Medical Mycology December 2004, 42, 485 /498

Review

Toll-like receptors as key mediators in innate antifungal immunity

ALEXANDER ROEDER*, CARSTEN J. KIRSCHNING$, RUDOLF A. RUPEC*, MARTIN SCHALLER%, NTHER WEINDL* & HANS CHRISTIAN KORTING* GU *Department of Dermatology and Allergology, Ludwig-Maximilian University, Munich, $Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich and %Department of Dermatology, Eberhard-Karl University, Tuebingen, Germany

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The Toll protein of Drosophila is a transmembrane receptor involved in dorsoventral polarization during embryonic development and recognition of infection. In mammals, Toll-like receptors (TLRs) constitute a novel protein family involved in innate immunity and respond to a wide spectrum of microorganisms, including fungi, bacteria, viruses, and protozoa. Specific agonists for nine of the ten members of the human TLR family have been described to date. TLRs as well as the TLR-associated adaptor molecule MyD88 have been implicated in the recognition of the fungal pathogens Candida albicans , Aspergillus fumigatus , Cryptococcus neoformans and Pneumocystis carinii . Moreover, several pathogen associated molecular patterns (PAMPs) located in the cell wall or cell surface of fungi have been identified as potential ligands. Yeast zymosan activates TLR2/ TLR6 heterodimers, whereas Saccharomyces cerevisiae - and C. albicans- derived mannan seems to be detected by TLR4. Phospholipomannan, present in the cell surface of C. albicans has been shown to be recognized by TLR2, while TLR4 mainly interacts with glucuronoxylomannan, the major capsular polysaccharide of C. neoformans . MyD88 has been implicated in TLR signalling of linear (1 03)-bD-glucan, and of b-glucan from P. carinii . These data point towards the ability of the innate immune system to utilize TLRs that are specific to different types and components of pathogenic fungi. Recent evidence further suggests that TLRs cooperate with other immune receptors involved in fungal recognition and that the selective induction of adaptor proteins finally leads to distinct signalling events upon fungal challenge.
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Keywords Aspergillus fumigatus , Candida albicans , Cryptococcus neoformans , innate immunity, toll-like receptors

Introduction
In recent years, fungi have played a dominant role, with increasing medical importance, among clinicians as

Correspondence: Gu nther Weindl, Ludwig-Maximilian University, Department of Dermatology and Allergology, Frauenlobstrae 9 /11, D-80337 Munich, Germany. Tel.: '/49 (0)89 5160 6151; Fax: '/49 (0)89 5160 6007; E-mail: [email protected]

well as microbiologists. In patients who are immunosuppressed or debilitated in some other way (e.g. due to chemotherapy or HIV-infection), the polymorphic yeast Candida albicans is a causative agent in mycoses of the skin, oral cavity and oesophagus, gastrointestinal tract, vagina and vascular system. Even with modern antimycotic agents, the attributable mortality rate of patients with invasive candidiasis approaches 40% [1]. Moreover, resistance to azole antifungals such as
DOI: 10.1080/13693780400011112

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fluconazole, clotrimazole, itraconazole and ketoconazole continues to be a significant problem among hospitalized patients. Hence new ways to treat or overcome fungal infections are strongly awaited. Modulation of the antifungal immune response might be a promising strategy owing to recent findings in the field of innate immunity. A rapid immunological response ensures survival upon infection. The adaptive immune system generates a random and wide variety of antigen receptors by gene rearrangement, followed by selection and expansion of cell clones expressing receptors with relevant specifities, as well as establishment of immunological memory, a process requiring several days. Randomly generated antigen receptors mediate responses upon interaction with exogenous antigens or even self-antigens potentially inappropriately. Such misconduction can lead to allergy or autoimmune disease. Prior to the adaptive immune system, the innate immune system confers rapid recognition of microbia through a limited repertoire of germline-encoded receptors. The agonists of these receptors represent conserved molecular patterns produced by broad groups of microbial species. Toll-like receptors (TLRs) are homologues of the Drosophila receptor Toll and constitute a novel protein family of cellular receptors that mediates recognition of microbial pathogens and subsequent inflammatory response in vertebrates. This article reviews major aspects of the respective cellular recognition system including molecular activation mechanisms that mediate the initiation of immune responses towards microbial challenge. Finally, the recent findings on fungal recognition by mammalian TLRs are summarized.

Antimicrobial defence in Drosophila

Insects combat infections by rapidly producing antimicrobial peptides in the fat body and in haemocytes. Toll was identified as a receptor being essential for ontogenesis and antimicrobial resistance in Drosophila [2]. A lack of Toll prevents development of ventral or lateral cell types and embryonic patterning. Furthermore, Toll is involved in immunity towards fungal and Gram-positive bacterial challenge in the fruit fly [3], while defence against Gram-negative bacteria is mainly dependent on a parallel pathway, the so-called immune deficiency (IMD) pathway [4]. However, both signal transduction pathways have recently been implicated in the recognition of fungi and Gram-positive bacteria [5]. The association of the Toll pathway and immune response is based on studies of regulation of genes encoding antimicrobial peptides. The activation of proteolytic cascades by Gram-positive bacteria func-

tionally downstream of a circulating peptidoglycan recognition protein (PGRP /SA) and of fungi, which trigger a cascade involving the serine protease persephone, lead to the generation of activated Spaetzle, the putative ligand of Toll in Drosophila [6,7]. Tolldependent immune responses result in activation of the Dorsal/Dif cascade [8]. Three transcription factors / Dorsal, Dif and Relish, members of the Rel/NF-kB family of proteins / have been identified. They are specifically activated in cells of the fat body of larvae and adult animals by translocation into the nucleus in response to infection. Toll was found to induce antifungal peptide genes. Thus, mutation of the Toll gene led to impaired defence against Aspergillus fumigatus infection [2]. In response to infection Toll induces activation of nuclear factor kappa (NF-k) B family transcription factors and produces antifungal peptides such as drosomycin and attacin. The analysis of the Drosophila genome has revealed the existence of nine proteins belonging to the Toll family of receptors [9]. The sequence similarity of the cytoplasmic portion of Drosophila Toll and mammalian interleukin-1 receptor (IL-1R) intracellular domains suggested similarities in Toll- and IL-1R signalling and illustrates the evolutionary conservation of both cellular signalling systems. Indeed, two recent studies used Drosophila melanogaster as a model to study host /pathogen interactions of C. albicans and Cryptococcus neoformans [10,11]. Infection of wild-type and Toll-deficient flies revealed that the Toll mutants are highly susceptible to C. albicans , whereas wild-type Drosophila is highly resistant. Additionally, utilizing specific C. albicans mutants, the authors could demonstrate a strong correlation between the importance of virulence factors in mammals and Drosophila [10]. Another group investigated the antifungal pathway of D. melanogaster upon challenge with C. neoformans [11]. While all flies survived after ingestion with non-pathogenic Cryptococcus spp. or Saccharomyces cerevisiae , flies fed with C. neoformans died within 2 or 3 days. As mentioned, the Toll and the IMD pathway have been implicated in antifungal defence; however, flies bearing a mutation in either the Toll or the IMD pathway were not more susceptible to killing than wild-type flies, indicating that the immune system is not capable of mounting an immune response to this fungus, or C. neoformans is able to successfully escape immune recognition surveillance. Interestingly, when the fungus was injected into the haemolymph of wild-type and IMD mutant flies, most of the flies survived the systemic infection. In contrast, spaetzle mutant flies, as well as spaetzle; IMD double mutants were highly susceptible
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to injected C. neoformans . These data suggest a considerable difference in systemic and intestinal immune responses of Drosophila to fungal pathogens and demonstrate the complexity of the Drosophila immune system.

TLRs and their agonists

To date, the sequences of 11 human and 12 murine TLRs have been described, with TLR11, TLR12 and TLR13 being the newest members of this receptor family [18,19]. However, ligands and specific functions have not yet been described for all receptors (Fig. 1 shows some of the known TLR agonists).

Vertebrate Toll-like receptors and involvement in host defence

The innate immune system recognizes conserved pathogen associated molecular patterns (PAMPs), which represent broad groups of microbial species rather than a single specific species, through germ lineencoded proteins, such as pattern recognition receptors (PRRs) [12]. The receptors of the innate immune system are expressed by cells playing crucial roles in immunity (e.g. monocytes, macrophages, dendritic cells (DC), B- and T-cells, as well as endothelial cells) [13,14]. TLRs confer PAMP recognition and their signalling triggers synthesis followed by release of proinflammatory cytokines and induces expression of co-stimulatory molecules for promoting activation of adaptive immunity during antigen presentation. PAMPs such as lipopolysaccharide (LPS, endotoxin), peptidoglycan (PGN), lipoteichoic acid (LTA), bacterial lipopeptide and unmethylated CpG-DNA contribute to the integrity of microbial cells. Mutation or loss of PAMPs on mutation might be either lethal for the microbial cell or reduce its viability. PAMP structures vary only to a limited extent from species to species and thus represent microorganisms of broad groups of species to the host organism. Recognition of infection is based on a limited number of PRRs expressed by the host organism. TLR signalling activates immune cells, for example macrophages, to produce cytokines, further inflammatory mediators, as well as effector substances, for instance, nitric oxide or reactive oxygen species. Furthermore, it could be shown that activation of the TLR signalling pathway by bacteria regulates phagocytosis at multiple steps including internalization and phagosome maturation [15]. Efficient host defence is mediated by potent antigen-presenting cells, such as DC which play a key role in control and induction of acquired immunity upon recognition of immunostimulatory PAMPs (e.g. PGN or LTA) [16]. DC that encounter invading microbial pathogens capture antigens and migrate to lymphoid organs, where they home to the T-cell areas. While migrating to the lymph nodes, they mature from an endocytic/phagocytic stage to a stage of antigen presentation and efficient T- and B-cell stimulation [17]. This process exemplifies the importance of TLRs not only in direct early immune responses, but also in activation of adaptive immunity.
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TLR4
Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria most extensively characterized in terms of its structural and functional properties within the group of known PAMPs. TLR4 has been identified as the prime signal transducer for bacterial LPS [20]. LPS is bound by LPS-binding protein (LBP) which delivers LPS to the cellular CD14 receptor. Subsequently, CD14 seems to load LPS onto the TLR4-MD-2 complex and enhances the formation of LPS-TLR4-MD-2 complexes [21,22]. MD-2 lacks a transmembrane domain and is presented on the cell surface in association with the ectodomain of TLR4 [20]. The expression of wild-type MD-2 in CHO cells bearing a mutation in MD-2 that abrogated LPS-induced signalling restores LPS sensitivity [23]. Another study revealed that MD-2 must be bound to TLR4 before MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes which then initiates subsequent signalling cascades [22]. Recently, a slightly different model has been proposed, in which a stable MD-2/LPS complex directly activates TLR4 after soluble MD-2 has bound LPS in a CD14 dependent manner [24]. Considering the existence of further MD-2-like molecules that might regulate specific functions of TLRs, pattern recognition through TLRs might be of rather large complexity in terms of differential specificity. TLR4 not only mediates recognition of LPS but also of non-PAMP agonists such as the diterpene from the tree Taxus brevifolia and anticancer drug Taxol [25], the fusion protein (F Protein) of respiratory syncytial virus (RSV) [26], heat-shock protein (HSP) 60 [27] and the extra domain A (EDA) region of the extracellular matrix protein fibronectin [28]. However, although the list of non-LPS agonists is rapidly increasing, one has to take precaution concerning potential residual amounts of LPS in the corresponding protein preparations. Recent studies provided evidence for the activation of TLR4 by minute amounts of LPS present in commercial HSP60 preparations [29].

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Fig. 1 Toll-like receptors (TLRs) and their ligands with respect to recognition of fungi. The Toll receptor of Drosophila , TLR1 to TLR11, and the IL-1 receptor (IL-1R) are lined up schematically. Leucine-rich repeats (LRRs) of the extracellular domains of Toll and TLRs, as well as transmembrane domains are represented as boxes: cystein-rich clusters are drawn as half ovals; the IgG-like domains (IgG) of the IL-1R extracellular domain are symbolized as open circles; cytoplasmically located Toll-IL-1R-plant resistance domains (TIR) are depicted as ovals; exemplary endogenous ligands as well as microbial agonists are indicated directly above the referable TLR utilized. Arrows depict functional cooperation in pattern recognition, for instance TLR2 cooperates with TLR1 and TLR6 to distinguish between triacyl and diacyl lipopeptides and zymosan activates heterodimers consisting of TLR2 and TLR6. Fungal species, as well as zymosan, from which distinct molecular patterns have not yet been identied, are presented above the regarding Toll/TLR implicated in mediation of their cellular recognition. Abbreviations: c.p., cytoplasma; CpG DNA, unmethylated DNA containing CG motifs; e.m., extracellular milieu; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MALP, macrophage-activating lipopeptide representing diacyl lipopeptides; OspA, outer surface protein A of Borrelia burgdorferi representing triacyl lipopeptides; PGN, soluble peptidoglycan; poly I:C, polyinosine-polycytidylic acid representing double-stranded RNA; Spaetzle, endogenous ligand of Toll; ssRNA, single-stranded RNA; t.m., transmembrane domain; UPEC, uropathogenic Escherichia coli .

TLR2, TLR1 and TLR6

A variety of agonistic molecules has been implicated in cell activation through TLR2 including peptidoglycan and lipoteichoic acid from Gram-positive bacteria, bacterial lipoproteins, mycobacterial lipoarabinomannan, glycosylphosphatidylinositol anchored proteins of Trypanosoma cruzi, a phenol soluble modulin produced by Staphylococcus epidermidis , and yeast zymosan [30]. While classical Gram-negative bacterial endotoxin is sensed through TLR4, specific LPS species, such as LPS of Leptospira interrogans [31] or Porphyromonas gingivalis [32] have been reported to be sensed through TLR2. The microbial product and antifungal drug amphotericin B induced activation of NF-kB and the release of cytokines via TLR2, CD14, and MyD88 in murine macrophages and human cell lines. Given the acute toxicity of this drug, which may result from production of proinflammatory cytokines by innate immune cells, it could be possible to reduce this severe side effect through neutralizing specific receptors or adaptor proteins of the TLR signalling

pathway [33]. One basis of the specificity for such a broad spectrum of PAMPs might be agonist specific heterodimerization of TLR2 with TLR1 and TLR6 leading to activation of NF-kB and cytokine production (e.g. TNF-a or IL-12) [34]. Moreover, inactivation through gene targeting of either TLR2 or TLR6 diminished the reactivity of immune cells to a mycoplasmal lipopeptide, implying the dependence of macrophage-activating lipopeptide (MALP)-2 recognition from TLR2 and TLR6 dimers [34]. On the other hand, TLR1 is required for TLR2-mediated responses to triacylated bacterial lipoproteins, indicating ligand specificity of the complex of each of both TLRs [35].

TLR9
Analysis of TLR9-deficient mice has revealed the TLR9 dependent mediation of cell activation by bacterial CpG-DNA. The immunostimulatory activity of bacterial DNA is attributed to unmethylated CpG motifs in genomic DNA. Unmethylated bacterial CpG- DNA induced cytokine production, B-cell
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proliferation, DC maturation, and induction of systemic shock were absent in TLR9-deficient cells and mice. Furthermore, sensitivity to CpG-DNA could be reconstituted by ectopic overexpression of TLR9 in a CD14 and MD-2 independent manner [36]. DNAPKcs have also been implicated in recognition of CpGDNA [37].

TLR5
TLR5 mediates cellular stimulation by bacterial flagellin, the monomeric 55-kDa subunit of flagella localized in the outer membrane of Gram-positive and Gramnegative bacteria [48]. Flagellin like other PAMPs induces IkB degradation, NF-kB activation, expression of IL-8 and recruitment of signalling molecules, such as MyD88 and IRAK (IL-1 receptor associated kinase) [49].

TLR3
A difference of TLR3 as compared to other mammalian TLRs is the lack of the conserved proline residue in the position equivalent to proline-712 of mouse TLR4, indicating specifities of TLR3 signalling [38]. Furthermore, concerning tissue distribution, it is of note that TLR3 is expressed predominantly, though not exclusively, in dendritic cells [32]. Analysis of TLR3 knockout mice has revealed involvement of TLR3 in the recognition of double-stranded RNA (dsRNA) [39]. TLR3 dependent production of interferon (IFN)-b is mediated by the Toll-interleukin-1 receptor (TIR) domain-containing adapter inducing IFN-b (TRIF), also known as TIR-containing adaptor molecule (TICAM)-1, independently of the adaptor molecules MyD88 and TIR domain-containing adaptor protein (TIRAP) or MyD88-adaptor-like (MAL) [40].

TLR11
Very recently, Zhang et al . [18] described a new member of the TLR family, termed TLR11, which is particularly abundant in the kidney and bladder of mice and recognizes uropathogenic bacteria, in particular Escherichia coli . Simultaneously this new receptor has been also discovered by Tabeta et al . [19] and named TLR12. Several stop codons in the putative open reading frame indicate that humans seem to lack a functional receptor. Thus, humans might be more susceptible to urinary tract infections than mice, as speculated by the authors [18]. However, the stop codons in TLR11 may represent a form of genetic polymorphism, which has already been demonstrated for TLR5 [50].

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TLR7 and TLR8

Until recently, the identity of natural agonists for TLR7 and TLR8 remained unknown, albeit synthetic compounds have already been implicated as agonists for TLR7 and human but not murine TLR8 [41 /44]. The low-molecular-weight imidazoquinoline compounds imiquimod and its derivative R-848 (resiquimod), as well as the synthetic adjuvant loxoribine, have potent immunoactivating properties by inducing synthesis of IFN-a and other cytokines in various cell types. After exposure to imiquimod and loxoribine immune response was severely impaired and signalling was completely abolished in both MyD88- and TLR7-deficient mice [41,42]. Furthermore, R-848 has also been reported to activate immune cells via human TLR8 independently of TLR7 in human cells (HEK293 cells) in a dose-dependent manner [43]. Lee et al . [44] showed that several guanosine analogues activate immune cells through TLR7 and that this activation requires endosomal maturation. Taken these findings together, it may be speculated that viral RNA or RNA fragments are likely candidates for TLR7 ligands. Indeed, lately three groups independently reported single-stranded RNA (ssRNA) as a natural ligand for TLR7 [45 /47] and human TLR8 [45].
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TLR family signalling pathways

Analysis of IL-1 and TNF signalling provided a basis for analysis of TLR signalling [51]. The proinflammatory cytokine IL-1 is a regulator of immune and inflammatory responses and is recognized by nearly all cell populations in the body. More than 100 genes are regulated by IL-1, including genes encoding cytokines and cytokine receptors, growth factors, adhesion molecules and various cell types of the skin, including keratinocytes, endothelial cells, and fibroblasts. Pathway analyses have focused on IL-1 induced activation of transcription factors NF-kB and c-Jun/AP-1 that activate transcription of several cytokine genes. IL1-R and TLR signalling leading to induction of NF-kB starts with recruitment of MyD88 to the receptor, followed by activation of the IL-1 receptor associated kinases IRAK-1 to IRAK-4 complexing with the adaptor molecule tumour necrosis factor receptorassociated factor (TRAF)6 [52]. The TLR/IL-1 receptor/plant resistance (TIR) domain of adaptor MyD88 interacts via a homophilic interaction with the TIR domain of the receptor. The 35-kDa protein MyD88 is located cytosolically. Its carboxy-terminal module binds the receptor and contains a TIR domain; the amino-terminal portion contains a death domain (dd)

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that mediates apoptosis. The dd module of MyD88 recruits IRAKs to the IL-1 receptor and to TLRs. Like MyD88, the adaptor protein TOLLIP (Toll-interacting protein) and TRIF also associate with IRAK and the TIR domains of the receptors and recruit IRAK to the receptor complex [53,54]. Further downstream, IRAKs subsequently bind TRAF6 that activates the transforming growth factor b-activated kinase (TAK1). TAK1 both induces the activator protein-1 (AP-1) and the IkB kinase (IKK) complex which phosphorylates IkB and initiates its degradation thereby unmasking the nucleus localization signal (NLS) of NF-kB enabling nuclear translocation and gene activation. In vitro studies showed that TRAF6 functions as a ubiquitin ligase that, together with cofactors Ubc13 and Uev1A (TRIKA1: TRAF6 regulated IKK activator 1), mediate the assembly of polyubiquitin chains to activate IKK. The components of TRIKA2 have been identified as TAK1, TAB1 and TAB2 and the kinase activity of TAK1 is dependent on the ubiquitination of TRAF6 following IL-1 stimulation [55]. Two IKK-related kinases, TANK-binding kinase-1 (TBK1) and the inducible IkB kinase (IKK-i or IKKo), have been implicated in NF-kB activation. In vitro studies suggest now that both kinases phosphorylate and activate the transcription factor IFN-regulatory factor 3 (IRF-3), which in turn regulate the expression of IFN-b, a type I IFN produced during viral infections and after challenge with LPS [56 /59].

TIR domain-containing adaptors

As mentioned above, MyD88 associates with the intracellular domains of members of the IL-1R and TLR families upon ligand binding. Various studies established MyD88 as a critical adaptor molecule involved in TLR signalling and release of cytokines [60]. However, MyD88 is not a requisite for effective signalling downstream of TLRs since NF-kB and MAPK activation in response to LPS was reduced and delayed, but not absent in MyD88-deficient cells, whereas it was completely suppressed in TLR4-deficient cells [61]. Further investigation of the MyD88independent pathway revealed that cells from MyD88 knockout mice failed to produce inflammatory cytokines in response to LPS, but induced IRF3 and activated IFN-inducible genes such as the glucocorticoid attenuated response gene 16 and interferoninducible protein 10 [62]. Genome database searches led to the identification of at least five TIR domain-containing adaptor proteins [63]. The second TIR domain-containing molecule discovered after MyD88 was termed TIRAP or

MAL [64,65]. Initial studies indicated that TIRAP/ MAL associates with the TIR domain of TLR4, but not with TLR9, and inhibition of TIRAP/MAL blocks LPS-induced maturation of dendritic cells from both wild-type and MyD88-deficient mice-results that strongly suggest that this adaptor is responsible for MyD88-independent TLR4 signalling. However, stimulation of macrophages from TIRAP/MAL knockout mice still showed activation of IFN-inducible genes by LPS and also in cells from double knockout (TIRAP/ MAL and MyD88) mice. Moreover, the observation of an impaired response to TLR2 ligands in mice lacking TIRAP/MAL led to the conclusion that TIRAP/MAL is involved in the MyD88-dependent signalling pathway via TLR2 and TLR4 [66,67] and an alternative TIR domain-containing adaptor molecule may be involved in activation of IRF3. Two groups independently described the next adaptor molecule, TRIF [68] or TICAM-1 [40]. Both studies indicate that TRIF/TICAM-1 is involved in the TLR3mediated MyD88-independent pathway and activates NF-kB and IRF3. By generating a chemically induced mutation in the TRIF gene, called Lps2, it turned out, that mice failed to induce type I IFN, such as IFN-a and IFN-b, when infected with mouse cytomegalovirus and they showed a defective response after challenge with LPS, indicating the additional involvement of TRIF/TICAM-1 in the MyD88-independent pathway via TLR4 [69]. Simultaneously, Yamamoto et al . [54] observed an impaired expression of IFN-inducible genes in TRIF-deficient mice mediated by TLR3 and TLR4. A fourth TIR domain-containing adaptor similar to TRIF has recently been identified, the TRIF-related adaptor molecule (TRAM) [70] or TICAM-2 [71] which activates IRF-3, IRF-7, and NF-kB, but its function seems to be restricted to the TLR4 pathway [72]. These findings demonstrate that selective utilization of adaptor proteins can lead to distinct signalling events caused by different microbial species. Individual TLRs may activate alternative signalling pathways in order to fine-tune the immune response to microbial challenge. The MyD88-dependent and -independent signal pathways are shown in Figs. 2 and 3.

TLRs involved in fungal recognition

While the major focus of research has been on bacterial and viral infections, less is known about the function of TLRs against fungal pathogens and fungal PAMPs. Most studies so far addressed the two major fungal pathogens, C. albicans and A. fumigatus , and only few
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Fig. 2 MyD88-dependent Toll-like receptor (TLR) signalling pathway with proximal signalling mediators. Signalling by TLRs involves signalling intermediates, such as MyD88, TIRAP/MAL, IRAK, and TRAF6, which are also associated with pathways activated by the IL-1 receptor complex resulting in NF-kB (early-phase) and AP-1 activation. TIRAP/MAL is implicated in the signalling pathway via TLR2 and TLR4. The involvement of key signalling mediators downstream of the TLRs is shown here. Abbreviations: AFT2, activating transcription factor 2; AP-1, activator protein-1; ERK, extracellular signal-regulated kinase; IKK complex, IkB kinase complex; IRAK, interleukin-1 receptor associated kinase; JNK, Jun N-terminal kinase; MAL, MyD88-adaptor-like; MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MyD88, myeloid differentiation marker 88; NF-kB, nuclear factor kappa B; TAK1, transforming growth factor b-activated kinase; TIRAP, TIR domain-containing adaptor protein; TLR, Toll-like receptor; TRAF6, tumour necrosis factor receptor-associated factor 6.

reports dealt with specific fungal PAMPs (Table 1) and their involvement in innate immunity.

Fungal pathogens
Netea et al . [73] demonstrated the involvement of TLR2 and TLR4 in host defence to C. albicans . Although growth of C. albicans was increased in TLR4-defective mice as compared to wild-type mice, TLR4 did not affect the level of TNF-a and IL-1b production in mouse macrophages upon stimulation with C. albicans . TNF-a and IL-1b release by human mononuclear cells stimulated with C. albicans is rather induced through TLR2 since the expression of these
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cytokines was significantly lower after blocking TLR2 with a specific anti-TLR2 antibody. On the other hand, the release of neutrophil chemokines KC and macrophage inhibitory protein (MIP)-2 was demonstrated to be induced through TLR4 by impairment of chemokine expression in macrophages of TLR4-defective mice. However, in a subsequent study TLR2-deficient mice surprisingly showed an increased resistance to disseminated candidiasis as compared to wild-type mice [74]. The mice showed normal production of the proinflammatory cytokines TNF-a, IL-1a and IL-1b, but a strong reduction of IL-10 and decrease of CD4 'CD25 ' regulatory T cells, both dependent on TLR2. In contrast, another group

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Fig. 3 MyD88-independent Toll-like receptor (TLR) signalling pathway with proximal signalling mediators. Signalling by TLRs involves signalling intermediates, such as TRIF/TICAM-1 and TRAM/TICAM-2, resulting in NF-kB (late-phase) and IRF3 activation. TRIF/TICAM-1 is implicated in the MyD88-independent pathway mediated by TLR3 and TLR4, whereas TRAM/TICAM-2 is restricted to the TLR4 pathway. The involvement of key signalling mediators downstream of the TLRs is shown here. Abbreviations: IKK, IkB kinase; IRF3, interferonregulatory factor 3; NF-kB, nuclear factor kappa B; TBK1, TANK-binding kinase-1; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TICAM, TIR-containing adaptor molecule.

reported impaired survival and reduced production of TNF-a and MIP-2 of mice lacking TLR2 compared to control mice [75]. In a later study TLR2-deficient mice were capable of mounting vaccine-induced resistance to C. albicans and an acquired specific humoral response likewise to control mice [76].

In our own laboratory we investigated whether the recognition of viable C. albicans by macrophages differs from recognition of antimycotic-treated C. albicans [77]. Macrophages from wild-type mice activated NF-kB in a TLR2 and TLR4 dependent manner, whereas C. albicans treated with a mixture of three

Table 1 Role of Toll-like receptors (TLRs) in recognition of fungal pathogen associated molecular patterns (PAMPs) Fungal species Candida albicans Cryptococcus neoformans Pneumocystis carinii Saccharomyces cerevisiae / PAMP Mannan Phospholipomannan Glucuronoxylomannan b-glucan Mannan Zymosan Linear (1 0/3)-b-D-glucan Involved TLRs or adaptor proteins TLR4 TLR2 (partially TLR4, TLR6) TLR2, TLR4 MyD88 TLR4 TLR2, TLR6 (heterodimers) MyD88 References [87] [86] [88] [94] [87] [34] [93]

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antifungal drugs (i.e. amphotericin B, nystatin and itraconazole) solely employed TLR2, implicating that treatment of C. albicans infections with antimycotics releases otherwise covered PAMPs and results in a TLR2-mediated stimulation of macrophages in addition to the direct effect of these drugs on C. albicans . Furthermore, analysis of signal transduction pathways upon stimulation of wild-type macrophages with antimycotic-treated C. albicans demonstrated the activation of the MAP kinases JNK, ERK, and p38, as well as the transcription factors c-Jun/AP-1 and NF-kB. However, Deva et al . [78] reported the involvement only of the MAP kinase p38 and NF-kB in HeLa cells stimulated by viable C. albicans . The reason for this difference is not clear at the moment, though it may be due to the different C. albicans preparations or cell types. Nonetheless, it may be speculated that a crosstalk between the signal transduction pathways exists that makes a more specified gene regulation possible. As for A. fumigatus , an ubiquitous saprophytic fungus, several studies with sometimes contradictory results have been published. Wang et al . [79] first implicated TLR4 and CD14 in host defence to A. fumigatus . Blockade of TLR4 and CD14 slightly reduced the release of TNF-a after stimulation of human monocytes with hyphal fragments. In contrast, TLR2 and MyD88 contributed to the production of TNF-a by A. fumigatus conidia and hyphae in both murine and human cells, whereas CD14 was only involved in human cells [80]. Another report provided evidence that both, TLR2 and TLR4, play a central role in sensing conidia and hyphae of A. fumigatus and a nonpathogenic A. niger isolate [81]. The authors suggested that the similar recognition of pathogenic and nonpathogenic aspergilli indicates that the specific pathogenic potential of A. fumigatus is not due to a potential escape mechanism that prevents recognition by the innate immune system. However, a study by Netea et al . [82] demonstrated that Aspergillus hyphae might escape innate immune recognition through TLRs: both Aspergillus conidia and hyphae were capable of inducing cytokines via TLR2, whereas only conidia were able to stimulate TNF-a and IL-1 through TLR4. Furthermore, solely Aspergillus hyphae enhanced the release of IL-10 in a TLR2-dependent manner owing to the loss of TLR4-mediated signals, implying phenotypic switching as an escape mechanism by Aspergillus during germination. The reasons for these partially varying results may be speculative and due to functional differences between human and murine TLRs or just the experimental design itself, e.g. by using TLR4defective versus TLR4-deficient mice by using different fungal strains.
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In a recent comprehensive study the influence of the IL-1R-, TLR- and MyD88-dependent signalling pathway after challenge with C. albicans or A. fumigatus was evaluated in vivo [83]. Bellocchio et al . could show that the MyD88-dependent pathway is essential for the innate resistance to both fungi. The susceptibility of MyD88-deficient mice to candidiasis and aspergillosis was associated with a defective Th1 response most likely due to defective DC activation. In addition, polymorphonuclear neutrophils (PMN) showed impaired antifungal effector function in terms of phagocytosis and fungicidal activity. Interestingly, mice lacking MyD88 survived the A. fumigatus infection, pointing to the involvement of MyD88-independent mechanisms. Furthermore, it was demonstrated that the involvement of MyD88 may occur through signalling by distinct TLRs depending on fungal species, morphotypes, and site of infection. Primary infection with Candida required IL-1RI and MyD88 for resistance, whereas TLR4 and MyD88 were indispensable for Aspergillus . The assessment of fungal growth in the kidneys of mice after intravenous (i.v.) infection with Candida yeast and hyphae revealed that the fungal burden after infection with Candida yeast was significantly higher in IL-1RI- and MyD88-deficient mice as compared to control mice, while no increase of fungal growth in the kidneys was observed after infection with Candida hyphae. Analysis of fungal growth in the stomach after intragastric infection with Candida hyphae compared to the kidneys upon i.v. infection showed a differential involvement of TLR2 and TLR4 in the control of disseminated or mucosal infections. The authors also demonstrated that the contribution of TLRs to innate and adaptive Th1 immunity to each fungus may vary, consistent with the ability of each individual TLR to activate specialized antifungal effector functions on PMN and DC [83]. Investigation of the signalling pathway downstream of TLRs revealed a different role for MyD88 in terms of regulating phagocytosis and cytokine production in response to C. albicans and A. fumigatus [84]. Macrophages of MyD88-deficient mice showed reduced intracellular killing and phagocytosis, as well as an impaired production of TNF-a for C. albicans yeast and hyphae, as compared to wild-type mice, whereas no effect on ingestion and killing, and cytokine release was observed with A. fumigatus conidia and hyphae, respectively.

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Cell wall components and fungal PAMPs

The fungal cell wall is complex and harbours a variety of molecules, including glucan, chitin, mannoproteins,

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various other cell wall enzymes, and proteins [85], being additional potential PAMPs. Yeast zymosan, a cell wall component of S. cerevisiae , has been shown to activate heterodimers consisting of TLR2 and TLR6 [34]. Phospholipomannan (PLM), a phylogenetically unique glycolipid present in the cell surface of C. albicans , has recently been identified as a likely PAMP for TLR2 and partially TLR4 and TLR6 [86]. Incubation of murine macrophages with PLM activated NF-kB and TNF-a in a TLR-dependent manner. Cells lacking TLR2 showed almost no production of TNF-a, whereas TLR4- and TLR6-deficient macrophages led to a decreased cytokine production compared to macrophages from wild-type mice, indicating the involvement of several TLRs for a single molecule. Another recent study concerning fungal cell wall components discusses activation of TLR4 in recognition of S. cerevisiae - and C. albicans -derived mannan in human monocytes [87]. Although the mannan fractions partially suffered from heavy contamination with LPS, the secretion of TNF-a was not inhibited by polymyxin B, indicating that endotoxin contamination was not responsible for the observed biological effects of the mannan fractions. TLR4, as well as LPS-binding protein and CD14 were essential for the TNF-a production by human monocytes. Glucuronoxylomannan (GXM), the major capsular polysaccharide of C. neoformans has been shown to bind TLR2, TLR4 and CD14 in vitro [88]. GXM stimulated NF-kB translocation in PMBC as well as macrophages, and activated an NF-kB-dependent reporter construct in fibroblasts transfected both with TLR4 and CD14 but not after transfection with TLR2. However, induction of NF-kB failed to release TNF-a or activate MAP kinase pathways. As GMX is the major virulence factor of C. neoformans being present in high concentrations in patients with cryptococcosis, the partial activation of TLR signalling pathways might be an immunodysregulatory effect triggered by GMX as proposed by the authors. At the moment, it is not clear whether C. neoformans and LPS share the same signalling pathway downstream of TLR4. However, another explanation for the differential responses to GXM and LPS upon TLR4 binding might be the utilization of different adaptor proteins, thus triggering different cellular responses. Furthermore, studies suggest that, instead of inducing secretion of cytokines, CD14 and TLR4 might have a crucial role in mediating the cellular internalization and phagocytosis of GXM by monocytes and neutrophils [89,90]. Recently, the role of TLRs has been investigated in vivo following infection with C. neoformans [91]. Mice deficient for MyD88 were significantly more susceptible compared

to wild-type mice after i.v. and intranasal (i.n.) infection with live C. neoformans , while TLR2 knock-out mice succumbed earlier solely by the i.n. route. Following i.n., i.v., and intraperitoneal infection, the mortality rate of TLR4-defective mice was similar to wild-type mice. The decreased survival of MyD88 knock-out mice correlated with an increased fungal burden in the lung together with higher serum and lung GXM concentrations as compared to wild-type mice. Interestingly, analysis of lung and brain cytokine levels revealed no major differences between uninfected and infected mice, further indicating that TLRs might not be involved in secretion of cytokines during cryptococcal infections. Linear (1 0/3)-b-D-glucans, accounting for more than half of the fungal cell wall [92], activated NF-kB in macrophages, with MyD88 being involved as an essential component for this activation [93]. The adaptor protein MyD88 has also been implicated in the recognition of Pneumocystis carinii cell wall b-glucans [94]. Besides the potential involvement of TLRs in the mediation of b-glucan, Dectin-1, a new lectin receptor for b-glucan containing particles including zymosan and C. albicans , releases in cooperation with TLR2, TNF-a and IL-12 in response to fungalderived b-glucans [95]. Moreover, binding and nonopsonic phagocytosis and killing of P. carinii by alveolar macrophages could be inhibited by blocking of Dectin-1 [96]. Recognition of P. carinii by Dectin-1 led to secretion of the proinflammatory chemokine macrophage inflammatory protein-2. In contrast, mice lacking the mannose receptor (MR) showed a similar internalization of P. carinii as compared to macrophages isolated from wild-type mice, indicating that the MR is not involved in the macrophage-mediated killing of P. carinii [96]. Interestingly, Brown and co-workers could recently show that the internalization mechanism of -glucan ligands, including yeast particles, by Dectin-1 is distinct from any other known phagocytotic receptor such as complement or Fc receptors [97]. Overall, the majority of these studies implicated mainly TLR2 and TLR4 in the elicitation of immune responses against the two major fungal pathogens C. albicans and A. fumigatus infections. Most investigations coped with the interaction of fungal pathogens with macrophages, and systemic infections, respectively. The majority of Candida infections are mucosal [98] and the first line of innate defence is the epithelial cell. However, at present, little is known about the role of oral or vaginal epithelial TLRs in the mediation of immune responses against Candida , though one has to bear in mind that C. albicans is a harmless colonizer of mucosal surfaces in healthy individuals. During the
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period of colonization, extensive fungal growth is limited through release of antimicrobial peptides from keratinocytes, or due to existence of other bacteria of the microbial flora, such as lactobacilli in the vagina. In this stage of colonization without clinical symptoms and signs of inflammation neither the facultative pathogen nor the host might induce a (TLR mediated) inflammatory cytokine response. However, when these conditions get out of balance, for instance due to antibiotic therapy or immunosuppression, superficial or even systemic infections may occur. The putative role of keratinocytes in innate immunity against C. albicans has recently been addressed, as human keratinocytes express TLR2 and TLR4 as well as MyD88. Moreover, Candida -killing activity of keratinocytes required NF-kB activation and a microbial induced upregulation of IL-8 could be inhibited by antibodies raised against TLR2 and TLR4 [99]. Further studies are needed to clarify the function of TLRs in epithelial cells.

molecule-3-grabbing non-integrin) was shown to bind and internalize C. albicans and is able to function as an adhesion receptor as well as a phagocytic receptor, similar to TLRs [105]. On resident peritoneal macrophages, a DC-SIGN homolog, termed SIGNR1 (SIGNrelated 1), was identified as a major MR involved in recognition of zymosan and C. albicans [106]. Although previous investigations focused mainly on individual receptors or signalling pathways, new aspects of cooperation between innate immune receptors and their signal transduction pathways are now gradually emerging and may improve our knowledge of the complex innate immune responses.

Perspectives
The initial description of Drosophila Toll as a signal transducer in antifungal response points towards a TLR function in mammalian antifungal defence. The above mentioned studies clearly demonstrate that TLRs contribute to the signal transduction induced by many PAMPs, to the induction of inflammation, and to the activation of adaptive immunity. The simultaneous activation of multiple PRRs by one fungal pathogen endow the immune system with a broad range of possibilities for a specific and effective immune response. However, with regard to the TLRs, several studies could demonstrate that pathogens are able to manipulate or escape innate immune recognition [107]. The apparent phenotypic switching of A. fumigatus as a mechanism to escape TLR recognition [82] implies that fungal pathogens may have evolved to selectively recognize and exploit TLRs. Furthermore, it has been shown that mice lacking TLR2 are more susceptible to C. albicans infection [75], in contrast to the similar [83] or even increased resistance [74] of TLR2-knockout mice compared to control mice [74]. Although the reason for this discrepancy is not clear, it is tempting to speculate whether C. albicans has developed structures to exploit TLRs and induce suppression of innate immunity [74]. More specifically, it may be possible that structures of C. albicans interacting with TLR2 are not actually PAMPs, as PAMPs are microbial components against which TLRs have evolved the ability to recognize and respond to pathogens. Further understanding of the fungal escape mechanisms and of the cooperation of multiple innate immune receptors will undoubtedly offer novel therapeutic strategies for immunomodulation and pharmacological targeting and might be a new and promising way to treat or overcome fungal infections.

Other immune receptors involved in fungal recognition

Various other receptors are involved in recognition of fungal microorganisms, indicating that TLRs may not be the only essential signal transducers in recognition of fungal microorganisms. Immune receptors recognizing opsonized fungi include, for instance, complement (CR) and opsonic Fcx (FcxR) receptors. CR3 (also known as CD11b/CD18, Mac-1, and 2-integrin) has originally been identified as a receptor for fungal-derived glucans besides playing a critical role in phagocytotic processes of opsonized microbes together with FcxR. Recent studies revealed a potential cooperation in terms of immune response between CR3 and TLR4/CD14 after stimulation with LPS [100,101]. PCSC, a polysaccharide purified from the sclerotum of the fungus Poria cocos Wolf, induced p38 and nitric production via CR3 and TLR4/CD14, providing evidence for receptor collaboration in defence against fungal molecules [102]. Optimal phagocytosis of pathogens requires opsonization, although unopsonized yeast can be internalized and killed through other receptors such as the MR and Dectin-1 [97,103]. Romani et al . conducted an extensive study to determine the impact of fungal recognition through different recognition receptors on DC [104]. The relative contributions of MR, Dectin-1, CR3 and FcxR to phagocytosis, cytokine release and expression of co-stimulatory molecules have been evaluated utilizing live unopsonized or opsonized Candida yeasts or hyphae. Two other PRRs have recently been implicated in recognition of fungi. The C-type lectin receptor DC-SIGN (dendritic cell-specific intercellular adhesion
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