. We incubated human neutrophils with A. fumigatus hyphae opsonized in 10% human serum or heat-inactivated human serum and found similarly, that in the presence of human serum, killing and/or growth inhibition of A. fumigatus hyphal growth was increased in the presence of serum

(Figure 6.3A). Heat-inactivated serum performed equally to non-inactivated serum, indicating that complement activation was not required. However, one study describes a role for the classical complement pathway in recognition, phagocytosis and intracellular killing of A. fumigatus (333). In contrast to the study previously described, this work found no role for antibodies alone in A. fumigatus uptake. Furthermore, C5a deficient mice were more susceptible to A

.fumigatus infection, therefore a role for complement also warrants further investigation.

The requirement for antibodies or FcRs in fungal keratitis could be assessed using antibody or B-cell deficient mice and FcR knock-out mice. Some studies have used the common Fcγ chain knockout mice, which presents a problem given that this adapter protein is also used for signaling by Dectin-2 and

Dectin-3. Mice may need to be sensitized to fungi prior to infection to recapitulate normal humoral responses developed in humans through environmental exposure to fungal antigens. Furthermore a role of antibodies in

NET formation could be assessed by measuring NET release in the presence or absence of serum and blocking antibodies to Fc receptors. Since the cornea is a

181 mucosal tissue it would also be interesting to look at a role for IgA in fungal keratitis.

In addition to antibodies, neutrophils contain a molecule called pentraxin

3, which is an “innate antibody” that can interact with complement and FcRs.

PTX3 was first shown to be elevated in plasma of patients with invasive aspergillosis and PTX3-/- mice were more susceptible to A. fumigatus pulmonary infection (334). Although initial studies identified macrophages and dendritic cells as the key source of PTX3 it was later demonstrated that neutrophils contain

PTX3 in secondary and tertiary granules and release it into NETs and that PTX3-

/- neutrophils show impaired A. fumigatus phagocytosis and conidial killing (335).

The mechanism of PTX3 activity on neutrophils was shown to involved

CR3/FcγR cross-talk. FcγR engagement by PTX3 resulted in activation of CR3 and increased phagocytosis of complement-opsonized A. fumigatus conidia.

Interestingly, A. fumigatus secreted proteases were found to mediate degradation of PTX3, a virulence factor that was implicated in chronic pulmonary infections of cystic fibrosis patients (336). It would be interesting to investigate a role for PTX3 in A. fumigatus keratitis and extracellular hyphal killing of the fungus, by infecting WT vs. PTX3-/- mice and evaluating fungal burden. Since

PTX3 can activate CR3 it would be interesting to determine if this activation also led to enhanced binding to β glucan and ROS production and/or NET formation.

This could be tested by comparing WT vs PTX3-/- neutrophils or by adding exogenous PTX3 to neutrophils and measuring ROS production and NET formation in response to β glucan in the presence or absence of blocking

182 antibodies to CR3 and FcgRs. If PTX3 is protective, recombinant PTX3 could be used as a therapy for keratitis, possibly in conjunction with inhibitors of A. fumigatus proteases to prevent PTX3 degradation.

Modulating NET formation: Implications for Therapy

Neutrophils have both a protective and damaging role during infection and inflammation which is why an understanding of how individual effector functions contribute to protection vs. damage is imperative, so that responses can be modulated to maximize protection from infection and reduce tissue damage.

If NET formation proves to be essential for control of A. fumigatus infection in vivo, NETosis could be promoted by increasing immune system exposure to A. fumigatus antigens, such as β glucan, that stimulate NETosis. Increasing cell wall

β glucan by treatment with the anti-fungal echinocandin drugs increased neutrophil recognition and killing of of Aspergillus in vitro(280). Preventing GAG production or removing GAG from the hyphal cell wall, as discussed earlier in this chapter, could also increase β glucan exposure to promote NET formation.

If NET formation is not protective in fungal infection and promotes harmful inflammation, blocking NETosis could be an important therapeutic intervention.

While preventing neutrophil migration altogether may not be ideal, due to the essential role of these cells in limiting fungal growth, specifically inhibiting NETs while preserving other activity, including phagocytosis and calprotectin or AMP secretion could be beneficial. The PAD4 inhibitors discussed in Chapter 4 have potential as NET inhibitors, although we only observed partial inhibition of NET

183 formation in vitro. These inhibitors can be tested in vivo in our corneal infection model for effects on fungal killing and corneal damage. Syk activation is likely involved in NET formation downstream of CR3 signaling and numerous studies have identified chemical inhibitors of Syk with biological activity that could potentially be used as anti-inflammatory therapies (337, 338). Syk inhibitors could be tested for their ability to inhibit NETosis in vitro and in vivo. Because

Syk has widespread involvement in immune cell signaling it is possible that Syk inhibition would disrupt other neutrophil functions that may be beneficial for clearing infection.

Several inhibitory receptors have been implicated in regulating NETosis and may represent targets for therapies to reduce the damaging effects of NETs in infection or autoimmune disease. Engagement of Siglec-9, a sialic acid binding receptor, was shown to inhibit NET formation in the presence of P. aeruginosa with attached surface sialoglycoproteins(339). Another inhibitory receptor, signal inhibitory receptor on leukocytes-1 (SIRL-1) was shown to inhibit NET formation induced by both S. aureus and autoantibodies from SLE serum. Interestingly,

SIRL-1 suppressed Fc-receptor, but not S. aureus-induced ROS production and bacterial killing was preserved upon SIRL-1 engagement (340, 341). Finally, another ITIM-containing receptor, leukocyte associated Ig-like receptor 1 (LAIR-

1), was identified on neutrophils activated with TNFα, GM-CSF or fMLP (342). It is possible that LAIR-1 may have a role in regulating neutrophil activation and

NETosis. Antibodies or molecules that activate Siglec-9, SIRL-1 or LAIR-1 could be developed for therapeutic use in infection and inflammatory diseases.

184

Furthermore, an understanding of signaling pathways downstream of these receptors in neutrophils could provide more targets of anti-inflammatory therapies.

Interestingly, the ligand for LAIR-1 is collagen, which could mean that the cornea is a naturally inhibitory environment for neutrophils and other immune cells and that inhibiting LAIR-1 signaling could actually improve clearance of infection if NETs show a protective effect in vivo (343). LAIR-1-collagen interaction can be inhibited by either a soluble form of LAIR-1 or related receptor

LAIR-2, which can be produced as recombinant proteins for therapeutic use, or by non-stimulatory monoclonal antibodies (344, 345).

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Concluding Remarks

In this work we have furthered our understanding of how neutrophils control fungal infections, by demonstrating a role for calprotectin-mediated Zn and Mn chelation. We investigated NET formation as a means of CP release and identified a CR3-ROS-PAD4 dependent mechanism of NET release in response to β-Glucan. Furthermore, we have found that fungal Zn uptake is required for virulence during corneal infection. In an effort to disrupt fungal Zn homeostasis we found that the anti-malarial agent atovaquone inhibits fungal growth through disrupted metal homeostasis and mitochondrial function. These studies open up new avenues for treatment of fungal infection, in particular using the principles of nutritional immunity. An urgent need for new and effective anti-fungal agents means that future studies should focus on both modulating immune responses for optimal anti-microbial activity without detrimental inflammation and inhibiting fungal virulence factors that promote infection.

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Neutrophils

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