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C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation

Wevers, B.A.

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Citation for published version (APA):Wevers, B. A. (2014). C-type lectin signaling in dendritic cells: molecular control of antifungal inflammation.

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16 Chapter one

C-type lectin receptors orchestrate antifungal immunity

one.

Brigitte A. Wevers, Teunis B.H.

Geijtenbeek and Sonja I.

Gringhuis

Department of Experimental Immunology, Academic Medical

Center, University of Amsterdam, Amsterdam, NL.

Future Microbiology, 8; 839-854 (2013) – published in modified form

Fungal infections are an emerging threat for human

health. A coordinated host immune response is

fundamental to successful elimination of an invading

fungal microbe. A panel of C-type lectin receptors

expressed on dendritic cells enables innate recognition

of fungal cell wall carbohydrates and tailors adaptive

responses by presenting antigen as well as instruction of

CD4+ T helper cell fates. Well-balanced T helper cell type

1 and interleukin-17-producing T helper cell responses

are crucial in antifungal immunity and facilitate

phagocyte clearance of fungal encounters. Strikingly,

different classes of fungi trigger distinct sets of C-type

lectin receptors to evoke a pathogen-specific T helper

response. In this chapter we have outlined the key roles

of several C-type lectin receptors during the generation

of protective antifungal immunity, with special emphasis

on the distinct signaling pathways and transcriptional

programs triggered by these receptors, which collaborate

to orchestrate polarization of the T helper response.

- an introduction

C-type lectin receptors orchestrate antifungal immunity 17

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Fungi are ubiquitous in the environment. Some fungi, including Malassezia species (spp.),

Candida spp., and Pneumocystis jirovecii (formerly Pneumocystis carinii) have successfully

established life-long commensal relationships with the human host, and colonize cutaneous

and mucosal surfaces without necessarily causing disease1. Even, fungal microbes are being

recognized as intestinal commensals (referred to as the mycobiome) that strongly interact

with the gut immune system2. Pathogenic fungi take advantage of an altered state of host

immunity to cause (lethal) opportunistic infections, with a rapidly growing population of

immunosuppressed patients at risk3. Commensal fungal-derived ligands (i.e. β-1,6-glucans)

can also drive chronic clonal expansion of mature B cells, and, in doing so, might contrib-

ute to the pathogenesis of B cell chronic lymphocytic leukemia (B-CLL)4. Although largely

unrecognized, this view of fungal epidemiology is dramatically challenged by the grow-

ing incidence of fungal diseases in seemingly healthy individuals. Emerging pathogenic

fungi, such as Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis,

have developed many elaborate mechanisms to overcome host immune surveillance

and establish primary and life-threatening infections1. Highly virulent Cryptococcus gattii

genotypes have gained recent prominence following a major and ongoing outbreak of fatal

cryptococcal meningitis in western North America5.

Resisting fungal infection. The human immune system is equipped with effective defense

mechanisms to mediate protection to fungal infection, yet activation of these responses

requires the coordinated activation and complex interplay of specialized types of immune

cells. Hence, host immunity has to accomplish a challenging task: maintaining tissue

homeostasis by eradicating invading fungi that can cause harm, while preventing immu-

nopathology and tolerating the commensal fungal strains being important for our health6,7.

The human immune system comprises two arms that complement one another: innate

immunity (‘natural’ immunity) and adaptive immunity (‘acquired’ immunity). The innate

system facilitates immediate but non-specific host defense mechanisms against microbial

infection8. Skin and epithelial surfaces act as physical barriers of protection, and at mucosal

tissues, mucus layers and immunoglobulin A (IgA) secreted by plasma B cells work in concert

to prevent tissue invasion by fungal pathogens9-11. Innate effector cells residing in skin and

mucosa, such as interleukin (IL)-17-producing innate lymphoid cells (ILCs) and epithelial

cells, further contribute by producing antimicrobial peptides12. When fungal pathogens

successfully breach host barriers, rapidly recruited phagocytic cells, including neutrophils

and macrophages, facilitate immune protection during the earliest stages of infection and

mediate local fungal elimination13,14. Despite these effective immune mechanisms, the innate

system lacks specificity or the ability to generate immunological memory and life-long pro-

tection. These tasks are accomplished by the effector B and T lymphocyte populations from

FUNGAL PATHOGENS: FAR BEYOND COMMENSALISM & OPPORTUNISTIC INFECTION

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18 Chapter one

1. Pattern recognition

IL-12

IL-27IL-21

IL-1βIL-23IL-6

Mature DC

Dendritic cell

Costimulation

GranzymePerforin

IL-10TGF-β

MHC II

MHC I

IL-2IFN-γ

IFN-I

IL-2IL-12

IL-22IL-17

IL-4IL-13

NaiveCD4+

T cell

TH2 cell(GATA3)

T reg(Foxp3)

TH17 cell(RoRγT)

TH cell(T-bet)

Memory cells

CD8CD8

EffectorCTL

NaiveCD8+

T cell

IL-2(TH1)

Phagocyte activation

Mucosal homeostasis

TGF-β

NaiveB cell

Memory cellsB cell

B cell

Plasmacell

IgG2a IFN-γ (TH1)

IgG1

IgE

IL-4 (TH2)

Immunity to fungi and bacteria

Immunity to fungi,intracellular pathogens

Regulation,tolerance

Immunity to extra-cellular parasites

B cell help

TFH cell(Bcl-6)

IL-4

3. Migration to central lymphoid organs

2. Maturation

PAMPsDAMPs

Cytokines

TGF-βIL-2

Box 1. Pathogen-specific lympocyte populations.

Upon delivery of their cognate antigen by DCs,

in addition to receiving co-stimulatory and

cytokine signals, naive T cells in secondary

lymphoid organs become activated and

differentiate into effector lymphocyte

populations -each with specific functions and

gene expression programs for appropriate

elimination of different types of microbes.

Th1 and Th2 cells are the founding members

of the effector or ‘helper’ CD4+ T cell subset

family, discovered more than 25 years ago150.

Ever since, numerous other heterogeneous

Th subsets have been characterized – the

three most prevalent being Th17 cells that

produce IL-17151, T regulatory (Treg) cells33

and T follicular helper (Tfh) cells152. Through

actions of lineage-specific transcription

factors, CD4+ T cells differentiate into effector


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the adaptive immune system, characterized

by lineage-specific effector molecules and

regulate host immunity in a pathogen-specif-

ic manner (Box 1). Not surprisingly therefore,

B and T cells play a central role in providing

optimal protection to insults by fungal patho-

gens15,16, with the fundamental importance

of effector CD4+ T cells dramatically exem-

plified by the exceptionally high frequency

of life-threatening cryptococcal infections

in HIV-1/AIDS patients with declined CD4+ T

cell numbers17.

Two functionally distinct CD4+ T cell

subsets are considered key to effective fun-

gal microbe elimination: T helper cell type

1 (Th1) and IL-17-producing T helper (Th17)

cells. Th1 and Th17 responses can be induced

in parallel, yet, although still considerably

uncertain, their degree of contribution is pre-

sumably context dependent, e.g. pathogen-

and/or tissue-specific18: Th1 cells take part

in the cellular defense, important during dis-

seminated disease, and orchestrate optimal

macrophage activation, whereas Th17 cells

predominantly maintain barrier immunity

at mucosal surfaces and act on neutrophils.

These effector T cells secrete cytokines to

mediate their influence on other immune

cells during an antifungal immune response.

Th1 cells secrete interferon-γ (IFN-γ), which

triggers a plethora of systemic effector mech-

anisms, such as antibody class switching to

opsonizing subtypes, upregulation of MHC

molecules for enhanced antigen presenta-

tion, and stimulation of macrophage effector

functions (e.g. production reactive oxygen

intermediates)19. IFN-γ might also directly

affect fungal growth, as it inhibits the yeast-

to-hyphal transition in C. albicans20. The Th17

subsets that secrete restricted patterns of

cytokines and express different chemokine

receptors, ensuring tailored responses to the

type of threat encountered153,154. Th1 cells are

dedicated to efficiently combat intracellular

bacteria and viruses, by producing IFN-γ,

while Th2 cells produce IL-4 and IL-13 for

defense to extracellular parasites155. The Th17

cell subset, on the other hand, selectively

produces IL-17, providing protection to fungal

and bacterial threats21. The population of

Treg cells, producing cytokines TGF-β and

IL-10, plays a crucial role in the maintenance

of immune homeostasis156. Tfh cells represent

another effector CD4+ T cell population

with a specialized function: Tfh cells help

B cells generate antibody responses to T

cell-dependent antigens for clearance of

pathogens by phagocytes or the complement

system157. Effector CD8+ T cells, cytotoxic T

cells -in contrast to helper CD4+ T cells not

further subdivided- produce multiple effector

molecules, such as perforin and granzyme,

and are specialized in destroying virally

infected cells and/or tumor cells158. About

10% of the effector CD4+ and CD8+ T cells

acquire a memory phenotype: quiescent

long-lived central memory T cell (Tcm) and

effector memory T cell (Tem) populations

that are able to quickly respond to antigen

re-exposure159. Memory B cell generation,

in addition to isotype switching to IgG, IgA

and IgE subtypes, depends on cytokine

present in the local environment (i.e. Th2-

derived IL-4 induces IgE class-switching)160.

Although Th cells have been considered

terminally differentiated immune cells, this

view is considerably challenged in recent

years, with many examples of Th cells

flexible in their cytokine production profile,

and hence their effector phenotype153.

Intr

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20 Chapter one

effector molecule is an important mediator of tissue inflammation: IL-17 acts on a broad range

of immune and non-immune cells and is key to the recruitment, migration and activation

of neutrophils21. Th17 cells are also an important source for IL-22, which promotes, together

with IL-17, production of protective antimicrobial peptides to mediate mucosal microbial

resistance22,23 (Box 1). The protective role of Th17 responses during fungal infection is under-

scored by the severe recurrent and chronic Candida infections in patients with genetic defects

in the Th17 axis, including individuals suffering from chronic granulomatous disease (CGD)

and hyper IgE syndrome24-30. Similarly, Th17 cells and IL-17 have been shown to mediate

protection in numerous experimental mouse models of fungal infection31,32. Paradoxically,

exaggerated antimicrobial Th17 responses are often associated with tissue damage. The

magnitude of pathogenic Th17 cell activity as well as unwanted Th17 responses directed

against commensal fungi can be kept in check by regulatory T (Treg) cells, the natural

gatekeepers of immune homeostasis33. In any case, finely tuned Th1 and Th17 responses

probably maximize fungal elimination and, at the same time, minimize host tissue damage

during inflammation (recent reviews on this topic have been published elsewhere:16,34,35).

Instruction of the adaptive (antifungal) response is coordinated through the actions of

specialized antigen-presenting cells (APCs), principally dendritic cells (DCs), which provide

all signals necessary for naive T cells to acquire an effector phenotype: T cell receptor (TCR)

stimulation, co-stimulation and cytokines36, thereby contributing to T cell-dependent B cell

help and generation of antibody responses.

DCs reside in the periphery or circulate through blood and monitor for signs of microbial

attack, but also host derived danger signals released in response to stress, tissue damage

and necrotic cell death. Being specialized in sensing conserved microbial structures termed

pathogen-associated molecular patterns (PAMPs) and damage-associated molecular

patterns (DAMPs), through pattern recognition receptors (PRRs), DCs can discriminate

between different classes of potential danger37,38. PAMPs refer to molecules associated

with pathogenic and non-pathogenic microbes, such as cell wall components and nucleic

acids of fungal, bacterial and viral origin, while DAMPs are endogenous molecules released

upon stress or damage to the host: amongst others high-mobility group protein 1 (HMGB1),

heat-shock proteins (HSPs), extracellular ATP and uric acid crystals. Immature DCs capture

and internalize pathogens or self-molecules, simultaneous encounter with PAMPs/DAMPs

induces a cascade of phenotypical changes. This so-called maturation process entails

upregulation of costimulatory molecules, lymphoid tissue homing receptor CCR7, and major

histocompatibility complex (MHC)-antigen complexes, allowing DCs to activate naive T cells

in central lymphoid organs. Responding T cells start to proliferate and differentiate, and, as

DENDRITIC CELLS AND THE GENERATION OF ANTIFUNGAL IMMUNITY


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distinct effector populations, rapidly enter sites of local inflammation were they perform

their effector function to aid in pathogen-specific clearance (Box 1).

Pattern recognition receptors. Instruction of the adaptive response by DCs is subject to

tight regulation, and this process is dependent on a large panel of germ-line encoded

PRRs39. DCs express a large variety of membrane- and cytoplasmic-localized PRRs,

including the archetypical Toll-like receptors (TLRs) and C-type lectin receptors (CLRs)

and NOD-like receptors (NLRs) (further discussed in Box 2). To coordinate DC-induced

inflammatory responses, PRRs control four crucial processes. First, PRRs allow DCs

to discriminate between different classes of PAMPs and DAMPs, and as such ‘license’

them to drive pathogen-specific responses37. Moreover, DC-expressed PRRs facilitate

internalization and processing of pathogen-derived antigens for subsequent antigen

presentation in the context of MHC molecules. In addition, a selective set of PRRs induc-

es intracellular signaling for activation of two additional processes: transcriptional

activation of a core set of innate response genes, leading to expression of co-stimula-

tory molecules, chemokines and cytokines40,41; and the assembly of cytosolic protein

complexes, inflammasomes, for posttranslational processing of IL-1β family members42

(Figure 1). Since the local cytokine milieu created by dendritic cells is instrumental to

the fate lineage decision of differentiating CD4+ T helper cells36,43, PRR-induced signal-

ing is crucial for clonal expansion and differentiation of a responding antigen-primed

T cell population.

Fundamental to the expression of many inflammatory cytokine and chemokine genes

is the activation of the nuclear factor-κ B (NF-κB) family of transcription factors, which are

designated as central coordinators of the innate immune response. NF-κB homo- and

heterodimers are retained inactive within the cytoplasm by inhibitory proteins of the IκB

family. Upon a PRR-mediated signal, the IκB inhibitory complex is degraded, and subse-

quently initiates release and nuclear translocation of NF-κB dimers44. In addition, PRRs also

activate other transcription factors, such as transcription factor activator protein-1 (AP-1)

for expression of cytokines and chemokines, as well as numerous interferon regulatory

factors (IRFs) for induction of type I interferon (IFN-I) responses.

Tailoring T helper responses to fungal infection. The local cytokine milieu created by DCs

is instrumental to the fate lineage decision of differentiating CD4+ T helper cells36,43; cyto-

kine actions involve direct induction or repression of a lineage-specific transcription

factor or essential growth factor(s). Regarding activation and maintenance of human

antifungal Th1 and Th17 effector subsets, several cytokines are considered of crucial

importance. Th1 cells differentiate from naive T cells in response to DC-derived IL-1245.

IL-12 binding to its cognate receptor (IL12R) on activated CD4+ T cells triggers, via STAT4,

transcription of the lineage-specific transcription factor T-bet46, which mediates the

Intr

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22 Chapter one

ii Antigen presentationi Pattern recognition

TLR5 TLR4

Endosome

CpG DNA

dsRNA ssRNA

TLR3

MincleSap1

30

TLR9 TLR7/TLR8

Dectin-1

LPS β-glucanMannose

Dectin-2CLRsTLRs

PAMPsFungi

Bacteria

Viruses

Necrosis

DAMPs

RLRs NLRs

AIM2IFI16

RIG-I MDA5

ds/ssRNA

NOD2

MDP

NLRP3

dsDNA

PYHINs

Flagellin

Host cell death

Golgi

Surface

Surface

Lysosome

Endosome

MHC IIloading

Endogenousproteins

MHC I loading

MHC I

PhagosomePhagosytosis

MHC II

Proteasome MHC I MHC II

ER

Figure 1. Four principal roles of pattern recognition receptors (PRRs). (i) Innate immune cell-associated

PRRs recognize distinct types of pathogen associated molecular patterns (PAMPs) or damage-associated

molecular patterns (DAMPs), allowing instruction of tailored adaptive immune responses. Numerous

PRR families have been characterized; while some are stationed at cell membranes, such as Toll-like

receptors (TLRs) and C-type lectin receptors (CLRs), others are located within the cytosol. RIG-I-like

receptors (RLRs), NOD-like receptors (NLRs) and PYHIN sensors. Prominent PRR family members and

their cognate ligands are depicted in the figure. (ii) PRRs facilitate internalization and/or processing of

peptide-derived antigens for presentation in the context of major histocompatibility (MHC) class I and II I

secretion of IFN-γ. Human Th17 cell fate determination involves multiple cytokines: IL-6,

IL-23, IL-1β, IL-21 and TGF-β21, although debate continues regarding requirement and

primary source of the latter. Th17 development is dependent on transcription factors

STAT3 and RORγt. Signaling by DC-derived IL-6 and IL-23 directly activates STAT3

and subsequently RORγt. IL-21, another STAT3 activator, is expressed by Th17 cell and

promotes maintenance of Th17 differentiation via an autocrine route. IL-1β functions

during early and late stages of Th17 cell commitment, possibly by counteracting the

inhibitory effects of IL-12 and IL-10 on Th17 differentiation47. Th17 cells produce the


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iii Gene transcription iv Inflammasome activation

Pro-IL-1βPro-IL-18

C/B/Mscaffold

SykROSK+ efflux

Canonical

Cytokinerelease

Non-cannonical

Pro-caspase-1

NLRP3ASC

ASC

Pro-caspase-8

Dectin-1

Mytochondria

Stress signals

Processing

IL-18

IL-1βCaspase-1Caspase-8

PAMPsDAMPs

TLR4

Myd88TRIF

CD14

TRIF

MAVS RIP2

Syk

IRFsType I IFNs

TAK1TBK1

ChemokinesCytokines

Antimicrobial peptidesCostimulatory molecules

AP1NF-kB

IRAK

RIG-I

MDA5

NOD1/2

Dectin-1

TLR3

TRAFs

molecules to naive CD4+ and CD8+ T cells, respectively. (iii) Several PRRs transduce intracellular

signaling upon their activation, leading to transcriptional activation of numerous innate response

genes. TRAF adaptor proteins account for integration and diversification of PRR signaling for activation

of different transcription factors. (iv) Furthermore, PRRs can mediate the assembly and activation of

cytosolic protein complexes -caspase-1-containing canonical or caspase-8-containing non-canonical

inflammasomes- for posttranslational processing and maturation of cytokines from the IL-1β family.

C/B/M, CARD9-Bcl-10-MALT1; dsDNA/RNA, double-stranded DNA/RNA; MDP, muramyl dipeptide; IFNs,

interferons; ROS, reactive oxygen species; ss-RNA, single-stranded RNA.

signature cytokines IL-17A (referred to as IL-17), IL-17F and IL-2221 (Box 1, with Figure

1 in Chapter 6 providing a more comprehensive overview). Foxp3-expressing Tregs

restrain uncontrolled chronic Th1 and Th17 effector responses deleterious to the host

and exist as a mature T cell subpopulation in the periphery (natural (n)Tregs), but can

also be induced from naive CD4+ T cells by IL-2. Induced regulatory T cells (iTregs)

acquire suppressive activity in response to transforming growth factor-β (TGF-β)48.

Thus, DCs are masters in command of an army of lymphocytes and hence shape the

adaptive arm of an ensuing antifungal inflammatory response. Depending on the

Intr

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24 Chapter one

fungal species encountered as well as the host cell type, specific PRRs will be activated,

which elicit distinct downstream signaling events that collectively determine the overall

adaptive response tailored to the encountered microbes.

The fungal cell wall composition is dynamic and highly variable, yet consists of a multitude

of putative and unique PRR ligands49. The core structure is dominated by polysaccharides,

comprising mainly β-1,3-glucan, β-1,6-glucan and chitin polymers, surrounded by a layer

enriched in mannosylated glycoproteins50. Phospholipomannan, α-glucans, and galac-

tomannan constitute cell wall components found in only a minority of fungi51. Fungal

components can be recognized by more than one receptor (e.g. β-glucan recognition by

both langerin and dectin-1), resulting in differential responses, and certain PRRs transduce

divergent intracellular signaling pathways upon binding distinct ligands; exemplified by

the mannose- and fucose-based signaling induced by DC-specific ICAM-3-grabbing non-in-

tegrin (DC-SIGN) with pro- or anti-inflammatory outcomes, respectively (further discussed

below). Notably, as we demonstrate in Chapter 2 of this thesis, even closely related strains

within one taxonomic group can differentially trigger innate receptors on dendritic cells52.

In sum, expression of a plethora of innate PRRs permits the host immune system to mount

an effective, and above-all, tailored antifungal adaptive response.

TLRs, among the most well characterized PRRs, have been assigned function in the anti-

fungal immune response. Fatal Aspergillus fumigatus infections observed in Toll-deficient

Drosophila provided an initial link between TLR components and antifungal immunity53. In

mammalian studies with murine infection models, TLRs were found to have critical roles in

both innate recognition and driving protective responses54,55. Strikingly, however, the control

of antifungal defense in men is not dominated by any of the TLR members. Humans with

genetic defects in the universal TLR adaptor molecule -shared as well by the IL-1 receptor

(IL-1R) and IL-18R- MyD88 are highly susceptible to bacterial, but not fungal infections56,57.

Strictly under conditions of severe immunosuppression, single nucleotide polymorphisms

(SNPs) in human TLR1 and TLR4 genes predispose to infection with fungi57,58. Also, human

TLRs are considered incapable to autonomously elicit robust Th17 skewing52. During fungal

infection, engagement of most TLRs potentiates strong IL-12p70 production, and thus favors

Th1 polarization16,59, although Treg activation by TLR2 is considered an exception60. Murine

TLRs exhibit some potential to augment Th17 responses61, possibly reflected by their ability

to activate transcription factor NF-κB subunit c-Rel (discussed below) in some instances, in

marked contrast to their counterparts in men52,62. Thus, at least in the human setting, TLRs

cannot be held solely responsible for the control of fungal elimination. Presumably, TLRs

contribute to fungal binding and operate as co-stimulators that promote or repress signals

SENSING FUNGAL INVASION: C-TYPE LECTIN RECEPTORS TAKE CENTER STAGE


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by other PRRs to shape the overall antifungal response35.

Emerging evidence indicates that the control of human antifungal defense is instead

dominated by C-type lectin receptors (CLRs; Box 2). Langerin and mannose receptor (MR) are

CLR sensors for fungi with endocytic activity and facilitate or, in the case of MR, contribute

to phagocytosis of fungal particles63-66. These receptors subsequently direct delivery of the

fungal cargo into the appropriate phagosomal route; internalized fungi are either processed

for antigen presentation, intracellular NLRP3/caspase-1 inflammasome activation, or, alter-

natively, for degradation in an attempt to clear the fungal threat. It is becoming evident that

several myeloid CLRs act as PRRs that exhibit potential to transduce intracellular signaling

to direct transcription of innate response genes40. Among these signaling CLRs, several have

been implicated in antifungal immunity, all with distinct mechanisms of action: dectin-1,

dectin-2, mincle, and DC-SIGN67-70. Whether langerin and MR, besides promoting fungal

uptake and processing, transduce intracellular signaling has not been formally proven.

It is, however, likely that both CLRs modulate intracellular signaling indirectly, simply by

influencing recruitment of signaling receptors to the phagocytic synapse71.

It is of particular interest that CLR signaling through downstream assembly of a complex

containing CARD9 is indispensable for the generation of a Th17-dominated response52,72,73,

and additionally induces Th1 polarization72,74. Dectin-1 represents the prototype antifungal

CLR, which renders DCs fully competent to direct Th1 and Th17 immunity after exposure

to fungal β-glucan72,74. Recent genetic studies in humans signify the importance of the

dectin-1/CARD9 axis, as signaling defects have been connected to susceptibility to fungal

infection. A SNP in dectin-1 (Y238X) that introduces a premature stop codon and prevents

functional dectin-1 expression, predisposes to chronic mucocutaneous candidiasis (CMC),

due to limited production of IL-17 and low numbers of Th17 cells in peripheral blood75.

Even, in patients receiving immunosuppressive medication, the dectin-1 Y238X SNP has

been associated with enhanced susceptibility to invasive aspergillosis76-78. Patients with a

loss-of-function CARD9 mutation similarly display greatly reduced numbers of circulating

Th17 cells and CMC, but with far more severe clinical symptoms and manifestations of lethal

systemic disease79,80. This discrepancy has been observed likewise in murine infection

models: deficiency of CARD9 or its upstream effector PKC-δ, results in invasive infection

and rapid lethality, rather than the lack of dectin-1 alone72,81. In sum, these studies strongly

suggest redundancy between CARD9-coupled receptors, whereas CARD9 dysfunction is

detrimental for host control of fungal infection.

Apart from dectin-1, CLRs dectin-2 and mincle also transmit signals via the CARD9

module73,82. Indeed, dectin-1 is thought to be dispensable for protection of mice against

infection with certain subtypes of fungi, with induction of Th17 responses to systemic C.

albicans predominated by dectin-2, not dectin-173,83, and likewise, several studies have sug-

gested contribution of mincle to establishment of protective immunity in mice69,84,85. The

studies described in this thesis aimed at the functional characterization of both dectin-2

Intr

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26 Chapter one

The innate immune system senses presence of

potential danger via recognition of molecular

structures unique to different types of microbes

(PAMPs) or damaged-self (DAMPs), through

pattern recognition receptors (PRRs)39. Different

PRRs recognize different types of PAMPs,

while a given pathogen or self-molecule can be

recognized by multiple PRRs simultaneously.

This allows PRRs -in specific combinations- to

instruct the adaptive immune system how to

respond best: providing information about

the initiation, type, duration and magnitude

of the response161. Hence, PRRs dictate the

outcome of an ensuing immune response.

Numerous classes of PRRs have been identified,

functioning in distinct extra- and intracellular

compartments but also cell types. Most

prominent PRR families are listed below, along

with some of their best-studied members:

Membrane bound receptors.

Toll-like receptors (TLRs), amongst the best-

studied PRRs, are glycosylated type I membrane

proteins, comprising a leucine-rich repeat

(LRR) ectodomain for recognition of well-

defined PAMPs. The 10 known human TLRs

are stationed at the cell surface for sensing

(myco)bacterial, fungal and parasitic PAMPs

(e.g. lipopolysaccharide from gram-negative

bacteria by TLR4 and bacterial lipoproteins

by TLR1/TLR2 and TLR2/TLR6 complexes),

or signal from intracellular vesicles upon

detection of double-stranded (ds)RNA (TLR3),

single-stranded microbial (ss)RNA (TLR7) or

CpG-rich methylated microbial DNA (TLR9).

TLRs recruit a single or distinct set of Toll-IL-1

receptor TIR-domain-containing adaptor

molecules, such as Myd88, TRIF, TIRAP

and TRAM, to their cytosolic domains for

induction of downstream signaling events

and expression of genes encoding cytokines,

chemokines and antimicrobial peptides162.

Expression of TLRs is cell-type specific.

The C-type lectin receptor (CLR) superfamily

is a large group of proteins characterized

by the presence of one or more C-type

lectin-like domain(s) (CTLDs)163. CLRs

primarily sense carbohydrate moieties

such as mannose and fucose (e.g. by DC-

SIGN) as well as β-glucan (i.e. by dectin-1) on

pathogens and host-derived glycoproteins,

but some recognize F-actin filaments (i.e.

DNGR-1)164,165, and ribonucleoproteins82

released by necrotic host cells (i.e. mincle).

CLRs are implicated in cell adhesion and

communication processes, detection of cell

death, and uptake of (altered-) self and non-

self-antigens145,166. In addition, several CLRs are

able to transduce Syk-dependent signaling,

thereby predominating the antifungal immune

response. This occurs directly through an

intracellular ITAM-like domain (i.e. dectin-1),

or indirectly via association with an ITAM-

containing adaptor molecule (e.g. dectin-2

and mincle)40, resulting in induction and/or

modulation of cytokine and type I interferon

(IFN) responses via transcription factors

NF-κB, IRF1 and IRF5 (findings described

in Chapters 2, 3, 4 and 5 of this thesis).

Cytoplasmic sensors.

NOD-like receptors (NLRs) constitutes the

largest family of the cytoplasmic localized

PRRs. Similar to the TLR family, NLRs contain

LRR motifs for detection of wide variety of

PAMPs and DAMPs, while, in contrast to TLRs,

they are expressed by a wide variety of cell

types. A central nucleotide-binding domain

controls NLR oligomerization, while their

Box 2. Pattern Recognition Receptors.


on

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caspase-recruitment (CARD), pyrin (PYD) and

baculovirus-inhibitory repeat (BIR) domains

facilitate intracellular signal transduction.

The NLR family comprises more than 20

members, which can be further divided into

NLRPs (previously NALPs) and NODs (also

known as NLRCs). NLRPs are well-known

for their ability to sense viral infection, upon

which they assemble into cytoplasmic

protein complexes (inflammasomes) together

with ASC and caspase-1 for maturation

of IL-1β cytokines. NLRP3 assembles into

inflammasome complexes upon indirect

sensing of a wide variety of microbial and

endogenous stress signals (i.e. RNA viruses,

bacteria and mitochondria-derived stress

signals: mROS, mtDNA and cadiolipin),

presumably via detecting a commonly induced

K+ efflux. NLRC4 assembles inflammasomes

upon recognition of bacterial flagellin and a

type III and IV secretion system components167.

Despite containing a CARD-domain, NOD1 and

NOD2 are non-inflammasome forming NLRs;

NOD1 and NOD2 sense bacterial peptidoglycan

motifs and as oligomers associate with

adaptor RIP2 for induction of signaling leading

to expression of cytokines, chemokines

and reactive oxygen species (ROS)42.

RIG-I-like receptors (RLRs), including RIG-I

and MDA5, are a family of DExD/H box RNA

helicases, sensing ssRNA and dsRNA from viral

origin or processed-self within the cytoplasmic

compartment. RIG-I is also capable of sensing

dsDNA indirectly, after it has been processed

into ssRNA structures via RNA polymerase

III (RNAP III). RIG-I and MDA5 signal via an

IPS-1 signalosome for induction of type I IFN

responses via transcription factors IRF1, IRF3

and IRF7 or cytokines via CARD9/Bcl-10-

dependent NF-κB activation, but also mediate

NLRP3 inflammasome assembly (RIG-I). The

third RLR family member, LGP2, lacks a CARD

motif for type I IFN signaling and is thought to

function as a modulator of RIG-I and MDA5168.

RLR family members are well known for their

ability to crosstalk with TLRs and other PRRs

for modulation of adaptive immune responses;

aberrant or dysregulated RLR signaling has

been linked to development of autoimmunity.

The PYHIN protein family is a group of IFN-

inducible proteins, sensing cytosolic dsDNA

from viruses and bacteria through their PYD

and/or HIN200 domain(s). AIM2 is a PYHIN

member with established PRR function,

mediating inflammasome assembly but

not gene transcription, while numerous

PYHINs, including IFI16, are putative dsDNA

sensors signaling for STING-dependent

induction of cytokines and type I IFNs169.

Intr

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28 Chapter one

and mincle in human antifungal immunity, and in accordance with the above notions, we

provide evidence for key roles for both dectin-2 (Chapter 2) and mincle (Chapters 3, 4 and 5).

DC-SIGN signals independent of the CARD9 axis, yet binds several fungi and dynamically

regulates assembly of its signalosome to amplify or inhibit T helper polarization86. Thus,

multiple CLRs (i.e. dectin-1, DC-SIGN, dectin-2 and mincle) signal collaboratively to yield

the most optimal antifungal response; the mechanisms of which are now beginning to be

elucidated at the molecular level.

The remainder of this chapter focuses on the distinct signaling pathways and transcriptional

programs by which CLRs dectin-1, DC-SIGN, dectin-2 and mincle can influence the adaptive

outcome of an antifungal response. We explore the hypothesis that, in terms of effector

mechanisms, signaling CLRs implicated in human antifungal immunity can be classified

into two distinct groups: (i) receptors that act individually and provide all transcriptional

signals required to bridge innate and adaptive responses -that is by instructing CD4+ T

helper cell polarization, and (ii) receptors that respond cooperatively and modulate signals

from other PRRs to fine-tune a particular adaptive response. Dectin-1 is the prototype of

an inducer, specialized in directing both Th1 and Th17 cell responses, whereas DC-SIGN

is a prominent member of the second class that distinctively modulates Th1 responses. In

fact, our current studies (described in Chapters 2, 3, 4 and 5 of this thesis) reveal that CLRs

dectin-2 and mincle can also be classified as modulating CLRs.

Dectin-1, the central paradigm for a signaling CLR, is expressed primarily in cells from

myeloid origin, including DCs, macrophages, monocytes, neutrophils, langerhans cells

(LCs) and eosinophils, yet also found on B cells and mouse innate γδ T cells63,87,88. By means

of its β-1,3-glucan and β-1,6-glucan carbohydrate specificity, dectin-1 is capable of binding

most if not all fungi, due to the abundance of β-glucans (polymers of D-glucose linked by

β-glucosidic bonds) in nearly all fungal cell walls50. Ligand binding by dectin-1 occurs in a

Ca2+-independent manner, which is divergent from most other CLRs. Studies in both human

and mice have documented recognition of numerous pathogenic species by dectin-1,

including Aspergillus spp., Candida spp., Coccidiodides spp., capsule-deficient C. neoformans,

Fonsecaea pedrosoi, H. capsulatum, and P. jirovecii85,89-95. Also, dectin-1 interacts with myco-

bacteria, albeit via recognition of a yet unknown ligand96,97.

Not surprisingly, fungal cell wall β-glucan abundance influences initial innate detec-

tion by dectin-1. The opportunistic pathogen C. albicans has a dimorphic appearance, and

β-glucans become more accessible during transition from the commensal yeast form into

the invasive filamentous form98. Although the ability to undergo phase transition is strong-

DECTIN-1: THE CORNERSTONE OF HUMAN ANTIFUNGAL IMMUNITY


on

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ly associated with fungal pathogenicity98, the host might use this event to discriminate

invasion from colonization and provoke an antifungal inflammatory response99. Dectin-1

presumably is an important player in local tissue immunosurveillance, with commensal

fungi being important constituents of the host skin, oral, and gut microbiota2. Interestingly, a

dectin-1 gene variant has been associated with aggravation of inflammatory bowel disease

(IBD) severity2, suggesting that altered sensing of fungi by dectin-1 contributes to aberrant

immune responses in IBD. Nevertheless, some pathogenic fungal strains strategically mask

their β-1,3-glucans to prevent immune recognition, even phagocytosis, and succeed in gain-

ing virulence. The immunologically inert capsule of Cryptocuccos spp. and hydrophobic,

RodA-rich, layer of Aspergillus conidia are considered most extreme examples100,101.

In myeloid cells, dectin-1 transduces downstream signaling via a unique intracellular

signaling domain, which delivers activation signals to Src and Syk family kinases. This

domain resembles an immunoreceptor tyrosine-based activation motif (ITAM), termed

hemITAM, but differs from a conventional ITAM in that it possesses only one of the two Tyr-

x-x-Leu (YxxL) sequences102. Binding of the tandem Src homology domain 2 (SH2) domains

of Syk to dually phosphorylated ITAMs is crucial for Syk activation. Because of its unusual

ITAM, dectin-1 is thought to dimerize to provide such a docking site103,104, analogous to the

hemITAM-containing receptor Clec-2105. Syk undergoes autophosphorylation at numerous

tyrosines upon binding to dectin-1 in order to initiate downstream signal transduction106

(Figure 2). Two related membrane-associated tyrosine phosphatases (i.e. CD45 and CD148)

mediate Src family kinase activation, but need to be quickly segregated from the dectin-1

synapse to avoid dephosphorylation of the ITAM tyrosine residues, and permit productive

signaling107. Notably, soluble, β-glucan polymers have been found incapable of excluding

CD45 and CD148 activity, even though they bind dectin-1107. This may ensure that dectin-1

signaling is activated solely upon encountering an invading fungus, which should be elim-

inated, and not harmless shed β-1,3-glucan fragments.

Dectin-1 mediates phagocytic uptake of fungal particles107, directs fungal destruction

(through production of toxic reactive oxygen intermediates) by macrophages, neutrophils

and DCs108, and controls, via Ca2+-dependent NFAT transcription, the microbicidal activity

of neutrophils (e.g. degranulation)109. In addition to these immediate antimicrobial effector

responses, dectin-1 signaling promotes efficient MHC class II presentation of fungal-derived

antigens to CD4+ T cells110, and activates nuclear translocation of transcription factor NF-κB

to mediate release of innate response mediators that shape the overall adaptive response.

Syk-dependent signaling. In human DCs, dectin-1 autonomously orchestrates activation

of all NF-κB subunits, through activation of both the classical and noncanonical NF-κB

pathways, and accordingly expression of Th1 and Th17 polarizing cytokines52, via

induction of two independent signaling pathways. Syk-dependent signaling induces

assembly of a trimolecular signaling complex consisting of CARD9, Bcl-10 and MALT1111.

Intr

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30 Chapter one

Inducing receptor

p

1 p

p

p

p p

pSYK

CARD9K63

IL1BIL23A

pro-IL-1β

inactivation

p65 RelB

p

p65 p50a p

IL12AIL6 IL12B

ITAM-like motif

β-glucanFungi

Dectin-1

y y

DC-SIGN

Mannose

LSP1

KSR

1CNK1

Raf-1pp

Raf-1

Fucose

LSP1

Virus

Ras

2. Reduced transcription

KSR1

CNK1Raf-1

1. Enhancedp65 activity

Raf-1

Nucleus

(+ Signal 2)

RelB

p100

MALT1 Bcl-10

NIK

Non canonical

TH17 promoting

TAK1

CanonicalIκBα

NF-κB

p52 RelB

NF-κB

Modulating receptor

2ROS

TH1 promoting

IL-1β

Inflammasomes

Casp 1ASC

NLRP3

Caspase 1c-

Rel

p50

Casp 8

CARD9

ASC

Caspase 8

MALT1 Bcl-10

Figure 2. Dectin-1 provides all transcriptional signals to generate Th1- and Th17- polarizing cytokine

profiles, while signaling induced by DC-SIGN modulates CD4+ T cell responses. (a) Upon β-glucan sens-

ing, dectin-1 activates two independent signaling cascades that integrate at the level of nuclear factor-kB

(NF-κB) activation: Syk- and Raf-1-based pathways. Recruitment of Syk to the phosphorylated (P) dual

tyrosine (Y) motifs of dectin-1 facilitates, via an intermediate kinase (possibly PKCδ; not shown), the as-

sembly of a complex consisting of CARD9, Bcl-10 and MALT1. This CARD9/Bcl-10/MALT1 scaffold then

presumably undergoes non-degenerative Lys63 (K63)-linked poly-ubiqitination (polyUb)170, which can

be recognized by cofactors such as TAK1 and TRAF proteins. This leads to the activation of IKK subunit β

(IKKβ; not shown), which phosphorylates the NF-κB inhibitor protein IkBα, and targets it for proteasomal

degradation. Following IkBα degradation, canonical NF-κB subunits (depicted as p65-p50 and c-Rel-p50

dimers) can enter the nucleus, to drive expression of, among other inflammatory mediators, IL-6, IL-1β

and IL-23, which induce Th17, and the Th1 polarizing cytokine IL-12p70. Syk-dependent signaling also I


on

e.

initiates an alternative or non-canonical NF-κB pathway that culminates in the activation of RelB: IKKα

(not depicted) is activated by NIK, and initiates processing of NF-κB inhibitor p100 into p52, which enters

the nucleus together with RelB. CARD9 and Bcl-10 are involved in recruitment of all NF-κB subunits,

whereas the paracaspase MALT1, through its proteolytic activity, strictly targets c-Rel. Besides initiation

of canonical and non-canonical NF-κB signaling (1), Syk mediates generation of reactive oxygen species

(ROS) production for NLRP3/caspase-1 inflammasome activation (2). Raf-1-dependent signaling culmi-

nates in phosphorylation at serine 276 (Ser276) and acetylation (a) of p65. Phopshorylated p65 restrains

RelB activity, while acetylated p65 has prolonged nuclear activity and enhances transcription of IL6 and

IL12 genes. The CARD9/Bcl-10/MALT1 scaffold also initiates formation of an alternative caspase-8 inflam-

masome in which the CARD9/Bcl-10/MALT1 triad is linked to caspase-8 and the adaptor protein ASC for

proteolytic processing of pro-IL-1β. (b) DC-SIGN signaling is ligand-specific and affects Th1 polarization.

(left) A pre-assembled trimeric complex consisting of KSR1, CNK and Raf-1 is, via the adaptor molecule

LSP1, constitutively associated with the cytoplasmic tail of DC-SIGN. Binding of mannose-containing

pathogens (such as fungi) to DC-SIGN induces activation of the serine/threonine kinase Raf-1, which

in turn mediates phosphorylation and acetylation of NF-κB subunit p65. These modifications prolong

the nuclear activity of p65, resulting in increased transcription rates at specific genes, including those

encoding IL-12p70, crucial for antifungal Th1 polarization. (right) Fucose-containing pathogens trigger

an alternative, and LSP1-dependent, pathway, accompanied by disassembly of the KSR1/CNK/Raf-1 triad.

Activation of this cascade attenuates pro-inflammatory cytokine production, thus negatively affects Th1

responses, via a yet unknown mechanism. Please note that the example given here concerns dectin-1

as an inducer of antifungal Th1 and Th17 responses, but DC-SIGN can influence antifungal responses

induced by any other innate receptor.

Downstream intermediate PKCδ is likely to couple Syk activity directly to CARD9

phosphorylation and recruitment81. The CARD9/Bcl-10/MALT1 scaffold subsequently

activates oligomerization of the IκB kinase (IKK) complex to allow nuclear translocation

of canonical NF-κB subunits p65 and c-Rel111. It is not entirely clear how the CARD9/

Bcl-10/MALT1 scaffold targets assembly of the IKK complex, but it presumably occurs

indirectly and involves cofactors such as TRAF proteins (e.g. TRAF2 and TRAF6)112

and TAK1, analogous to TCR/BCR signaling81,112. Classical NF-κB signaling by dectin-1

via CARD9 leads to transcription of genes such as IL1B, IL6, IL23A (encoding the p19

subunit of IL-23), IL12A (IL-12p35), and IL12B (IL-12/IL-23 p40 subunit)74, promoting

both Th1 and Th17 responses52,74. Unlike most PRRs, dectin-1-Syk signaling simulta-

neously activates the noncanonical NF-κB subunit RelB, which involves NIK. This

pathway partly antagonizes responses induced by dectin-1 through classical NF-κB

signaling, since RelB suppresses IL12B and IL1B transcription, and hence p65- and

c-Rel-mediated IL-12p70 and pro-IL-1β expression, by preventing RNA polymerase II

recruitment74 (Figure 2). Strikingly, dectin-1 activates a second pathway through Raf-

1 that integrates with Syk-dependent signaling at the level of NF-κB activation, and is

crucial for further fine-tuning of cytokine transcription by dectin-1.

Intr

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32 Chapter one

The road to Th17 induction via MALT1. As mentioned, dectin-1 is a PRR specialized in

propagation of Th17 responses. Recent studies have given major new insights into two

molecular processes utilized by dectin-1 in human dendritic cells to drive this Th17-

polarizing cytokine profile. These involve alternative processing of pro-IL-1β97 and,

as our data in Chapter 2 of this thesis point out, selective activation of NF-κB subunit

c-Rel52. Strikingly, both rely on the MALT1 scaffold protein, underlining a dual role for

MALT1 in shaping Th17 immunity by dectin-197.

MALT1 has been found crucial for pro-IL-1β processing via a noncanonical caspase-8

inflammasome. Production of bioactive IL-1β is strictly regulated and requires proteolytic

processing of pro-IL-1β, formerly thought solely attributable to the NLRP3/caspase-1 inflam-

masome. Indeed, internalization of some pathogenic fungi by dectin-1 and subsequent

Syk-dependent signaling triggers a conventional NLRP3/caspase-1 inflammasome for

the processing of inactive pro-IL-1β into its 17-kDa mature form. Dectin-1, however, directs

formation of an alternative noncanonical caspase-8 inflammasome for pro-IL-1β cleavage,

without interference of additional cytosolic sensors. This inflammasome consist of the

CARD9/Bcl-10/MALT1 scaffold linked to caspase-8 and the adaptor protein ASC, and is

assembled in response to all (fungal-) pathogens bound by dectin-197. Indirectly, MALT1

has a crucial and indispensable role in caspase-8 inflammasome activity, given that it

directly interacts (presumably dimerization through their respective caspase domains113)

with caspase-8 to prevent autocleavage of caspase-8, which would lead to apoptosis. The

intermediate processing of caspase-8 allows targeted processing of IL-1β97 (Figure 2). Thus,

MALT1 enables dectin-1 to autonomously elicit IL1B transcription as well as rapid IL-1β

maturation, to orchestrate induction of Th17 immunity.

In addition, as we demonstrate in Chapter 2 of this thesis, MALT1 has a specialized

function in regulating expression of Th17-polarizing cytokines IL-23p19 and pro-IL-1β. Specifically, we demonstrate that MALT1, through the selective targeting of NF-κB subunit

c-Rel, controls a transcriptional subprogram for efficient induction of IL23A and IL1B gene

transcription52, indicating that MALT1 activity is crucial for optimal Th17 effector responses

induced by dectin-1; a notion further discussed in Chapter 6.

Fine-tuning T helper responses by Raf-1. Dectin-1 is also capable of relaying YxxL- and

Syk-independent signaling via the serine/threonine kinase Raf-174, a pathway originally

identified downstream of DC-SIGN (further described later on)62. Dectin-1-mediated

Raf-1 activation induces selective phosphorylation and subsequent acetylation of p65,

which has two important functional consequences. First, phosphorylated p65 can

restrain RelB into inactive p65-RelB dimers to partially reverse the repressing effects

of RelB on IL12B and IL1B transcription. RelB is, however, not completely sequestered

and neutralized by p65; residual RelB can induce moderate production of Th2-related

chemokines CCL17 and CCL22. Acetylation of p65 typically prolongs its activity and


on

e.

transcription rate, and, downstream dectin-1, results in enhanced expression of IL6,

IL12A and IL10 74 (Figure 2). Overall, the Raf-1 pathway permits and fine-tunes induc-

tion of Th1 and Th17 immunity by dectin-1-Syk signaling by balancing p65 and RelB

activities. Clearly, dectin-1-induced NF-κB activation is subject to tight regulation by

distinct pathways which are induced separately but are cooperating at multiple layers

to shape the overall immune response.

DC-SIGN is a striking example of a signaling CLR that affects adaptive responses induced

by other PRRs at the level of NF-κB activation. Activation of DC-SIGN signaling can have

profound effects on immune responses directed against several pathogens (e.g. mycobac-

teria)86, and is even exploited by HIV-1 to facilitate productive DC infection and subsequent

in trans infection of T cells - the primary HIV-1 target cells114. Although the role of DC-SIGN in

the host defense against fungi has been studied less extensively, DC-SIGN has the potential

to modulate antifungal effector mechanisms when activated by fungal pathogens. Most

notably, the immunological outcome is dependent on the carbohydrate composition of

the ligand involved; DC-SIGN transduces divergent signaling cascades upon ligation with

mannose- or fucose- containing ligands.

DC-SIGN is predominantly expressed on DC subsets, yet also found on a subpopulation

of macrophages115. Tetrameric surface expression enables DC-SIGN binding to mannose,

fucose, N-acetyl-glucosamine (GlcNAc) and mannan moieties with high-avidity, which

occurs in a Ca2+-dependent manner116. DC-SIGN is involved in the recognition of endogenous

carbohydrates (e.g. ICAM-3 on T cells) as well as ligands derived from numerous pathogens,

including viruses, bateria, helminths, and fungi117. DC-SIGN harbours several internaliza-

tion motifs in its cytoplasmic tail118, which allow for robust endocytic activity and fungal

uptake; among the fungal pathogens bound via exposed mannose residues are C. albicans,

A. fumigatus, capsule-deficient C. neoformans, and Chrysosporium tropicum70,119,120.

Despite bearing a classical intracellular YxxL motif, DC-SIGN is an exceptional signaling

CLR in that it does not transduce downstream signals via the Syk-CARD9 axis, and, moreover,

is incapable of activating transcriptional programs (e.g. cytokine expression) on its own62.

A pre-assembled complex consisting of KSR1, CNK and Raf-1 is constitutively attached to

the cytoplasmic domain of DC-SIGN via the adaptor molecule LSP186. Mannose-containing

pathogens such as mycobacteria and HIV-1, and most likely comprising several species of

fungi, induce Raf-1 activation upon DC-SIGN binding. Signaling downstream Raf-1 then leads

to modulation of the transcriptional activity of NF-κB subunit p65, yet only when nuclear

translocation of p65 is induced by any other PRR. Raf-1 phosphorylates p65 at serine (Ser)

276 and controls subsequent acetylation of p65 by two histone acetyltransferases: CBP and

DC-SIGN: LIGAND-SPECIFIC SIGNALING FOR TH1 MODULATION

Intr

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34 Chapter one

p300. These post-translational modifications functionally affect p65, defined by prolonged

nuclear activity and increased transcription rates particularly at the IL12A, IL12B, and IL6

genes62. Thus, depending on the PRR coactivated by the fungus, triggering of DC-SIGN/

Raf-1 signaling by mannose-expressing fungi will increase expression of IL-12p70, crucial

for activation of a Th1 polarization program (Figure 2). DC-SIGN-Raf-1 signaling can also

abolish the RelB-dependent suppression of IL12B and IL1B transcription and thereby

directly influence Th17 immunity, as would be the case when dectin-1 is co-ligated. In

contrast, in response to fucose-bearing ligands (i.e. the gastric pathogen Helicobacter pylori

and endogenous Lewis antigens) the KSR1/CNK/Raf-1 triad is selectively disassembled

from the cytoplasmic tail of DC-SIGN for initiation of an alternative, and Raf-1-independent,

signaling cascade that attenuates production of pro-inflammatory cytokines (Figure 2),

with the exact molecular mechansims still being elusive86. This modulatory pathway can

potentially be targeted by pathogenic fungal strains for evading an immune response, with

fucosylated moieties found in selected species of fungi121. Overall, these findings support

the hypothesis that carbohydrate-specific signaling by DC-SIGN can effectively modulate an

antifungal immune response induced by other PRRs, and customize it to the fungal strain

encountered. Whether DC-SIGN indeed affects fungus-specific T helper cell differentiation

awaits further investigation.

Apart from being typified as a LC marker122,123, human dectin-2 is more generally expressed

among cells from myeloid origin, specifically DC subtypes, macrophages, monocytes,

and neutrophils14,52,123. Dectin-2 possesses a typical Ca2+-dependent CTLD with affinity for

high-mannose structures (Man9

GlcNAc2

)124. Consistently, dectin-2 has been implicated in

recognition of α-mannose from a number of fungi: Candida spp., A. fumigatus, capsule-de-

ficient C. neoformans, H. capsulatum, Microsporum audouinii, Malassezia spp. Paracoccoides

brasiliensis, and Trichophyton rubrum52,68,124-126, but also unknown components from house

dust mite allergens, the parasitic worm Schistosoma mansoni, and Mycobacterium tuberculo-

sis124,125,127. In contrast to what its name suggest, dectin-2 is only 27% homologous to dectin-1.

The short cytoplasmic tail of dectin-2 lacks an obvious signaling motif but associates with

the ITAM-bearing Fc receptor common-γ (FcRγ) chain for signal transduction and solid cell

surface expression68. In addition, dectin-2 likely forms a heterodimeric PRR with another,

less well-characterized, ITAM-coupled CLR128,129: MCL (also referred to as dectin-3)130. Their

respective homodimers were found to bind α-mannose structures less efficiently, suggesting

that MCL acts as a subunit of a high-affinity PRR complex for sensing fungal infection130.

Engagement of dectin-2 induces phosphorylation of the dual tyrosine residues within

the coupled FcRγ ITAM and, subsequently, recruitment of Src family kinases and Syk68,73. Syk

DECTIN-2


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activation by dectin-2 is a prerequisite not only for CARD9-dependent induction of NF-κB-

mediated gene transcription, but also for activation of MAP kinase pathways83. Similar to

dectin-1 signaling, canonical NF-κB activation by dectin-2 requires assembly of the CARD9/

Bcl-10/MALT1 scaffold73, and probably involves many of the same intermediate players,

including TRAFs and TAK181. However, as our data in Chapter 2 of this thesis demonstrate,

human DC-expressed dectin-2, in stark contrast to dectin-1, does not equally activate all

NF-κB subunits. We demonstrate that dectin-2-Syk signaling culminates in selective c-Rel

activation via MALT1 for selective production of Th17-polarizing cytokines IL-1β and IL-23

subunit p19 (IL-23p19). Thus, dectin-2 is a representative of a class of signaling CLRs that

modulate antifungal immunity; the implications for Candida albicans-specific Th17 responses

are further discussed in Chapter 6.

Interestingly, dectin-2 has been assigned function in the (dys-) regulation of pulmonary

Th2 responses in a mouse allergy model, further emphasizing a role for dectin-2 as a modu-

lator of T helper cell polarization. Through Syk-dependent generation of proinflammatory

lipid mediators, such as cysteinyl leukotrienes, dectin-2 signaling contributes to patho-

logical airway inflammation in response to extracts derived from house dust mite and A.

fumigatus125,131. Th2 immunity, characterized by alternatively activated macrophages and

antibody class switching to non-opsonizing and IgE subclasses, controls parasitic infec-

tions but is considered deleterious during the course of fungal infection16. A. fumigatus is

an exceptional fungus in being a successful opportunistic pathogen and major allergen101,

and might therefore utilize sophisticated strategies to avoid destruction. It remains to be

determined whether dectin-2 signaling in general contributes to Th2-biased immunity,

given its ability to recognize the parasite S. mansoni, and whether dectin-2, in doing so, is

targeted by (virulent) fungal strains for immune evasion. However, consistent with the

immune modulatory functions of dectin-2, this might in fact be dictated by the additional

PRR(s) co-activated by these fungi.

MINCLE

Mincle expression predominates on myeloid cell types, including DCs, macrophages and

neutrophils, yet is also found on B cells132. Through recognition of an ill-defined mannose-rich

ligand, possibly α-mannose, present on glycolipids, mincle recognizes several pathogens

from fungal origin, including Candida spp., Malassezia spp., F. pedrosoi69,84,126,133. Additionally,

mincle exhibits potential to bind the mycobacterial glycolipid trehalose-6,6-dimycolate

(TDM, also known as mycobacterial cordfactor), as well as its synthetic derivate TDB (treha-

lose-6,6-dibehenate), both well-known for their therapeutic adjuvancy134,135. As such, mincle

is held responsible for the Th1/Th17 adjuvancy of TDM and TDB in mice135,136, yet it is still

unclear whether mincle has a role in controlling Mycobacterium tuberculosis infection137,138.

Intr

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36 Chapter one

In addition, mincle has been implicated in anti-bacterial defenses; mincle appears to have

a non-redundant role in the protective response to Klebseilla pneumonia infection in mice,

by preventing hyperinflammation, although direct binding of mincle to these bacteria has

not been proven139. While mincle is not directly involved in the phagocytic engulfment of

particulate cargo82,84, it does localize to the phagocytic synapse when macrophages interact

with C. albicans84.

Mincle possesses a single extracellular CRD for the recognition of microbial carbo-

hydrates in a Ca2+-dependent fashion69,140. The mincle CRD contains a primary glutamic

acid/proline-asparagine (EPN) motif, predictive for its mannose specificity, flanked by a

hydrophobic groove, for binding branched acyl chains (fatty acids)140,141. This additional

binding domain probably fine-tunes the specificity of mincle towards the sugar-proximal

parts of lipid-moieties. Its cytoplasmic tail is devoid of any classic signaling motifs, and via

a positively charged arginine residue, mincle associates with the FcRγ chain for transduc-

tion of ITAM-coupled signaling, in analogy to dectin-282. Moreover, and similar to dectin-2,

mincle heterodimerizes with MCL for cell surface expression142. Although structural anal-

yses have demonstrated that MCL also contains a hydrophobic domain for binding the

branched acyl chains present in TDM and TDB, its CRD lacks a primary EPN motif140, critical

for TDM binding by mincle134. Given that MCL binds TDM with much lower affinity142, and

that heterodimer formation with MCL is not unique to mincle, the mincle/MCL interaction

possibly confers additional function to each of the molecules. Most likely, heterodimerization

with MCL enhances the ligand binding affinity, allowing mincle to detect small numbers

of glycolipids on the fungal surface. Furthermore, coupling to MCL might ensure efficient

signal transduction, and could also reflect a mechanism by which mincle acquires endo-

cytic receptor function143, with MCL demonstrated to exhibit endocytic activity128, in stark

contrast to mincle82,84. Whether mincle and MCL indeed function cooperatively, also in the

context of human antifungal immunity, remains to be demonstrated.

Next to its role as a microbial sensor, mincle is involved in the innate recognition of dam-

aged-self. As such, mincle binds the ribonuceoprotein SAP-130, derived from necrotic cells

that have lost their membrane integrity82. The mincle-mediated response to damaged-self

comprises infiltration of neutrophils for cellular clean-up82, with a possible role for mincle in

the induction of pathological inflammatory events following ischemic stroke144. Strikingly,

mincle recognizes SAP-130 in the absence of Ca2+ and independent of the CRD residues

involved in fungal binding82, implying dual ligand specificity. Also, the proinflammatory

response to necrotic cell death by mincle can be considered a sterile reaction, linked to tis-

sue repair, rather than infection control and full-blown activation of adaptive immunity145.

It remains to be established if mincle, analogous DC-SIGN, transduces divergent signaling

pathways to influence these distinct proinflammatory processes, or whether this is actually

dependent on the presence or absence of co-stimulated PRRs.

Ligation of murine mincle activates the highly conserved Syk-CARD9 signaling axis81,82,134,


on

e.

whereas its activity in the context of murine Fonsecaea infection has been shown inadequate

for induction of protective antifungal responses85. Although costimulation of other PRRs

was demonstrated sufficient for subsequent clearance of the pathogen, these protective

responses were dependent on mincle signaling as well, indicative of a modulatory func-

tion for mincle during establishment of antifungal immunity85. Our work on the functional

characterization of human mincle described in this thesis confirmed and extended these

previous findings, by demonstrating that mincle-dependent Syk signaling in human DCs

culminates in assembly of the complete CARD9/Bcl-10/MALT1 signaling module (Chapter

3). Most strikingly, though, we found that mincle does not signal for NF-κB activation (Chapter

4), and that mincle does not exhibit the potential to activate cytokine responses on its own

(Chapters 3 and 4). Instead, our data uncover an alternate mechanism of action: mincle cou-

ples Syk-CARD9 signaling to a PI(3)K-PKB pathway for modulation of cytokine responses

induced by other PRRs (Chapters 3, 4 and 5), having serious implications for the ability of

human DCs to promote concurrent Th1 and Th17 responses during fungal infection, an

issue further discussed in Chapter 6 of this thesis.

CONCLUDING REMARKS AND OUTLOOK

As defined and discussed here, immunity to fungal infection is orchestrated by multiple CLRs

expressed on dendritic cells. Two types of antifungal CLRs can be recognized: (i) receptors

that evoke a T helper polarization program autonomously and (ii) receptors that modulate

signals from other receptors to fine-tune a particular response. Unbalanced inflammation

can have deleterious effects on host immunity, and it is becoming evident that these types

of CLRs together transduce collaborative signaling to ensure tightly controlled and tailored

effector responses upon fungal intrusion. Further characterization of the molecular mech-

anisms by which CLRs control these processes may therefore give important insights into

defense mechanisms deployed against pathogenic fungi, and will facilitate tailor made

vaccines and novel targets to combat fungal infections.

Antiviral effectors for antifungal control? Our understanding of antifungal immune

responses has been expanded recently by studies supporting a possible role for type

I IFNs -classic antiviral effectors- during fungal-induced inflammation. Type I IFNs are

produced by murine DCs in response to cytoplasmic fungal nucleic acids 146,147, but also

it has been demonstrated that murine dectin-1 directly signals for IFN-β expression148,

though precise functional contribution to the overall antifungal immune response

remains enigmatic. Moreover, defective expression of human type I IFN genes is

associated with susceptibility to CMC, albeit through unknown mechanisms149. Our

study described in Chapter 5 corroborates these initial findings by providing evidence

Intr

od

uct

ion

38 Chapter one

for contribution of a type I IFN response to

human antifungal inflammation, which

also sheds new light on the molecular prin-

ciples of CLR cooperation.

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