Neutrophils versus Pathogenic Fungi through the magnifying glass of nutritional immunity

Maria Joanna Niemiec

Doctoral thesis

Department of Molecular Biology

Molecular Infection Medicine Sweden (MIMS)

Umeå University

Umeå, 2015

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-261-1 ISSN: 0346-6612 Elektronisk version tillgänglig på http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden 2015

To all those who were always convinced this moment would come.

i

Table of Contents

Table of Contents i Publications included in this thesis iii Publications not included in this thesis iv Abstract v Clarifications vii Introduction & Background 1

1. Neutrophils 1 1.1 The innate immune system - overview 1 1.2 Professional phagocytes 3 1.3 Neutrophils – from bone marrow to the site of infection 5 1.4 NETosis – neutrophil functionality post mortem 10 1.5 Neutrophil disorders 12 2. Human fungal pathogens 14 2.1 The fungal threat 14 2.2 The yeast Candida albicans and other Candida ssp. 15 2.3 The mold Aspergillus nidulans and other Aspergillus ssp. 16 2.4 Fungal defense strategies against neutrophil killing 17 2.5 Impact of fungal morphology during pathogenesis 19 3. Nutritional immunity 22 3.1 The third branch of the human immune system – major concepts 22 3.2 Zinc during fungal infections 23 3.3 Iron, copper, and manganese during fungal infections 25

Methodological remarks 28 Neutrophil isolation 28 Fungal strains 28 Metal consistency 28 Synchrotron radiation X-ray fluorescence 29 In vitro infections for RNA-sequencing 29

Aims 31 Results & Discussion 33

Paper I 33 NET calprotectin is an effector during A. nidulans inhibition 33 NET inhibition is reversible by Zn supplementation 34 Paper II 36 NETosis reduces Zn availability in the surrounding 36 NETs contain Fe, but not Zn 37 Neutrophils are exceptionally high in Fe 37 Neutrophil nucleus, granules and void vacuoles show characteristic element

profiles 39

ii

Paper III 42 Neutrophil antimicrobial effectors are not induced by C. albicans 42 Neutrophil transcriptional response to C. albicans contains few metal retaining

genes 44 C. albicans response to neutrophils and NETs includes metal acquisition genes 46

Conclusions & Outlook 49 Acknowledgements 51 References 53

iii

Publications included in this thesis

Paper I

Restoration of anti-Aspergillus defense by neutrophil extracellular traps in

human chronic granulomatous disease after gene therapy is calprotectin-

dependent.

Bianchi M* & Niemiec MJ*, Siler U, Urban CF and Reichenbach J.

Journal of Allergy and Clinical Immunology, 2011.

* equal contribution

Paper II

Trace element landscape of resting and activated human neutrophils on the

sub-micrometer level.

Niemiec MJ, De Samber B, Garrevoet J, Vergucht E, Vekemans B, De

Rycke R, Björn E, Sandblad L, Wellenreuther G, Falkenberg G, Cloetens P,

Vincze L & Urban CF.

Metallomics, 2015.

Paper III

RNA-Seq transcription profile of the neutrophil – Candida albicans in vitro

interaction.

Niemiec MJ, Grumaz C, Mills I, Ermert D, Desel C, Sohn K & Urban CF.

Manuscript.

Those articles will be referred to in this thesis as paper I, II, and III.

iv

Publications not included in this thesis

Paper IV

Candida albicans escapes mouse neutrophils.

Ermert D* & Niemiec MJ*, Röhm M, Glenthøj A, Borregaard N, Urban CF.

Journal of Leukocyte Biology, 2013.

* equal contributions

Paper V

Malaria-enhanced neutrophil-dependent clearance of S. pneumoniae in an

in vivo co-infection model.

Nelson M, Niemiec MJ, Fahlgren A, Nahrevanian S, Urban CF, Bergström

S & Normark J.

Manuscript.

v

Abstract

Neutrophils are among the first white blood cells recruited to the site of in-

fection once microbial pathogens enter the host organism. At site, they per-

form a well-orchestrated chain of processes that aims to kill the microbial

invader. Most prominent, neutrophils engulf microbes to inactivate them

intracellularly, a process called phagocytosis. Alternatively, neutrophils can

release neutrophil extracellular traps (NETs). NETs consist of chromatin

decorated with antimicrobial effector proteins – a structure that can entan-

gle bacteria and fungi. Neutrophils are crucial during fungal infections. This

is reflected in the increased risk of fungal infections resulting of neutropenia.

The concept of nutritional immunity describes every infection as a battle for

resources. Those are mostly metal trace elements.

While neutrophils were seen for a long time as powerful, but “mindless”,

killers with a limited set of actions and no transcriptional capacity, this view

is in the flux.

In the presented thesis, it was my goal to gain new insights into the interplay

of neutrophils and fungi – with special attention to metal nutritional aspects.

We compared human neutrophils lacking the ability to undergo NETosis,

due to a non-functional NADPH complex, and neutrophils from the same

person that were “cured” by gene therapy. We investigated those NETs and

found that their inhibitory activity towards the mold Aspergillus nidulans

depends on calprotectin, a known zinc-chelator.

Considering the high influx of neutrophils, we wanted to unravel the neutro-

phils’ contribution to the metal milieu at the site of infection and trace ele-

ment changes resulting from NETosis. By combining synchrotron radiation

XRF and ICP-MS, we analyzed the neutrophil metallome and the spatial

element distribution in activated neutrophils and NETs. Most strikingly, we

found neutrophils to be exceptionally high in Fe and the process of NETosis

to be reducing available Zn in the surrounding and the early phagosome,

possibly by the formation of Zn-rich vesicles.

vi

Using RNA-sequencing, we analyzed the interplay of the Candida albicans

and neutrophils face-to-face. We dissected their transcriptional profile and

revealed a complex response in neutrophils that include cytokine induction

and cellular rearrangement. We further were the firsts to explore the tran-

scriptional response of C. albicans to NETs. Our data indicates a distinct

response compared to intact neutrophils or other known stress triggers.

Metal homeostasis was affected in Candida in both set-ups.

In summary, this thesis provides new insights into the interaction of fungal

pathogens with neutrophils and emphasizes the impact of nutritional aspects

on this interplay. A deeper understanding of the nutritional immunity during

fungal infection might open up new strategies to tackle fungal infections – a

growing threat worldwide.

vii

Clarifications

CAT catalase CD cluster of differentiation CGD chronic granulomatous disease CLR C-type lectin receptor CR complement receptor Ctr Cu transporter DC dendritic cell DEG differentially expressed gene ERK extracellular signal-regulated kinases, = MAPK ET extracellular traps FcγR Fc (fragment crystallizable) γ receptor Fet ferrous transporter FPR formylated peptide receptor Fre ferric reductase Ftr Fe transporter G-CSF granulocyte-colony stimulating factor GPX glutathione peroxidase GUT gastrointestinally induced transition ICAM intracellular adhesion molecule ICP-MS inductively coupled plasma mass spectrometry Ig immunoglobulin IL interleukin IRAK interleukin-1 receptor-associated kinase LAD leukocyte adhesion deficiency MAC membrane attack complex MBL mannan-binding lectin MEK = MAPKK or MAP2K, Mitogen-activated protein kinase kinase MMP matrix metalloproteinase MPO myeloperoxidase MR mannose receptor MYD88 myeloid differentiation primary response gene 88 NADPH nicotinamide adenine dinucleotide phosphate NETs neutrophil extracellular traps NLR nod-like receptor NLRP3 NOD-like receptor family, pyrin domain containing 3

NO nitric oxide p.i. post infection PAD peptidylarginine deiminases PMN polymorphonuclear cell PSGL-1 P-selectin glycoprotein ligand-1 RLH RIG-like helicases RNS reactive nitrogen species ROS reactive oxygen species SCN severe congenital neutropenia SOD superoxide dismutase SR-XRF synchrotron radiation X-ray fluorescence TLR toll-like receptor TNF tumor necrosis factor WHIM Warts, Hypogammaglobulinemia, Infections, and Myelokathexis

syndrome ZnT Zn transporter Zrt Zn-regulated transporter

viii

1

Introduction & Background

1. Neutrophils

Neutrophils are the most abundant white blood cells. With numbers up to

1011 generated every day, they constitute 60-80 % of all nucleus-containing

blood cells. Neutrophils mature in the bone marrow under the control of G-

CSF and belong to the myeloid lineage of hematopoietic cells. After leaving

the bone marrow to enter circulation, 95 % of mature neutrophils are post-

mitotic. As professional phagocytes, they are assigned to be part of the innate

immune system [1].

1.1 The innate immune system - overview

The human immune system is traditionally divided into two major branches:

the adaptive and the innate immune system. As indicated by their names, the

innate immune system is present from the very beginning of life and needs

no further training, while the adaptive immune system can learn and, very

importantly, memorize. Due to these properties, the innate immune re-

sponse is fast, but rather unspecific, and the adaptive immune system is

slower, but pathogen-specific. While the innate immune response can occur

immediately, the adaptive immune response takes between 3-4 days to be

fully engaged [2]. Both branches of the immune system communicate with

each other, and new findings of recent years indicate that the strict separa-

tion of the two is not entirely correct [3]. For instance, activated innate im-

mune cells produce cytokines that are recognized by cells from the adaptive

immune system. In addition, innate immune cells present microbial antigens

to T cells [2]. On top of this cross-talk between the branches, trainable as-

pects of the innate immune system in organisms lacking an adaptive im-

mune system (plants, invertebrates), but also in mammals, are observed and

debated [3].

The first line of defense of the human body against invaders are mechanical

and chemical barriers, composed and produced by skin, hair, nail, and mu-

2

cosal tissues. While hair and nail, consisting of dead cells, exclusively pro-

vide mechanical resistance, skin and mucosal tissues have functions way

beyond. Skin and mucosal cells as well as glands contribute to a chemical

barrier. Well-known examples are the very low pH in the stomach, the en-

zyme lysozyme in tears or fatty acids on the skin. Epithelial cells are so-

called non-professional immune cells and provide dual function. They are

tightly connected, and therefore provide mechanical resistance. Most im-

portantly, they sense microbial invaders and cell damage leading to secretion

of peptides and proteins. Those weaken microbes or recruit professional

immune cells, like neutrophils, to the site of infection. Finally, the normal

flora on body surfaces contributes to this first hurdle for invading pathogens

[2, 4].

In areas below skin or mucosa, tissue-residing macrophages patrol to fend

off invading microbes. Upon encounter of an intruder, they will not only

initiate the killing of the pathogen, but also release cytokines to promote

inflammation and chemokines to recruit neutrophils and monocytes [2]. In

addition to macrophages, tissue-residing mast cells contribute to inflamma-

tion. Upon activation, they secrete pro-inflammatory mediators that are

stored ready-made in granules or synthesized on demand. Archetypical mast

cell effectors are histamine, heparin, or TNF-α [2]. Inflammation is charac-

terized by four hallmarks: pain, redness, heat, and swelling. Heat and red-

ness are caused by an increased blood flow. Swelling and pain result from

increased permeability of the blood vessels to allow white blood cells to enter

the tissue – accompanied by influx of fluid and proteins. The deliberate clot-

ting of local blood vessels, to reduce the chance of a pathogen spreading sys-

temically, contributes to the phenotype of inflammation [2].

Other tissue-residing immune cells are dendritic cells. Dendritic cells are

often assigned to be part of the innate immune system, but at the same time

known to be crucial for the cross-talk with the adaptive immune system. Like

macrophages, they are antigen-presenting cells.

In contrast, a number of other innate immune cells are present mostly in

circulation. They are basophils, eosinophils, and NK cells. Basophils and

eosinophils are closely related to neutrophils, since they all contain charac-

3

teristic granules and stem from the myeloid lineage in the bone marrow. NK

cells originate from the lymphoid lineage and attack host cells that present

markers of cancer or viral infection. Together with DCs, NK cells act at the

interface of innate and adaptive immune system. Activated by certain patho-

gens, both cells produce cytokines that cause proliferation of CD4 T cells [2,

5].

The complement system supports the function of the cell-mediated immune

system. It consists of 20-30 different proteins synthesized mostly by hepato-

cytes. Three complement cascades are known: the classical, the alternative

pathway and the lectin pathways. While activated differently, they all have

three major functions: 1) opsonization and clumping of foreign material to

enhance phagocytosis; 2) induction of chemotaxis to the site of infection; 3)

lysis of microbes by the membrane attack complex (MAC) [2, 4]. Due to

chemotactic and phagocytosis-inducing capacities, the complement system is

tightly connected to phagocytes, and therefore to neutrophil functionality.

1.2 Professional phagocytes

Phagocytes are classified into professional and non-professional [6]. This

classification remains debatable though, as it is solely based on phagocytosis

efficacy.

Uptake of microbes is a crucial weapon in the armory of the innate immune

system. During internalization, microbes are engulfed and enclosed by plas-

ma membrane until a vesicle segregates, which is then called phagosome.

This phagosome fuses with the lysosomal or granular vesicles to form the

phagolysosome – a very hostile intracellular compartment in which the mi-

crobe is exposed to low pH, nutrient starvation, lytic enzymes and reactive

oxygen species (ROS) [7].

In addition to neutrophils and monocytes/macrophages, DCs and mast cells

are also sometimes considered professional phagocytes. Since discussing

these two cell types in detail lies beyond the scope of this thesis, the term

‘professional phagocyte’ will in the following only refer to the neutrophils

and monocytes/macrophages.

4

Like neutrophils, monocytes are generated during myelopoesis in the bone

marrow and from there released into the blood stream. Around 10 % of all

white blood cells are in fact monocytes [8]. Unlike neutrophils, monocytes

can leave the blood stream without an acute infection signal. Monocytes that

enter the tissue mature subsequently into macrophages. Remarkably, the

type of tissue and the type of stimulation determine the ‘activation pheno-

type’ of these macrophages. In general, macrophages can be either M1 or M2

macrophages, which corresponds to classically or alternatively activated,

respectively. M1 macrophages are described as the ‘pro-inflammatory’ type,

as they produce nitric oxide more effectively and secrete large amounts of

pro-inflammatory cytokines and chemokines. M2 macrophages are the ‘in-

flammation-limiting’ or ‘wound-healing’ type. They secrete extracellular

matrix proteins and express cytosolic arginase. Examples for tissue-specific

macrophages are the alveolar macrophages of the lung or microglia of the

brain [8].

Even though both cells types are often used interchangeably, neutrophils and

macrophages show some important differences – not only in the interaction

with other immune cells, but also at their core function: phagocytosis. While

both cell types actively engulf microbes, the phagolysosome maturation and

the processes inside show some distinct features [8, 9].

Both cell types take up foreign material, e.g. microbes, in the same two ways.

Either by FcγR-mediated extension of the cell as a pseudopodium to take up

IgG-opsonized particles, or by complement receptor-mediated uptake that is

described as a ‘sinking into the cell’ to internalize complement-opsonized

particles [10]. The mechanisms are very similar, but the speed of internaliza-

tion is different. IgG-opsonized targets were described to be taken up faster

by neutrophils than by macrophages [9].

In macrophages, the phagosome fuses with endosomes and lysosomes to

mature gradually to the phagolysosome [11]. In neutrophils, neutrophil-

specific granules rapidly fuse with the phagosome [12]. It therefore seems to

be more suitable to refer to a ‘mature phagosome’ to describe the hostile,

granule-infused phagosome in neutrophils, instead of calling it a

phagolysosome as in macrophages. Due to their distinct formation process,

5

the proteins in the phagolysosome and the ‘mature phagosome’ are not the

identical.

And neither is the pH. While the pH in the ‘mature phagosome’ stays more

or less at around neutral pH 6-7, the phagolysosomal pH decreases down to

pH 4-5 during maturation.

The last major difference of neutrophil and macrophage phagocytosis is the

oxidative burst. The NADPH complex, assembled in activated neutrophils

and macrophages, is present in the cytoplasmic membrane and generates

superoxide towards the extracellular milieu, and accordingly in the

phagosome towards the microbe inside. Superoxide (O2-) is converted to

hydrogen peroxide (H2O2) by superoxide dismutases (SODs). Hydrogen per-

oxide can react with present cations via the Fenton reaction to create hy-

droxyl radicals (OH˙). The oxidative burst in neutrophils is overall stronger

than in macrophages, because neutrophils can recruit more NADPH to the

‘mature phagosome’ than dictated by the pure membrane internalization

event. Further, neutrophils possess the enzyme myeloperoxidase (MPO),

which is absent in macrophages [13]. MPO converts H2O2 to hypochlorous

acid (HOCl), one of the most reactive oxidative agents. Both professional

phagocytes, neutrophils and macrophages, produce reactive nitrogen species

(RNS). First, the production of nitric oxide (NO˙) is catalyzed by NO syn-

thases that convert L-arginine to L-citrulline during the reaction. NO˙ can

subsequently react with O2- to generate harmful reactive nitric oxide species

[14]. Additionally, NO˙ possesses immunomodulatory functions [8]. Even

though neutrophils are capable of producing NO˙ and subsequently RNS,

the role of RNS in neutrophils is less well understood than in macrophages.

1.3 Neutrophils – from bone marrow to the site of infection

Neutrophils are remarkable immune cells that were for a long time under-

studied. Traditionally perceived as strong, but simple, killers lacking exten-

sive regulation schemes, an increasing body of seminal studies has consider-

ably changed the general view of these cells.

6

Neutrophils mature in the bone marrow during a process called

granulopoesis to be released into the blood stream [1].

Large numbers are produced and removed every day. Neutrophil life span is

still a matter of debate. Generally described to be between 6 and 8 h, recent

studies have suggested that neutrophils could live up to 5-6 days [15-17].

Nevertheless, the lifetime is short, compared to other immune cells.

In the body, neutrophils reside either in circulation or in so-called marginat-

ed pools. In addition to the bone marrow itself, the liver, the spleen, and the

lung were described as locations to contain mature neutrophils that can be

quickly released, if so required [1].

Neutrophils, hence potentially harmful, do not leave the blood stream and do

not migrate into the surrounding tissue randomly. Upon microbial invasion

or tissue damage, the affected epithelial or tissue cells, as well as residing

immune cells, will release cytokines like TNF-α or IL-1β that induce the ex-

pression of P-selectin, E-selectin or integrins on the luminal surface of the

blood vessel. Constitutively expressed ligands for those on the neutrophil,

namely PSGL-1 and L-selectin, will bind to P- and E-selectin. Hence, the

neutrophil is captured and rolls on the endothelial surface. Firm adhesion is

mediated by neutrophil integrins and ICAM-1 and ICAM-2 on endothelial

cells. A complex chain of events then allows the paracellular or even

transcellular passaging of the neutrophils through the endothelial lining, a

process known as extravasation.

In the tissue underneath, the neutrophil is confronted with a number of new

stimuli. Chemokines or chemotactic factors are sensed by neutrophils, which

are able to migrate towards the infection site along a concentration gradient.

Typical chemotactically active factors are IL-8, bacterial formylated peptides,

or Candida albicans β-glucan [1, 15, 18]. Local G-CSF triggers cytokine pro-

duction, while pro-inflammatory cytokines and pathogen-associated molecu-

lar patterns (PAMPs) can induce ‘priming’. Neutrophils present only few

pattern recognition receptors (PRRs) on their surface per se, although in

tissue, ‘primed’ neutrophils increase these numbers. This conversion is cru-

cial for precise microbial location in infected tissue [19].

7

Once at the site of infection, neutrophils recognize and distinguish the mi-

crobial invader by the binding of PAMPs to their PRRs. Neutrophil-attached

PPRs include toll-like receptors (TLR), C-type lectin receptors (CLR), NOD-

like receptors (NLR), RIG-like helicases (RLH) as well as formylated peptide

receptors (FPR) and the yet uncategorized TREM1[20]. Neutrophils also

produce secreted PRRs. From all known fungal-sensing PRRs, approximate-

ly half are present on neutrophils (Tab. 1). These are the surface TLRs TLR2

and TLR4, the intracellular TLR9, the CLRs Dectin-1, Mincle and Galectin-3,

the NOD-like receptor NLRP3, the FcγR and the complement receptor 3 [21-

29].

The recognition of a microbe immediately causes a chain of events aimed at

killing the pathogen. Most frequently, neutrophils will attempt to engulf the

invader and inactivate it intracellularly by toxication in the ‘mature

phagosome’ (see 1.2). Executing effectors are primarily ROS and antimicro-

bial peptides and proteins previously stored in granules; reduction of the pH

seems to be less important in neutrophils [7].

Granules are filled with hundreds of different proteins and are traditionally

subdivided into 3+1 classes. They vary in size, content and even origin [30,

31]. Instead of understanding granules as clearly distinguishable, “granules

can be viewed as a continuum where subsets are defined based on a selection

of marker proteins” [19]. The three archetypical granule classes are prima-

ry/azurophil, secondary/specific, and tertiary/gelatinase granules. Their

marker proteins are MPO, lactoferrin, and gelatinase MMP9, respectively.

Only during granulocyte differentiation in the bone marrow, granules are

generated and filled – a process referred to as the ‘first transcriptional burst’.

The granule content varies according to the ‘targeting-by-timing’ model,

since not all granules accrue at the same time. Interestingly, the granules

formed last, the tertiary granules, are the ones to be mobilized first [18, 19].

Degranulation is directed towards the phagosome, but also to the extracellu-

lar surrounding.

8

Table 1: PRRs involved in fungal immune recognition

PRRs involved in the recognition of fungal pathogens are listed together with their ligands and related to their presence in human neutrophils [20-29].

PRR class PRR PAMP

Expressed /

functional in

neutrophils

TLR

TLR2* Phospholipomannan yes

TLR3 RNA (Aspergillus) no

TLR4 Mannan yes

TLR7 RNA (Candida ssp.) not in humans

TLR9 CpG-oligodeoxy-nucleotides yes

CLR

MR N-linked-mannan no

Dectin-1 (CLEC7A) β-1,3-Glucan yes

Dectin-2* (CLEC6A) High-mannan structures

+ α-mannan unclear

DC-SIGN/SIGNR1 Mannan no

Mincle** (CLEC4E) Unknown yes

Galectin-3 (soluble) β-mannosides (α?) yes, in humans

CR3 β-glucan yes

MBL (soluble) Mannan no

Scavenger

receptor

CD36 Unknown no

SCARF Unknown no

NLR NLRP3 unknown yes

* With or without TLR1 or TLR6, ** with FcγR

Secretory vesicles are classified as granules, too. In simple terms, secretory

vesicles are subcellular compartments storing a number of receptors that are

quickly translocated to the plasma membrane during ‘priming’ [19].

Largely underestimated, neutrophils participate in shaping the inflammatory

response at the site of infection and prepare the ground for immune cells

that are recruited at later time points – from both the innate and adaptive

immune system.

9

Figure 1: Major antimicrobial processes performed by neutrophils during innate immune defense Neutrophils enter the site of infection following a chemokine gradient. Upon micro-bial encounter, they engulf and intracellularly toxify the microbe, secrete antimicro-bial proteins into the surrounding, or release cytokines to recruit other immune cells. Upper left: chemotaxis, upper right: phagocytosis, lower left: degranulation, lower right: cytokine secretion.

Interaction occurs with macrophages, DCs, NK cells, and T cells via the re-

lease of cytokines and chemokines, which are expressed on demand, not

stored like most antimicrobial effectors [1]. This protein expression event is

sometimes referred to as the ‘second transcriptional burst’ – with the first

burst being the one in the bone marrow and no transcriptional activity in

circulation [15].

During intraphagosomal toxication of a microbe, bacterial or fungal, ROS are

generated that will induce neutrophil cell death eventually. Most prominent-

ly, ROS induce apoptosis, but it is also proposed that very high levels of ROS

promote necrosis [32]. ROS-dependence of NETosis was demonstrated with

different stimuli [33, 34]. Apoptosis is in many respects the cell death route

to prefer, since it prevents release of harmful neutrophil content into the

surrounding. Apoptotic neutrophils are removed by tissue-residing macro-

phages in liver, spleen, and bone marrow during normal homeostasis. Dur-

10

ing infection, they are taken up by macrophages, but also DCs, at the respec-

tive site [32]. Remarkably, this so-called ‘efferocytosis’ dampens a cascade

that induces G-CSF production – thus, high numbers of apoptotic neutro-

phils in tissue reduce the G-CSF controlled release of neutrophils into circu-

lation [18].

1.4 NETosis – neutrophil functionality post mortem

Since their discovery about a decade ago, neutrophil extracellular traps have

been the subject and inspiration for many studies. The list of pathogens de-

scribed to be either inducing NET formation and/or sensitive to NETs has

become long, and includes organisms as different as the bacteria Mycobacte-

rium tuberculosis and Shigella flexneri, the fungi Candida albicans, Crypto-

coccus neoformans, and Aspergillus fumigatus, but also parasites like Plas-

modium falciparum [35-40]. NETs might even play a role during viral infec-

tions [41]. Since NETs fibers have a DNA backbone, it is not surprising that

many bacterial pathogens possess extracellular DNAses to escape entrap-

ment by NETs [42, 43].

Phagocytes of many organisms produce NETs or ETs: from mammals, birds,

fish, but also invertebrates, such as acoelomates, oysters or shrimp [36, 44-

48]. In humans, not only neutrophils, but also mast cells and eosinophils

release DNA traps [49, 50].

Although several NETosis models were proposed in the last years, only one

will be described and referred to in this thesis: the ROS-dependent NET

formation involving DNA from the nucleus [34, 51]. According to current

understanding, this is most likely the NETosis mechanism induced by fungal

pathogens [52, 53].

In this model (Fig. 2), an external stimulus triggers the induction of the pro-

teinase kinase C (PKC), which leads to the formation and activation of the

NADPH complex. The produced ROS induce, over yet not fully understood

routes, the loss of integrity of nuclear and granular membranes. At this

point, the plasma membrane is still intact, allowing intracellular components

to get in contact and to mix – even though originating from formally sepa-

rated compartments. Granular proteins promote chromatin decondensation.

11

Eventually, the plasma membrane ruptures and the neutrophil DNA is re-

leased “decorated” with a number of antimicrobial proteins [34, 38, 54].

Remarkably, these proteins are not randomly NET-associated, but defined,

and reflect the NETosis process well: the NETome includes indeed proteins

from the nucleus, from granules, and from the cytoplasm.

Figure 2: ROS-dependent NETosis

Neutrophils undergo NET formation upon external stimulation. Four stages are

characteristic in this process (from left to right): 1) Neutrophils sense an activating

stimulus. The NADPH oxidase complex is formed and superoxide is generated. 2)

Membranes of nucleus and granules disintegrate. 3) Content of nucleus, granules

and cytoplasm mix. 4) Plasma membrane ruptures, NETs are released [51].

[Re-used with permission of the Nature Publishing Group, License 3601390788934]

NETosis is distinct from apoptosis and necrosis, and accordingly considered

an additional cell death mechanism [34, 55].

NET induction and the responsible intracellular events are not fully under-

stood, yet. Several pathways are supposedly involved in NETosis: the Raf-

MEK-ERK pathway, PAD4-mediated citrullination of histones, elastase-

mediated chromatin decondensation, and autophagy [54, 56-58]. Numerous

stimuli, biotic and abiotic, induce NETs. The most prominent abiotic stimu-

lus used to trigger NET formation very effectively in vitro is phorphol 12-

myristate 13-acetate [36].

When considering the versatile armory of neutrophils, it is obvious that one

of the most intriguing questions is, how neutrophils “decide” to undergo

NETosis instead of other antimicrobial defense mechanisms. While the one

trigger and the one pathway are not identified yet, neutrophil heterogeneity

might hold some clues. A recent study demonstrated a possible link between

NETosis and an olfactomedin 4-expressing neutrophil subtype [59]. In addi-

tion, a preferential release of NETs towards bulky pathogens was proposed

[60].

12

It should be mentioned here as well that NETs do not only play a role during

microbial infections, but also during sterile inflammation, e.g. in autoim-

mune diseases [1].

The presented thesis’ focus lies on the antimicrobial mode of action of NETs,

especially towards fungal pathogens.

1.5 Neutrophil disorders

Neutrophil disorders can be divided in inherited genetic defects and non-

inherited disturbances of neutrophil homeostasis. Neutropenia, the reduced

count of neutrophils, is in some cases the result of accelerated neutrophil

death and is amongst others caused by chemotherapy or bone marrow trans-

plantation. Neutrophilia, the pathologically increased number of neutro-

phils, can be the result of repressed neutrophil death, and occurs e.g. in in-

fections and auto-immune diseases [32]. A number of genetic neutrophil

defects are known today – in all of them, the patient is facing an increased

risk of microbial infection.

Severe congenital neutropenia (SCN) is a disease caused by the blockage of

full bone marrow maturation of neutrophils. Life-long G-CSF treatment is

necessary to counteract the increased risk of bacterial and fungal infections.

In leukocyte adhesion deficiencies (LADs), neutrophils are not able to leave

the blood stream and enter the site of infection, because the adhesion cas-

cade is corrupt. In patients suffering from the WHIM syndrome, neutrophils

are less prone to leave the bone marrow, reducing the number of neutrophils

in circulation [1].

In addition to diseases mostly affecting neutrophil homeostasis, direct neu-

trophil antimicrobial efficacy can be abrogated by genetic defects, too.

Chronic granulomatous disease (GGD) is characterized by a non-functional

NADPH complex that restrains the neutrophil’s superoxide production, im-

pairing its ability to kill microbial pathogens effectively. This in turn affects

the patient’s life expectancy and requests life-long surveillance [1]. In addi-

tion, CGD patients suffer from sterile inflammation causing predominantly

kidney damage. The mechanisms of the hyper-inflammatory state are less

13

well understood. Most likely, macrophage activation and regulation of neu-

trophil apoptosis play a role [61, 62].

Mutations of down-stream mediators of TLR signaling, namely MYD88 and

IRAK4, increase the risk of infection predominantly in young children. In-

terestingly, IRAK4-deficient neutrophils are capable of killing S. aureus, E.

coli, and C. albicans [63]. Subtotal or total MPO-deficiency increases the risk

of life-threatening infections. Still, an inherited MPO deficiency can also be

asymptomatic [64]. Similarly, a lack of specific granules, affecting antimi-

crobial proteins from secondary and tertiary granules, subsequently, leads to

recurrent microbial infections [1].

14

2. Human fungal pathogens

Human fungal pathogens account for high number of superficial and system-

ic infections worldwide [65]. Nevertheless, they lead a shadowy existence

compared to bacterial or viral infectious agents. Not only in basic research,

but also in training of medical professionals, fungi are often undervalued.

Initiatives like LIFE (Leading International Fungal Education, life-

worldwide.org), the Fungal Infection Trust, or the ‘Mycotic Diseases Branch’

of the CDC aim to change perception worldwide.

2.1 The fungal threat

Even though the exact numbers vary from source to source, it is clear that

only a small group of all fungal species can cause disease in humans. A dozen

of those account for 90 % of all mycoses [4]. A group of 4 genera is responsi-

ble for 90 % of all fungal-related deaths: Candida, Aspergillus, Cryptococcus

and Pneumocystis [65].

According to LIFE, fungal diseases can be assigned to five different classes:

invasive fungal infections, chronic lung and deep tissue infections, allergic

fungal disease, mucosal infections, and skin, hair, and nail infections. The

last two groups are often paired and as such called ‘superficial infections’.

With approximately 25 % of all humans worldwide affected, these infections

lead to an immense physical burden [65]. Invasive fungal infections are

scarce in comparison, but accompanied by relatively high mortality. Recent

estimations suggest 1.5 million deaths per year. Individual mortality rates

vary due to fungus and location. Extremes are 95 % mortality in

A. fumigatus infections, 75 % in C. albicans infections, 70 % in

C. neoformans infections, or 80 % for Pneumocystis jirovecii [65]. While

transmission from human to human does not play a major role for fungal

infections in general, treatment of fungal pathogens is challenging. Limited

choices of antifungals, drug resistances, and severe side effects complicate

successful therapy [4].

15

2.2 The yeast Candida albicans and other Candida ssp.

Candida ssp. are the most important cause of opportunistic fungal infections

worldwide. They are the fourth most common nosocomial blood stream iso-

lates in the U.S. In Europe, Candida causes systemic infections just as often

as the prominent bacteria E. coli or S. aureus [66, 67]. Like most fungi, Can-

dida ssp. are opportunistic, meaning they are pathogenic only under certain

circumstances, namely if the host immune defense is impaired. Noteworthy,

the number of immuno-compromised has increased over the past decades

[68, 69]. The reasons for immunodeficiency are as versatile as its extent. A

healthy human life withholds passages of weaker immune defense, since the

extremes of age and pregnancy decrease the system’s force. In addition,

many diseases and disorders impair the immune defense. Risk factors are

e.g. organ transplantation, gastrointestinal surgery, autoimmune diseases,

treatment with corticosteroids, blood cancers, and HIV. Further, the protec-

tive microflora can be out of order due to long-term antibiotic treatment.

Fungi are normally outcompeted by commensal bacteria colonizing the re-

spective niche in the body. If the equilibrium is destroyed, resistant bacteria

and non-bacterial invaders are at chance [4]. Therefore, it seems fair to claim

that advanced medical care paradoxically favors fungal infections to some

extend - not only Candida.

As an archetypical opportunist, C. albicans causes the majority of all invasive

fungal infections in humans, while simultaneously being carried by approx-

imately every other human without doing any harm [70, 71].

When C. albicans infects the immuno-compromised, the spectrum of tissues

and organs targeted is broad. Mucosal tissue in mouth and gut, lung, and

nails can be infected. In systemic candidiasis, Candida smites kidneys, heart,

and even the central nervous system [4]. Severe candidiasis obtained in a

medical care unit has a 30-50 % chance of being fatal [67, 72].

The most remarkable feature of C. albicans is to grow as round budding

yeasts or as long filamentous hyphae. Both morphotypes are essential for

virulence: The yeast is highly proliferative while hyphae are considered im-

portant for invasion and destruction of tissues [73-75]. Additionally,

16

C. albicans grows as pseudo-hyphae of which the contribution to patho-

genicity is largely unknown. Primary patient samples contain usually a mix-

ture of yeasts and filamentous fungal cells [4].

Not only amongst all fungal pathogens, but also amongst the Candida ssp.,

C. albicans is the dominating species [71]. Nevertheless, other Candida ssp.

are on the rise – mostly due to their natural resistance to common antifun-

gals. They are e.g. C. glabrata, C. kruzei or C. parapsilosis.

2.3 The mold Aspergillus nidulans and other Aspergillus ssp.

Unlike Candida ssp., Aspergillus ssp. are no natural colonizers of the human

body. Nevertheless, contact with Aspergillus is inevitable, since spores are

omnipresent in the environment worldwide. Aspergilli are molds, growing

primarily on rotting biological material and spread as small and light spore

forms via airborne routes. Spores, or conidia in Aspergillus, are dormant

until certain environmental conditions (temperature, moisture) allow them

to revive. Conidia swell, germinate to germlings, which grow further into

somatic hyphae, and those eventually give rise to conidiophores that produce

new conidia [4, 76]. The infectious capacity of Aspergillus ssp. is often de-

scribed as an evolutionary accident [77]. The live style in rotting biological

material with its occasional high temperatures might allow this mold to tol-

erate 37° C body temperature better than other pathogens [78]. On the con-

trary, Aspergilli favor carbon-rich substrates.

The primary infectious route of Aspergillus, due to airborne spread, is the

respiratory tract, but conidia can enter the body also through wounds. Re-

sulting diseases are allergic bronchopulmonary aspergillosis, invasive

aspergillosis, and aspergilloma [79]. Invasive aspergillosis can be either

chronical, e.g. in cystic fibrosis patients, or acute, which occurs e.g. due to

neutropenia. Some Aspergillus ssp. produce mycotoxins, which are taken up

in contaminated food and resist cooking and extremes in pH [4].

A. fumigatus is the most common causative agent of aspergillosis. Other

pathogenic species are A. flavus, A. niger, and A. nidulans.

17

Even though A. nidulans is not remarkably important in other immuno-

compromised risk groups, including neutropenic patients, it is highly rele-

vant for CGD patients. In these patients, following A. fumigatus, A. nidulans

is the second most encountered fungal species. Interestingly, virulence of

A. nidulans in CGD patients exceeds that of A. fumigatus [80]. Because

comparative studies of both molds and their interplay with the CGD host are

rare, it is not yet clearly understood how this shift of power occurs.

2.4 Fungal defense strategies against neutrophil killing

Microbes avoid neutrophil killing by directly interfering with neutrophil

functionality on different levels: recognition and contact, phagocytosis, in-

tracellular toxication, host cell survival, and NETosis [81].

Neutrophils enter the site of infection by following gradients of chemokines.

There they sense pathogens by their PRRs (see 1.3). Therefore interfering

with neutrophil recruitment will diminish the risk of being attacked by neu-

trophils. Cryptococcus neoformans secretes glucuroxylomannan, a compo-

nent of its capsule, that reduces chemotaxis of neutrophils, even though mi-

croglia produce neutrophil-attracting IL-8 [82]. Capsule polysaccharides,

like the secreted mannoprotein MP-4 of C. neoformans are also capable to

trigger the shedding of L-selectin and the loss of the TNF-α receptor on neu-

trophils, rendering them less able to leave circulation and desensitized for

this pro-inflammatory cytokine, respectively [83, 84]. The surface protein

RodA, or hydrophobin, of A. fumigatus has a rather indirect effect on neu-

trophils: RodA forms the outer layer of conidia, and masks them for recogni-

tion by DCs or alveolar macrophages [76]. This leads to reduced recruitment

of neutrophils in vivo [85]. C. albicans circumvents its detection by masking

its β-glucan with mannoproteins [86].

The major strategy of most fungi to avoid phagocytosis is most probably the

sheer size of some morphotypes, such as for instance long, filamentous hy-

phae (see 2.5) or titan cells [87]. In addition, fungi manipulate the function

of the complement system. Host complement regulator Factor H is bound by

the C. albicans protein Pra1p, a process normally used by the host to protect

18

itself from the complement system [88]. Similar observations were made for

A. fumigatus conidia [89].

Once fully taken up into a phagocyte, fungi induce intracellular survival

mechanisms. The knowledge about intracellular persistence strategies of

fungi in neutrophils is limited – most research in the past focused on macro-

phages. Even though neutrophils and macrophages share similarities, differ-

ences predominate. Therefore, conclusions from macrophages should be

applied to neutrophils with caution (see 1.2).

According to transcription studies conducted with C. albicans, carbon star-

vation and the subsequent induction of the glyoxylate metabolism seem to

occur in both phagocytes, while amino acid starvation was only observed in

neutrophils [90-93]. Then again, a strong response to oxidative stress was

observed in both neutrophils and macrophages [91, 92, 94]. In neutrophils,

the Candida response included the up-regulation of ROS-detoxifying super-

oxide dismutases (SODs), catalase (CAT), and glutathione peroxidases

(GPXs). Interestingly, a direct comparison of intra- and extracellular stress

response of neutrophil-encountering C. albicans demonstrated that the ROS

stress response occurs inside and outside of the neutrophil. The RNS stress

response on the contrary, was only observed in internalized Candida cells

[93]. Additionally, there are species-specific responses from different fungal

pathogens towards the attack of phagocytes. The transcriptional response of

C. neoformans in macrophages, for instance, is characterized by a carbon

starvation response and additionally by expression of extracellular lipases.

However, it lacks the induction of the glyoxylate cycle [95]. An alternative

strategy to endure starvation upon internalization is employed by

C. neoformans and C. glabrata. They activate autophagy within the

phagolysosome [96, 97]. Conidia of A. fumigatus exposed to human neutro-

phils reprogram their metabolism in a similar fashion as C. albicans. Conidia

induce genes from the glyoxylate cycle, but also from fatty acid metabolism.

Both, conidia and hyphae induce an oxidative stress response upon neutro-

phil encounter [98].

19

Interestingly, the cell surface protein RodA, present on A. fumigatus conidia,

but absent on hyphae, serves to diminish a general immune recognition,

rendering conidia nearly immunologically inert [99].

With all this said, it should be mentioned that most pathogenic fungi are

indeed susceptible to neutrophil killing. Of all three fungi discussed above,

only C. neoformans is able to resist neutrophil killing [81]. The escape of

C. albicans from neutrophils via hyphal growth has only been observed in

mouse, but not in human neutrophils [100].

2.5 Impact of fungal morphology during pathogenesis

Fungal morphology is very versatile. C. albicans can grow as budding yeast,

as pseudohypha, or as a true hypha. The life cycle of A. nidulans includes

conidia, hyphae, and even conidiophores [101]. The exact mechanisms re-

sponsible for these many “faces” are complex and a detailed discussion goes

beyond the scope of this thesis. Nevertheless, different morphologies influ-

ence virulence greatly, and will therefore be shortly introduced here – with

special emphasis on filamentous growth.

Yeasts are the harmless and proliferative, hyphae the harmful and invasive

growth form of C. albicans. While tempting, this simplification needs to be

expanded by a number of observations: First, not the one-way switch from

yeast to hyphae, but the transition between all growth forms is necessary for

full virulence. More than one C. albicans morphotype is usually isolated

from primary patient samples or infected animals. Biofilms consist of heter-

ogeneous C. albicans communities. Second, the host immune status affects

the in vivo morphology profiles of C. albicans, so the proportions of the indi-

vidual growth form. Third, C. albicans mutants unable to form hyphae in

vitro might still form hyphae in vivo. Forth, the act of hyphal formation itself

is not the only crucial factor for host damage and immune recognition. More

important are the specific properties of the hypha, like hypha-specific

adhesins or invasions, e.g. Hwp1p and Als3p, respectively, as well as the lack

of β-glucan exposure on hyphae. Finally, a recent study proposed an addi-

20

tional gastro-intestinal C. albicans phenotype optimized for this particular

niche, the GUT cell [4, 22, 101-104].

While C. albicans yeast and hyphae are easily “produced” in vitro, the gener-

ation of uniform pseudohyphae without buds or true hyphae is still challeng-

ing [101]. Consequently, very little is known about cell wall properties and

immune recognition of this morphotype [22].

The life cycle of Aspergillus ssp. is not tightly bound to the human host. Still,

the moist and warm environment of the body can wake up conidia that will

then form hyphae. Even more than C. albicans yeasts, Aspergillus conidia

stay under the radar of the immune system. Shielded by immunologically

inert hydrophobin RodA, resting conidia are hardly detected by tissue-

residing DCs or macrophages in the lung [76]. Only upon germination, fun-

gal PRRs are eventually exposed and the germlings and hyphae are recog-

nized and attacked [85, 99].

Figure 3: Neutrophils engulfing and enwrapping of C. albicans Neutrophils engulf C. albicans yeasts successfully (left), but fail to do so with hy-phae. Hyphae are enwrapped (right), preferentially beginning from the hypha end. Mother cells often stick out.

Fungal hyphae easily reach lengths that exceed the dimensions of phagocytes

(Fig. 3). Even though neutrophils can stretch considerably, large hyphae

cannot be fully engulfed. Interestingly, NETosis toward A. fumigatus was

demonstrated to be more common towards hyphae than to resting and swol-

len conidia [99]. In line with this notion, a recent study including C. albicans

suggested that NET formation occurs predominantly towards large patho-

genic structures [60].

Due to their very different dimensions and the aforementioned differential

surface composition, comparative studies of fungal morphotypes must be

interpreted cautiously. A recently introduced dry mass adjustment for

21

C. albicans yeasts and hyphae proposes to infect not with the same number

of fungal cells, but with the same dry mass – which eventually matches in-

teracting fungal surface areas [105].

22

3. Nutritional immunity

The term ‘nutritional immunity’ was introduced in 1975 by Eugene D. Wein-

berg as he reviewed host mechanisms to withhold Fe from microbes during

infection [106]. Since then, numerous studies have been conducted on this

topic. In recent years, the field gained increasing interest. Searching the

NCBI library for ‘nutritional immunity’ in spring 2015 results in little over 60

articles since 1975, out of which more than 65 % were younger than 5 years.

The renaissance of nutritional immunity (and not just the term) is likely the

result of improved detection methods, especially for metal elements, and the

urgent need for new, non-antibiotic, strategies to fight microbial infections.

3.1 The third branch of the human immune system

– major concepts

Originally, nutritional immunity denoted host-depletion of nutrients from

invading pathogens. Nowadays, the perception has widened to the two-sided

battle for valuable resources. Nutritional immunity refers to the transition

metals Zn, Fe, Cu, and Mn. Metals are crucial, as they act as co-factors in

enzymes and stabilize three-dimensional protein structures [107, 108]. Nu-

tritional immunological actions occur in parallel to the innate and adaptive

immune response, more tightly connected to the innate immune system.

Consequently, nutritional immunity can be considered either part of the

innate immune system, or alternatively as a third branch.

Every aspect of nutritional immunity, from the host or pathogen perspective,

exemplifies the co-evolutionary nature of infections in a remarkable manner.

Best studied is the fight for Fe, but most concepts apply for all transition

metals in eukaryotes.[106, 109] In the host, Fe is mainly kept intracellular -

free Fe is essentially unavailable in blood or other extracellular liquids. It is

bound to transferrin in plasma and lactoferrin in external secretions. How-

ever, in order to grow within the host, microbial pathogens cope with these

limitations. In case of direct contact between the host Fe source and the

pathogen, host Fe-retaining proteins are modified to release their Fe load

into the extracellular environment for uptake. Secondly, many bacteria and

23

fungi secrete siderophores, small molecules with exceptionally high Fe affini-

ty that free Fe from host proteins followed by resorption into the pathogen’s

cell. In response, mammals produce lipocalin-2, to intercept siderophores.

This can in turn be counteracted by many pathogens which disguise their

siderophores by glycosylation [109]. Since metal limitation is a dominant

antimicrobial strategy, it is not surprising that some pathogens were found

to be able to sequester and store higher levels of specific essential metal than

their non-pathogenic relatives [110].

3.2 Zinc during fungal infections

Zn is a special element. Unlike other transition metals, Zn is environmentally

very abundant, water-soluble at +II, and redox-stable. It is the strongest

Lewis acid amongst the +II cations. Consequently, Zn is a co-factor for many

catalytic reactions. Of all metal-containing enzymes, 9 % use Zn – so Zn is

the second most abundant metal cofactor after Mg. Zn enzymes catalyze

reactions from all classes of enzymes (EC 1-6), but the most abundant is EC

3, the hydrolases [107]. Further, Zn stabilizes Zn-finger domains, a function

that is especially important in eukaryotic organisms. Bioinformatic ap-

proaches comparing genomes predict that approximately 7 % of all proteins

in eukaryotes are Zn proteins, which is in the same range as in prokaryotes.

Differences appear in the usage of Zn-finger proteins. In prokaryotes, only

0.2-0.4 % of the proteome are Zn-fingers. In eukaryotes, this increases to an

average of 3 % [111]. Considering the connection of Zn availability and gene

regulation, it is not surprising that a lack of Zn is potentially harmful, and

leads to apoptosis – as demonstrated for mammalian cells [112]. Several

studies have demonstrated the importance of Zn in pathogenic and non-

pathogenic fungi. C. albicans is very sensitive to Zn starvation. Zn availabil-

ity does not only affect morphogenesis, it can actually be the limiting factor if

all other nutrients are available [113, 114]. A role in virulence is indicated by

the relevance of Zn in biofilm formation, but also tolerance to intrinsic oxi-

dative stress by the cytoplasmic Sod1p [115]. Sod1p is a Cu/Zn superoxide

dismutase. Notably, the cell surface superoxide dismutases Sod4p and Sod6p

24

that are crucial to survive host-derived ROS, have Cu/Zn as their active site

[116].

Zinc acquisition in fungal pathogens is mediated by a small number of pro-

teins and is conserved in C. albicans and A. fumigatus. Zn enters the cyto-

plasm via Zrt plasma membrane transporter (CaZrt1p/AfZrfC,

CaZrt2p/AfZrfB). From there, Zn can be transported further into the vacuole

through ZnT transporters (Zrc1p/Cot1p). In mammalian cells, ZnT trans-

porters are responsible for cellular Zn efflux. Zn can be mobilized from the

vacuolar pool by Zrt exporters (CaZrt3p). Interestingly, no cellular Zn ex-

porter has been described in fungi, yet. The homeostasis of Zn is regulated

on two levels: the expression of Zrt importers, controlled by Zap1p in yeasts

and Zaf1p in Aspergilli, and Zn import and export from subcellular orga-

nelles [117]. Vacuolar storage might be especially important during infection,

since it allows proliferation even under Zn restricting conditions [118]. In

addition, C. albicans uses a zincophore system – a secreted protein that se-

questers Zn from host cells and re-associates with CaZrt1p to deliver Zn to

the fungal cell: Pra1p [119]. The expression of Pra1p is repressed under acidic

conditions, but induced under zinc starvation or at neutral/alkaline pH

[119]. A similar zincophore system might exists in A. fumigatus [120]. Re-

markably, Pra1p is not only beneficial for the fungus, it is actually immuno-

genic and immune-modulatory [120]. Amongst many functions, Pra1p can

serve as a ligand to neutrophil integrin receptor αMβ2 and promote neutro-

phil killing [121]. Probably due to this “side effect”, Pra1p in not conserved in

all pathogenic fungi.

As for other transition metals, the host restricts also Zn availability tightly.

Zn concentrations are kept low in the extracellular milieu, the cytosol and

the phagosome [117, 122]. Remarkably, zinc poisoning has been observed in

the phagosome of Mycobacterium-infected macrophages [123].

There have now been attempts to disturb Zn homeostasis as an antifungal

treatment [124].

25

3.3 Iron, copper, and manganese during fungal infections

The transition metals Fe, Cu, and Mn also act as co-factors in

metalloproteins. The respective metal enzymes are less abundant than Zn

enzymes, but still exceed those with elements like Co and Ni. Of all enzymes,

8 % contain Fe, 6 % contain Mn, and 1 % contains Cu. The dominant catalyt-

ic classes are oxidoreductases (EC 1) for Fe and Cu, and transferases for Mn

(EC 2)[107]. Nevertheless, the general concepts of nutritional immunity ap-

ply to Fe, Cu, and Mn.

Fe uptake in pathogenic fungi is quite similar in C. albicans, C. neoformans,

and A. fumigatus and occurs in two ways, reductive and the non-reductive

[107, 125]. Briefly summarized, the high affinity reductive uptake system

functions by the interplay of three enzymes. A reductase (Fre) converts ferric

Fe3+ to ferrous Fe2+. The actual uptake is mediated by a permease (Ftr) and a

multicopper ferroxidase (Fet). Probably due to gene expansion (and func-

tional redundancy) of the reductases and multicopper oxidases, only the

permease Ftr1 has been shown to be crucial in blood stream infections of

C. albicans. Of note, the pigment melanin can reduce ferrous iron. In

C. neoformans, melanin biosynthesis and Fe uptake are closely connected.

In C. albicans, the retrieval of Fe from both host proteins, ferritin and trans-

ferrin, is dependent on the reductive pathway [126, 127].

Non-reductive Fe uptake is based on the import of Fe-containing proteins,

namely hemoglobin and siderophores. C. albicans has receptors for heme

(e.g. Rbt5p), but also for ferritin (reductive uptake). The ferritin receptor

Als3p is expressed only by hyphae [127]. Siderophores are not produced by

C. albicans, but they can be utilized as an Fe source [125]. The production,

secretion, and uptake of siderophores plays an important role in

A. fumigatus, and the reductive Ftr-based uptake system is less important

for A. fumigatus virulence. A. fumigatus is also not able to retrieve Fe from

heme [128]. The gut is probably the only site in the body where microbes

encounter high Fe levels. Indeed, C. albicans is, as a colonizer of this niche,

able to resist Fe toxicity. The transcriptional regulator Sfu1p represses Fe

uptake, and represses the Fe-uptake and virulence regulator Hap43p. Sfu1

26

homologues exist in C. neoformans and A. fumigatus [128]. Finally, the vac-

uole was demonstrated to play a role in Fe homeostasis in Saccharomyces

cerevisiae., which could indicate a similar system in pathogenic yeasts [128]

Fe uptake is amongst those processes that are modulated in the recently

identified GUT morphology, a C. albicans phenotype specialized in gastro-

intestinal persistence [103]. One of the most important direct functions of Fe

during host-pathogen interactions is likely the detoxification of H2O2 by the

iron-dependent heme-enzyme catalase.

Uptake of Fe and Cu are very tightly connected and conserved in fungi. Most

obviously, the ferroxidase (Fet) is actually a Cu-dependent enzyme, and the

Fe reductases (Fre) are indeed ferric/cupric reductases that reduce Cu2+ to

Cu1+ through the oxidation of Fe2+ to Fe3+, and vice-a-versa [129]. Reduced

Cu1+ is taken up by Cu transporters (Ctr). Ctr1p and Ctr3p are plasma mem-

brane-localized, Ctr2p is vacuolar – and all three transport towards the cyto-

sol. Interestingly, the number of CTR copies varies between fungal species

and might affect virulence due to an effect on e.g. hyphal growth. Melanin

formation in C. neoformans is Cu-dependent. The export of Cu from the

cytosol occurs actively through the Golgi-located ATPase Ccc2p. A

C. neoformans mutant lacking CCC2 was shown to be attenuated in viru-

lence, likely due to the impaired melanin formation in this mutant [128]. The

same mutation had no effect in C. albicans, a fungal species for which mela-

nin production has not been reported. Ccc2 receives Cu from the Cu chaper-

one Atx1 [129]. Homologues of the Ccc2/Atx1 system exist in A. fumigatus

[128]. During an infection, pathogens face Cu starvation as well as Cu intoxi-

cation. Cu “poisoning” was described as a mode of action against

intraphagosomal Mycobacteria in macrophages [130]. Because of this, Cu

export and Cu detoxifying metallothioneins are very important.

Metallothioneins are conserved in prokaryotes and eukaryotes. A potential

role in virulence was demonstrated in C. neoformans, a known macrophage

persister, but not yet in C. albicans and A. fumigatus. However, C. albicans

is known to export excess Cu, indicating that fungi are primed to resist

potential Cu poisoning in vivo [128]. At the same time, Cu is essential for

pathogenic survival. Most fungal SODs, crucial to withstand superoxide

27

stress, have Cu in their active site. In C. albicans, SOD1, SOD4, and SOD6

are Cu/Zn-dependent, SOD5 was recently described to be a Cu-only SOD

[131]. C. albicans extracellular SOD4 and SOD5 were demonstrated to detox-

ify superoxides produced during interactions with macrophages. Interesting-

ly, secreted SODs from A. fumigatus are recognized by the human immune

system [128].

Manganese plays a decisive in photosynthesis. In non-plant eukaryotes, its

function is restricted to mitochondrial enzymes, including SOD2 and SOD3.

Nevertheless, the human host restricts access to Mn by releasing calprotectin

that is capable of binding Zn and Mn [132, 133]. The macrophage phagosome

is actively depleted of Fe and Mn upon infection. Phagosomal Mn intoxica-

tion, as demonstrated for Cu and Zn, has not been observed [134]. Mn re-

striction is important during bacterial infections, especially since some bac-

terial pathogens have substituted Fe metalloproteins by Mn metalloproteins,

but its role during fungal pathogenesis requires more investigation [132, 135,

136].

28

Methodological remarks

In the following, major remarks regarding the methods used in paper I, II,

and III are briefly discussed.

Neutrophil isolation

In paper I, II and III presented in this thesis, neutrophils were isolated from

human blood by Histopaque/Percoll purification. The resulting cell suspen-

sion of this protocol contains > 90 % neutrophils. Contaminating cells are

granulocytes, mostly eosinophils, and T-cells. Monocytes and B-cells are

consistently present with an abundancy < 0.1 %. The later is especially im-

portant for the expression profiling in paper III. Other white blood cells are

very potent cytokine producing cells, and especially the discrimination of

monocyte and neutrophil functionality crucial – due to their close relation-

ship and yet striking differences.

Fungal strains

The A. nidulans strains used for the in vitro experiments presented in paper

I is the original isolate derived from the CGD patient.

The C. albicans strain SC5314 was used in the experiments of paper III.

Originally a patient isolate in the 1970s, it is a widely used strain for in vitro

and in vivo research. No virulence defects are known today [71].

Metal consistency

Amongst the trace elements, Zn is the environmentally most abundant. Due

to this, special precautions were taken for quantitative analyses. Water was

always taken directly from a Millipore water suspensor and stored in plastic

vessels. Purchased media and buffers were used consistently from one batch

throughout all experiments. Salts, acids, and bases were purchased as clean

as possible (e.g. ‘TraceSELECT’). No glass ware was used – only plastic ma-

terials got in contact with the solutions. Finally, no autoclaved goods were

29

used, due to possible contamination via metal-enriched steam during this

process.

Synchrotron radiation X-ray fluorescence

Synchrotron radiation X-ray fluorescence (SR-XRF) is a method originally

utilized in material science. Nowadays, its applications are very versatile.

Very simplified, a strong beam is pointed at a sample. In every atom, the

electrons are excited to an extent where ionization occurs, so an electron is

abstracted. This causes instability, which is compensated by other electrons

“falling” on lower energy levels. As a result of this “fall”, energy is emitted. If

the beam is strong enough, even the lowest orbitals are affected. Every ele-

ment will emit energy specific for its electron profile. The beam strength and

focusing, which is, in technical terms, by far the most challenging factor for

the methodology, correlate with sensitivity. As of today, investigations of

biological samples are only possible at synchrotron facilities. For the meas-

urements of paper II, we used the P06 beamline at DESY (Germany) and the

ID22NI beamline at the ESRF (France). When conducting such an experi-

ment, three essentials must be considered. Firstly, it is not possible yet to

analyze living cells or wet samples. Attempts are made to allow the analyses

of frozen samples, or cells in a liquid drop, but those are not technically ma-

tured enough. At the moment, the analysis of single cells with subcellular

resolution is only possible in fixed and dehydrated samples. Secondly, the

element integrity of the samples needs to be preserved as much as possible.

This can be challenging when using biological samples. Finally, the number

of samples is limited due to long measurement times (3-8 h per scan) and

limited access to SR-XRF facilities (usually maximum one week at a time).

Nevertheless, SR-XRF is the only method as of today that allows element

mapping of single cells at this resolution.

In vitro infections for RNA-sequencing

The aim of the study presented in paper III was to elucidate the influence of

C. albicans morphotypes on the interaction with neutrophils and NETs. To

30

do so, we complemented the assessment of pathogen amounts infecting host

cells. We utilized the well-known multiplicity of infection (MOI) and addi-

tionally a dry mass correlation set up [105]. Using this correlation of dry

mass and metabolic activity of Candida, we infected the neutrophils with

MOI 1 of yeasts, and the same dry mass of hyphae. By doing so, the infec-

tious load like e.g. the interacting surface, should be more comparable in the

yeast and hypha infection.

31

Aims

Neutrophils are potent killers of fungal pathogens. Much is known about

their antimicrobial weapon arsenal and the survival strategies of the fungal

counterparts. At the same time, relatively little is known about the neutro-

phils’ potential to contribute to the events of ‘nutritional immunity’.

The aim of this thesis was therefore to investigate mechanisms of neutro-

phils to interfere with fungal metal homeostasis.

Paper I

Is the Zn chelator calprotectin, whose release from neutrophils is dependent

on NETosis, a universal antifungal effector?

Paper II

Do neutrophils indeed have the potential to starve fungal pathogens from

crucial trace element?

Paper III

Does the fight for trace element resources reflect in the transcription profile

of C. albicans and neutrophils during infection? Do NETs actually affect

metal homeostasis in C. albicans?

32

33

Results & Discussion

Paper I

NET calprotectin is an effector during A. nidulans inhibition

Infections with A. nidulans occur almost exclusively in CGD patients. As a

very last course of treatment, the defective gene of the NADPH complex can

be temporarily substituted with a functioning gene through gene therapy.

Besides lacking the ability to produce sufficient superoxide, CGD neutrophils

cannot undergo NETosis. After gene therapy, both functions were restored in

the cured gp91 phox+ neutrophils. A. nidulans was sensitive to NETs derived

from gp91 phox+ neutrophils [53].

Figure 4: NET-calprotectin-induced growth inhibition of A. nidulans A+B: NETosis was triggered neutrophils isolated from peripheral blood by stimula-tion with PMA. To create NETs extracts, NETs were digested with MNase/DNAse and concentrated. NETs extracts were supplemented with an α-S100A9 antibody or an isotype control. Extracts were infected with A. nidulans conidia (A) or pre-grown hyphae (B). Fungal growth was scored by metabolic activity using XTT. C: Neutro-phils from WT or S100A9-/- mice were triggered with PMA to undergo NET for-mation. NETs were infected with A. nidulans conidia or pre-grown hyphae. Fungal growth was scored by metabolic activity using XTT. [Re-produced from [137] with permission from Elsevier]

34

Earlier studies demonstrated that calprotectin is the dominant NET protein

to inhibit C. albicans [38]. The sensitivity of C. albicans to calprotectin is

long known [138]. We therefore aimed to analyze whether calprotectin was

the key effector in NET inhibition of A. nidulans when NETs were derived

from gp91 phox+ neutrophils.

And indeed, both growth forms of A. nidulans, conidia and hyphae, were

inhibited in NET extracts and not affected if NET extracts were treated with

an α-S100A9 antibody masking the protein (Fig. 4A + 4B). Further, conidia

and hyphae were inhibited by NETs from C57BL/6 mouse neutrophils, but

not or only fairly, by NETs from S100A9-/- mice (Fig. 4C).

NET inhibition is reversible by Zn supplementation

Calprotectin chelates Zn and this function is acknowledged as the major

antifungal mode of action. C. albicans could be rescued from NET inhibition

by the addition of Zn2+.[38]. Similarly, A. nidulans conidia and hyphae were

not growth inhibited by gp91 phox+ NETs, if the infection was supplemented

with additional Zn2+ (Fig. 5A + 5B). It should be noted that gp91 phox+ NETs

were as efficient as control NETs from healthy donors throughout all exper-

iments, while gp91 phox- NETs failed to inhibit A. nidulans. Considering the

incapability of gp91 phox- the undergo NETosis, those “NETs” should be

probably better referred to as long-term PMA-stimulated neutrophils. In

spite of this, long-term PMA-stimulated gp91 phox- neutrophils did not have

an inhibitory power towards A. nidulans either.

In conclusion, these in vitro experiments demonstrate the functionality of

NET-associated calprotectin as an inhibitor of A. nidulans – by the chelation

of Zn. This indicates a universal importance of calprotectin in NET-mediated

inhibition of pathogenic fungi. Of note, NET formation was demonstrated in

vivo towards C. albicans and ex vivo towards A. fumigatus [38, 99]. In a

Aspergillus lung infection model, NET formation was also dependent on the

function of the NADPH complex [62].

35

Figure 5: Rescue of fungal growth from NET inhibition by Zn

NETosis was triggered in neutrophils isolated from peripheral blood by stimulation

with PMA. NETs were infected with A. nidulans conidia (A) or pre-grown hyphae (B)

and supplemented with one µM Zn2+. Fungal growth was scored by metabolic activi-ty using XTT. [Re-produced from [137] with permission from Elsevier].

36

Paper II

NETosis reduces Zn availability in the surrounding

NETs inhibit fungal growth in a calprotectin-dependent manner and this is

reversible by Zn supplementation. Calprotectin release occurs in neutrophils

only during NETosis and significant proportions of calprotectin stay NET-

bound [38]. At the same time, NET formation releases the entire intracellu-

lar content. This event is not necessarily favorable, since this includes also

metal ions carefully kept inside the immune cell. It was therefore important

to determine whether the process of NET formation actually reduces Zn

availability for the fungal pathogen. To answer this, NETosis was induced in

cell culture medium supplemented with increasing concentrations of Zn2+

and the resulting Zn concentration was measured before and after NETosis

by ICP-MS. Indeed, NET release affected Zn in the NET supernatant (Fig. 6).

Figure 6: NETosis-mediated reduction of zinc availability. NET formation was induced in neutrophils with PMA for 4 h in the presence of dif-ferent Zn2+ concentrations. The remaining zinc concentration in protein-filtered supernatants was quantified by ICP-MS. Zinc-binding is saturated at higher concen-trations at 62 % (grey dash line). Zinc reduction is stronger at concentrations be-low ca. 4-5 µM indicating specific binding (grey dotted line). [Re- produced from [139] with permission from The Royal Society of Chemistry]

37

The strongest reduction was detected if the Zn concentration was below 3-6

µM. Here, only 0-55 % of unbound Zn remained. At higher concentration,

the Zn reduction capacity reached saturation, but the remaining Zn never

exceeded 62 % of the original concentration.

This indicates that available Zn can be diminished through NET formation

by binding specifically to Zn-binding proteins, like calprotectin, but can also

be loosely associated to negatively charged biomolecules, like e.g. DNA.

NETs contain Fe, but not Zn

NET formation is characterized by a stage during which granular, cytoplas-

mic and nuclear contents are allowed to mix. Since neutrophils are not per se

Zn free, the low Zn content of NETs upon release was put to the test.

NETosis was induced in low Zn cell culture medium and the resulting NETs

were analyzed by SR-XRF (Fig. 7). In this in vitro set-up, NETs contained

only Fe clearly co-localizing with their filamentous structure, while no Zn,

Cu, or Mn were detected. Occasional hotspots of these four elements were

most likely due to cell debris accumulations. Cell residues lining the circular

area of the former cell are typical. They are visible in simple light microsco-

py, but also in the element maps.

Neutrophils are exceptionally high in Fe

At the site of infection, neutrophils that have phagocytosed a pathogen will

eventually undergo apoptosis and be removed by other immune cells [18,

32]. While no neutrophil content is released in this process, NET formation,

but also necrosis are characterized by the loss of plasma membrane integrity.

Considering the high influx of neutrophils to the infection site, their presence

could easily affect the local metal milieu. To avoid providing trace element

nutrients to microbial pathogens, neutrophils might be exceptionally low in

these elements. We analyzed neutrophil lysates and compared their

metallome, full metal content, to other cells widely used in in vitro experi-

ments: macrophage-like J774 cells and HeLa cells (Fig. 8).

38

Figure 7: Element distributions (P, S, Ca, Zn, Fe, Cu, and Mn) of NETs Neu-trophils were seeded on a Si3N4 membrane, stimulated with PMA for 4 h and subse-quently freeze-dried. Image size: 100 μm x 100 μm, step size: 1 μm and 3 s scan-ning time/point. The RGB image represents a colored overlay of the elemental in-tensities corresponding to Ca (red), Zn (green) and Fe (blue). An optical image was taken at the beamline microscope (2500x magnification). Beamline: P06, DESY, Hamburg. [Re-produced from [139] with permission from The Royal Society of Chemistry]

Figure 8: Metallome comparison of neutrophils with J774 and HeLa cells Cultured J774 and HeLa cells were alkaline-lyzed and the resulting lysates were quantified by ICP-MS. The average content was related to the average content of neutrophils, n = 3, and normalized by cell volume. [Re-produced from [139] with permission from The Royal Society of Chemistry]

39

Our analyses revealed that J774 cells contain more Zn, Cu, and Mn, but less

Fe than neutrophils. HeLa cells displayed higher intracellular abundance of

Cu and Mn, and lower abundance of Fe. The Zn content was very similar to

that of neutrophils. In other word, neutrophils were consistently lower in

Mn, but higher in Fe. The Zn and Cu content was very similar to the other

cells, indicating that neutrophils are not purposely low in Zn. Similar

tendencies were observed when our results were compared to a comparable

analysis conducted with numerous cell lines [140]. While the low Mn content

can likely be explained by the low mitochondria count in neutrophils, con-

taining e.g. Mn-SODs, the high Fe content is probably resulting from the

enzyme MPO, which contains Fe-binding heme [13, 141].

Neutrophil nucleus, granules and void vacuoles show character-

istic element profiles

SR-XRF allows the mapping of elements at subcellular level. When analyzing

resting and activated neutrophils, typical morphological changes characteris-

tic of NETosis were reflected in the element maps of these cells. In resting

neutrophils, two major features stood out. The lobulated nucleus contained

higher P, Ca, Zn, and Fe than the cytoplasm, while it contained lower density

of S and Cu. Especially in the Fe map, subnuclear structures resembling po-

tential nucleoli as well as eu- and heterochromatin were clearly recognizable.

In the cytoplasm, Fe- and Zn-rich speckles were visible. Due to their size and

partial co-localization of both elements, those speckles most likely represent

granules. Expectedly, stimulation with PMA considerably affected neutrophil

morphology (Fig. 9). Among the most striking features were appearing ‘void

vacuoles’ – intracellular compartments that contained lower concentrations

of Ca, Zn, Fe, Cu, and potentially Mn. General low abundance of the latter

two elements impairs certainty though. Those ‘void vacuoles’ result from

pinocytotic events and are therefore likely to resemble features of an early

stage phagosome of engulfing neutrophils [142]. Morphological remodeling

of the neutrophil nucleus – loss of lobules and changing size - could be ob-

served in the element maps of activated neutrophils, even up to two-three h

40

post stimulation. Granular Fe/Zn speckles were detectable one h post infec-

tion, but nearly undetectable in later stages of NETosis, indicating the loss of

granular membrane integrity. Since SR-XRF detects elements independent

of their chemical status, we investigated the fate of labile Zn, also known as

loosely- bound or associated Zn, by FluoZin-staining neutrophils and follow-

ing them after PMA stimulation by life cell microscopy until they release

NETs (ca. three h)*. Again, the nucleus and cytoplasm were distinguishable.

Void vacuoles, low in labile Zn, appeared as well. Most interestingly, we ob-

served Zn-rich small and round structures appearing that increased in num-

ber and intensity (i.e. concentration of Zn) over time. They stayed partially

associated with the cell debris, even after NET release. Therefore might

therefore contribute to the typical shape of cell residue after NET formation.

NETs themselves were not detectable by FluoZin. Like in the SR-XRF analy-

sis of NETs, NETosis was induced in a low-Zn cell culture medium, so NETs

were likely not loaded with Zn upon release. Altogether, this data indicates

that neutrophils aim to starve microbial pathogens, intra- and

extracellularly. Further, the Zn-rich vesicles appearing in later stages of

NETosis might contribute to an active removal in Zn from the cytoplasm,

before neutrophils open up to release NETs.

* www.rsc.org/suppdata/mt/c4/c4mt00346b/c4mt00346b1.avi

41

Figure 9: Element distributions (P, S, Ca, Zn, Fe, Cu, and Mn) of two acti-vated human neutrophils Neutrophils were stimulated with PMA for 1 h, high pressure frozen, cryosubstitued in resin and sliced in 2 μm thin sections before deposition onto a Si3N4 wafer. Image area is 15 μm (hor.) x 11.2 μm (vert.); step size is 50 nm and 400 ms dwell time/point. The RGB image represents a colored overlay of the elemental intensities corresponding to Ca (red), Zn (green) and Fe (blue). The additional image is a corresponding TEM image from a similar but not exact same area of the grid (2500x magnification, lower right). Beamline: ID22NI, ESRF, Grenoble. [Re- pro-duced from [139] with permission from The Royal Society of Chemistry]

42

Paper III

Neutrophil antimicrobial effectors are not induced by C. albicans

Neutrophils are transcriptionally more active than widely believed. Their life

cycle rather contains a low-transcription phase in circulation. In the tissue,

stimulated miscellaneously, these phagocytes are capable of producing a

number of cytokines - the ‘second transcriptional burst’. Effector proteins

are produced during granulopoesis in the bone marrow [1, 18].

On these grounds, it was no surprise that most archetypical neutrophil effec-

tor genes were unaffected in an in vitro infection with C. albicans. Having a

closer look, some effector genes were actually transcribed to remarkable

extent, even in uninfected neutrophils. Most dominantly, the genes coding

for the heterodimer calprotectin were very strongly transcribed (Tab. 3).

Together, the transcription of only S100A8 and S100A9 corresponds to

> 6 % of the entire transcription (sum of rpkm). And indeed, calprotectin is

known as the most abundant cytosolic protein in neutrophils – it constitutes

60 % of total cytosolic protein. Other genes with high transcription rates are

coding for components of the NADPH complex (NCF2, NCF4 and RAC2),

extracellular proteases (LYZ and MMP9), and another S100 protein

(S100A12). In the group with medium transcription, we detected genes for

proteins of the NADPH complex (NCF1 and CYBA) and the heme-containing

catalase (CAT). Genes transcribed with a low rate were coding for additional

components of the NADPH complex (RAC1 and CYBB), the antimicrobial

peptide cathelicidin (CAMP) and the alarmin amphoterin (HMGB1). No, or

nearly no transcripts were detected for most granular proteins. Amongst

those are e.g. genes for elastase, myeloperoxidase, lactotransferrin,

defensins, or collagenase. In other words, the only granular proteins that are

continuously transcribed in circulating neutrophils were lysozyme C and

gelatinase B.

43

Table 3: Consistency in neutrophil effector protein transcription

Neutrophils were infected with C. albicans yeasts and hyphae. The transcription

rate of typical neutrophil effector genes, reflected by the average rpkm of resting and infected cells, is displayed and ranked: ++++/dark grey = very high, +++/grey = high, ++/pale grey = normal, +/light grey = low, -/white = not de-tected or irrelevant.

gene effector protein average rpkm rated

S100A9 S100A9 17890 ++++

S100A8 S100A8 4721 ++++

NCF2 NADPH oxidase, p67 669 +++

LYZ lysozyme C 435 +++

S100A12 S100A12 372 +++

NCF4 NADPH oxidase, p40 293 +++

MMP9 gelatinase B 258 +++

RAC2 NADPH oxidase, Rac2 236 +++

CYBA NADPH oxidase, p22-PHOX 115 ++

NCF1 NADPH oxidase, p47 62 ++

CAT catalase 51 ++

RAC1 NADPH oxidase, Rac1 25 +

CAMP cathelicidin, LL-37 25 +

CYBB NADPH oxidase, gp91-PHOX 16 +

HMGB1 high-mobility group protein 1/amphoterin 8 +

TCN1 transcobalamin-I 4 -

CRISP3 cysteine-rich secretory protein-3 1 -

LTF lactotransferrin 1 -

ELANE neutrophil elastase 0 -

DEFA1B defensin 1 0 -

DEFA1 neutrophil defensin 1 0 -

MMP8 neutrophil collagenase 0 -

CTSG cathepsin G 0 -

MPO myeloperoxidase 0 -

PRTN3 proteinase 3 0 -

BPI bactericidal/permeability-increasing protein 0 -

DEFA3 neutrophil defensin 3 0 -

DEFA4 neutrophil defensin 4 0 -

DEFA5 defensin 5 0 -

DEFA6 defensin 6 0 -

44

This observation correlates with the fact that gelatinase B is expressed very

late in granulopoesis, at the band cell stage and in the segmented neutrophil.

MPO, elastase, defensins and lactotransferrin are produced at earlier stages

[143].

Neutrophil transcriptional response to C. albicans contains few

metal retaining genes

In paper III, we investigated the impact of the C. albicans morphotype on

the interaction with neutrophils and NETs. In neutrophils infected with

Candida, we identified a total of 318 DEGs throughout the course of infec-

tion. The majority was regulated similarly in response to yeasts or hyphae.

Still, a group of 36 genes was identified to be induced or repressed stronger

in the presence of Candida hyphae. We dissected the neutrophil transcrip-

tion response with respect to genes that play a role in nutritional immunity.

Since oxidative stress is very tightly connected to metal homeostasis, genes

from both groups were considered. As described in paper III, the gene func-

tion was assigned using the online databases GeneCards and NCBI.

As stated earlier (see page 42), genes for antimicrobial effector proteins were

either not transcribed or constitutively expressed at high levels. Remarkably,

we identified a confined number of DEGs in Candida-infected neutrophils

that are functionally related to nutritional immunity (Tab 4). Four induced

DEGs are involved in the detoxification of ROS. They are namely the oxida-

tive stress response 1 gene OXSR1, the glutathione peroxidase 1 gene GPX1,

the oxLDL receptor gene OLR1, and the gene for the iron-sulfur cluster as-

sembly 1 ISCA1. Especially the protein coded by OLR1 is a very versatile pro-

tein. One of its functions is to bind oxidizes lipids resulting from intracellular

ROS and mediate a cell response, but is also described to have a role in cell

adhesion. The hemoglobin delta gene HBD was repressed in neutrophils

infected with C. albicans hyphae. The coded protein is part of hemoglobin

A2, which is the rarer form of hemoglobin A in adult humans. Other hemo-

globin genes (HBA1, HBA2, and HBB) were transcribed at high levels, but

not differentially expressed (data not shown). Interestingly, we found a Zn

transporter gene to be induced in the response of neutrophils to C. albicans

45

hyphae: SLC30A6. The gene product, the solute carrier family 30 member 6,

transports Zn from the cytosol into the trans-Golgi network and vesicular

compartments. The up-regulation of SLC30A6 is especially interesting con-

sidering the findings from paper I and II. We showed that cytosolic

calprotectin, which is released during NETosis, is the key effector protein

during NET-mediated inhibition of A. nidulans confirming our earlier study

with C. albicans.

Table 4: Nutritional immunity DEGs in Candida-infected neutrophils

Neutrophils were infected with C. albicans yeasts and hyphae. The fold change of

transcription (log2) related to unstimulated neutrophils is displayed. Genes with a fold change above the threshold of 21 are highlighted with a grey background (DEGs). Fold changes excluded due to statistical uncertainties (cpm threshold) are not considered: n.c.

Fold change in neutrophils (log2)

Gene 15 min 30 min 60 min 15 min 30 min 60 min Me/Ox

HBD 0.06 -0.12 -0.07 -1.88 -2.58 -2.30 Fe

GPX1 0.03 0.37 1.59 -0.11 0.54 n.c. Ox

ISCA1 -0.73 0.29 1.90 -0.74 0.47 1.83 Ox

OLR1 n.c. n.c. 5.45 n.c. n.c. 5.20 Ox

OXSR1 0.23 0.25 1.76 0.18 0.20 1.73 Ox

SLC30A6 n.c. 0.61 0.51 3.29 3.45 2.94 Zn

C. albicans yeasts C. albicans hyphae

Both fungi were rescued from NET inhibition by supplementation of Zn [38,

137]. In paper II, the Zn landscape of resting and activated neutrophils was

analyzed by XRF, ICP-MS and FluoZin-based life cell imaging. Interestingly,

we identified vesicular compartments containing labile Zn in neutrophils

upon stimulation with PMA. Further, we demonstrated the actual reduction

of available Zn in the supernatant of NETs for the first time. Considering that

the up-regulation of SL30A6 is a specific response towards hyphae of

C. albicans and that NETs are preferentially released towards hyphae, this

data suggests the following: Upon NET-inducing stimulation, neutrophils

remove labile Zn from their cytosol via Zn transporters like the product of

SL30A6 and store it in vesicular compartments, possibly zincosomes, to

reduce Zn release during NETosis cell burst. In resting cells, free Zn is very

limited. Yet during the major intracellular rearrangements that accompany

NETosis, precaution might be necessary. For full functionality, it is crucial

that calprotectin remains unsaturated with Zn before NET release. Only

46

then, the extracellular calprotectin can chelate Zn from the surroundings and

possibly from cell residues, rendering it less available for microbial patho-

gens [60, 99, 137, 139, 144-146].

C. albicans response to neutrophils and NETs includes metal ac-quisition genes

The transcriptional response of C. albicans yeasts and hyphae was analyzed

for genes whose products are likely to be involved in aspects of nutritional

immunity (NI) - relevant during the interaction of the pathogen and the

phagocyte. In paper III, we only considered genes with a fold change > 4 as

DEGs. In the following, genes with a fold change > 2 will be discussed, too.

It is evident, that most of the NI genes affected are only differentially ex-

pressed in yeasts (Tab. 5). Among those, the genes of the C. albicans

zincophore system were induced in yeasts very early in the infection of neu-

trophils (PRA1, ZRT1), but also when challenged with NETs. The orf19.6555

was only induced in neutrophil-encountering yeasts. Orthologues of this

gene code for mitochondrial Zn transporters. In addition to Zn, Cu homeo-

stasis was clearly affected in neutrophil-encountering yeasts. Ferric

reductase genes, catalyzing the initial step of Cu uptake, were induced in

yeasts (FRE3, CFL11, orf19.7077). ATX1, the gene for the intracellular Cu

metallochaperone, is strongly repressed in yeasts. In the secretory Cu path-

way of eukaryotic cells, Atx1p delivers Cu to ATPases of the trans-Golgi net-

work to mediate export from the cytosol [129]. Two such ATPase genes were

affected in neutrophil-challenged Candida yeasts: CCC2 and CRP1 were in-

duced and repressed, respectively. Ccc2p is a described as an ATPase pump-

ing Cu into the Golgi network, while Crp1p is considered to pump Cu out of

the cell. The metallothionein gene CRD2 was induced in yeast, but repressed

in hyphae. In summary, although Cu-related gene regulation is not an explic-

it reflection of a high or low Cu concentration, it certainly documents a dis-

turbance in Cu homeostasis and oxidative stress, the latter being indicated

by the presence of three different ferric reductases (personal correspondence

with Prof. Simon Labbé, Université de Sherbrooke, Canada; and [147]).

47

A third important metal homeostasis is concerning Fe. Four related genes

were differentially expressed in the infection. Three of those genes code for

proteins involved in heme uptake and metabolism; they either were only

induced in hyphae (PGA7) or repressed in yeasts (orf19.1034, HEM15). In-

terestingly, FTH1, the gene for a putative vacuolar Fe transporter, was in-

duced only in hyphae, but not in yeasts. Finally, a number of oxidative stress

detoxification genes were also identified. Most of them were induced in

yeasts and hyphae (SOD3, SOD4, SOD5, GCS1, IFR1, and OYE32). Of note,

IFR1 and OYE32 are described to be induced upon nitric oxide stress. TRP99

is the only potential ROS detoxifier gene repressed in neutrophil-challenged

Candida yeasts.

Table 5: Nutritional immunity DEGs in neutrophil-challenged C. albicans

Neutrophils were infected with C. albicans yeasts and hyphae. The fold change of

transcription (log2) related to Candida incubated in the same medium for the same time w/o neutrophils is displayed. Genes with a fold change above the threshold of 22 are highlighted with grey background (DEGs); genes with a fold change > 21 are highlighted in light grey.

Fold change in yeasts (log2) Fold change in hyphae (log2)

Gene

15

min

30

min

60

min

30

min

15

min

30

min

60

min

30

min Me/Ox

ATX1 -5.46 -5.56 -5.82 -0.12 -0.59 -0.62 -0.27 0.09 Cu

CCC2 2.68 2.74 3.36 -1.03 1.07 0.93 0.75 -0.09 Cu

CRD2 1.42 2.49 2.85 1.02 -1.69 -2.11 -2.13 0.80 Cu

CRP1 -4.76 -5.36 -5.54 0.63 -1.94 -2.22 -2.04 0.52 Cu

SOD5 2.25 2.61 1.84 0.95 0.91 0.83 0.56 0.15 Cu

CFL11 4.91 4.70 5.87 -0.60 -0.73 -1.13 -1.53 0.18 Cu/Fe

FET3 -1.45 -2.56 -1.19 -0.77 -0.24 0.32 0.83 -0.64 Cu/Fe

FRE3 1.68 2.13 2.40 -0.43 0.03 0.08 0.01 -0.65 Cu/Fe

orf19.7077 1.24 1.12 2.53 0.04 -0.65 -0.26 -0.12 0.24 Cu/Fe

SOD4 1.18 2.24 2.53 0.57 0.65 1.26 1.49 0.31 Cu/Zn

FTH1 0.47 0.48 0.24 0.19 1.41 1.60 2.31 0.85 Fe

HEM15 -1.28 -1.64 -2.09 -0.03 -0.35 -0.19 0.12 -0.11 Fe

orf19.1034 -2.01 -2.66 -2.77 0.65 -0.96 -1.15 -0.77 0.52 Fe

PGA7 0.33 0.35 1.04 0.27 1.49 1.32 2.19 1.17 Fe

SOD3 1.75 -0.68 2.98 0.55 -0.24 0.30 1.19 1.04 Mn

TRP99 -0.99 -1.51 -2.03 0.08 -0.49 -0.84 -0.33 0.57 Ox

GCS1 0.81 1.36 2.09 -1.36 0.27 0.17 0.34 -0.62 Ox

IFR1 2.12 2.36 2.46 1.07 2.97 1.76 1.21 1.50 Ox

OYE32 2.56 2.06 1.69 1.05 4.07 2.64 2.44 1.81 Ox

orf19.6555 2.92 3.03 2.30 0.02 1.07 0.94 0.86 -0.04 Zn

PRA1 3.32 1.03 1.49 1.77 -0.27 0.35 0.63 0.90 Zn

ZRT1 2.01 -0.04 0.34 1.45 -0.23 0.26 0.37 0.84 Zn

neutrophils NETs neutrophils NETs

48

Of all genes presented, only a small group was differentially expressed in

Candida challenged by NETs – none of those beyond the DEG threshold

used in paper III (4-fold). Weakly induced in yeasts were oxidative stress

response genes IFR1 and OYE32, the zincophore genes PRA1 and ZRT1, and

the metallothionein gene CRD2. Slightly repressed in yeasts were the Golgi-

ATPase gene CCC2 and the glutathione synthesis gene GCS1. Of those, IFR1

and OYE32 were slightly induced in NETs-challenged hyphae. The heme

acquisition gene PGA7 was found to be slightly induced in NETs-triggered

hyphae.

In summary, this transcription profile indicates that C. albicans encounter-

ing neutrophils and NETs faces not only oxidative stress, but also disturb-

ances in metal homeostasis. The urge to acquire Zn is evident; the Cu and Fe

homeostases need to be restored. Interestingly, NETs seem to trigger Zn

acquisition and an oxidative stress response in C. albicans yeasts.

49

Conclusions & Outlook

Neutrophils can effectively kill C. albicans yeasts and hyphae, by either di-

rect interaction or post mortem as NETs. Calprotectin was identified as the

key effector protein in NETs, and Zn chelation is most probably the main

mode of action [38, 81]. This indicates that neutrophils and in particular

NETs control fungi by applying the armory of nutritional immunity. In the

present thesis, the interaction of C. albicans with neutrophils and NETs was

investigated by RNA-sequencing with respect to metal acquisition and

transport. The trace element landscape of resting and activated neutrophils

and NETs was analyzed by combining SR-XRF, ICP-MS, and life cell imag-

ing. Finally, the universal antifungal potential of NET-associated

calprotectin was confirmed for A. nidulans. In conclusion, the results of pa-

per I, II, and III point to an important role of nutritional immunity aspects of

the Candida-neutrophil interplay. Neutrophils show potential to starve fungi

for metals in two ways: intracellularly after phagocytosis and extracellularly

by NETosis (Fig. 10). Further, hints were collected indicating the formation

and packing of intracellular zinc-retaining vesicles, possibly with the in-

volvement of the zinc transporter SLC30A6, that could drain the cytoplasm

of labile Zn before cells open up to release NETs. These vesicles might even

stay intact after NETosis and keep Zn separated from microbes after the

neutrophil lost integrity.

As of today, there are still many open questions that need to be addressed in

order to support our model. What exactly are the Zn-rich vesicles appearing

in stimulated neutrophils? Do they contain the transporter SLC30A6? Do the

same events occur in neutrophils infected with C. albicans yeasts and hy-

phae? Do these vesicles appear during phagocytosis of fungi, or only during

NETosis? Can phagocytosis and NETosis be separated, or do cells release

NETs even after fungi engulfment of fungal cells?

The importance of Zn during neutrophil interaction with pathogenic fungi is

supported by the findings of the papers included in this thesis. They shed

50

new light on the impact of ‘nutritional immunity’ during fungal infection, an

emerging field of research.

Figure 10: Model of intra- and extracellular Zn starvation by neutrophils

During infection, neutrophils encounter C. albicans yeasts and hyphae (upper left,

upper right). Yeast cell are preferentially engulfed into a low-Zn phagosome. Upon contact with hyphae, neutrophils can induce NETosis. During this, Zn-containing vesicles appear that potentially drain the cytoplasm from labile Zn. After NET re-lease, Zn might be retained in vesicle-withholding cell debris, while NETs are deco-rated with calprotectin to sequester Zn from invading microbes.

51

Acknowledgements

To reach this point in my life, it took the support and understanding of many

companions. The most important are recognized here, in (almost)

chronological order.

Zu allererst möchte ich meiner Kernfamilie danken. Liebe Mama, lieber

Papa, liebe Tini, lieber Bohni, lieber Opa! Ohne Euch wäre ich nicht da,

wo ich bin.

Dasselbe gilt für meine Freunde aus Nicht-Umeå, die mir über all die Jahre

die Stange gehalten haben. Katja, Sprinkie und Dani aus B-1162; Herr

Klein und Ulli aus der alma mater LSP; Hanna, Markus, Rhea,

Schweizi und Alex aus dem Storchen in TÜ!

Danken will ich auch den Mentoren, die meine ersten Gehversuche in der

academia begleitet haben: Julia Frick, Timo Birkenstock, Andreas

Peschel.

Und nun zu dir, Doktorvater Constantin! Es fällt natürlich schwer, die

vergangenen Jahre kurz und bündig zusammen zu fassen. Nichtsdestotrotz

gibt es ein paar Dinge, die besonderen Dank meinerseits verdienen. Allem

voran danke ich dafür, dass du deine wissenschaftlichen Ansprüche auch in

schweren Zeiten nicht verraten hast. Das ist beeindruckend. Außerdem

gebührt dir Dank für die viele Freiheit, die ich hatte und habe, und das große

Vertrauen, das du mir vom ersten Tag an entgegen gebracht hast. Und

natürlich für das Verständnis und Interesse an all dem privaten Kram, der

sonst noch so passiert ist in sechs Jahren Nordschweden.

Which brings me to the CU lab. Thank you guys for quite a ride. David for

really being there in the beginning, smoothing the transition from the PCI to

MolBiol, and teaching me. Thank you, Marios, for being the sunshine of the

group. Thx, Pedro, for being the superglue of the group. Thank you, Ava,

for raising the bar. And thank you Marc, Anna, Erik, Madhu, and

Sandra for completing the gang.

Thanks to my co-supervisor Bernt Eric Uhlin and the journal clubs

SvB/UvPR/CL/LS and AS for honest interest in my projects.

Thanks to all collaborators involved in the projects mentioned in this little

book. Chrissi, Jan, and Björn, you have been true allies.

52

Frau Dr. Meyer (Fräulein Meyer a.D.), du bist die beste Freundin-

Kollegin-MitMutter-Hybridin, die ich mir hätte wünschen können. Danke

dafür.

Barbara, Sophie, Michaela, Katrin, fellow DAs Maria and Mari,

Artur und Kristin! Me reaching this point in life mentally sane is also your

merit. DANKE/MERCI/TACK.

Zu guter Letzt noch ein paar Worte für den ganz harten Kern: Moritz, ABER

ich liebe Sie! Herja und Warin, ihr seid der schönste Grund, meine

heiligen frühen Arbeitsstunden zu opfern und die beste Motivation, die

verbleibenden Stunden sinnvoll zu nutzen. Euch allen, danke für Eure

Geduld und die Freude beim Nach-Hause-Kommen.

HORRIDO!

53

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