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Interactions of Toll-like receptors with fungi
Microbes and Infection 6 (2004) 1351–1355 www.elsevier.com/locate/micinf
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Interactions of Toll-like receptors with fungi Stuart M. Levitz * Evans Memorial Department of Medicine, Boston Medical Center, Boston University School of Medicine, Room X626, 650 Albany Street, Boston, MA 02118, USA Available online 22 October 2004
Abstract The role of Toll-like receptors (TLRs) in signaling immune responses to fungal pathogens is reviewed. TLR2 and TLR4, acting via the adapter protein MyD88, signal responses to Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans in vitro, although the relative significance of these TLRs to host defenses in vivo is unclear. © 2004 Published by Elsevier SAS. Keywords: Mycoses; CD14 antigen; MyD88-adapter-like protein; Toll-like receptor; Zymosan
1. Introduction It is estimated that there are between 100,000 and 1,000,000 species of fungi inhabiting our planet. The ubiquitous presence of fungi in the environment has led to the evolution of innate and acquired host defense antifungal mechanisms in multicellular animals. Indeed, few fungal species regularly cause invasive disease in people, and most of these act as opportunistic pathogens in individuals with specific immune defects. Concomitant with the vast increase in the numbers of immunocompromised persons, there has been a corresponding increase in the incidence of life-threatening mycoses. Thus, fungal infections, particularly aspergillosis, candidiasis, cryptococcosis, and pneumocystosis, have emerged as some of the top few infectious killers in patients with AIDS and certain malignancies. Fungi are eukaryotic, and as such share many of the basic cellular features of mammalian cells such as a nuclear membrane, 80S ribosomes, endoplasmic reticulum, and Golgi apparatus. The major distinguishing feature is that fungi have a rigid cell wall. The cell wall imparts upon the fungus physical protection and structural support [1]. Importantly, the cell wall contains distinctive molecules, particularly polysaccharides that are rarely, if ever, present in mammalians. These pathogen-associated molecular patterns (PAMPs) can decorate the surface of the fungus, and include b-glucans, chitin, * Corresponding author. Tel.: +1-617-638-7904; fax: +1-617-638-7923. E-mail address: [email protected] (S.M. Levitz). 1286-4579/$ - see front matter © 2004 Published by Elsevier SAS. doi:10.1016/j.micinf.2004.08.014
and mannoproteins (mannans). b-Glucans and mannans will be discussed in more detail below, as each has been shown to be recognized by mammalian pattern-recognition receptors. The basic cell wall structure of fungi consists of a linear b-glucan backbone from which there are covalently attached branches of additional b-glucan, chitin and mannoproteins. The mannoproteins tend to be highly glycosylated; N-linkages often contain hundreds of mannose residues, which may be extensively branched [2]. O-linkages tend to be shorter but still consist predominantly of mannose [3]. In contrast, fully processed mammalian glycoproteins are rarely mannosylated. This enables exposed cell wall mannose and circulating mannoproteins to be recognized as foreign by host mannose receptors. Many immunodominant fungal antigens have been shown to be mannosylated, with the immunodominance dependent upon the presence of mannosylation [3–5]. Although the cell walls of only a few fungi have been defined in any great detail, it is becoming apparent that there is considerable interspecies (and even intraspecies) variation. For example, in some fungi, the mannans are surface exposed, whereas in others the mannans are confined to the inner cell wall and thus not recognized by mannose receptors. Aspergillus species modifies its mannans by the addition of galactofuranose to form galactomannan [6]. Cryptococcus neoformans has a thick polysaccharide capsule that masks cell wall ligands [7]. The immunological consequences of these variations are increasingly becoming appreciated.
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This review focuses on the current state of knowledge of Toll-like receptors (TLRs) in immune recognition of fungi. The original association of Toll with antimicrobial defenses in Drosophila was made by Lemaitre et al. [8]. Flies deficient in Toll rapidly succumbed to infection when pricked with a needle that had been dipped in a suspension of Aspergillus fumigatus conidia. Toll signals rapid synthesis of antifungal peptides by a signaling pathway that includes induction of Dorsal. Lemaitre et al. astutely noted that Toll and Dorsal shared structural and functional similarity to IL-1 receptors and NF-jB. This led others to search for Toll homologs in mammalian cells. There exist at least 11 TLRs, each of which shares an extracellular domain containing leucine-rich repeats and a cytoplasmic Toll/IL-1 receptor homology (TIR) domain [9].
2. Zymosan Zymosan is a cell wall preparation predominantly composed of b-glucans and mannan derived from the yeast Saccharomyces cerevisiae. Underhill et al. [10] expressed hemagglutinin A-tagged murine TLR2 in the mouse macrophage cell line, RAW-TT10, and challenged the cells with zymosan particles. Within 5 min, TLR2 underwent redistribution from the cell surface to the phagosome. Transfection with a dominant-negative form of TLR2 abolished zymosan-stimulated TNF-a production but had no effect on phagocytosis. This led to the concept that TLRs signal inflammatory responses to particulate stimuli but other receptors were responsible for actual phagocytosis, a concept later validated with other fungi [11,12]. Recently, the b-glucan receptor dectin-1 was identified as the receptor responsible for phagocytosis of zymosan particles [12,13]. Moreover, signaling inflammatory responses requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of dectin1 acting cooperatively with TLR2 and MyD88.
3. C. neoformans 3.1. Capsule as a virulence factor C. neoformans, the etiological agent of cryptococcosis, is an encapsulated yeast that has a strong predilection for causing infections in persons with defects in T-cell-mediated immunity, particularly those with AIDS. The capsule is the organism’s major virulence factor, with acapsular organisms being essentially avirulent. Capsule surrounds the fungus, masking potential ligands on the cell wall, and is also shed. Unopsonized C. neoformans is poorly recognized by phagocytes. However, the fungus is a potent activator of the complement system, and C3-opsonized C. neoformans is recognized by the complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on phagocytes [14]. Capsule tends to elicit poor B-cell responses, although
under certain conditions, anticapsular antibody may also contribute to opsonization. Approximately 90% of capsular material is composed of glucuronoxylomannan (GXM). Shed GXM has a molecular weight estimated at approximately 106 Da and circulates in the blood and cerebrospinal fluid of patients with cryptococcosis at µg/ml concentrations and is likely to be present in tissues at mg/ml amounts. 3.2. In vitro studies Shoham et al. [7] demonstrated specific binding of cryptococcal GXM to Chinese hamster ovary (CHO) fibroblasts transfected with human TLR2, TLR4 and/or CD14. However, GXM stimulated nuclear translocation of NF-jB only in the CHO cells that were transfected with both TLR4 and CD14. GXM also stimulated nuclear NF-jB translocation in peripheral blood mononuclear cells (PBMCs) and the RAW264.7 cell line. Interestingly though, challenge of these cells with GXM resulted in neither TNF-a secretion nor activation of the ERK1/2, p38 and SAPK/JNK mitogenactivated protein (MAP) kinase pathways. These findings suggest that the interaction between GXM and CD14/ TLR4 may represent a mechanism of immune dysregulation whereby the organism incompletely activates cascades leading to TNF-a production. In support of a role for TLR4 and CD14 as receptors for GXM, Ellerbroek et al. [15] demonstrated that the inhibitory effect of GXM on neutrophil rolling could be blocked using monoclonal antibodies directed against CD14 and TLR4. 3.3. In vivo mouse models Mice deficient in CD14, TLR2, TLR4, and MyD88 were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans [16]. The MyD88–/– mice had significantly reduced survival compared with wild-type C57BL/6 mice after pulmonary and intravenous challenge with C. neoformans. In contrast, the phenotype was less pronounced with the other mouse groups. TLR2–/– mice died significantly sooner following pulmonary challenge but not intravenous challenge, while for the CD14–/– mice, there was a trend towards reduced survival only following intravenous challenge. Finally, mortality was similar comparing TLR4-mutant C3H/HeJ mice and control C3H/HeOuJ mice following either pulmonary or intravenous challenge.
4. Candida albicans 4.1. Clinical manifestations of infection Infections due to Candida species are among the most common of the mycoses. Clinical manifestations range from mucocutaneous infections to life-threatening deep-seated infection. Interestingly, T-cell dysfunction predisposes to mu-
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cocutaneous infections, while qualitative and quantitative disorders of neutrophil function greatly increase the risk of disseminated disease. Published work examining the role of TLRs in candidiasis has focused on C. albicans, which is the most common species seen, although non-albicans candidiasis has emerged in recent years and now accounts for approximately 50% of bloodstream isolates. Most Candida species are dimorphic, with both yeast and hyphal forms observed in infected tissues. 4.2. In vitro studies In vitro studies have identified a role for both TLR2 and TLR4 in responses to C. albicans. Macrophages from TLR4mutant C3H/HeJ mice had impaired chemokine expression and neutrophil recruitment, but produced levels of proinflammatory cytokines that were similar to those seen with wild-type mice [17]. Antibodies directed against TLR2, but not TLR4, blocked TNF-a production from human PBMCs stimulated with C. albicans [17]. Consistent with a role for TLR2, in vitro production of TNF-a and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2–/– mice in response to yeast cells and hyphae of C. albicans was significantly lower than in wild-type mice [18]. Bellocchio et al. [19] examined the candidacidal activity of neutrophils from wild-type, TLR2–/–, TLR4–/–, TLR9–/–, IL-1R–/– and MyD88–/– mice. Activity against both yeast and hyphae was diminished in neutrophils from MyD88- and IL-1R1deficient mice. In contrast, the antifungal activity of neutrophils from the TLR2-, TLR4- and TLR9-deficient mice was either unaffected, or in some cases, even slightly increased. MyD88–/– murine bone marrow-derived dendritic cells also had impaired phagocytosis and killing of C. albicans yeast cells [20]. In another study, neutralization of TLR2 and TLR4 inhibited the synthesis of PGE2 by HeLa cells stimulated with C. albicans [21]. While the above experiments examined responses to whole C. albicans, some investigators have focused on defining the molecules responsible for stimulating TLR responses. Tada et al. [22] demonstrated TNF-a production from human monocytes stimulated with mannans isolated from C. albicans and S. cerevisiae. The TNF-a production required the presence of LPS-binding protein, and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. A potential problem with these experiments was the possible presence of endotoxin contamination in the mannan preparations. Polymyxin B did not inhibit TNF-a production in response to the S. cerevisiae mannan, but polymyxin B controls were not reported for the C. albicans mannan preparation. Jouault et al. [23] demonstrated that a cell wall surface glycolipid, phospholipomannan, stimulates murine macrophages to produce TNF-a via a TLR2-dependent mechanism. b-Glucans from C. albicans also stimulate TNF-a via a mechanism dependent upon dectin-1 and TLR2 [12].
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4.3. Role of TLR in vivo Following an intravenous challenge with C. albicans, TLR4-defective C3H/HeJ mice were more susceptible to infection than wild-type C3H/HeN mice [17]. However, when TLR4–/– mice on a C57BL6 background were challenged with C. albicans hyphae, the knockout mice survived longer than the wild-type controls [19]. Netea et al. [24] found that TLR2–/– mice were paradoxically more resistant to disseminated Candida infection. The mechanism appeared to be impaired IL-10 release in the TLR–/– mice, which resulted in an accompanying decrease in CD4+CD25+ regulatory T cells. However, in two other models of disseminated candidiasis, TLR2–/– mice had significantly impaired survival, in one model, and no difference in survival in the other model [18,19]. Following a gastric challenge with C. albicans hyphae, Bellocchio et al. [19] found a decrease in IFN-c-producing Th1 cells and an increase in IL-4producing Th2 cells in TLR2-, TLR4-, TLR9-, IL1-RI-, and MyD88-deficient mice, compared with wild-type mice. In humans, TLR polymorphisms have been associated with altered risks for certain infectious and cardiovascular diseases. However, a study of Dutch women found that the TLR4 Asp299Gly polymorphism does not seem to play a role in the susceptibility to, and severity of, human urogenital C. albicans infection [25]. Moreover, in a small study of patients with chronic mucocutaneous candidiasis, there was no obvious association with TLR2 or TLR4 polymorphisms [26].
5. A. fumigatus
5.1. Spectrum of aspergillosis Fungi of the genus Aspergillus are ubiquitous in the environment. Exposure is common and generally occurs following inhalation of metabolically inactive (“resting”) airborne conidia (spores). In the absence of an effective host defense, the conidia swell and then germinate, forming hyphae, the invasive form of the fungus. Thus, following exposure, the host has opportunities to kill three morphotypes of the fungus: resting conidia, swollen conidia and hyphae. The clinical spectrum of aspergillosis ranges from allergic disease in atopic individuals to invasive disease in the severely immunocompromised. Despite advances in therapy, the mortality of invasive aspergillosis remains very high. Interestingly, amphotericin B, which is one of the main drugs used to treat invasive aspergillosis (and other mycoses), frequently causes infusion-related side effects including fever and chills, which are thought to be due to the release of proinflammatory cytokines. Sau et al. [27] demonstrated that amphotericin B stimulates proinflammatory cytokine release by a mechanism dependent upon TLR2, CD14, and MyD88.
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5.2. A. fumigatus and TLR Studies examining the role of TLR in aspergillosis have focused mostly on A. fumigatus, the most common species to cause human disease. The results, which are summarized below, are at times seemingly conflicting. Some of the disparities are undoubtedly due to experimental variation, including which A. fumigatus strain was utilized, as well as whether the fungus was killed, and if so, by what method. There are also well-defined distinctions in the responses to the three morphotypes of the fungus, and in some studies rigorous controls to rule out endotoxin contamination were not undertaken. Regarding the host response, speciesspecific and cell type-specific differences also exist. Taken together though, the published data, summarized below, point to involvement of TLR2, TLR4, and CD14 in immune responses to A. fumigatus. Mambula et al. [11] utilized knockout mice and transfected human cells to examine the contribution of TLR2, TLR4, MyD88, and CD14 to signaling in response to resting conidia, swollen conidia, and hyphae of A. fumigatus. Optimal signaling responses in both the mouse and human cells required TLR2 acting through the central adapter protein MyD88. CD14 was important for responses to the human cells but not the murine cells. However, despite their contribution to NF-jB activation and cytokine release, TLR2, MyD88, and CD14 were not required for phagocytosis. TLR4-deficient macrophages made less TNF-a compared with wild-type cells when stimulated by A. fumigatus swollen conidia but not A. fumigatus hyphae. Netea et al. [28] also found a role for TLR4 in response to conidia but not hyphae, whereas Meier et al. [29] found a role for TLR2 and TLR4 in signaling responses to both conidia and hyphae. Wang et al. [30] found that hyphal-stimulated TNF-a production from human monocytes was inhibited by monoclonal antibodies directed against TLR4 or CD14, but not by anti-TLR2 antibodies [30]. Conflicting data also exist with regard to phagocytosis and killing. Mambula et al. [11] found no role for TLR2, TLR4, MyD88, and CD14 in phagocytosis of A. fumigatus conidia. Marr et al. [20] observed that incubation of MyD88–/– murine bone marrow-derived dendritic cells with A. fumigatus conidia resulted in phagocytosis and fungal killing at levels comparable to those seen in wild-type cells. However, Bellochhio et al. [19] found a defect in phagocytosis and killing of conidia by MyD88–/– and TLR4–/– (but not TLR2–/– and TLR9–/–) murine PMNs. Consistent results were obtained in vivo using a model in which mice were immunosuppressed with cyclophosphamide and then challenged via the pulmonary route with A. fumigatus conidia. The wild-type, TLR2–/– and TLR9–/– mice all had similar survival curves, but the MyD88–/– and TLR4–/– mice died significantly sooner. The A. fumigatus antigens responsible for TLR-mediated stimulation have not been purified. Braedel et al. [31] examined two crude preparations derived from A. fumigatus cul-
tures, one derived from a glass-bead-disrupted cellular extract and the other from culture supernatants. The cellular extract stimulated IL-12 p40 production from murine bone marrow-derived dendritic cells in a TLR2-dependent, TLR4independent manner, whereas the culture supernatant stimulated IL-12 p40 in the absence of TLR2 or TLR4 (but not in cells which lacked both TLR2 and TLR4). Interestingly, the cellular extract stimulated IL-6 secretion in a TLR2independent manner, while the culture supernatant required TLR4 for IL-6 production.
6. Conclusion While much progress has been made elucidating the contribution of TLRs in the host response to fungi, much still needs to be learned. Thus far, only three fungal ligands, b-glucan, GXM, and phospholipomannan that stimulate TLR responses have been identified on the molecular level. In addition, there are published data on only a few of the pathogenic fungi. As noted above, some of these data are conflicting, and the reasons for the disparate results need to be established. Finally, the downstream effects of TLR stimulation need to be defined, including the signaling pathways utilized and the nature of the inflammatory response elicited. The inflammatory response seen in fungal infections can be a “two-edged sword”. While necessary for effective host defenses, an overly exuberant or misdirected inflammatory response can lead to deleterious effects due to bystander tissue destruction. Ultimately, the challenge will be to apply our understanding of how fungi stimulate TLR-mediated immune responses to therapeutic strategies in the at-risk patient population.
References [1]
[2] [3] [4]
[5]
[6] [7]
F.M. Klis, P. Mol, K. Hellingwerf, S. Brul, Dynamics of cell wall structure in Saccharomyces cerevisiae, FEMS Microbiol. Rev. 26 (2002) 239–256. J.E. Cutler, N-glycosylation of yeast, with emphasis on Candida albicans, Med. Mycol. 39 (Suppl. 1) (2001) 75–86. M.K. Mansour, S.M. Levitz, Fungal mannoproteins: the sweet path to immunodominance, ASM News 69 (2003) 595–600. S.M. Levitz, S. Nong, M.K. Mansour, C. Huang, C.A. Specht, Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans, Proc. Natl. Acad. Sci. USA 98 (2001) 10422–10427. M.K. Mansour, L.S. Schlesinger, S.M. Levitz, Optimal T-cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors, J. Immunol. 168 (2002) 2872–2879. M. Bernard, J.P. Latge, Aspergillus fumigatus cell wall: composition and biosynthesis, Med. Mycol. 39 (Suppl. 1) (2001) 9–17. S. Shoham, C. Huang, J.M. Chen, D.T. Golenbock, S.M. Levitz, Toll-like receptor 4 mediates intracellular signaling without TNFalpha release in response to Cryptococcus neoformans polysaccharide capsule, J. Immunol. 166 (2001) 4620–4626.
S.M. Levitz / Microbes and Infection 6 (2004) 1351–1355 [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
B. Lemaitre, E. Nicolas, L. Michaut, J.M. Reichhart, J.A. Hoffmann, The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults, Cell 86 (1996) 973–983. D. Zhang, G. Zhang, M.S. Hayden, et al., A Toll-like receptor that prevents infection by uropathogenic bacteria, Science 303 (2004) 1522–1526. D.M. Underhill, A. Ozinsky, A.M. Hajjar, et al., The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens, Nature 401 (1999) 811–815. S.S. Mambula, K. Sau, P. Henneke, D.T. Golenbock, S.M. Levitz, Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus, J. Biol. Chem. 277 (2002) 39320–39326. G.D. Brown, J. Herre, D.L. Williams, J.A. Willment, A.S. Marshall, S. Gordon, Dectin-1 mediates the biological effects of beta-glucans, J. Exp. Med. 197 (2003) 1119–1124. B.N. Gantner, R.M. Simmons, S.J. Canavera, S. Akira, D.M. Underhill, Collaborative induction of inflammatory responses by dectin1 and Toll-like receptor 2, J. Exp. Med. 197 (2003) 1107–1117. S.M. Levitz, A. Tabuni, Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin, J. Clin. Invest. 87 (1991) 528–535. P.M. Ellerbroek, L.H. Ulfman, A.I. Hoepelman, F.E. Coenjaerts, Cryptococcal glucuronoxylomannan interferes with neutrophil rolling on the endothelium, Cell. Microbiol. 6 (2004) 581–592. L.E. Yauch, M.K. Mansour, S. Shoham, J.B. Rottman, S.M. Levitz, Involvement of CD14, Toll-like receptor (TLR) 2, TLR4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo, Infect. Immun. 72 (2004) 5373–5382. M.G. Netea, C.A. Van der Graaf, A.G. Vonk, I. Verschueren, J.W. Van der Meer, B.J. Kullberg, The role of Toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis, J. Infect. Dis. 185 (2002) 1483–1489. E. Villamon, D. Gozalbo, P. Roig, J.E. O’Connor, D. Fradelizi, M.L. Gil, Toll-like receptor-2 is essential in murine defenses against Candida albicans infections, Microbes Infect. 6 (2004) 1–7. S. Bellocchio, C. Montagnoli, S. Bozza, et al., The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo, J. Immunol. 172 (2004) 3059–3069. K.A. Marr, S.A. Balajee, T.R. Hawn, et al., Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens, Infect. Immun. 71 (2003) 5280–5286.
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[21] R. Deva, P. Shankaranarayanan, R. Ciccoli, S. Nigam, Candida albicans induces selectively transcriptional activation of cyclooxygenase2 in HeLa cells: pivotal roles of Toll-like receptors, p38 mitogenactivated protein kinase, and NF-kappa B, J. Immunol. 171 (2003) 3047–3055. [22] H. Tada, E. Nemoto, H. Shimauchi, et al., Saccharomyces cerevisiaeand Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner, Microbiol. Immunol. 46 (2002) 503– 512. [23] T. Jouault, S. Ibata-Ombetta, O. Takeuchi, et al., Candida albicans phospholipomannan is sensed through Toll-like receptors, J. Infect. Dis. 188 (2003) 165–172. [24] M.G. Netea, R. Sutmuller, C. Hermann, et al., Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells, J. Immunol. 172 (2004) 3712–3718. [25] S.A. Morre, L.S. Murillo, J. Spaargaren, H.S. Fennema, A.S. Pena, Role of the Toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection, J. Infect. Dis. 186 (2002) 1377–1379 (author reply 1379). [26] C.A. Van der Graaf, M.G. Netea, I.P. Drenth, R.H. Te Morsche, J.W. Van der Meer, B.J. Kullberg, Candida-specific interferon-gamma deficiency and Toll-like receptor polymorphisms in patients with chronic mucocutaneous candidiasis, Neth. J. Med. 61 (2003) 365– 369. [27] K. Sau, S.S. Mambula, E. Latz, P. Henneke, D.T. Golenbock, S.M. Levitz, The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism, J. Biol. Chem. 278 (2003) 37561–37568. [28] M.G. Netea, A. Warris, J.W. Van der Meer, et al., Aspergillus fumigatus evades immune recognition during germination through loss of Toll-like receptor-4-mediated signal transduction, J. Infect. Dis. 188 (2003) 320–326. [29] A. Meier, C.J. Kirschning, T. Nikolaus, H. Wagner, J. Heesemann, F. Ebel, Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages, Cell. Microbiol. 5 (2003) 561–570. [30] J.E. Wang, A. Warris, E.A. Ellingsen, et al., Involvement of CD14 and Toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae, Infect. Immun. 69 (2001) 2402–2406. [31] S. Braedel, M. Radsak, H. Einsele, et al., Aspergillus fumigatus antigens activate innate immune cells via Toll-like receptors 2 and 4, Br. J. Haematol. 125 (2004) 392–399.
Forum
Interactions of Toll-like receptors with fungi Stuart M. Levitz * Evans Memorial Department of Medicine, Boston Medical Center, Boston University School of Medicine, Room X626, 650 Albany Street, Boston, MA 02118, USA Available online 22 October 2004
Abstract The role of Toll-like receptors (TLRs) in signaling immune responses to fungal pathogens is reviewed. TLR2 and TLR4, acting via the adapter protein MyD88, signal responses to Cryptococcus neoformans, Aspergillus fumigatus and Candida albicans in vitro, although the relative significance of these TLRs to host defenses in vivo is unclear. © 2004 Published by Elsevier SAS. Keywords: Mycoses; CD14 antigen; MyD88-adapter-like protein; Toll-like receptor; Zymosan
1. Introduction It is estimated that there are between 100,000 and 1,000,000 species of fungi inhabiting our planet. The ubiquitous presence of fungi in the environment has led to the evolution of innate and acquired host defense antifungal mechanisms in multicellular animals. Indeed, few fungal species regularly cause invasive disease in people, and most of these act as opportunistic pathogens in individuals with specific immune defects. Concomitant with the vast increase in the numbers of immunocompromised persons, there has been a corresponding increase in the incidence of life-threatening mycoses. Thus, fungal infections, particularly aspergillosis, candidiasis, cryptococcosis, and pneumocystosis, have emerged as some of the top few infectious killers in patients with AIDS and certain malignancies. Fungi are eukaryotic, and as such share many of the basic cellular features of mammalian cells such as a nuclear membrane, 80S ribosomes, endoplasmic reticulum, and Golgi apparatus. The major distinguishing feature is that fungi have a rigid cell wall. The cell wall imparts upon the fungus physical protection and structural support [1]. Importantly, the cell wall contains distinctive molecules, particularly polysaccharides that are rarely, if ever, present in mammalians. These pathogen-associated molecular patterns (PAMPs) can decorate the surface of the fungus, and include b-glucans, chitin, * Corresponding author. Tel.: +1-617-638-7904; fax: +1-617-638-7923. E-mail address: [email protected] (S.M. Levitz). 1286-4579/$ - see front matter © 2004 Published by Elsevier SAS. doi:10.1016/j.micinf.2004.08.014
and mannoproteins (mannans). b-Glucans and mannans will be discussed in more detail below, as each has been shown to be recognized by mammalian pattern-recognition receptors. The basic cell wall structure of fungi consists of a linear b-glucan backbone from which there are covalently attached branches of additional b-glucan, chitin and mannoproteins. The mannoproteins tend to be highly glycosylated; N-linkages often contain hundreds of mannose residues, which may be extensively branched [2]. O-linkages tend to be shorter but still consist predominantly of mannose [3]. In contrast, fully processed mammalian glycoproteins are rarely mannosylated. This enables exposed cell wall mannose and circulating mannoproteins to be recognized as foreign by host mannose receptors. Many immunodominant fungal antigens have been shown to be mannosylated, with the immunodominance dependent upon the presence of mannosylation [3–5]. Although the cell walls of only a few fungi have been defined in any great detail, it is becoming apparent that there is considerable interspecies (and even intraspecies) variation. For example, in some fungi, the mannans are surface exposed, whereas in others the mannans are confined to the inner cell wall and thus not recognized by mannose receptors. Aspergillus species modifies its mannans by the addition of galactofuranose to form galactomannan [6]. Cryptococcus neoformans has a thick polysaccharide capsule that masks cell wall ligands [7]. The immunological consequences of these variations are increasingly becoming appreciated.
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This review focuses on the current state of knowledge of Toll-like receptors (TLRs) in immune recognition of fungi. The original association of Toll with antimicrobial defenses in Drosophila was made by Lemaitre et al. [8]. Flies deficient in Toll rapidly succumbed to infection when pricked with a needle that had been dipped in a suspension of Aspergillus fumigatus conidia. Toll signals rapid synthesis of antifungal peptides by a signaling pathway that includes induction of Dorsal. Lemaitre et al. astutely noted that Toll and Dorsal shared structural and functional similarity to IL-1 receptors and NF-jB. This led others to search for Toll homologs in mammalian cells. There exist at least 11 TLRs, each of which shares an extracellular domain containing leucine-rich repeats and a cytoplasmic Toll/IL-1 receptor homology (TIR) domain [9].
2. Zymosan Zymosan is a cell wall preparation predominantly composed of b-glucans and mannan derived from the yeast Saccharomyces cerevisiae. Underhill et al. [10] expressed hemagglutinin A-tagged murine TLR2 in the mouse macrophage cell line, RAW-TT10, and challenged the cells with zymosan particles. Within 5 min, TLR2 underwent redistribution from the cell surface to the phagosome. Transfection with a dominant-negative form of TLR2 abolished zymosan-stimulated TNF-a production but had no effect on phagocytosis. This led to the concept that TLRs signal inflammatory responses to particulate stimuli but other receptors were responsible for actual phagocytosis, a concept later validated with other fungi [11,12]. Recently, the b-glucan receptor dectin-1 was identified as the receptor responsible for phagocytosis of zymosan particles [12,13]. Moreover, signaling inflammatory responses requires the cytoplasmic tail and immunoreceptor tyrosine activation motif of dectin1 acting cooperatively with TLR2 and MyD88.
3. C. neoformans 3.1. Capsule as a virulence factor C. neoformans, the etiological agent of cryptococcosis, is an encapsulated yeast that has a strong predilection for causing infections in persons with defects in T-cell-mediated immunity, particularly those with AIDS. The capsule is the organism’s major virulence factor, with acapsular organisms being essentially avirulent. Capsule surrounds the fungus, masking potential ligands on the cell wall, and is also shed. Unopsonized C. neoformans is poorly recognized by phagocytes. However, the fungus is a potent activator of the complement system, and C3-opsonized C. neoformans is recognized by the complement receptors CR1 (CD35), CR3 (CD11b/CD18), and CR4 (CD11c/CD18) on phagocytes [14]. Capsule tends to elicit poor B-cell responses, although
under certain conditions, anticapsular antibody may also contribute to opsonization. Approximately 90% of capsular material is composed of glucuronoxylomannan (GXM). Shed GXM has a molecular weight estimated at approximately 106 Da and circulates in the blood and cerebrospinal fluid of patients with cryptococcosis at µg/ml concentrations and is likely to be present in tissues at mg/ml amounts. 3.2. In vitro studies Shoham et al. [7] demonstrated specific binding of cryptococcal GXM to Chinese hamster ovary (CHO) fibroblasts transfected with human TLR2, TLR4 and/or CD14. However, GXM stimulated nuclear translocation of NF-jB only in the CHO cells that were transfected with both TLR4 and CD14. GXM also stimulated nuclear NF-jB translocation in peripheral blood mononuclear cells (PBMCs) and the RAW264.7 cell line. Interestingly though, challenge of these cells with GXM resulted in neither TNF-a secretion nor activation of the ERK1/2, p38 and SAPK/JNK mitogenactivated protein (MAP) kinase pathways. These findings suggest that the interaction between GXM and CD14/ TLR4 may represent a mechanism of immune dysregulation whereby the organism incompletely activates cascades leading to TNF-a production. In support of a role for TLR4 and CD14 as receptors for GXM, Ellerbroek et al. [15] demonstrated that the inhibitory effect of GXM on neutrophil rolling could be blocked using monoclonal antibodies directed against CD14 and TLR4. 3.3. In vivo mouse models Mice deficient in CD14, TLR2, TLR4, and MyD88 were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans [16]. The MyD88–/– mice had significantly reduced survival compared with wild-type C57BL/6 mice after pulmonary and intravenous challenge with C. neoformans. In contrast, the phenotype was less pronounced with the other mouse groups. TLR2–/– mice died significantly sooner following pulmonary challenge but not intravenous challenge, while for the CD14–/– mice, there was a trend towards reduced survival only following intravenous challenge. Finally, mortality was similar comparing TLR4-mutant C3H/HeJ mice and control C3H/HeOuJ mice following either pulmonary or intravenous challenge.
4. Candida albicans 4.1. Clinical manifestations of infection Infections due to Candida species are among the most common of the mycoses. Clinical manifestations range from mucocutaneous infections to life-threatening deep-seated infection. Interestingly, T-cell dysfunction predisposes to mu-
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cocutaneous infections, while qualitative and quantitative disorders of neutrophil function greatly increase the risk of disseminated disease. Published work examining the role of TLRs in candidiasis has focused on C. albicans, which is the most common species seen, although non-albicans candidiasis has emerged in recent years and now accounts for approximately 50% of bloodstream isolates. Most Candida species are dimorphic, with both yeast and hyphal forms observed in infected tissues. 4.2. In vitro studies In vitro studies have identified a role for both TLR2 and TLR4 in responses to C. albicans. Macrophages from TLR4mutant C3H/HeJ mice had impaired chemokine expression and neutrophil recruitment, but produced levels of proinflammatory cytokines that were similar to those seen with wild-type mice [17]. Antibodies directed against TLR2, but not TLR4, blocked TNF-a production from human PBMCs stimulated with C. albicans [17]. Consistent with a role for TLR2, in vitro production of TNF-a and macrophage inhibitory protein-2 (MIP-2) by macrophages from TLR2–/– mice in response to yeast cells and hyphae of C. albicans was significantly lower than in wild-type mice [18]. Bellocchio et al. [19] examined the candidacidal activity of neutrophils from wild-type, TLR2–/–, TLR4–/–, TLR9–/–, IL-1R–/– and MyD88–/– mice. Activity against both yeast and hyphae was diminished in neutrophils from MyD88- and IL-1R1deficient mice. In contrast, the antifungal activity of neutrophils from the TLR2-, TLR4- and TLR9-deficient mice was either unaffected, or in some cases, even slightly increased. MyD88–/– murine bone marrow-derived dendritic cells also had impaired phagocytosis and killing of C. albicans yeast cells [20]. In another study, neutralization of TLR2 and TLR4 inhibited the synthesis of PGE2 by HeLa cells stimulated with C. albicans [21]. While the above experiments examined responses to whole C. albicans, some investigators have focused on defining the molecules responsible for stimulating TLR responses. Tada et al. [22] demonstrated TNF-a production from human monocytes stimulated with mannans isolated from C. albicans and S. cerevisiae. The TNF-a production required the presence of LPS-binding protein, and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. A potential problem with these experiments was the possible presence of endotoxin contamination in the mannan preparations. Polymyxin B did not inhibit TNF-a production in response to the S. cerevisiae mannan, but polymyxin B controls were not reported for the C. albicans mannan preparation. Jouault et al. [23] demonstrated that a cell wall surface glycolipid, phospholipomannan, stimulates murine macrophages to produce TNF-a via a TLR2-dependent mechanism. b-Glucans from C. albicans also stimulate TNF-a via a mechanism dependent upon dectin-1 and TLR2 [12].
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4.3. Role of TLR in vivo Following an intravenous challenge with C. albicans, TLR4-defective C3H/HeJ mice were more susceptible to infection than wild-type C3H/HeN mice [17]. However, when TLR4–/– mice on a C57BL6 background were challenged with C. albicans hyphae, the knockout mice survived longer than the wild-type controls [19]. Netea et al. [24] found that TLR2–/– mice were paradoxically more resistant to disseminated Candida infection. The mechanism appeared to be impaired IL-10 release in the TLR–/– mice, which resulted in an accompanying decrease in CD4+CD25+ regulatory T cells. However, in two other models of disseminated candidiasis, TLR2–/– mice had significantly impaired survival, in one model, and no difference in survival in the other model [18,19]. Following a gastric challenge with C. albicans hyphae, Bellocchio et al. [19] found a decrease in IFN-c-producing Th1 cells and an increase in IL-4producing Th2 cells in TLR2-, TLR4-, TLR9-, IL1-RI-, and MyD88-deficient mice, compared with wild-type mice. In humans, TLR polymorphisms have been associated with altered risks for certain infectious and cardiovascular diseases. However, a study of Dutch women found that the TLR4 Asp299Gly polymorphism does not seem to play a role in the susceptibility to, and severity of, human urogenital C. albicans infection [25]. Moreover, in a small study of patients with chronic mucocutaneous candidiasis, there was no obvious association with TLR2 or TLR4 polymorphisms [26].
5. A. fumigatus
5.1. Spectrum of aspergillosis Fungi of the genus Aspergillus are ubiquitous in the environment. Exposure is common and generally occurs following inhalation of metabolically inactive (“resting”) airborne conidia (spores). In the absence of an effective host defense, the conidia swell and then germinate, forming hyphae, the invasive form of the fungus. Thus, following exposure, the host has opportunities to kill three morphotypes of the fungus: resting conidia, swollen conidia and hyphae. The clinical spectrum of aspergillosis ranges from allergic disease in atopic individuals to invasive disease in the severely immunocompromised. Despite advances in therapy, the mortality of invasive aspergillosis remains very high. Interestingly, amphotericin B, which is one of the main drugs used to treat invasive aspergillosis (and other mycoses), frequently causes infusion-related side effects including fever and chills, which are thought to be due to the release of proinflammatory cytokines. Sau et al. [27] demonstrated that amphotericin B stimulates proinflammatory cytokine release by a mechanism dependent upon TLR2, CD14, and MyD88.
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5.2. A. fumigatus and TLR Studies examining the role of TLR in aspergillosis have focused mostly on A. fumigatus, the most common species to cause human disease. The results, which are summarized below, are at times seemingly conflicting. Some of the disparities are undoubtedly due to experimental variation, including which A. fumigatus strain was utilized, as well as whether the fungus was killed, and if so, by what method. There are also well-defined distinctions in the responses to the three morphotypes of the fungus, and in some studies rigorous controls to rule out endotoxin contamination were not undertaken. Regarding the host response, speciesspecific and cell type-specific differences also exist. Taken together though, the published data, summarized below, point to involvement of TLR2, TLR4, and CD14 in immune responses to A. fumigatus. Mambula et al. [11] utilized knockout mice and transfected human cells to examine the contribution of TLR2, TLR4, MyD88, and CD14 to signaling in response to resting conidia, swollen conidia, and hyphae of A. fumigatus. Optimal signaling responses in both the mouse and human cells required TLR2 acting through the central adapter protein MyD88. CD14 was important for responses to the human cells but not the murine cells. However, despite their contribution to NF-jB activation and cytokine release, TLR2, MyD88, and CD14 were not required for phagocytosis. TLR4-deficient macrophages made less TNF-a compared with wild-type cells when stimulated by A. fumigatus swollen conidia but not A. fumigatus hyphae. Netea et al. [28] also found a role for TLR4 in response to conidia but not hyphae, whereas Meier et al. [29] found a role for TLR2 and TLR4 in signaling responses to both conidia and hyphae. Wang et al. [30] found that hyphal-stimulated TNF-a production from human monocytes was inhibited by monoclonal antibodies directed against TLR4 or CD14, but not by anti-TLR2 antibodies [30]. Conflicting data also exist with regard to phagocytosis and killing. Mambula et al. [11] found no role for TLR2, TLR4, MyD88, and CD14 in phagocytosis of A. fumigatus conidia. Marr et al. [20] observed that incubation of MyD88–/– murine bone marrow-derived dendritic cells with A. fumigatus conidia resulted in phagocytosis and fungal killing at levels comparable to those seen in wild-type cells. However, Bellochhio et al. [19] found a defect in phagocytosis and killing of conidia by MyD88–/– and TLR4–/– (but not TLR2–/– and TLR9–/–) murine PMNs. Consistent results were obtained in vivo using a model in which mice were immunosuppressed with cyclophosphamide and then challenged via the pulmonary route with A. fumigatus conidia. The wild-type, TLR2–/– and TLR9–/– mice all had similar survival curves, but the MyD88–/– and TLR4–/– mice died significantly sooner. The A. fumigatus antigens responsible for TLR-mediated stimulation have not been purified. Braedel et al. [31] examined two crude preparations derived from A. fumigatus cul-
tures, one derived from a glass-bead-disrupted cellular extract and the other from culture supernatants. The cellular extract stimulated IL-12 p40 production from murine bone marrow-derived dendritic cells in a TLR2-dependent, TLR4independent manner, whereas the culture supernatant stimulated IL-12 p40 in the absence of TLR2 or TLR4 (but not in cells which lacked both TLR2 and TLR4). Interestingly, the cellular extract stimulated IL-6 secretion in a TLR2independent manner, while the culture supernatant required TLR4 for IL-6 production.
6. Conclusion While much progress has been made elucidating the contribution of TLRs in the host response to fungi, much still needs to be learned. Thus far, only three fungal ligands, b-glucan, GXM, and phospholipomannan that stimulate TLR responses have been identified on the molecular level. In addition, there are published data on only a few of the pathogenic fungi. As noted above, some of these data are conflicting, and the reasons for the disparate results need to be established. Finally, the downstream effects of TLR stimulation need to be defined, including the signaling pathways utilized and the nature of the inflammatory response elicited. The inflammatory response seen in fungal infections can be a “two-edged sword”. While necessary for effective host defenses, an overly exuberant or misdirected inflammatory response can lead to deleterious effects due to bystander tissue destruction. Ultimately, the challenge will be to apply our understanding of how fungi stimulate TLR-mediated immune responses to therapeutic strategies in the at-risk patient population.
References [1]
[2] [3] [4]
[5]
[6] [7]
F.M. Klis, P. Mol, K. Hellingwerf, S. Brul, Dynamics of cell wall structure in Saccharomyces cerevisiae, FEMS Microbiol. Rev. 26 (2002) 239–256. J.E. Cutler, N-glycosylation of yeast, with emphasis on Candida albicans, Med. Mycol. 39 (Suppl. 1) (2001) 75–86. M.K. Mansour, S.M. Levitz, Fungal mannoproteins: the sweet path to immunodominance, ASM News 69 (2003) 595–600. S.M. Levitz, S. Nong, M.K. Mansour, C. Huang, C.A. Specht, Molecular characterization of a mannoprotein with homology to chitin deacetylases that stimulates T cell responses to Cryptococcus neoformans, Proc. Natl. Acad. Sci. USA 98 (2001) 10422–10427. M.K. Mansour, L.S. Schlesinger, S.M. Levitz, Optimal T-cell responses to Cryptococcus neoformans mannoprotein are dependent on recognition of conjugated carbohydrates by mannose receptors, J. Immunol. 168 (2002) 2872–2879. M. Bernard, J.P. Latge, Aspergillus fumigatus cell wall: composition and biosynthesis, Med. Mycol. 39 (Suppl. 1) (2001) 9–17. S. Shoham, C. Huang, J.M. Chen, D.T. Golenbock, S.M. Levitz, Toll-like receptor 4 mediates intracellular signaling without TNFalpha release in response to Cryptococcus neoformans polysaccharide capsule, J. Immunol. 166 (2001) 4620–4626.
S.M. Levitz / Microbes and Infection 6 (2004) 1351–1355 [8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
B. Lemaitre, E. Nicolas, L. Michaut, J.M. Reichhart, J.A. Hoffmann, The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults, Cell 86 (1996) 973–983. D. Zhang, G. Zhang, M.S. Hayden, et al., A Toll-like receptor that prevents infection by uropathogenic bacteria, Science 303 (2004) 1522–1526. D.M. Underhill, A. Ozinsky, A.M. Hajjar, et al., The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens, Nature 401 (1999) 811–815. S.S. Mambula, K. Sau, P. Henneke, D.T. Golenbock, S.M. Levitz, Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus, J. Biol. Chem. 277 (2002) 39320–39326. G.D. Brown, J. Herre, D.L. Williams, J.A. Willment, A.S. Marshall, S. Gordon, Dectin-1 mediates the biological effects of beta-glucans, J. Exp. Med. 197 (2003) 1119–1124. B.N. Gantner, R.M. Simmons, S.J. Canavera, S. Akira, D.M. Underhill, Collaborative induction of inflammatory responses by dectin1 and Toll-like receptor 2, J. Exp. Med. 197 (2003) 1107–1117. S.M. Levitz, A. Tabuni, Binding of Cryptococcus neoformans by human cultured macrophages. Requirements for multiple complement receptors and actin, J. Clin. Invest. 87 (1991) 528–535. P.M. Ellerbroek, L.H. Ulfman, A.I. Hoepelman, F.E. Coenjaerts, Cryptococcal glucuronoxylomannan interferes with neutrophil rolling on the endothelium, Cell. Microbiol. 6 (2004) 581–592. L.E. Yauch, M.K. Mansour, S. Shoham, J.B. Rottman, S.M. Levitz, Involvement of CD14, Toll-like receptor (TLR) 2, TLR4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo, Infect. Immun. 72 (2004) 5373–5382. M.G. Netea, C.A. Van der Graaf, A.G. Vonk, I. Verschueren, J.W. Van der Meer, B.J. Kullberg, The role of Toll-like receptor (TLR) 2 and TLR4 in the host defense against disseminated candidiasis, J. Infect. Dis. 185 (2002) 1483–1489. E. Villamon, D. Gozalbo, P. Roig, J.E. O’Connor, D. Fradelizi, M.L. Gil, Toll-like receptor-2 is essential in murine defenses against Candida albicans infections, Microbes Infect. 6 (2004) 1–7. S. Bellocchio, C. Montagnoli, S. Bozza, et al., The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo, J. Immunol. 172 (2004) 3059–3069. K.A. Marr, S.A. Balajee, T.R. Hawn, et al., Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens, Infect. Immun. 71 (2003) 5280–5286.
1355
[21] R. Deva, P. Shankaranarayanan, R. Ciccoli, S. Nigam, Candida albicans induces selectively transcriptional activation of cyclooxygenase2 in HeLa cells: pivotal roles of Toll-like receptors, p38 mitogenactivated protein kinase, and NF-kappa B, J. Immunol. 171 (2003) 3047–3055. [22] H. Tada, E. Nemoto, H. Shimauchi, et al., Saccharomyces cerevisiaeand Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner, Microbiol. Immunol. 46 (2002) 503– 512. [23] T. Jouault, S. Ibata-Ombetta, O. Takeuchi, et al., Candida albicans phospholipomannan is sensed through Toll-like receptors, J. Infect. Dis. 188 (2003) 165–172. [24] M.G. Netea, R. Sutmuller, C. Hermann, et al., Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells, J. Immunol. 172 (2004) 3712–3718. [25] S.A. Morre, L.S. Murillo, J. Spaargaren, H.S. Fennema, A.S. Pena, Role of the Toll-like receptor 4 Asp299Gly polymorphism in susceptibility to Candida albicans infection, J. Infect. Dis. 186 (2002) 1377–1379 (author reply 1379). [26] C.A. Van der Graaf, M.G. Netea, I.P. Drenth, R.H. Te Morsche, J.W. Van der Meer, B.J. Kullberg, Candida-specific interferon-gamma deficiency and Toll-like receptor polymorphisms in patients with chronic mucocutaneous candidiasis, Neth. J. Med. 61 (2003) 365– 369. [27] K. Sau, S.S. Mambula, E. Latz, P. Henneke, D.T. Golenbock, S.M. Levitz, The antifungal drug amphotericin B promotes inflammatory cytokine release by a Toll-like receptor- and CD14-dependent mechanism, J. Biol. Chem. 278 (2003) 37561–37568. [28] M.G. Netea, A. Warris, J.W. Van der Meer, et al., Aspergillus fumigatus evades immune recognition during germination through loss of Toll-like receptor-4-mediated signal transduction, J. Infect. Dis. 188 (2003) 320–326. [29] A. Meier, C.J. Kirschning, T. Nikolaus, H. Wagner, J. Heesemann, F. Ebel, Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages, Cell. Microbiol. 5 (2003) 561–570. [30] J.E. Wang, A. Warris, E.A. Ellingsen, et al., Involvement of CD14 and Toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae, Infect. Immun. 69 (2001) 2402–2406. [31] S. Braedel, M. Radsak, H. Einsele, et al., Aspergillus fumigatus antigens activate innate immune cells via Toll-like receptors 2 and 4, Br. J. Haematol. 125 (2004) 392–399.