Spherules Derived from Coccidioides posadasii Promote Human Dendritic Cell Maturation and Activation
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感染与免疫杂志 2006年第4期
Valley Fever Center for Excellence Department of Microbiology and Immunology Arizona Cancer Center
Department of Medicine and Medical and Research Services, Southern Arizona Veterans Affairs Medical Center, Tucson, Arizona
ABSTRACT
Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E2. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.
INTRODUCTION
Coccidioidomycosis is a fungal infection endemic in the southwestern United States, including Arizona and the San Joaquin Valley of California, and in parts of Central and South America (20, 29). While the immunologic response to infection with Coccidioides has not been fully defined, an increase in the severity of infection correlates with decreased cellular immunity in response to coccidioidal antigens (6, 19). Experimental murine coccidioidomycosis models demonstrate that cellular immunity is important for control of infection (1, 14, 25), as do human in vitro studies (4, 6, 7, 10, 19, 32, 33). Additionally, patients with human immunodeficiency virus infection who have depleted CD4 counts are predisposed to Coccidioides infection (2, 3, 8, 24, 40).
Coccidioides exists in the soil as a mold and produces barrel-shaped arthroconidia, which upon inhalation by a susceptible host differentiate into spherules. Dendritic cells (DC) serve as both initiators and modulators of immune responses and bridge the innate and acquired arms of the immune system (13). DC-mediated antifungal responses have been reported for Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans (15, 28, 38), and we have previously demonstrated that DC, when loaded with the coccidioidal antigen preparation T27K, can induce lymphocyte transformation in peripheral blood mononuclear cells (PBMC) obtained from Coccidioides-nonimmune and Coccidioides-anergic donors (33). Little is known about the early events involved in the innate immune response against Coccidioides spherules. The present study evaluates the interaction between spherules and DC with regard to binding, uptake, maturation, and antigen presentation in order to examine the early immune response to coccidioidal infection in the human system.
MATERIALS AND METHODS
Cytokines and antibodies. The following cytokines and antibodies (purchased from Pharmingen, San Diego, CA, unless otherwise noted) were used: anti-CD1a-fluorescein isothiocyanate (FITC), CD14-TC (Caltag, Burlingame, CA), CD40-FITC, CD80-phycoerythrin, CD83-phycoerythrin, CD86-cytochrome c, HLA-DR-FITC, all corresponding isotype control antibodies, recombinant human interleukin 4 (IL-4) (Peprotech, Rocky Hill, NJ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex, Seattle, WA).
Preparation of spherules and labeling with FITC. Coccidioides spherules were prepared by a modification of the methods described by Sun and Huppert (37). Silveira strain C. posadasii arthroconidia were inoculated into 1 liter of modified converse medium in a 2-liter flask at a concentration of 7 x 106 and incubated for 96 h at 39°C with orbital shaking (180 rpm, 8% CO2). Spherules were harvested by centrifugation at 5,100 rpm for 40 min in a Beckman Allegra centrifuge with a TS-5.1 rotor and washed three times with sterile distilled water. The spherule pellet was resuspended in 7.5 ml sterile water and autoclaved at 15 lb/in2 for 20 min at 121°C. To ensure that spherules were not viable, 0.1 ml of the autoclaved suspension was added to glucose yeast extract plates in duplicate and incubated at room temperature for 2 weeks. No growth was observed, and spherules were considered nonviable. The methods used to generate spherules result in very pure cultures. All arthroconidia differentiate into spherules and no mycelial remnants are present. All manipulations of potentially viable C. posadasii organisms were accomplished under biosafety level 3 conditions in laboratories registered with the CDC for possession of this select agent. All glassware used for spherule preparation was baked prior to use to prevent the introduction of exogenous lipopolysaccharides. The spherule preparation was tested for endotoxin by use of a Limulus amoebocyte lysate assay (Clongen Laboratories, Germantown, MD) and was found to contain less than 20 endotoxin units/μg.
Spherules were stored in 1x sterile phosphate-buffered saline (PBS) at 4°C until use and then diluted into AIM-V medium (Invitrogen, Carlsbad, CA). A total of 1 x 104 spherules/well were added to lymphocyte transformation assay mixtures. For DC maturation studies and spherule antigen presentation proliferation assays, a 1:4 spherule-to-DC ratio was used. In some cases, fluorescein-labeled spherules were used in experiments. Spherules (6 x 107) were suspended in 0.1 M carbonate buffer (pH 9.0) and incubated with FITC (Sigma, St. Louis, MO) at a final concentration of 0.16 mg/ml at 4°C overnight. Spherules were thoroughly washed with PBS and stored at 4°C in the dark until use.
Identification of Coccidioides-immune and nonimmune donors. Isolation of PBMC from healthy individuals was performed with the approval of the Human Subjects Protection Committee at the University of Arizona. Donor immunity status was determined by performing lymphocyte proliferation assays with the coccidioidal antigen preparation T27K, a soluble, aqueous supernatant derived by mechanical disruption of thimerosal-killed coccidioidal spherules followed by centrifugation at 27,000 x g (41). Briefly, 5 x 105 PBMC were added to flat-bottomed wells of 96-well plates in AIM-V medium alone, with 12.5 μg/ml of T27K, or with 10.0 μg/ml phytohemagglutinin (mitogen used as a positive control for cellular proliferation) and incubated in 5% CO2 at 37°C for 5 days. Subsequently, cells were pulsed with 1.0-μCi/well [3H]thymidine (NEN Life Science Products, Perkin-Elmer, Wellesley, MA) for 18 h. Cells were harvested onto Unifilter plates (Perkin-Elmer) by use of a cell harvester (Perkin-Elmer). [3H]thymidine incorporation was measured using a scintillation counter (TopCount; Perkin-Elmer). T27K has been previously demonstrated to specifically distinguish Coccidioides-immune donors from nonimmune donors (11, 12, 33). PBMC from all individuals tested were activated by phytohemagglutinin. A nonimmune donor had a stimulation index of <5-fold cpm greater than that of the control, while an immune donor had an index of 5-fold cpm above that of the control. Lymphocyte transformation assays using spherules (1 x 104 spherules/well) were performed as described above.
Generation of dendritic cells. DC were generated from nonimmune PBMC by a modification of the methods described by Romani et al. (34). Briefly, whole blood was layered on Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) to obtain PBMC. PBMC were plated in T-75 flasks in AIM-V medium (Life Technologies, Grand Island, NY) and allowed to adhere for 2 h in a 37°C incubator containing 5% CO2. To remove the nonadherent PBMC fraction, flasks were washed several times with sterile PBS. X-Vivo 15 medium (BioWhittaker, Walkersville, MD) supplemented with 1,000 IU/ml GM-CSF and 500 IU/ml IL-4 was added to adherent PBMC, which were cultured for 5 days. Day 5 immature DC were cultured with either tumor necrosis factor alpha (TNF-) (Peprotech, Rocky Hill, NJ) and prostaglandin E2 (PGE2) (Sigma, St. Louis, MO) to induce maturation or with spherules at concentrations determined not to inhibit cell growth or viability (1:4 ratio of spherules to DC).
Confocal microscopy. Immunofluorescence was examined with a Zeiss confocal laser scanning microscope (510 Meta LNO). Immature DC (1 x 106) were cultured with FITC-labeled spherules (5 x 105) for 24 or 72 h. DC were harvested and stained both intracellularly and extracellularly with mouse anti-human HLA-DR-allophycocyanin (APC) (Pharmingen) with a Fix & Perm kit (Caltag). Cells were illuminated with an argon ion multiline laser tuned to 488 nm to excite FITC and with a He/Ne ion laser tuned to 633 nm to excite APC. Standard filters were placed in front of detectors to collect light. Pairs of images were superimposed for colocalization analysis.
Binding assay of FITC-labeled spherules on DC. Immature DC (5 x 105) were incubated with the indicated number of FITC-labeled spherules at 37°C and 4°C in 200 μl sterile PBS. For dose-dependent experiments, DC were mixed with 0, 3.1 x 104, 1.25 x 105, 2.5 x 105, or 5.0 x105 spherules for 1 hour. For time-dependent experiments, DC were mixed with 2.5 x 105 spherules for 1, 2, 4, or 6 h. Cells were immediately analyzed by flow cytometry. A total of 10,000 gated DC events were collected. Spherule binding was quantified as mean fluorescence intensity (MFI). Inhibition of spherule uptake by immature DC (iDC) was performed by pretreating iDC (5 x 105) with 3.0 mg/ml mannan (Sigma Chemical Co., St. Louis, Mo.) for 30 min before the addition of FITC-labeled spherules (2.5 x 105).
Phenotypic analysis of DC. DC were stained with fluorochrome-conjugated antibodies specific for DC markers CD1a, 14, 40, 80, 83, and 86 and HLA-DR in PBS for 30 min at 4°C and fixed in 2% paraformaldehyde prior to flow cytometric analysis (FACScan; Becton Dickinson, Franklin Lakes, NJ). A gate was set on large cells, and 10,000 gated events were collected for analysis.
Cytokine production by DC. A Flex Set cytometric bead array kit (IL-, IL-6, IL-10, IL-12p70, and TNF-; Pharmingen) and an IL-8 enzyme-linked immunosorbent assay kit (Pharmingen) were used to determine cytokine concentrations present in DC supernatant according to the manufacturer's instructions.
Functional DC activity. To assay autologous lymphocyte proliferation, DC (2 x 104 cells/well; iDC, mature DC [mDC], or DC cultured with spherules) were plated in triplicate in 96-well flat-bottom plates in AIM-V medium and irradiated with 50 Gy from a 60Co source. Nonadherent autologous PBMC (2 x 105 cells/well) were added to the DC in a total volume of 200 μl of medium. [3H]thymidine incorporation was assessed as described above.
Statistical analysis. Results for spherule lymphocyte transformation assays were analyzed using the Mann-Whitney U test, with data presented as median (minimum, maximum). Phenotypic analysis of DC surface markers, cytokine production, and DC antigen presentation were analyzed using one-tailed Mann-Whitney U tests. A P value of <0.050 was considered to be statistically significant.
RESULTS
Spherules activate PBMC from Coccidioides-immune donors. Immune and nonimmune donor identification was performed by culturing PBMC with the T27K coccidioidal antigen preparation and measuring [3H]thymidine uptake. When PBMC (5 x 105 cells/well) from both donor groups (five immune donors and six nonimmune donors) were cultured with 1.0 x 104 spherules, there was specific activation of PBMC isolated from immune donors but not from nonimmune donors (Table 1).
DC bind spherules in a dose- and time-dependent manner. Spherules labeled with FITC were incubated with DC at 37°C or at 4°C for 1 hour. This mixture was subjected to flow cytometric analysis to observe cellular fluorescence indicating association of spherules with DC. Spherule binding to DC was greater at 37°C than at 4°C as measured by MFI, suggesting an active binding mechanism (Fig. 1A). DC-spherule association was dose dependent at 37°C, as increasing numbers of spherules resulted in increased MFI. Additionally, spherule binding to DC increased over time (Fig. 1B). When iDC were incubated with mannan (3.0 mg/ml), an inhibitor of the mannose receptor (MR) (35), for 30 min prior to the addition of FITC-labeled spherules, spherule binding was inhibited by 49.01% ± 7.26%. When DC were incubated with zymosan, the ligand for Dectin-1, no inhibition of spherule binding to DC was observed (data not shown).
In addition, we incubated iDC with FITC-labeled spherules for either 24 or 72 h at 37°C. Subsequently, DC were fluorescently labeled with an APC-conjugated HLA-DR-APC antibody. Cells were fixed, permeabilized, and intracellularly labeled with anti-HLA-DR and were then analyzed by confocal microscopy (Fig. 2). We observed that after 24 h of culture, an average of 60% of DC ingested from one to six spherules (Fig. 2A), and that after 72 h, intact spherules could no longer be detected within the DC (Fig. 2B). Moreover, processed spherule antigens colocalize with HLA-DR both within and on the surface of cells, suggesting antigen presentation. When we saturated the system by adding an abundance of FITC-labeled spherules to DC, more than 90% of DC took up spherules. There was a direct correlation between the number of spherules added to DC cultures and the number of DC that take up spherules (as demonstrated in Fig. 1A). Overall, increasing the number of FITC-labeled spherules incubated with DC led to increased fluorescence, and DC were observed to take up more than one spherule.
Spherules induce DC activation. Since we observed spherule-specific PBMC responses from immune donors and the ability of DC to bind, internalize, and process spherules, we investigated the role that spherules may play in the initiation of DC maturation. iDC were generated from PBMC and cultured with spherules (iDC-sph) for 48 h. As shown in Fig. 3A to C, morphological differences were observed between iDC and iDC-sph, with the latter appearing morphologically similar to DC matured with TNF- and PGE2. Frame A depicts characteristic immature DC (smaller and less differentiated than mature DC). DC matured with TNF- and PGE2 are depicted in frame B. Mature DC appear larger than immature DC and have "veiled" structures. No spherules were observed in DC cultures after 48 h (frame C), demonstrating uptake and processing by DC.
DC were harvested and stained with monoclonal antibodies to analyze cell phenotype (Fig. 4). The number of DC cultured with spherules expressing costimulatory molecules (CD40, CD80, and CD86) was significantly increased compared to that of immature DC. The number of cells expressing CD83, a classic maturation marker, and HLA-DR was also significantly increased on iDC-sph compared to that of immature DC. Conversely, the numbers of cells expressing CD1a, an iDC marker, and CD14, a monocyte marker, were decreased for both mature DC and iDC-sph (data not shown). The maturation effect of iDC cultured with spherules was similar to that of classical DC maturation agents, TNF- and PGE2, albeit not to the same degree. In addition, endocytosis of FITC-labeled dextran was diminished in iDC-sph compared to that in iDC (data not shown), and there was an increase in responses in allogeneic mixed lymphocyte reaction assays for iDC-sph (data not shown). Both of these observations demonstrate the maturation status of these cells.
Supernatants from iDC alone or iDC cultured with spherules were harvested after 48 h of culture. We observed an increase in proinflammatory cytokine production by iDC cultured with spherules (Table 2.). Immature DC cultured with spherules for 48 h produced significantly increased amounts of TNF-a, IL-1-, IL-8, and IL-10 compared to iDC cultured without spherules.
DC cultured with spherules process and present spherule antigens to nonimmune PBMC. To determine if iDC-sph could present spherule-associated antigens and prime lymphocytes, DC were used to stimulate autologous nonadherent PBMC from nonimmune individuals. DC-sph induced proliferation of PBMC at levels significantly higher than iDC and mDC (Fig. 5). Nonimmune PBMC were not activated by T27K or spherules in the absence of DC.
DISCUSSION
Human fungal infections have increased over the past decade, resulting in increased morbidity and treatment costs. Elucidation of DC-fungus interaction will aid in understanding fungal virulence and persistence in the host. This report describes that DC are capable of Coccidioides spherule binding, internalization, and presentation of Coccidioides-associated antigens, indicating that fungal capture is important in the initiation of effective antifungal cellular responses.
We generated monocyte-derived DC from healthy, nonimmune individuals for these studies. We have previously published the findings that monocyte-derived DC can be generated from individuals with disseminated disease and that these DC are identical in phenotype and function to DC generated from nonimmune individuals (33). In addition, monocyte-derived DC from immune individuals are identical phenotypically and functionally to nonimmune DC, as these individuals are healthy with no evidence of disease.
Earlier work described the ability of human monocytes to phagocytose and kill Coccidioides arthroconidia, suggesting that an early immune response may be mounted in vivo after infection (5, 9). However, arthroconidia not cleared from the host may differentiate into spherules capable of endosporulating, resulting in the production of a new generation of spherules. If spherules are not effectively cleared from the lungs, this process can continue, thereby increasing fungal burden. This is the first study to describe uptake and processing of C. posadasii spherules by human DC. Immature DC bound spherules within 1 hour; this was followed by spherule internalization within 24 h and spherule processing was detected by 72 h. The ratio of spherules to DC used was 0.5:1, and we observed that not all DC had phagocytosed spherules. We have also saturated the system in which we added an abundance of FITC-labeled spherules to DC; typically, greater than 90% of DC take up spherules. While we do not believe that there are two different DC populations, it is possible that there are. Future experiments using flow cytometry and cell sorting might resolve this issue.
It has been suggested that coccidioidal spherules are too large to be phagocytosed by neutrophils and macrophages (23). However, we demonstrate that DC can readily take up spherules and we have in fact frequently observed multiple spherules inside of human DC. The average size of these spherules was approximately 14 μm. Spherules were mature, as defined by the presence of classical thick cell walls and the development of endospore formation as observed by phase-contrast microscopy. However, the spherules used in our in vitro model are smaller than spherules observed in vivo (average size, 80 to 100 μm). It is possible that these larger spherules would not be susceptible to phagocytosis.
A study by Bozza et al. demonstrated that murine DC internalization of A. fumigatus is mediated by the MR (15). Additionally, mannoproteins derived from C. neoformans bind to mannose receptors and are processed by murine APC (26). Furthermore, blockage of the MR resulted in inhibition of T-cell activation. In our study, we demonstrate that the mannose receptor might also play a role in Coccidioides internalization, as spherule binding was inhibited by mannan. Chieppa et al. showed that cross-linking of the MR on immature DC with an MR-specific antibody for 24 h resulted in DC maturation (18). We pretreated DC with mannan for 30 min prior to the addition of spherules. We did not investigate the effects of mannan treatment on DC but believe that DC maturation did not occur in this short period of time and that spherule uptake was inhibited due to the blocked MR on DC, not due to cellular maturation.
Morphological and phenotypic differences were observed in DC cultured with spherules. Spherule uptake resulted in DC functional maturation, as determined by decreased endocytic capacity and stimulation of allogeneic PBMC activation compared to iDC. The induction of maturation of human DC by fungal agents, including Aspergillus conidia, Candida yeast cells, Malassezia yeast cells, and mannoproteins derived from Cryptococcus, has been described in other studies (16, 17, 30, 31, 36). DC-fungus interactions in these studies have been linked to the recognition of pathogens by pathogen recognition receptors such as DC-SIGN and the mannose receptor. Viriyakosol et al. have demonstrated that both Toll-like receptor 2 (TLR-2) and Dectin-1 are important in the recognition of C. posadasii by use of an in vitro murine macrophage model (39).
We did not empirically define the mechanism of spherule-induced maturation of immature DC. We surmise that maturation is induced in a multifactorial manner. It is highly probable that spherule antigen engagement to C-type lectin receptors and TLRs on DC induces intracellular signaling cascades, resulting in DC maturation and cellular activation. Antigen uptake by immature DC is primarily mediated by C-type lectin receptors (e.g., mannose receptor). Pietrella et al. demonstrated that mannoproteins from C. neoformans promote DC maturation and activation (31). Inhibition of mannoprotein internalization inhibited the upregulation of CD40 and CD80 (classical DC costimulatory molecules). They also showed that treatment of DC with mannoproteins resulted in the degradation of phosphorylated IB (downstream effect is NF-B translocation to the nucleus, resulting in the transcription of genes important in cellular activation). NF-B activation plays a role in proinflammatory cytokine production. We observed TNF- production by DC cultured with spherules. TNF- is a cytokine widely used to mature DC in vitro. Therefore, the combination of antigen recognition and cytokine production likely contributed to DC maturation in our model. While we did not investigate the contribution of TLRs in spherule recognition, pathogen recognition by TLRs on DC also leads to maturation (21, 27). For example, Hertz et al. demonstrated that microbial lipopeptides induced DC phenotypical and functional maturation by TLR-2 interaction and that this maturation could be blocked using an anti-TLR-2 antibody (22).
T-cell activation requires phagocytosis and processing of coccidioidal spherules by DC. DC processed internalized spherules, and these spherule-derived antigens activated autologous PBMC in nonimmune donors. Coculture of lymphocytes and spherules in the absence of DC yielded negligible cellular activation in individuals not previously exposed to Coccidioides. However, the introduction of nonimmune autologous nonadherent PBMC into DC cultures in which the DC had been exposed to spherules for 48 h resulted in cellular activation of PBMC. This has also been described by a C. neoformans study in which DC were highly efficient at yeast engulfment and degradation for presentation to T cells compared to macrophages used as APC (38). This is the first report showing that human DC are able to bind, internalize, process, and present Coccidioides posadasii spherules to nonimmune PBMC, indicating that spherules are an excellent source of antigen.
We do not believe spherule activation of PBMC to be a mitogenic response because the data presented in Table 1 demonstrated that only immune donor PBMC and not nonimmune donor PBMC are activated by spherules. Bulk PBMC were cultured with spherules, and if spherule-derived mitogens were present we would have observed activation of PBMC from nonimmune individuals as well. Only when we cultured lymphocytes together with DC loaded with spherules did we observe nonimmune activation, reinforcing the power of DC antigen presentation.
It is not known if the autoclaving of spherules results in the denaturation of carbohydrates, glycolipids, and glycoproteins such that DC recognize cryptic epitopes not naturally found on spherules. However, we will be addressing this question in subsequent studies by exposing DC to live spherules and arthroconidia.
In conclusion, the results presented here provide insight into the role that immature DC play in the response to infection with Coccidioides. Further characterization of the receptors involved in spherule recognition and binding to DC will help us understand the innate immune response and the pathogenesis of Coccidioides infection.
ACKNOWLEDGMENTS
We thank D. Pappagianis and K. Simmons for providing T27K and K. Orsborn and L. Lewis for spherule preparation.
This work was supported by a grant from the NIH NIAID (IPO1AI061310-01) and grants from the Arizona Biomedical Research Commission #9008 and Merit Review from the Department of Veterans Affairs.
Present address: Center for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ 85287.
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Department of Medicine and Medical and Research Services, Southern Arizona Veterans Affairs Medical Center, Tucson, Arizona
ABSTRACT
Previous studies have shown that dendritic cells (DC) pulsed with T27K, an antigenic preparation derived from spherules (of Coccidioides posadasii), activate peripheral blood mononuclear cells (PBMC) from nonimmune subjects as well as from patients with disseminated coccidioidomycosis. In this study, we have assessed the interaction between human DC and C. posadasii spherules in order to better understand the initial response between Coccidioides and the human host. Whole autoclaved spherules induced lymphocyte transformation in PBMC obtained from immune but not from nonimmune donors. Immature DC (iDC) bound fluorescein isothiocyanate-labeled spherules in a time- and temperature-dependent manner. This binding was blocked by the addition of mannan, suggesting mannose receptor involvement in the DC-Coccidioides interaction. Binding was subsequently associated with ingestion and intracellular processing of spherules. Coculturing of spherules with iDC was associated with the development of mature DC that were morphologically, phenotypically, and functionally similar to those induced by tumor necrosis factor alpha and prostaglandin E2. Finally, spherules incubated with iDC induced activation of PBMC from nonimmune donors. These data indicate that human DC are capable of binding, internalizing, and presenting antigens from Coccidioides spherules and suggest that DC may play a critical early role in the formation of a cellular immune response in human coccidioidomycosis.
INTRODUCTION
Coccidioidomycosis is a fungal infection endemic in the southwestern United States, including Arizona and the San Joaquin Valley of California, and in parts of Central and South America (20, 29). While the immunologic response to infection with Coccidioides has not been fully defined, an increase in the severity of infection correlates with decreased cellular immunity in response to coccidioidal antigens (6, 19). Experimental murine coccidioidomycosis models demonstrate that cellular immunity is important for control of infection (1, 14, 25), as do human in vitro studies (4, 6, 7, 10, 19, 32, 33). Additionally, patients with human immunodeficiency virus infection who have depleted CD4 counts are predisposed to Coccidioides infection (2, 3, 8, 24, 40).
Coccidioides exists in the soil as a mold and produces barrel-shaped arthroconidia, which upon inhalation by a susceptible host differentiate into spherules. Dendritic cells (DC) serve as both initiators and modulators of immune responses and bridge the innate and acquired arms of the immune system (13). DC-mediated antifungal responses have been reported for Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans (15, 28, 38), and we have previously demonstrated that DC, when loaded with the coccidioidal antigen preparation T27K, can induce lymphocyte transformation in peripheral blood mononuclear cells (PBMC) obtained from Coccidioides-nonimmune and Coccidioides-anergic donors (33). Little is known about the early events involved in the innate immune response against Coccidioides spherules. The present study evaluates the interaction between spherules and DC with regard to binding, uptake, maturation, and antigen presentation in order to examine the early immune response to coccidioidal infection in the human system.
MATERIALS AND METHODS
Cytokines and antibodies. The following cytokines and antibodies (purchased from Pharmingen, San Diego, CA, unless otherwise noted) were used: anti-CD1a-fluorescein isothiocyanate (FITC), CD14-TC (Caltag, Burlingame, CA), CD40-FITC, CD80-phycoerythrin, CD83-phycoerythrin, CD86-cytochrome c, HLA-DR-FITC, all corresponding isotype control antibodies, recombinant human interleukin 4 (IL-4) (Peprotech, Rocky Hill, NJ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) (Immunex, Seattle, WA).
Preparation of spherules and labeling with FITC. Coccidioides spherules were prepared by a modification of the methods described by Sun and Huppert (37). Silveira strain C. posadasii arthroconidia were inoculated into 1 liter of modified converse medium in a 2-liter flask at a concentration of 7 x 106 and incubated for 96 h at 39°C with orbital shaking (180 rpm, 8% CO2). Spherules were harvested by centrifugation at 5,100 rpm for 40 min in a Beckman Allegra centrifuge with a TS-5.1 rotor and washed three times with sterile distilled water. The spherule pellet was resuspended in 7.5 ml sterile water and autoclaved at 15 lb/in2 for 20 min at 121°C. To ensure that spherules were not viable, 0.1 ml of the autoclaved suspension was added to glucose yeast extract plates in duplicate and incubated at room temperature for 2 weeks. No growth was observed, and spherules were considered nonviable. The methods used to generate spherules result in very pure cultures. All arthroconidia differentiate into spherules and no mycelial remnants are present. All manipulations of potentially viable C. posadasii organisms were accomplished under biosafety level 3 conditions in laboratories registered with the CDC for possession of this select agent. All glassware used for spherule preparation was baked prior to use to prevent the introduction of exogenous lipopolysaccharides. The spherule preparation was tested for endotoxin by use of a Limulus amoebocyte lysate assay (Clongen Laboratories, Germantown, MD) and was found to contain less than 20 endotoxin units/μg.
Spherules were stored in 1x sterile phosphate-buffered saline (PBS) at 4°C until use and then diluted into AIM-V medium (Invitrogen, Carlsbad, CA). A total of 1 x 104 spherules/well were added to lymphocyte transformation assay mixtures. For DC maturation studies and spherule antigen presentation proliferation assays, a 1:4 spherule-to-DC ratio was used. In some cases, fluorescein-labeled spherules were used in experiments. Spherules (6 x 107) were suspended in 0.1 M carbonate buffer (pH 9.0) and incubated with FITC (Sigma, St. Louis, MO) at a final concentration of 0.16 mg/ml at 4°C overnight. Spherules were thoroughly washed with PBS and stored at 4°C in the dark until use.
Identification of Coccidioides-immune and nonimmune donors. Isolation of PBMC from healthy individuals was performed with the approval of the Human Subjects Protection Committee at the University of Arizona. Donor immunity status was determined by performing lymphocyte proliferation assays with the coccidioidal antigen preparation T27K, a soluble, aqueous supernatant derived by mechanical disruption of thimerosal-killed coccidioidal spherules followed by centrifugation at 27,000 x g (41). Briefly, 5 x 105 PBMC were added to flat-bottomed wells of 96-well plates in AIM-V medium alone, with 12.5 μg/ml of T27K, or with 10.0 μg/ml phytohemagglutinin (mitogen used as a positive control for cellular proliferation) and incubated in 5% CO2 at 37°C for 5 days. Subsequently, cells were pulsed with 1.0-μCi/well [3H]thymidine (NEN Life Science Products, Perkin-Elmer, Wellesley, MA) for 18 h. Cells were harvested onto Unifilter plates (Perkin-Elmer) by use of a cell harvester (Perkin-Elmer). [3H]thymidine incorporation was measured using a scintillation counter (TopCount; Perkin-Elmer). T27K has been previously demonstrated to specifically distinguish Coccidioides-immune donors from nonimmune donors (11, 12, 33). PBMC from all individuals tested were activated by phytohemagglutinin. A nonimmune donor had a stimulation index of <5-fold cpm greater than that of the control, while an immune donor had an index of 5-fold cpm above that of the control. Lymphocyte transformation assays using spherules (1 x 104 spherules/well) were performed as described above.
Generation of dendritic cells. DC were generated from nonimmune PBMC by a modification of the methods described by Romani et al. (34). Briefly, whole blood was layered on Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden) to obtain PBMC. PBMC were plated in T-75 flasks in AIM-V medium (Life Technologies, Grand Island, NY) and allowed to adhere for 2 h in a 37°C incubator containing 5% CO2. To remove the nonadherent PBMC fraction, flasks were washed several times with sterile PBS. X-Vivo 15 medium (BioWhittaker, Walkersville, MD) supplemented with 1,000 IU/ml GM-CSF and 500 IU/ml IL-4 was added to adherent PBMC, which were cultured for 5 days. Day 5 immature DC were cultured with either tumor necrosis factor alpha (TNF-) (Peprotech, Rocky Hill, NJ) and prostaglandin E2 (PGE2) (Sigma, St. Louis, MO) to induce maturation or with spherules at concentrations determined not to inhibit cell growth or viability (1:4 ratio of spherules to DC).
Confocal microscopy. Immunofluorescence was examined with a Zeiss confocal laser scanning microscope (510 Meta LNO). Immature DC (1 x 106) were cultured with FITC-labeled spherules (5 x 105) for 24 or 72 h. DC were harvested and stained both intracellularly and extracellularly with mouse anti-human HLA-DR-allophycocyanin (APC) (Pharmingen) with a Fix & Perm kit (Caltag). Cells were illuminated with an argon ion multiline laser tuned to 488 nm to excite FITC and with a He/Ne ion laser tuned to 633 nm to excite APC. Standard filters were placed in front of detectors to collect light. Pairs of images were superimposed for colocalization analysis.
Binding assay of FITC-labeled spherules on DC. Immature DC (5 x 105) were incubated with the indicated number of FITC-labeled spherules at 37°C and 4°C in 200 μl sterile PBS. For dose-dependent experiments, DC were mixed with 0, 3.1 x 104, 1.25 x 105, 2.5 x 105, or 5.0 x105 spherules for 1 hour. For time-dependent experiments, DC were mixed with 2.5 x 105 spherules for 1, 2, 4, or 6 h. Cells were immediately analyzed by flow cytometry. A total of 10,000 gated DC events were collected. Spherule binding was quantified as mean fluorescence intensity (MFI). Inhibition of spherule uptake by immature DC (iDC) was performed by pretreating iDC (5 x 105) with 3.0 mg/ml mannan (Sigma Chemical Co., St. Louis, Mo.) for 30 min before the addition of FITC-labeled spherules (2.5 x 105).
Phenotypic analysis of DC. DC were stained with fluorochrome-conjugated antibodies specific for DC markers CD1a, 14, 40, 80, 83, and 86 and HLA-DR in PBS for 30 min at 4°C and fixed in 2% paraformaldehyde prior to flow cytometric analysis (FACScan; Becton Dickinson, Franklin Lakes, NJ). A gate was set on large cells, and 10,000 gated events were collected for analysis.
Cytokine production by DC. A Flex Set cytometric bead array kit (IL-, IL-6, IL-10, IL-12p70, and TNF-; Pharmingen) and an IL-8 enzyme-linked immunosorbent assay kit (Pharmingen) were used to determine cytokine concentrations present in DC supernatant according to the manufacturer's instructions.
Functional DC activity. To assay autologous lymphocyte proliferation, DC (2 x 104 cells/well; iDC, mature DC [mDC], or DC cultured with spherules) were plated in triplicate in 96-well flat-bottom plates in AIM-V medium and irradiated with 50 Gy from a 60Co source. Nonadherent autologous PBMC (2 x 105 cells/well) were added to the DC in a total volume of 200 μl of medium. [3H]thymidine incorporation was assessed as described above.
Statistical analysis. Results for spherule lymphocyte transformation assays were analyzed using the Mann-Whitney U test, with data presented as median (minimum, maximum). Phenotypic analysis of DC surface markers, cytokine production, and DC antigen presentation were analyzed using one-tailed Mann-Whitney U tests. A P value of <0.050 was considered to be statistically significant.
RESULTS
Spherules activate PBMC from Coccidioides-immune donors. Immune and nonimmune donor identification was performed by culturing PBMC with the T27K coccidioidal antigen preparation and measuring [3H]thymidine uptake. When PBMC (5 x 105 cells/well) from both donor groups (five immune donors and six nonimmune donors) were cultured with 1.0 x 104 spherules, there was specific activation of PBMC isolated from immune donors but not from nonimmune donors (Table 1).
DC bind spherules in a dose- and time-dependent manner. Spherules labeled with FITC were incubated with DC at 37°C or at 4°C for 1 hour. This mixture was subjected to flow cytometric analysis to observe cellular fluorescence indicating association of spherules with DC. Spherule binding to DC was greater at 37°C than at 4°C as measured by MFI, suggesting an active binding mechanism (Fig. 1A). DC-spherule association was dose dependent at 37°C, as increasing numbers of spherules resulted in increased MFI. Additionally, spherule binding to DC increased over time (Fig. 1B). When iDC were incubated with mannan (3.0 mg/ml), an inhibitor of the mannose receptor (MR) (35), for 30 min prior to the addition of FITC-labeled spherules, spherule binding was inhibited by 49.01% ± 7.26%. When DC were incubated with zymosan, the ligand for Dectin-1, no inhibition of spherule binding to DC was observed (data not shown).
In addition, we incubated iDC with FITC-labeled spherules for either 24 or 72 h at 37°C. Subsequently, DC were fluorescently labeled with an APC-conjugated HLA-DR-APC antibody. Cells were fixed, permeabilized, and intracellularly labeled with anti-HLA-DR and were then analyzed by confocal microscopy (Fig. 2). We observed that after 24 h of culture, an average of 60% of DC ingested from one to six spherules (Fig. 2A), and that after 72 h, intact spherules could no longer be detected within the DC (Fig. 2B). Moreover, processed spherule antigens colocalize with HLA-DR both within and on the surface of cells, suggesting antigen presentation. When we saturated the system by adding an abundance of FITC-labeled spherules to DC, more than 90% of DC took up spherules. There was a direct correlation between the number of spherules added to DC cultures and the number of DC that take up spherules (as demonstrated in Fig. 1A). Overall, increasing the number of FITC-labeled spherules incubated with DC led to increased fluorescence, and DC were observed to take up more than one spherule.
Spherules induce DC activation. Since we observed spherule-specific PBMC responses from immune donors and the ability of DC to bind, internalize, and process spherules, we investigated the role that spherules may play in the initiation of DC maturation. iDC were generated from PBMC and cultured with spherules (iDC-sph) for 48 h. As shown in Fig. 3A to C, morphological differences were observed between iDC and iDC-sph, with the latter appearing morphologically similar to DC matured with TNF- and PGE2. Frame A depicts characteristic immature DC (smaller and less differentiated than mature DC). DC matured with TNF- and PGE2 are depicted in frame B. Mature DC appear larger than immature DC and have "veiled" structures. No spherules were observed in DC cultures after 48 h (frame C), demonstrating uptake and processing by DC.
DC were harvested and stained with monoclonal antibodies to analyze cell phenotype (Fig. 4). The number of DC cultured with spherules expressing costimulatory molecules (CD40, CD80, and CD86) was significantly increased compared to that of immature DC. The number of cells expressing CD83, a classic maturation marker, and HLA-DR was also significantly increased on iDC-sph compared to that of immature DC. Conversely, the numbers of cells expressing CD1a, an iDC marker, and CD14, a monocyte marker, were decreased for both mature DC and iDC-sph (data not shown). The maturation effect of iDC cultured with spherules was similar to that of classical DC maturation agents, TNF- and PGE2, albeit not to the same degree. In addition, endocytosis of FITC-labeled dextran was diminished in iDC-sph compared to that in iDC (data not shown), and there was an increase in responses in allogeneic mixed lymphocyte reaction assays for iDC-sph (data not shown). Both of these observations demonstrate the maturation status of these cells.
Supernatants from iDC alone or iDC cultured with spherules were harvested after 48 h of culture. We observed an increase in proinflammatory cytokine production by iDC cultured with spherules (Table 2.). Immature DC cultured with spherules for 48 h produced significantly increased amounts of TNF-a, IL-1-, IL-8, and IL-10 compared to iDC cultured without spherules.
DC cultured with spherules process and present spherule antigens to nonimmune PBMC. To determine if iDC-sph could present spherule-associated antigens and prime lymphocytes, DC were used to stimulate autologous nonadherent PBMC from nonimmune individuals. DC-sph induced proliferation of PBMC at levels significantly higher than iDC and mDC (Fig. 5). Nonimmune PBMC were not activated by T27K or spherules in the absence of DC.
DISCUSSION
Human fungal infections have increased over the past decade, resulting in increased morbidity and treatment costs. Elucidation of DC-fungus interaction will aid in understanding fungal virulence and persistence in the host. This report describes that DC are capable of Coccidioides spherule binding, internalization, and presentation of Coccidioides-associated antigens, indicating that fungal capture is important in the initiation of effective antifungal cellular responses.
We generated monocyte-derived DC from healthy, nonimmune individuals for these studies. We have previously published the findings that monocyte-derived DC can be generated from individuals with disseminated disease and that these DC are identical in phenotype and function to DC generated from nonimmune individuals (33). In addition, monocyte-derived DC from immune individuals are identical phenotypically and functionally to nonimmune DC, as these individuals are healthy with no evidence of disease.
Earlier work described the ability of human monocytes to phagocytose and kill Coccidioides arthroconidia, suggesting that an early immune response may be mounted in vivo after infection (5, 9). However, arthroconidia not cleared from the host may differentiate into spherules capable of endosporulating, resulting in the production of a new generation of spherules. If spherules are not effectively cleared from the lungs, this process can continue, thereby increasing fungal burden. This is the first study to describe uptake and processing of C. posadasii spherules by human DC. Immature DC bound spherules within 1 hour; this was followed by spherule internalization within 24 h and spherule processing was detected by 72 h. The ratio of spherules to DC used was 0.5:1, and we observed that not all DC had phagocytosed spherules. We have also saturated the system in which we added an abundance of FITC-labeled spherules to DC; typically, greater than 90% of DC take up spherules. While we do not believe that there are two different DC populations, it is possible that there are. Future experiments using flow cytometry and cell sorting might resolve this issue.
It has been suggested that coccidioidal spherules are too large to be phagocytosed by neutrophils and macrophages (23). However, we demonstrate that DC can readily take up spherules and we have in fact frequently observed multiple spherules inside of human DC. The average size of these spherules was approximately 14 μm. Spherules were mature, as defined by the presence of classical thick cell walls and the development of endospore formation as observed by phase-contrast microscopy. However, the spherules used in our in vitro model are smaller than spherules observed in vivo (average size, 80 to 100 μm). It is possible that these larger spherules would not be susceptible to phagocytosis.
A study by Bozza et al. demonstrated that murine DC internalization of A. fumigatus is mediated by the MR (15). Additionally, mannoproteins derived from C. neoformans bind to mannose receptors and are processed by murine APC (26). Furthermore, blockage of the MR resulted in inhibition of T-cell activation. In our study, we demonstrate that the mannose receptor might also play a role in Coccidioides internalization, as spherule binding was inhibited by mannan. Chieppa et al. showed that cross-linking of the MR on immature DC with an MR-specific antibody for 24 h resulted in DC maturation (18). We pretreated DC with mannan for 30 min prior to the addition of spherules. We did not investigate the effects of mannan treatment on DC but believe that DC maturation did not occur in this short period of time and that spherule uptake was inhibited due to the blocked MR on DC, not due to cellular maturation.
Morphological and phenotypic differences were observed in DC cultured with spherules. Spherule uptake resulted in DC functional maturation, as determined by decreased endocytic capacity and stimulation of allogeneic PBMC activation compared to iDC. The induction of maturation of human DC by fungal agents, including Aspergillus conidia, Candida yeast cells, Malassezia yeast cells, and mannoproteins derived from Cryptococcus, has been described in other studies (16, 17, 30, 31, 36). DC-fungus interactions in these studies have been linked to the recognition of pathogens by pathogen recognition receptors such as DC-SIGN and the mannose receptor. Viriyakosol et al. have demonstrated that both Toll-like receptor 2 (TLR-2) and Dectin-1 are important in the recognition of C. posadasii by use of an in vitro murine macrophage model (39).
We did not empirically define the mechanism of spherule-induced maturation of immature DC. We surmise that maturation is induced in a multifactorial manner. It is highly probable that spherule antigen engagement to C-type lectin receptors and TLRs on DC induces intracellular signaling cascades, resulting in DC maturation and cellular activation. Antigen uptake by immature DC is primarily mediated by C-type lectin receptors (e.g., mannose receptor). Pietrella et al. demonstrated that mannoproteins from C. neoformans promote DC maturation and activation (31). Inhibition of mannoprotein internalization inhibited the upregulation of CD40 and CD80 (classical DC costimulatory molecules). They also showed that treatment of DC with mannoproteins resulted in the degradation of phosphorylated IB (downstream effect is NF-B translocation to the nucleus, resulting in the transcription of genes important in cellular activation). NF-B activation plays a role in proinflammatory cytokine production. We observed TNF- production by DC cultured with spherules. TNF- is a cytokine widely used to mature DC in vitro. Therefore, the combination of antigen recognition and cytokine production likely contributed to DC maturation in our model. While we did not investigate the contribution of TLRs in spherule recognition, pathogen recognition by TLRs on DC also leads to maturation (21, 27). For example, Hertz et al. demonstrated that microbial lipopeptides induced DC phenotypical and functional maturation by TLR-2 interaction and that this maturation could be blocked using an anti-TLR-2 antibody (22).
T-cell activation requires phagocytosis and processing of coccidioidal spherules by DC. DC processed internalized spherules, and these spherule-derived antigens activated autologous PBMC in nonimmune donors. Coculture of lymphocytes and spherules in the absence of DC yielded negligible cellular activation in individuals not previously exposed to Coccidioides. However, the introduction of nonimmune autologous nonadherent PBMC into DC cultures in which the DC had been exposed to spherules for 48 h resulted in cellular activation of PBMC. This has also been described by a C. neoformans study in which DC were highly efficient at yeast engulfment and degradation for presentation to T cells compared to macrophages used as APC (38). This is the first report showing that human DC are able to bind, internalize, process, and present Coccidioides posadasii spherules to nonimmune PBMC, indicating that spherules are an excellent source of antigen.
We do not believe spherule activation of PBMC to be a mitogenic response because the data presented in Table 1 demonstrated that only immune donor PBMC and not nonimmune donor PBMC are activated by spherules. Bulk PBMC were cultured with spherules, and if spherule-derived mitogens were present we would have observed activation of PBMC from nonimmune individuals as well. Only when we cultured lymphocytes together with DC loaded with spherules did we observe nonimmune activation, reinforcing the power of DC antigen presentation.
It is not known if the autoclaving of spherules results in the denaturation of carbohydrates, glycolipids, and glycoproteins such that DC recognize cryptic epitopes not naturally found on spherules. However, we will be addressing this question in subsequent studies by exposing DC to live spherules and arthroconidia.
In conclusion, the results presented here provide insight into the role that immature DC play in the response to infection with Coccidioides. Further characterization of the receptors involved in spherule recognition and binding to DC will help us understand the innate immune response and the pathogenesis of Coccidioides infection.
ACKNOWLEDGMENTS
We thank D. Pappagianis and K. Simmons for providing T27K and K. Orsborn and L. Lewis for spherule preparation.
This work was supported by a grant from the NIH NIAID (IPO1AI061310-01) and grants from the Arizona Biomedical Research Commission #9008 and Merit Review from the Department of Veterans Affairs.
Present address: Center for Innovations in Medicine, The Biodesign Institute, Arizona State University, Tempe, AZ 85287.
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