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* Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy;
Departments of Medicine and Microbiology, Boston University School of Medicine, Boston, MA 02118; and
Department of Immunology and Cell Biology, Mario Negri Institute, Milan, Italy
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Toll-like receptors (TLRs) are a family of conserved, mammalian cellular receptors that mediate cellular responses to structurally conserved pathogen-associated microbial products (11, 12). All TLRs activate a core set of stereotyped responses, such as inflammation (13). However, individual TLRs can also induce specific programs in cells of the innate immune system that are tailored for the particular pathogen (12, 13, 14). TLRs bear homology to the IL-1R type 1 (IL-1RI) and share a similar signaling cascade culminating in activation of NF-
B and mitogen-activated protein kinases (15, 16). This process facilitates the transcription of genes regulating the inflammatory and adaptive immune responses. The common signal pathways used by IL-1RI and TLRs involve recruitment of the adapter protein myeloid differentiation primary response gene 88 (MyD88) through the homophilic interaction of the Toll/IL-1R homology domain (17). MyD88, in turn, activates a series of IL-1R-associated kinases that are crucially involved in innate immunity. MyD88-deficient mice do not make TNF-
in response to activators of TLR2, TLR3, TLR4, or TLR9, and IL-1R-associated kinase-deficient mice are impaired in their response to LPS and IL-1 (18). However, in the case of TLR4-dependent LPS signaling, another protein (Mal/TIRAP) may also serve as an adapter molecule with or in place of MyD88 (19, 20). It is recognized that the intricacies of how TLRs signal will ultimately provide an explanation for the molecular basis of how cells involved in innate immunity dictate the processes of host defense specific to the provoking pathogen (14).
In the case of fungi, in vitro studies have shown that Cryptococcus neoformans, C. albicans, and A. fumigatus may signal through TLRs, particularly TLR2, TLR4, and TLR9, in a morphotype-specific fashion (21, 22, 23, 24, 25, 26, 27, 28). This will not come as a surprise, given the initial discovery of the Toll pathway in Drosophila as an essential mechanism of antifungal resistance (29). However, because of the complexity of TLR activation and function, including TLR cooperativity (30), the need for coreceptor (24), the sharing of common signal transduction pathways (31), and the dynamic regulation of TLR expression in vivo (32), the in vitro studies may not be sufficient to predict the impact of the total repertoire of TLRs on infections in vivo. In addition, the use of inactivated fungi in the in vitro assays cannot anticipate the impact on TLR functioning of fungal morphogenesis, which is known to be activated in vivo by a wide range of tissue-specific signals (33) and to affect host responses (4, 34).
In the present study we have taken a comprehensive approach to define the role of the IL-1R/TLR/MyD88-dependent signaling pathway in response to C. albicans and A. fumigatus. We evaluate the course and outcome of the infections together with parameters of innate and adaptive antifungal immunity in IL-1RI-, TLR2-, TLR4-, TLR9-, and MyD88-deficient mice with experimental models of infection in which the contributions of innate and adaptive immunities are well established (35). We found that the IL-1R/TLR/MyD88 system plays a strategic role at the interface between the host and fungi, having an impact on the generation of both host antifungal immunity and fungal infectivity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female, 8- to 10-wk-old C57BL6 mice were obtained from Charles River Breeding Laboratories (Calco, Italy) or The Jackson Laboratory (Bar Harbor, ME). Breeding pairs of homozygous TLR2- (36), TLR4- (37), TLR9- (38), IL-1RI- (39), and MyD88- (40) deficient mice, raised on the C57BL6 background, were bred under specific pathogen-free conditions at the breeding facilities of University of Perugia (Perugia, Italy) or Boston University Medical Center. All in vivo studies were performed in compliance with national, Perugia University, and Boston University animal care and use committee guidelines.
Microorganisms, culture conditions, and infections
Isogenic strains of C. albicans, obtained by mutagenesis in vitro and capable (Vir-13), or not (Vir-3), of yeast-to-hyphal transition, as assessed by germ-tube formation in vitro, were used (41). The A. fumigatus strain was obtained from a fatal case of pulmonary aspergillosis at the Infectious Diseases Institute of University of Perugia (41). Yeasts were harvested at the end of the exponential phase of growth and resuspended in IMDM (Life Technologies Italia, Milan, Italy) containing 5 µg/ml polymyxin B (Sigma-Aldrich, St. Louis, MO) and 50 µg/ml gentamicin. Conidia were harvested by extensive washing of the slant culture (on Sabouraud dextrose agar (Difco, Detroit, MI) supplemented with chloramphenicol for 4 days at room temperature) with 5 ml of 0.025% Tween 20 in saline. For generation of hyphae, Candida cells were allowed to germinate by culture at 37°C in 5% CO2 for 2 h in RPMI 1640 medium (by that time, >98% of cells had germinated). For Aspergillus infection, conidia were given intranasally (i.n.) for 3 consecutive days (2 x 107/20 µl saline/each injection) as previously described (6). Mice were anesthetized by i.p. injection of 2.5% avertin (Sigma-Aldrich). For primary disseminated candidiasis, mice were injected i.v. with 106/0.5 ml of C. albicans yeasts or hyphae; for reinfection, mice surviving the primary infection were injected i.v. with 106/0.5 ml of C. albicans hyphae 14 days later (41). For the gastrointestinal infection, 108 Candida hyphae were injected intragastrically (i.g.), via an 18-gauge, 4-cm-long plastic catheter in a volume of 0.2 ml saline/mouse as previously described (35). Cyclophosphamide (Sigma-Aldrich) was given at 150 mg/kg i.p. 1 day before challenge. Quantification of fungal growth in the organs of infected mice was performed by plating serial dilutions of homogenized organs in Sabouraud dextrose agar, and results (mean ± SE) were expressed as CFU per organ.
Cell purification
Polymorphonuclear neutrophils (PMNs) were isolated from the blood and peritoneal cavity of mice as previously described (35). Briefly, peritoneal PMNs were obtained 8 h after the i.p. injection of 1 ml of endotoxin-free 10% thioglycolate solution (Difco). Endotoxin was depleted from all solutions with Detoxi-gel (Pierce, Rockford, IL). To purify Gr-1+ PMN, 107 cells were incubated with biotin-conjugated anti-mouse Gr-1 mAbs (clone RB6-8C5; BD PharMingen, San Diego, CA) for 30 min at 4°C and then with avidin-conjugated magnetic MicroBeads (Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at 6°C, and magnetically separated with a positive selection column (Miltenyi Biotech) according to the manufacturer’s instructions. On FACS analysis, Gr-1+ PMN were >98% pure and stained positively for the CD11b myeloid marker. Cytospin analysis confirmed that the population consisted of polymorphonuclear cells (>98%). Murine CD11c+ DCs were isolated from spleens and lungs, and CD4+ T cells were isolated from mesenteric lymph nodes (MLN), thoracic lymph nodes (TLN), and spleens by magnetic cell sorting as previously described (5, 6, 8, 9).
Phagocytosis, cultures, and antifungal effector activity
For phagocytosis, peritoneal neutrophils from infected mice were incubated at 37°C with unopsonized Candida yeasts or Aspergillus conidia for 30 and 60 min, respectively, at an effector to fungal cells ratio of 1:3. After incubation, phagocytic cells were separated from nonphagocytosed fungal cells by centrifugation on an FBS gradient, and a 0.1-ml sample of the harvested phagocytic cells was used for cytospin preparation. The percentage of internalization was calculated on Giemsa-stained preparations as previously described (5, 6). For staining of degranulated PMNs, the cytospin preparations were subjected to methylene blue (for azurophil granules) or eosin (for nonazurophil granules) staining following standard procedures. For myeloperoxidase detection, the liquid diaminobenzidene substrate Pack Kit (BioGenex, Menarini Diagnostics, Florence, Italy) was used according to the manufacturer’s instructions on ematoxilin-counterstained preparations. For the fungicidal activity against Candida yeasts or Aspergillus conidia, PMNs were incubated with unopsonized fungal cells (at an effector to fungal cell ratio of 1:5) for 60 min (for yeasts) or 120 min (for conidia) at 37°C. Triton X was then added to the wells, and serial dilutions from each well were made in distilled water. Pour plates (four to six replicate samples) were made by spreading each sample on Sabouraud glucose agar. The number of CFUs and the percentage of CFU inhibition (mean ± SE), referred to as candidacidal or conidiocidal activity, were determined as previously described (5, 25). For the fungicidal activity against hyphae, a colorimetric MTT assay was used as previously described (42, 43). Briefly, graded numbers of PMNs (106, 5 x 105, and 105), either unexposed or pre-exposed for 2 h at 37°C to 10 ng/ml rTNF- (R&D Systems, Space Import-Export, Milan, Italy), were added to hyphae obtained from 105 yeasts in 96-well, flat-bottom microtiter plates. After 2 h at 37°C with occasional shaking (by that time, >98% of cells had germinated), the supernatants were aspirated, effector cells were lysed by adding sodium deoxycholate (0.5%), and hyphal viability was determined by MTT staining. Antifungal activity was calculated according to the following formula: percentage of hyphal damage [(1 - x)/C] x 100, where x represents the OD of test wells, and C represents the OD of control wells with hyphae only. Each set of conditions was tested in duplicate, and the results were averaged. PMN production of reactive oxygen intermediates (ROI) was performed by quantifying the superoxide anion (O2-) production by measuring the superoxide dismutase (SOD)-inhibitable reduction of cytochrome c as previously described (44). Peripheral PMNs (2 x 106) were incubated in the presence of 80 µM cytochrome c with 2 x 107 unopsonized yeasts or conidia for 60 or 120 min, respectively, at 37°C. A550 was measured in the culture supernatants in a Microplate Reader 550 (Bio-Rad, Rome, Italy), and the background absorbance in control tubes containing only buffer and cytochrome c was subtracted. Experiments were performed in triplicate, and the results were expressed as nanomoles of O2-. The effects of TLR stimulation on fungicidal activity, respiratory burst, and degranulation were assessed by preincubating PMNs for 120 min with the different TLR ligands (zymosan (10 µg/ml) and lipoteichoic acid (LTA; 1 µg/ml) from Staphylococcus aureus and LPS (10 µg/ml) from Salmonella minnesota Re 595 (all from Sigma-Aldrich) and unmethylated CpG oligonucleotide 1826 (2 µM) of proven immunostimulatory sequence (25)) before the addition of unopsonized yeasts or resting conidia. Photographs were taken using a high resolution microscopy color camera (AxioCam Color), using the AxioVision software Rel. 3.0 (Carl Zeiss, Milan, Italy). For cytokine determination, purified DCs were resuspended in IMDM with no serum, but with polymixin B, to avoid nonspecific activation by serum components and endotoxin and were pulsed with unopsonized Candida yeasts and hyphae or Aspergillus resting conidia for 2 h before the addition of amphotericin B to prevent fungal overgrowth as previously described (5, 6). Cytokine measurement was performed after an additional 22 h of coculture. The levels of cytokine production by DCs added with amphotericin B alone were below the detection limits of the assays (data not shown).
Proliferation assay by flow cytometric analysis
Proliferation of 1 x 106 splenic CD4+ T lymphocytes stimulated with 5 x 105 Ag-pulsed splenic DCs for 5 days at 37°C was assessed by labeling with CFSE (Molecular Probes, Eugene, OR) as previously described (9).
Cytokine and ELISPOT assays
The levels of cytokines in culture supernatants, lung, stomach, and kidney homogenates from infected mice were determined by ELISA kits (R&D Systems). The detection limits of the assays were <32 pg/ml for TNF- and <15 pg/ml for IL-10 and IL-12 p70. For enumeration of cytokine-producing CD4+ T cells, an ELISPOT assay was used on purified CD4+ T cells as previously described (5, 6, 9). Results are expressed as the mean number of cytokine-producing cells (±SE) per 105 cells, calculated using replicates of serial 2-fold dilutions of cells.
Statistical analysis
The log-rank test was used for paired data analysis of the Kaplan-Meier survival curves. Student’s t test or ANOVA and Bonferroni’s test were used to determine the statistical significance of differences in organ clearance and in vitro assays, as indicated in the figure legends Significance was defined as p < 0.05. In vivo groups consisted of four to six animals. The data reported were pooled from three to five experiments unless otherwise specified.
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Results |
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To assess the role of the IL-1R/TLR/MyD88-dependent pathway in the course and outcome of candidiasis and aspergillosis, TLR2-, TLR4-, TLR9-, IL-1RI-, and MyD88-deficient mice were infected with Candida yeasts or hyphae i.v., Candida hyphae i.g., or Aspergillus conidia i.n. Mice were then assessed for survival and fungal growth in the kidneys, stomach, and lungs, respectively. Similar to wild-type mice, TLR2-, TLR4-, and TLR9-deficient mice survived the infection with low virulence Candida yeasts, in contrast to IL-1RI- and MyD88-deficient mice, which succumbed to it (Fig. 1A). Susceptibility to virulent Candida hyphae given i.v. was not different among the different types of mice, in that all succumbed to the infection, although TLR4- and TLR9-deficient mice survived significantly longer (Fig. 1B). For aspergillosis, although each type of intact mouse survived the infection (data not shown), upon immunosuppression with cyclophosphamide there were important differences among strains, as TLR-4- and MyD88-deficient mice died sooner, and IL-1RI-deficient mice died later than the other strains (Fig. 1C). We also assessed the susceptibility to reinfection with Candida hyphae of mice surviving the primary infection with Candida yeasts and found that TLR-9-deficient and wild-type mice survived reinfection, whereas the majority of the TLR2- and TLR4-deficient mice succumbed (Fig. 1D). Resistance to reinfection of intact mice surviving the Aspergillus primary infection was also reduced in TLR2-deficient mice (data not shown).
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To correlate the above findings with patterns of innate antifungal resistance, we measured the levels of TNF- production early in the course of infection in kidneys and lungs of mice with candidiasis and aspergillosis, respectively, because this cytokine is required for resistance to both fungi (35) and is prototypically released through TLR-dependent pathways (12, 13, 17). We also assessed the antifungal effector activities of PMNs, as they are essential components of the innate resistance to fungi (45, 46). The levels of TNF- were particularly elevated in TLR2-deficient mice, similar to control mice in TLR9-deficient mice, and were ablated in MyD88-deficient mice infected with either fungus (Fig. 3). However, fungal species-specific differences did emerge, as TNF- levels were lower in TLR4- and IL-1RI-deficient mice infected with Candida, but not Aspergillus. Therefore, TNF- production was not predictive of resistance or susceptibility to infections, as neither was associated with a specific TLR repertoire. However, TNF- production was crucially dependent on the MyD88 pathway.
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is known to selectively impair the activity of PMNs against Candida hyphae (43), the effector function of PMNs was also assessed in the presence of TNF-. The ability of hyphal damage was greatly reduced by TNF- in TLR4-deficient PMNs and was slightly reduced in PMNs from TLR9-deficient or wild-type mice. No effect was observed on PMNs from the other types of mice, including MyD88-deficient mice which, similar to TLR4-deficient mice, showed defective TNF- production during the infection (Fig. 4A). Therefore, additional mechanisms, besides TNF- production, might regulate the expression of the antifungal effector functions of PMNs at the site of infection.
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Impact of the IL-1R/TLR/MyD88-dependent signaling pathway on antifungal adaptive immunity
To define the impact of the IL-1R/TLR/MyD88-dependent signaling pathway on the quality of the adaptive immunity to fungi, we determined the frequencies of IFN-
- or IL-4-producing CD4+T lymphocytes in uninfected mice or mice with gastrointestinal candidiasis or pulmonary aspergillosis. To this purpose, mice were infected i.g. with Candida hyphae or i.n. with Aspergillus conidia, and the number of Th1 or Th2 cytokine-producing cells was assessed in the MLN and TLN, respectively, 1 wk after the infection. No differences were observed in the frequencies of cytokine-producing cells among the different types of uninfected mice. However, the number of cells producing IFN- was significantly reduced and that of cells producing IL-4 was significantly increased in MyD88-deficient mice with either infection. A similar pattern of Th1/Th2 reactivity was observed in TLR9-deficient mice despite their remarkable antifungal resistance. Th2 cells predominated over Th1 cells in IL-1R-deficient mice with candidiasis, but not with aspergillosis. Finally, evidence of activation of both Th1 and Th2 cells was found in TLR2- or TLR4-deficient mice with candidiasis and aspergillosis, as the number of IL-4-producing cells was increased, and the number of cells producing IFN- was either decreased, in candidiasis, or remained unchanged, in aspergillosis, compared with that in control mice (Fig. 5).
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production and no IL-4 were detected in culture supernatants of proliferating cells (data not shown).
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Discussion |
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In line with the idea of the essential role played by MyD88-dependent signaling in the development of Th1 responses (17), the susceptibility of MyD88-deficient mice to candidiasis and aspergillosis was associated with the occurrence of a defective antifungal Th1 response, probably due to defective DC activation. However, as the antifungal effector functions of PMNs were also impaired, MyD88-dependent signaling has a crucial role in the expression of optimal antifungal innate resistance. For Aspergillus, the fact that intact MyD88-deficient mice survived the infection suggests that resistance to the fungus may also occur through MyD88-independent mechanisms. Intriguingly, Drosophila MyD88, although only needed for antifungal defense, is unable to induce expression of the antifungal peptide drosomycin in the absence of other adapters (50). Preliminary data showing the ability of LPS to activate antifungal effector functions in MyD88-deficient mice support this hypothesis. However, because TLR4 is known to signal through both the MyD88-dependent and -independent pathways (14), but TLR4-deficient mice are not more susceptible to aspergillosis than MyD88-deficient mice, further studies are needed to elucidate the role of MyD88-independent pathways in resistance to Aspergillus. It is of interest that the conidiocidal activity, more than the phagocytic ability, was almost completely ablated in TLR4-deficient PMNs, a finding suggesting that internalization of the fungus, in the absence of TLR4 signaling, may contribute to virulence by preventing fungal killing and thus favoring fungal survival.
In candidiasis, in line with the increased susceptibility to the infection seen in TLR4-defective C3H/HeJ mice (23), our study shows that TLR4-deficient mice mount a defective Th1-protective immunity to fungus in the face of an efficient innate antifungal resistance, particularly against hyphae. However, a more crucial role is played by IL-1R1, as both the innate and acquired Th1 resistances to Candida infections were severely impaired in its absence. The finding that, similar to MyD88-deficient mice, IL-1R-deficient mice were highly susceptible to low virulence Candida yeasts qualifies the IL-1RI/MyD88-dependent pathway as a major host determinant of fungal virulence in vivo. The killing ability of PMNs against Candida was also severely impaired in IL-1R-deficient mice, a finding consistent with the importance of IL-1-dependent (51), but more importantly IL-18-dependent (52), signaling in the activation of PMNs to an antifungal state. However, although anticandidal resistance was decreased in conditions of IL-1 (53) and/or IL-18 (54) deficiencies, neither deficiency was as severe as that of IL-1R1-deficient mice in terms of susceptibility to the infection.
TLR2 signaling by zymosan (55), 
-glucan (56), Candida phospholipomannan (26), Aspergillus conidia (24, 27), and hyphae (27, 28) led to the production of both inflammatory (TNF-) and anti-inflammatory (IL-10) cytokines depending on the cell type. Unopsonized zymosan also stimulated the respiratory burst of phagocytes, although opsonization greatly increased this capacity (57). However, the susceptibility of TLR2-deficient mice to primary candidiasis and aspergillosis was not different from that of control mice, a finding suggesting that the mice are fully competent at the level of innate antifungal resistance, as documented by reduced fungal growth in mice with primary disseminated candidiasis. In contrast, they failed to mount protective Th1 resistance, as evidenced by the high susceptibility to mucosal infection and reinfection, both known to be controlled by Th1 adaptive immunity (35). Both Th1 and Th2 lymphocytes were indeed activated in these mice despite the high level production of IL-12 p70 by DCs. The interesting finding was that IL-10 production was greatly reduced in DCs from these mice in response to fungi. Because IL-10-producing DCs are required for the expression of long-lasting memory antifungal immunity through the induction of regulatory T cells (58), it is possible that the Th1 hyporesponsiveness of TLR2-deficient mice is a consequence of the decreased IL-10 production by DCs. Whatever the mechanism is, our finding is consistent with the defective response to vaccination with Borrelia burgdorferi OspA seen in TLR2-deficient mice (36) as well as with the TLR2 dependency of IL-10 production in response to Aspergillus hyphae (27).
The findings with TLR9-deficient mice are interesting, although largely unexplained. As TLR9 stimulation by CpG enhances innate effector (59) and Th1 responses in experimental models of infection (60), including aspergillosis (25), one would reasonably expect decreased resistance to fungi. We found that TLR9-deficient mice were incapable of mounting an Ag-specific Th1 response, yet they were highly resistant to both mucosal candidiasis and reinfection. TLR9-deficient mice were particularly efficient in restricting fungal growth upon primary infection, particularly with Candida hyphae, against which they showed potent antifungal effector activity.
Consistent with their role in immune surveillance, TLRs are expressed at higher levels in tissues exposed to the external environment, such as lung and gastrointestinal tract (32). TLRs have also been shown to be expressed on human peripheral blood PMNs (61) and to mediate antimicrobial responses (59, 62). PMNs can exert antifungal activity by both oxidative and nonoxidative mechanisms (63). The former is characterized by the respiratory burst resulting in the generation of oxidants with potent antimicrobial activity. A multitude of mediators with nonoxidative antifungal activity has been also found in human PMN granules, with the primary (azurophil) granules containing, among others, myeloperoxidase and defensins (64). The quantity and specificity of delivery of these toxic neutrophil products ultimately will determine the relative efficiency of fungicidal activity vs inflammatory cytotoxicity to host cells.
It is well known that the various fungal forms not only elicit differing responses from PMNs and phagocytes, but also differ in their susceptibilities to cellular microbicidal mechanisms (47). We found in this study that murine PMN activation by the different fungal forms occurs through TLR signaling that affected the release of both granule constituents and oxidants more than phagocytosis. This is consistent with previous findings demonstrating roles for zymosan (57), LPS (65), CpG (59), and p38 mitogen-activated kinases (66) in regulating a variety of neutrophil functions, including the production of ROI and degranulation. Consistent with the expression of TLR2, TLR4, and TLR9 on PMNs upon exposure to fungi, zymosan, LPS, and, in part, CpG stimulated the fungicidal activity of PMNs. However, different mechanisms of antifungal activity were elicited. The production of ROI was higher in response to zymosan than LPS or CpG, but degranulation was maximally induced by LPS and less so with zymosan or CpG. Interestingly, azurophilic granules with myeloperoxidase activity were mobilized by LPS more than by zymosan or CpG. Experiments with Candida and Aspergillus hyphae have suggested that degranulation, more than ROI production, correlated with the maximum antifungal effector function of PMNs, an activity that occurs differently in the different types of mice and in response to the different TLR ligands (S. Bellocchio, C. Montegnoli, S. Bozzo, G. Rossi, and L. Romoni, manuscript in preparation). Together these findings, although highlighting the complexity and multitude of mechanisms underlying the different susceptibilities of TLR-deficient mice to the primary infections with each fungal morphotype, also suggest that different TLRs activate specialized antifungal effector functions on PMNs and support the idea that different PMN functions are mediated by divergent transduction pathways (63). Interestingly, stimulation with LTA abrogated the fungicidal activity of PMNs, and this was associated with stimulation of ROI production, but not with degranulation. This finding, although confirming that ROI stimulation without the concomitant activation of granule proteases is not sufficient to mediate antifungal activity (47, 48), also unmasks the effect of TLR cooperativity at the level of PMN activation.
In line with the idea that TLR2 ligands are recognized by heterodimers formed between TLR2 and other TLRs (30), host recognition of different configurations of lipoprotein/lipopeptides occurs through the heterodimeric association of TLR2 with either TLR1 or TLR6 (67). In this regard it is of interest that the capacity of PMNs to kill C. albicans was greatly reduced after exposure to medium, but not long, chain fatty acid-containing lipid emulsions in vitro (68) and in vivo (69). As similar results, in terms of TLR expression and functioning, have been obtained with human PMNs (S. Bellocchio, C. Montegnoli, S. Bozzo, G. Rossi, and L. Romoni, manuscript in preparation), together these data indicate that the expression of the innate response to fungi occurs through the involvement of distinct TLRs, each probably activating specialized antifungal effector functions on PMNs and implicating the MyD88-dependent signaling pathway. Importantly, the finding that TLRs affect the balance between fungicidal oxidative and nonoxidative mechanisms of PMNs suggests that TLR manipulation in vivo may be amenable to the induction of optimal microbicidal activity in the absence of inflammatory cytotoxicity to host cells.
It is still unclear whether pathogen-specific Th2 responses develop by default in the absence of the TLR/MyD88-dependent signal or whether a specialized class of recognition receptors activates Th2 responses upon recognition of Th2-specific pathogens (12, 14). This issue may be particularly relevant for the understanding of how Th2 immune responses are generated to fungal hyphae. We have recently shown that additional receptors of fungi, such as Fc, complement and lectin receptors, contributed to the generation of Th1/Th2 responses to the different fungal forms (4). The relation between TLRs and these receptors is largely unknown at the moment. The data from the present study show that the TLR repertoire contributes to the functional plasticity of DCs in response to the different fungal morphotypes, including hyphae. MyD88-deficient DCs were clearly impaired in their ability to activate Th1 cells. However, as no obvious correlation could be found between patterns of cytokine production by DCs and type of antifungal Th reactivity, such as in conditions of TLR2 or TLR4 deficiency, these findings indicate the existence of additional dysfunctional activities at the DC level, as previously suggested (70).
In conclusion, this study shows the diversity and complexity of the signaling pathways triggered by TLRs in response to fungi in vivo (summarized in Table I). Members of the TLR/IL-1R superfamily contributed to the generation of host immunity to C. albicans and A. fumigatus in a manner that is dependent on fungal species and morphotypes as well as the site of infection. Basically, the IL-1R1/MyD88-dependent pathway is essentially required for host resistance to Candida, whereas the TLR4/MyD88-dependent pathway is crucially involved in resistance to Aspergillus. However, as fungal morphogenesis in vivo is activated by a variety of tissue-dependent stimuli (33), this idea may accommodate the finding that TLRs are differently implicated depending on the site of the infection, as clearly shown in infections with Candida hyphae, and suggests that recruitment of multiple TLRs may occur during infection. Finally, the increased antifungal resistance observed under conditions of TLR deficiency not associated with impaired immune resistance suggests that TLRs may be exploited by fungi for survival in vivo.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luigina Romani, Department of Experimental Medicine and Biochemical Sciences, Microbiology Section, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail address: lromani{at}unipg.it
3 Abbreviations used in this paper: DC, dendritic cell; i.g., intragastrically; IL-1RI, IL-1R type 1; i.n., intranasally; LTA, lipotheicoic acid; MLN, mesenteric lymph node; MyD88, myeloid differentiation primary response gene 88; PMN, polymorphonuclear neutrophil; ROI, reactive oxygen intermediate; SOD, superoxide dismutase; TLN, thoracic lymph node; TLR, Toll-like receptor; Zym, zymosan.
Received for publication October 6, 2003. Accepted for publication December 23, 2003.
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References |
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H. Ramaprakash, T. Ito, T. J. Standiford, S. L. Kunkel, and C. M. Hogaboam Toll-Like Receptor 9 Modulates Immune Responses to Aspergillus fumigatus Conidia in Immunodeficient and Allergic Mice Infect. Immun., January 1, 2009; 77(1): 108 - 119. [Abstract] [Full Text] [PDF]
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 V. Balloy, J.-M. Sallenave, Y. Wu, L. Touqui, J.-P. Latge, M. Si-Tahar, and M. Chignard Aspergillus fumigatus-induced Interleukin-8 Synthesis by Respiratory Epithelial Cells Is Controlled by the Phosphatidylinositol 3-Kinase, p38 MAPK, and ERK1/2 Pathways and Not by the Toll-like Receptor-MyD88 Pathway J. Biol. Chem., November 7, 2008; 283(45): 30513 - 30521. [Abstract] [Full Text] [PDF]
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 E. G. Pamer TLR Polymorphisms and the Risk of Invasive Fungal Infections N. Engl. J. Med., October 23, 2008; 359(17): 1836 - 1838. [Full Text] [PDF]
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S. E. W. Grubb, C. Murdoch, P. E. Sudbery, S. P. Saville, J. L. Lopez-Ribot, and M. H. Thornhill Candida albicans-Endothelial Cell Interactions: a Key Step in the Pathogenesis of Systemic Candidiasis Infect. Immun., October 1, 2008; 76(10): 4370 - 4377. [Full Text] [PDF]
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O. S. Shin, R. R. Isberg, S. Akira, S. Uematsu, A. K. Behera, and L. T. Hu Distinct Roles for MyD88 and Toll-Like Receptors 2, 5, and 9 in Phagocytosis of Borrelia burgdorferi and Cytokine Induction Infect. Immun., June 1, 2008; 76(6): 2341 - 2351. [Abstract] [Full Text] [PDF]
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Z. G. Ramirez-Ortiz, C. A. Specht, J. P. Wang, C. K. Lee, D. C. Bartholomeu, R. T. Gazzinelli, and S. M. Levitz Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA Infect. Immun., May 1, 2008; 76(5): 2123 - 2129. [Abstract] [Full Text] [PDF]
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 S. C. Nance, A.-K. Yi, F. C. Re, and E. A. Fitzpatrick MyD88 is necessary for neutrophil recruitment in hypersensitivity pneumonitis J. Leukoc. Biol., May 1, 2008; 83(5): 1207 - 1217. [Abstract] [Full Text] [PDF]
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 A. Simon, B. J. Kullberg, B. Tripet, O. C. Boerman, P. Zeeuwen, J. van der Ven-Jongekrijg, P. Verweij, J. Schalkwijk, R. Hodges, J. W. M. van der Meer, et al. Drosomycin-Like Defensin, a Human Homologue of Drosophila melanogaster Drosomycin with Antifungal Activity Antimicrob. Agents Chemother., April 1, 2008; 52(4): 1407 - 1412. [Abstract] [Full Text] [PDF]
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S. Bozza, T. Zelante, S. Moretti, P. Bonifazi, A. DeLuca, C. D'Angelo, G. Giovannini, C. Garlanda, L. Boon, F. Bistoni, et al. Lack of Toll IL-1R8 Exacerbates Th17 Cell Responses in Fungal Infection J. Immunol., March 15, 2008; 180(6): 4022 - 4031. [Abstract] [Full Text] [PDF]
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C. Bretz, G. Gersuk, S. Knoblaugh, N. Chaudhary, J. Randolph-Habecker, R. C. Hackman, J. Staab, and K. A. Marr MyD88 Signaling Contributes to Early Pulmonary Responses to Aspergillus fumigatus Infect. Immun., March 1, 2008; 76(3): 952 - 958. [Abstract] [Full Text] [PDF]
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 S. Tsiodras, G. Samonis, D. T. Boumpas, and D. P. Kontoyiannis Fungal Infections Complicating Tumor Necrosis Factor {alpha} Blockade Therapy Mayo Clin. Proc., February 1, 2008; 83(2): 181 - 194. [Abstract] [Full Text] [PDF]
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V. Muller, D. Viemann, M. Schmidt, N. Endres, S. Ludwig, M. Leverkus, J. Roth, and M. Goebeler Candida albicans Triggers Activation of Distinct Signaling Pathways to Establish a Proinflammatory Gene Expression Program in Primary Human Endothelial Cells J. Immunol., December 15, 2007; 179(12): 8435 - 8445. [Abstract] [Full Text] [PDF]
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A. De Luca, C. Montagnoli, T. Zelante, P. Bonifazi, S. Bozza, S. Moretti, C. D'Angelo, C. Vacca, L. Boon, F. Bistoni, et al. Functional yet Balanced Reactivity to Candida albicans Requires TRIF, MyD88, and IDO-Dependent Inhibition of Rorc J. Immunol., November 1, 2007; 179(9): 5999 - 6008. [Abstract] [Full Text] [PDF]
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 T. M. Hohl and M. Feldmesser Aspergillus fumigatus: Principles of Pathogenesis and Host Defense Eukaryot. Cell, November 1, 2007; 6(11): 1953 - 1963. [Full Text] [PDF]
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A. Cassone, F. De Bernardis, and G. Santoni Anticandidal Immunity and Vaginitis: Novel Opportunities for Immune Intervention Infect. Immun., October 1, 2007; 75(10): 4675 - 4686. [Full Text] [PDF]
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C.-L. Ku, H. von Bernuth, C. Picard, S.-Y. Zhang, H.-H. Chang, K. Yang, M. Chrabieh, A. C. Issekutz, C. K. Cunningham, J. Gallin, et al. Selective predisposition to bacterial infections in IRAK-4 deficient children: IRAK-4 dependent TLRs are otherwise redundant in protective immunity J. Exp. Med., October 1, 2007; 204(10): 2407 - 2422. [Abstract] [Full Text] [PDF]
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 B. H. Segal Role of Macrophages in Host Defense Against Aspergillosis and Strategies for Immune Augmentation Oncologist, October 1, 2007; 12(suppl_2): 7 - 13. [Abstract] [Full Text] [PDF]
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V. Auerbuch and R. R. Isberg Growth of Yersinia pseudotuberculosis in Mice Occurs Independently of Toll-Like Receptor 2 Expression and Induction of Interleukin-10 Infect. Immun., July 1, 2007; 75(7): 3561 - 3570. [Abstract] [Full Text] [PDF]
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K. M. Empey, M. Hollifield, and B. A. Garvy Exogenous Heat-Killed Escherichia coli Improves Alveolar Macrophage Activity and Reduces Pneumocystis carinii Lung Burden in Infant Mice Infect. Immun., July 1, 2007; 75(7): 3382 - 3393. [Abstract] [Full Text] [PDF]
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 A. P. Phadke, G. Akangire, S. J. Park, S. A. Lira, and B. Mehrad The Role of CC Chemokine Receptor 6 in Host Defense in a Model of Invasive Pulmonary Aspergillosis Am. J. Respir. Crit. Care Med., June 1, 2007; 175(11): 1165 - 1172. [Abstract] [Full Text] [PDF]
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M. Feldmesser Role of Neutrophils in Invasive Aspergillosis{triangledown} Infect. Immun., December 1, 2006; 74(12): 6514 - 6516. [Full Text] [PDF]
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V. C. B. Bittencourt, R. T. Figueiredo, R. B. da Silva, D. S. Mourao-Sa, P. L. Fernandez, G. L. Sassaki, B. Mulloy, M. T. Bozza, and E. Barreto-Bergter An {alpha}-Glucan of Pseudallescheria boydii Is Involved in Fungal Phagocytosis and Toll-like Receptor Activation J. Biol. Chem., August 11, 2006; 281(32): 22614 - 22623. [Abstract] [Full Text] [PDF]
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 A. E. Medvedev, I. Sabroe, J. D. Hasday, and S. N. Vogel Invited review: Tolerance to microbial TLR ligands: molecular mechanisms and relevance to disease Innate Immunity, June 1, 2006; 12(3): 133 - 150. [Abstract] [PDF]
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A. K. Behera, E. Hildebrand, R. T. Bronson, G. Perides, S. Uematsu, S. Akira, and L. T. Hu MyD88 Deficiency Results in Tissue-Specific Changes in Cytokine Induction and Inflammation in Interleukin-18-Independent Mice Infected with Borrelia burgdorferi Infect. Immun., March 1, 2006; 74(3): 1462 - 1470. [Abstract] [Full Text] [PDF]
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 Y.-T.A. Teng Protective and Destructive Immunity in the Periodontium: Part 1--Innate and Humoral Immunity and the Periodontium Journal of Dental Research, March 1, 2006; 85(3): 198 - 208. [Abstract] [Full Text] [PDF]
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C. Murciano, E. Villamon, J.-E. O'Connor, D. Gozalbo, and M. L. Gil Killed Candida albicans Yeasts and Hyphae Inhibit Gamma Interferon Release by Murine Natural Killer Cells Infect. Immun., February 1, 2006; 74(2): 1403 - 1406. [Abstract] [Full Text] [PDF]
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K. J. Carpenter and C. M. Hogaboam Immunosuppressive Effects of CCL17 on Pulmonary Antifungal Responses during Pulmonary Invasive Aspergillosis Infect. Immun., November 1, 2005; 73(11): 7198 - 7207. [Abstract] [Full Text] [PDF]
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V. Balloy, M. Si-Tahar, O. Takeuchi, B. Philippe, M.-A. Nahori, M. Tanguy, M. Huerre, S. Akira, J.-P. Latge, and M. Chignard Involvement of Toll-Like Receptor 2 in Experimental Invasive Pulmonary Aspergillosis Infect. Immun., September 1, 2005; 73(9): 5420 - 5425. [Abstract] [Full Text] [PDF]
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 C. F. Benjamim, S. K. Lundy, N. W. Lukacs, C. M. Hogaboam, and S. L. Kunkel Reversal of long-term sepsis-induced immunosuppression by dendritic cells Blood, May 1, 2005; 105(9): 3588 - 3595. [Abstract] [Full Text] [PDF]
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S. Bozza, F. Fallarino, L. Pitzurra, T. Zelante, C. Montagnoli, S. Bellocchio, P. Mosci, C. Vacca, P. Puccetti, and L. Romani A Crucial Role for Tryptophan Catabolism at the Host/Candida albicans Interface J. Immunol., March 1, 2005; 174(5): 2910 - 2918. [Abstract] [Full Text] [PDF]
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 S. Bellocchio, R. Gaziano, S. Bozza, G. Rossi, C. Montagnoli, K. Perruccio, M. Calvitti, L. Pitzurra, and L. Romani Liposomal amphotericin B activates antifungal resistance with reduced toxicity by diverting Toll-like receptor signalling from TLR-2 to TLR-4 J. Antimicrob. Chemother., February 1, 2005; 55(2): 214 - 222. [Abstract] [Full Text] [PDF]
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A. Q. Khan, Q. Chen, Z.-Q. Wu, J. C. Paton, and C. M. Snapper Both Innate Immunity and Type 1 Humoral Immunity to Streptococcus pneumoniae Are Mediated by MyD88 but Differ in Their Relative Levels of Dependence on Toll-Like Receptor 2 Infect. Immun., January 1, 2005; 73(1): 298 - 307. [Abstract] [Full Text] [PDF]
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S. Bellocchio, S. Moretti, K. Perruccio, F. Fallarino, S. Bozza, C. Montagnoli, P. Mosci, G. B. Lipford, L. Pitzurra, and L. Romani TLRs Govern Neutrophil Activity in Aspergillosis J. Immunol., December 15, 2004; 173(12): 7406 - 7415. [Abstract] [Full Text] [PDF]
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L. Pitzurra, S. Bellocchio, A. Nocentini, P. Bonifazi, R. Scardazza, L. Gallucci, F. Stracci, C. Simoncelli, F. Bistoni, and L. Romani Antifungal Immune Reactivity in Nasal Polyposis Infect. Immun., December 1, 2004; 72(12): 7275 - 7281. [Abstract] [Full Text] [PDF]
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D. Serrano-Gomez, A. Dominguez-Soto, J. Ancochea, J. A. Jimenez-Heffernan, J. A. Leal, and A. L. Corbi Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin Mediates Binding and Internalization of Aspergillus fumigatus Conidia by Dendritic Cells and Macrophages J. Immunol., November 1, 2004; 173(9): 5635 - 5643. [Abstract] [Full Text] [PDF]
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L. E. Yauch, M. K. Mansour, S. Shoham, J. B. Rottman, and S. M. Levitz Involvement of CD14, Toll-Like Receptors 2 and 4, and MyD88 in the Host Response to the Fungal Pathogen Cryptococcus neoformans In Vivo Infect. Immun., September 1, 2004; 72(9): 5373 - 5382. [Abstract] [Full Text] [PDF]
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