Host recognition of fungal pathogens

Transcription

1 Drug Discovery Today: Disease Mechanisms Vol. 4, No DRUG DISCOVERY TODAY DISEASE MECHANISMS Editors-in-Chief Toren Finkel National Heart, Lung and Blood Institute, National Institutes of Health, USA Charles Lowenstein The John Hopkins School of Medicine, Baltimore, USA Infectious diseases Host recognition of fungal pathogens Helen S. Goodridge, David M. Underhill* Immunobiology Research Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA Fungal infection presents a serious risk to individuals with compromised immune systems. The development of novel therapeutic strategies to combat fungal infection requires a thorough appreciation of interactions between fungi and the host immune system. Our understanding of microbial recognition by the innate immune system has advanced considerably over the past decade with the discovery that pattern recognition receptors on the surface of myeloid cells detect microbial components. TLR2 and Dectin-1 are of particular importance for the recognition of pathogenic fungi. In this review, we discuss fungal detection by these receptors and their roles in the initiation of antifungal defense. Humans are constantly exposed to fungi, including the gut microflora, but fungal infections are relatively mild and uncommon in normal healthy individuals. However, in immunocompromised individuals (e.g. transplant recipients, cancer patients undergoing chemotherapy and AIDS patients) normally nonpathogenic fungi, including Candida, Cryptococcus, Aspergillus and Pneumocystis species, can cause potentially fatal infections. Microbial detection by the mammalian innate immune system initiates the host s response to invading microorganisms. Phagocytic cells, including macrophages, dendritic cells and neutrophils, survey their environment in the bloodstream and in tissues, and ingest, destroy and digest invading microbes. Antigen presenting cells, especially dendritic cells, present microbial antigens to T cells to initiate an adaptive *Corresponding author: D.M. Underhill (David.Underhill@cshs.org) Section Editor: W. Conrad Liles Division of Infectious Diseases, University of Toronto, Ontario, Canada immune response. Cytokine and chemokine production recruits other immune cells and directs the subsequent immune response. A panoply of pattern recognition receptors displayed on the surface of innate immune cells interact directly with microbial ligands or indirectly with microbes opsonized with serum components such as complement proteins and immunoglobulins. This review will focus on the two best-characterized fungal recognition receptors Toll-like receptor 2 (TLR2) and Dectin-1 and their roles in initiating antifungal responses (Fig. 1). Fungal receptors TLR2 A role for TLRs in antifungal responses has been clear from the earliest days of mammalian TLR immunology. TLRs were identified on the basis of their homology to the Drosophila protein Toll. The demonstration that Toll-deficient Drosophila are highly susceptible to Aspergillus infection [1], combined with the observation that mammals possess homologues of Drosophila Toll [2], was strongly indicative of a role for TLRs in mammalian antifungal responses. TLR2 has subsequently been demonstrated to be a key receptor for pathogenic fungi, including Candida albicans, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Pneumocystis carinii and Coccidioides posadasii (reviewed in [3 5]). TLR1 and TLR6, which form heterodimers with TLR2, have also been implicated in fungal recognition [6,7], though the precise requirement for these TLRs is currently unclear. Furthermore, TLR4 and TLR9 have been shown to play antifungal roles (reviewed in [4]) /$ ß 2007 Elsevier Ltd. All rights reserved. DOI: /j.ddmec

2 Drug Discovery Today: Disease Mechanisms Infectious diseases Vol. 4, No Figure 1. Roles of TLR2 and Dectin-1 in the antifungal response. TLR2 and Dectin-1 on the surface of myeloid phagocytes interact with fungi and their products. Dectin-1 ligation triggers internalization of yeast and induction of the oxidative burst, which contributes to the destruction of internalized yeast in the acidic, hydrolytic enzyme-rich environment of the phagolysosome. Degraded fungal antigen is presented to T cells to initiate adaptive immunity. TLR2 and Dectin-1 signaling is integrated to induce a repertoire of cytokines and chemokines that coordinate the inflammatory response and dictate the phenotype of the subsequent T cell response. The components of the yeast cell wall detected by TLR2 remain to be definitively identified. A variety of glycolipid components of nonfungal microbes have been identified as TLR2 ligands, including mycobacterial lipoarabinomannan, lipoteichoic acid from Staphylococcus, glycolipids from Treponema and glycosylphosphatidylinositol from trypanosomes [8 10]. Hence it would seem most likely that the fungal TLR2 ligand is also a glycolipid. One candidate is phospholipomannan; peritoneal macrophages from TLR2-deficient mice stimulated with phospholipomannan purified from C. albicans produce significantly less TNF-a than macrophages from wild-type mice [11]. However, it is not clear whether phospholipomannan is a cell wall component of other fungi and thus whether it could be a generic fungal TLR2 ligand. Dectin-1 Dectin-1 is a member of the NK-like C-type lectin family and is expressed widely on myeloid cells including macrophages, dendritic cells and neutrophils [12]. Dectin-1 has been demonstrated to recognize and mediate responses to a variety of fungi in vitro, and to date, four studies have directly explored the contribution of Dectin-1 during in vivo infection with pathogenic fungi. Steele et al. demonstrated that inflammatory responses to intratracheal infection with A. fumigatus conidia were significantly reduced by administration of a soluble form of Dectin-1 [13], Brown and colleagues found that Dectin-1-deficient mice are markedly more susceptible to C. albicans infection than wild-type mice [14] and Iwakura and colleagues observed that Dectin-1-deficient mice are significantly more susceptible to infection with P. carinii [15]. By contrast, Nakamura et al. have reported that Dectin-1-deficient mice are not significantly more susceptible to C. neoformans, probably because of the large capsule covering the cell wall [16]. Dectin-1 recognizes b-glucan in fungal cell walls. Brown and coworkers first demonstrated that Dectin-1 is a receptor for b- 1,3-linked glucans and that binding of fluorescently labeled zymosan (a cell wall preparation of S. cerevisiae), as well as intact S. cerevisiae and C. albicans, to Dectin-1-expressing NIH3T3 cells or to macrophages could be blocked by the soluble b- glucans laminarin and glucan phosphate, but not by mannan [17,18]. Oligosaccharide microarrays defined the minimum structure recognized as a b-1,3-glucan 10mer [19]. b-glucans are not produced by mammalian cells, making them ideal targets for innate immune receptors such as Dectin-1. Fungal cell morphology can be a key determinant in b- glucan recognition by Dectin-1 because of variation in b- glucan masking at the fungal surface. C. albicans can rapidly and reversibly switch between yeast and filamentous morphologies, and we have demonstrated that Dectin-1 can recognize b-glucan in the cell wall of the yeast form, but not the filamentous form [20]. The yeast-to-filament transition is important for pathogenicity, and the filamentous morphology is thought to provide some advantage during interaction with the mammalian immune system [21 23]

3 Vol. 4, No Drug Discovery Today: Disease Mechanisms Infectious diseases Similarly, cellular morphology of A. fumigatus determines whether b-glucan is accessible to recognition by Dectin-1. Aspergillus conidia (resting spores), which are ubiquitous in the environment, contain b-glucan that is shielded from recognition by a hard outer surface [13,24,25]. After inhalation, the spores swell and begin to grow as filaments. Swollen conidia and filaments are recognized by Dectin-1. Immune responses to fungi Phagocytosis of fungi When myeloid phagocytes bind to fungi, they are activated to eat them via an internalization process called phagocytosis. Once internalized, the fungi are destroyed and degraded in the acidic, hydrolytic enzyme-rich environment of the phagolysosome [26]. Although TLRs recognize fungi, they are not sufficient to trigger phagocytosis. TLR2 expression in nonphagocytic HEK293 cells is sufficient to confer some responsiveness to the model yeast cell wall particle zymosan (e.g. NF-kB activation), but is not sufficient to drive phagocytosis of the particle. Similarly, macrophages lacking TLR2, TLR4, or the TLR signaling adaptor MyD88 internalize yeast particles normally ([27] and personal observations). Dectin-1 is a major receptor for triggering phagocytosis of fungi (Fig. 1). Investigators established two decades ago that b-glucan recognition is important for macrophage phagocytosis of zymosan because it can be inhibited by the soluble b- glucan, laminarin [28,29]. Using an expression cloning strategy in which a macrophage cdna library was expressed in a nonphagocytic cell line, Brown and coworkers identified Dectin-1 as the b-glucan receptor responsible for phagocytosis of zymosan [17]. Subsequent studies have established that Dectin-1 is the primary (although certainly not the exclusive) receptor for phagocytosis of zymosan and live fungi by macrophages and dendritic cells [14,15,18,20]. It is important to note that phagocytosis of fungal cells is not completely blocked in the absence of Dectin-1. This is perhaps not too surprising because myeloid phagocytes will, given sufficient time, bind and internalize nearly any foreign particle. There are certainly receptors in addition to Dectin-1 that can bind to yeast and trigger phagocytosis. It is likely that receptors such as the mannose receptor participate in binding and internalization of yeast particles, though their relative contributions may vary with cell type and with fungal species and morphotypes. However, the data strongly suggest that for b-glucan-rich yeast cell wall particles, Dectin-1 is by far the most efficient phagocytic receptor. Dectin-1 triggers phagocytosis by activating tyrosine kinases (Fig. 2). The cytoplasmic tail of Dectin-1 contains a signaling motif that resembles the immunoreceptor tyrosinebased activation motifs (ITAMs) of lymphocyte antigen receptors (TCR and BCR) and Fc receptors. Although the structure of the ITAM-like motif of Dectin-1 is slightly unusual, Dectin-1 signaling triggers activation of Src family kinases and Syk like regular ITAMs [30,31]. Inhibitors of Src family kinases block Dectin-1-mediated phagocytosis in macrophages [32], though the particular Src family kinases that participate in Dectin-1-mediated phagocytosis have not been identified. The requirement for Syk is more varied; data suggest that Syk signaling plays a role in Dectin-1-mediated phagocytosis by dendritic cells but not macrophages [30 32]. Production of reactive oxygen species Production of reactive oxygen species (ROS) is coupled to phagocytosis of yeast by myeloid phagocytes, and is thought to be an important mechanism for killing internalized organisms. ROS is produced by the NADPH phagocyte oxidase, a multisubunit enzyme that assembles directly on the phagosomal membrane. Patients with Chronic Granulomatous Disease (CGD) lack specific components of this enzyme because of genetic defects, and are highly susceptible to a host of bacterial and fungal infections [33,34]. Similarly, mice engineered to mimic these defects are more susceptible to fungal infections than their wild-type counterparts [35,36]. Dectin-1 is a key receptor for triggering ROS production in response to fungi (Fig. 1). Dectin-1 knockdown and knockout technologies have shown a role for Dectin-1 in zymosaninduced ROS production [14,15,37], though serum-opsonization restores the activity [14]. We have also demonstrated by specifically crosslinking a tagged form of the receptor that Dectin-1 signaling is sufficient to drive ROS production in macrophages, and that Dectin-1-induced ROS induction is mediated by Src family kinases and Syk [31]. Although it is clear that Dectin-1 is capable of triggering phagocytosis and ROS production, the specific role of Dectin- 1 in the net ROS response of macrophages and neutrophils to live fungal pathogens is still being established [14,15,20,24]. Dectin-1 appears to play a partial role in the respiratory burst triggered by exposure to C. albicans and A. fumigatus, suggesting that other receptors may additionally recognize these fungi and trigger ROS production [14,15,24]. In the case of P. carinii, at least for alveolar macrophages, the role for Dectin-1 in triggering ROS appears dominant, because the response is completely lost in Dectin-1 / cells [15]. Bone marrow-derived macrophages from MyD88 / and TLR2 / mice have no defect in ROS production following zymosan stimulation, indicating that TLR signaling does not contribute to ROS induction [27]. Consistent with this, activation of TLR2 or TLR4 with pure agonists failed to trigger a significant ROS response. However, it has long been known that LPS can prime macrophages for an enhanced respiratory burst [38], and we have demonstrated that TLR2 activation primes macrophages for an enhanced Dectin-1-stimulated respiratory burst [27]. In these cases, TLR signaling must precede phagocytosis and the effect requires new protein synthesis, indicating that TLR signaling upregulates the cells capacity to produce ROS

4 Drug Discovery Today: Disease Mechanisms Infectious diseases Vol. 4, No Figure 2. Integration of TLR2 and Dectin-1 signals to tailor the antifungal transcriptional response. TLR2 signals via the Mal and MyD88 adaptors to activate NF-kB. Dectin-1 activates NF-kB in dendritic cells, but not macrophages, via the CARD9/Bcl10/Malt1 adaptor complex. Dectin-1 signaling also activates NFAT transcription factors in both macrophages and dendritic cells. Integration of these and other signals regulates transcription of inflammatory genes. Production of inflammatory mediators Macrophages and dendritic cells produce a variety of proinflammatory cytokines and chemokines, including TNF-a, IL- 6, IL-12 and MIP-2, following exposure to zymosan or live fungi, and both TLRs and Dectin-1 are implicated in their induction (reviewed in [39]) (Fig. 1). TLR2-MyD88 signals are sufficient to induce modest proinflammatory cytokine production by macrophages, but Dectin-1 signaling alone is not [27,31]. Furthermore, compared to TLR4, TLR2 is a relatively weak inducer of proinflammatory cytokine production [40]. However, analysis of the transcriptional responses of macrophages and dendritic cells to fungi has revealed collaboration between TLR2 and Dectin-1 signaling to amplify the TLR2 response and promote the induction of proinflammatory cytokines. HEK293 cell reconstitution assays, specific co-ligation of TLR2 and Dectin-1 in macrophages with pure ligands, and recent data from Dectin-1-deficient mice demonstrate that Dectin-1 signals collaborate with TLR2 signals to promote cytokine induction in macrophages [14,15,27]. TLR2 and Dectin-1 have also been implicated in the induction of IL-2 and the anti-inflammatory cytokine IL-10 production by dendritic cells following zymosan stimulation [15,30,37,41]. Production of inflammatory lipid mediators is another consequence of macrophage activation by zymosan and fungi. For example, zymosan stimulation triggers cpla 2 - mediated release of arachidonic acid, which is metabolized to generate eicosanoids including prostaglandins, prostanoids (prostacyclins and thromboxane) and leukotrienes. Arachidonic acid release and eicosanoid production by macrophages is observed within 30 min of intraperitoneal injection of mice with zymosan [42]. Leukotriene generation is catalyzed by 5-lipoxygenase, while inducible cyclooxygenase (Cox-2) is required for prostaglandin and prostanoid production. TLR2 and Dectin-1 collaborate in triggering arachidonic acid release, Cox-2 synthesis and prostaglandin generation following exposure of macrophages to zymosan and C. albicans yeast [37,43]. Many of the cytokines and chemokines induced following fungal recognition are known to be regulated by NF-kB transcription factors. TLR2-mediated activation of NF-kB is well established. In macrophages, Dectin-1 ligation in the absence of a TLR2 ligand fails to activate NF-kB [27,44], though Dectin-1 signals appear to collaborate with TLR2 to induce NF-kB activation and enhance cytokine production. By contrast, a recent study suggested that Dectin-1 signals can activate NF-kB directly in dendritic cells via the caspase recruitment domain (CARD)-containing adaptor CARD9 [45] (Fig. 2). Bone marrow-derived dendritic cells from CARD9-deficient mice exhibited defective cytokine responses (TNF-a, IL-6 and IL-2) to zymosan and C. albicans, but not to other TLR ligands, and NF-kB activation was reduced in CARD9 / cells. Furthermore, we recently demonstrated that Dectin-1, but not TLR2, signals trigger activation of nuclear factor of activated T cells (NFAT) transcription factors, which are classically associated with adaptive immune responses (TCR, BCR 250

5 Vol. 4, No Drug Discovery Today: Disease Mechanisms Infectious diseases and FcR ITAM signaling) and not innate antimicrobial responses [37]. NFAT signals appear to be responsible for Dectin-1-mediated Cox-2 induction, and consequently prostaglandin E 2 release, by zymosan-stimulated macrophages, as well as contributing to the induction of IL-2, IL-10 and IL-12 p70 by zymosan-stimulated dendritic cells. Initiation of the adaptive immune response Cytokine production following microbial exposure is a key determinant in the development of both innate inflammatory responses and the induction of adaptive immunity. In particular, the phenotype of the T cell response is crucially influenced by the cytokine profile of antigen presenting cells (Fig. 1). Fungal exposure has been widely reported to trigger IL-12 induction, and a dominant Th1 response has been considered crucial for protective immunity against fungal infection (reviewed in [5]). However, several groups have recently reported the generation of Th17 responses to C. albicans, A. fumigatus and P. carinii [46 49]. Acosta-Rodriguez et al. characterized human memory CD4+ T cells with specificity for C. albicans as mostly Th17 cells (IL-17 producing, RORgt-expressing) [46]. In vitro experiments suggested that the filamentous form of the fungus was responsible for directing the development of Th17 responses. Filaments stimulated monocyte-derived DCs to produce IL- 23 and primed Th17 responses, while yeast preferentially induced IL-12 production and the development of IFN-gproducing T cells. Such data would suggest that Dectin-1 plays no role in the induction of Th17 responses. By contrast, LeibundGut-Landmann et al. observed that the b-glucan preparation curdlan stimulated IL-23 production by murine DCs and primed Th17 responses, and that CARD9-deficient splenocytes failed to produce IL-17 in response to heat-inactivated C. albicans [50]. Huang et al. reported a protective role for IL-17 in systemic candidiasis [47]; following intravenous C. albicans injection, IL-17A receptor-deficient mice showed increased mortality and fungal burden, and decreased neutrophil recruitment. Furthermore, IL-17A expression by adenoviral delivery improved the survival of wild-type mice following systemic challenge with a lethal dose of C. albicans. Similarly, Rudner et al. observed that mice lacking the p19 subunit of IL-23, which maintains Th17 responses, were more susceptible to intratracheal P. carinii infection, and that neutralizing antibodies against p19 or IL-17 increased the fungal burden in wild-type mice [48]. By contrast, Zelante et al. reported that p19-deficient mice were somewhat protected from mucosal infection with C. albicans (intragastric) or A. fumigatus (intranasal) and have suggested that IL-23/Th17 responses may constrain protective IL-12/Th1 responses [49]. Effector T cell responses are controlled by IL-10-producing regulatory T (Treg) cells, which are thought to limit excessive inflammatory responses to prevent collateral damage to the host. However, Treg cells can also block antimicrobial effector responses, which may impair eradication of the pathogen. IL- 10 production in response to fungal exposure has been reported and Treg cells have been described in fungal infections [41,51], but it is not yet clear whether they represent a mechanism to restrict immunopathology or a fungal evasion strategy (reviewed in [52]). Conclusions Effective host defense against fungal infections requires the coordinated action of a variety of receptors and signaling networks to tailor innate and adaptive immune responses. In this review, we have highlighted the roles of TLR2 and Dectin-1 in the recognition of several clinically important fungi. 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