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The Journal of Immunology, 2006, 177: 459-467.
Copyright © 2006 by The American Association of Immunologists

Pneumocystis Cell Wall beta-Glucans Induce Dendritic Cell Costimulatory Molecule Expression and Inflammatory Activation through a Fas-Fas Ligand Mechanism1

Eva M. Carmona*, Robert Vassallo*, Zvezdana Vuk-Pavlovic*, Joseph E. Standing*, Theodore J. Kottom* and Andrew H. Limper2,*,{dagger}

* Thoracic Diseases Research Unit, Division of Pulmonary Critical Care and Internal Medicine, Department of Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905; and {dagger} Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN 55905


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Respiratory failure during Pneumocystis pneumonia is mainly a consequence of exaggerated inflammatory responses to the organism. Dendritic cells (DCs) are the most potent APCs in the lung and are key to the regulation of innate and adaptive immune responses. However, their participation in the inflammatory response directed against Pneumocystis infection has not been fully elucidated. Therefore, we studied the role of Pneumocystis carinii, as well as Saccharomyces cerevisiae, cell wall-derived beta-glucans, in DC costimulatory molecule expression. We further studied the impact of beta-glucans on subsequent T cell activation. Because cytokine secretion by DCs has recently been shown to be regulated by Fas ligand (FasL), its role in beta-glucan activation of DCs was also investigated. beta-Glucan-induced DC activation occurred in part through dectin-1 receptors. We demonstrated that DC activation by beta-glucans elicits T cell activation and polarization into a Th1 patterned response, but with the conspicuous absence of IL-12. These observations differed from LPS-driven T cell polarization, suggesting that beta-glucans and LPS signal DC activation through different mechanisms. We additionally determined that IL-1beta and TNF-{alpha} secretion by beta-glucan-stimulated DCs was partially regulated by Fas-FasL. This suggests that dysregulation of FasL could further enhance exuberant and prolonged cytokine production by DCs following DC-T cell interactions, further promoting lung inflammation typical of Pneumocystis pneumonia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Powerful and well-orchestrated immune responses are absolutely essential for the host to eradicate infections. With increasing numbers of immune suppressed patients, many once uncommon infections have emerged as significant threats to these populations. Among these, fungal pathogens now represent a serious concern to both immune suppressed individuals and other patient groups (1). Of particular interest, Pneumocystis species cause life-threatening pneumonia in susceptible hosts, with lung damage largely resulting from exaggerated lung inflammation secondary to infection, rather than from the pathogen itself (2, 3, 4). Our recent investigations have shown that the beta-glucan cell wall components of Pneumocystis are chiefly responsible for initiation of lung inflammation during this infection (5, 6).

Dendritic cells (DCs)3 are the most potent APCs present in lung. Pulmonary DCs are located near the portals of entry of the microorganisms, in contact with either alveolar or airway epithelial cells. These privileged locations position DCs to be one of the first cells to contact many inhaled microorganisms. Indeed, it has been shown that during Aspergillus infection, pulmonary DCs transport the organisms from the airways to the draining lymph nodes, where they trigger specific T cell responses. An adequate T cell immune response is subsequently necessary for the host to clear the infection. But, in the particular case of Pneumocystis, the exaggerated inflammatory response also causes substantial alveolar damage and respiratory failure.

CD4+ T lymphocytes are essential for effective host defense against Pneumocystis, functioning as memory cells that will recruit macrophages and monocytes to the site of infection (7). Mice with SCID suffer fatal Pneumocystis infections, despite the presence of functional neutrophils and macrophages (3, 8). In contrast, reconstitution of the immune system in lymphocyte-deficient mice results in a very intense T cell-mediated immune response, with attendant lung damage and impaired oxygen exchange (9, 10). Therefore, adequate regulation of T cell activation is required to eliminate Pneumocystis infection and still prevent exuberant lung damage. Because DCs are key regulators of both innate and adaptive immune activation, and because optimal T cell activation requires interactions with matured DCs, it is likely that the initiation and control of essential immune responses during Pneumocystis are critically mediated by DCs.

Accordingly, the present investigation was undertaken to determine the importance of DCs as initiators of the inflammatory responses following challenge with beta-glucans derived from Pneumocystis carinii or Saccharomyces cerevisiae. Recent reports have demonstrated the importance of Fas ligand (FasL) as a mechanism of DC activation, as well as participating in the regulation of IL-1beta production, not only by DCs, but also by macrophages. Therefore, we further sought to elucidate the role of FasL as a potential regulator of cytokine secretion by beta-glucan-challenged DCs. Because dysregulation of Fas-FasL interactions results in exuberant immune responses, such as during autoimmune diseases, we postulated that Fas-FasL mechanisms on DCs might contribute to the exaggerated inflammation observed during Pneumocystis infection.

In this study, we demonstrate that purified cell wall beta-glucans from Pneumocystis and Saccharomyces induce DC costimulatory molecule expression and cytokine production that further stimulate T cell activation and polarize these cells toward Th1 patterned responses. Although similar to LPS in inducing DC costimulatory molecule expression, fungal beta-glucans differed in their induced cytokine release patterns. In contrast with LPS, IL-1beta and TNF-{alpha} were found to be the major cytokines secreted by DCs challenged with beta-glucan, instead of the classic Th1-polarizing agent, IL-12. Moreover, beta-glucan stimulated expression of Fas by DCs, and subsequent inhibition of FasL impaired IL-1beta and TNF-{alpha} secretion during DC-T cell interactions in the presence of Pneumocystis beta-glucans. Thus, Fas-FasL interactions represent a novel mechanism mediating beta-glucan activation of DCs and DC-T cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Endotoxin-free buffers and reagents were scrupulously used in all experiments. LPS from Escherichia coli 026:B6, beta-glucan from S. cerevisiae, and other general reagents were obtained from Sigma-Aldrich, unless specified otherwise. P. carinii were derived originally from the American Type Culture Collection stock and have been subsequently passaged in our immunosuppressed rat colony (11).

Generation of a Pneumocystis glucan-rich cell wall isolate

The Mayo Institutional Animal Care and Usage Committee approved all animal experimentation. A glucan-rich cell wall fraction from Pneumocystis was prepared, as previously described (5, 12). Pneumocystis pneumonia was induced in dexamethasone-treated immunosuppressed Long-Evans rats (Harlan) (11). Pneumocystis organisms were isolated from lungs of heavily infected animals by homogenization and filtration through 10-µm filters. The organisms were autoclaved (120°C, 20 min) and disrupted by ultrasonication (200 W for 3 min, six times), and the glucans were isolated by NaOH digestion and lipid extraction, as previously detailed (5). The final product contained predominantly carbohydrate (95.7%) and released 82% of its content as D-glucose following hydrolysis (5). Extensive measures were used to ensure that the beta-glucan preparations were free of endotoxin. Before use in culture, the Pneumocystis cell wall fractions were washed with 0.1% SDS and then vigorously washed with distilled physiological saline to remove the detergent. The final preparations were assayed for soluble endotoxin with the Limulus amebocyte lysate assay method and found to consistently contain <0.125 U of endotoxin (5). Our prior studies have further demonstrated that host cell responses to Pneumocystis beta-glucans occur through mechanisms distinct from those induced by LPS (6). All Pneumocystis beta-glucan preparations were analyzed for their proinflammatory signaling capacities. The maximal stimulatory capacity of each Pneumocystis beta-glucan isolate varied somewhat from preparation to preparation. However, they all consistently stimulated monocytoid cell inflammatory markers such as TNF-{alpha} to a considerable degree.

Dendritic cells

Human blood mononuclear cells were isolated from buffy coats by Ficoll-Hystopaque density gradient centrifugation. Monocytes were purified from human PBMC using RosetteSep Monocyte Enrichment (StemCell Technologies), according to the manufacturer’s recommendations. Purity was confirmed by staining with FITC-conjugated anti-CD14 Ab (Sigma-Aldrich) and flow cytometry (FACScan; BD Immunocytometry Systems) and was routinely found to be >94%. Immature DCs were obtained by incubating monocytes at 2 x 106 cells/ml in RPMI 1640 medium supplemented with 5% heat-inactivated FBS (Invitrogen Life Technologies), 100 U/ml penicillin/streptomycin, and 2 mM L-glutamine and human rGM-CSF (800 IU/ml) (Berlex) and human rIL-4 (5 ng/ml) (R&D Systems) for 7–8 days. Fresh complete medium was replaced every 3 days. For some experiments, cells were left untreated or incubated with anti-human dectin-1 Ab or normal goat IgG as a control (R&D Systems) for 45 min before stimulation with beta-glucan.

T cells

Human peripheral blood T cells were isolated from buffy coats by Ficoll-Hystopaque density gradient centrifugation. CD3+ cells were purified by negative selection using RosetteSep T Cell Enrichment and RosetteSep (StemCell Technologies), according to the manufacturer’s recommendations. Purity was verified by staining with a FITC-conjugated anti-CD3 Ab (BD Pharmingen) and flow cytometry and was typically >97%.

Analysis of surface Ags

Flow cytometry analysis for surface staining was performed on DC suspensions (1 x 106 DCs/ml). Cells were washed with PBS containing 2% FCS, followed by a 30-min incubation at 4°C with human IgG to block Fc receptors. After washing, the cells were incubated for 30 min at 4°C with 1 µg of CD11c PE, HLA class II FITC, CD40 FITC, CD80 FITC, CD86 FITC (BD Pharmingen), and CCR7 FITC (R&D Systems) or mouse IgG1 PE isotype (BD Pharmingen). The DCs were then washed and fixed in 2% paraformaldehyde. Flow cytometry was performed and analyzed with CellQuest software (BD Biosciences). All experiments were performed in duplicate or triplicate and were repeated at least three times.

Cytokine detection

Cytokine production (IL-1beta, IL-2, IL-4, IFN-{gamma}, TNF-{alpha}, IL-10, IL-12 p70, and IL-12 p40) was measured in the medium of DC or T cell cultures by ELISA (IL-2, IL-4, IFN-{gamma}, TNF-{alpha}, IL-10, IL-12 p70 (Ready-Set-Go; BD Pharmingen); IL-1beta, IL-12 p40 (OptEIA; BD Biosciences)). DCs were stimulated in 5% FCS medium with either LPS (10 ng/ml to 10 µg/ml), S. cerevisiae beta-glucans (2.5 x 106 particles/ml), or Pneumocystis beta-glucans (2.5 x 106 particles/ml) in six-well plates at 2 x 106 cells/ml over various time periods (8–72 h).

In vitro T cell differentiation with allogeneic DCs

CD3+ T cells were cultured with immature DCs or with LPS, Pneumocystis beta-glucan, or Saccharomyces beta-glucan-stimulated DCs (DC:T cell ratios of 1:5, 1:10, or 1:20) for various periods of time (2–6 days) in 96-well plates using RPMI 1640 medium with 5% FBS. DCs were washed three times with fresh medium before incubation with T cells. For some experiments, T cells were preincubated for 1 h with Fas-FasL inhibitor (Kp7-6; Calbiochem), or with anti-human IL-1beta (R&D Systems) before culture with the DCs.

T cell proliferation assay

T cells were cultured with immature DCs or with either LPS, Pneumocystis beta-glucan, or Saccharomyces beta-glucan-stimulated DCs for 48 h. Cell proliferation was determined by BrdU incorporation (Amersham Biosciences).

Cell viability

Under each experimental condition, cell viability was confirmed using the XTT Assay Kit II (Roche Molecular Biochemicals). This system measures the conversion of sodium-3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzenesulfonic acid hydrate (XTT) to a formazan dye through electron coupling in metabolically active mitochondria using the coupling reagent N-methyldibenzopyrazine methyl sulfate. Only metabolically active cells are capable of mediating this reaction, which is detected by absorbance of the dye at 450–500 nm. All conditions were considered to exhibit adequate viability if assay measures were at least 80% or greater compared with controls.

Cell extract preparation and immunoblotting

To obtain total cellular proteins, cells were washed with cold PBS, resuspended in a modified whole cell extract phosphated Dulbecco’s buffer (40 mM Tris-HCl (pH 8), 0.3 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 10 mM NaF, 10 mM PNPP, 10 mM beta-glycerophosphate, 300 µM sodium orthovanadate, 1 mM DTT, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin), and centrifuged at 12,000 x g for 15 min at 4°C (13). The resultant supernatant contained total cellular protein. The amount of cellular protein present in the clarified supernatant was determined using the Bio-Rad protein assay (Bio-Rad).

For immunoblot assays, equal amounts of whole cell extract were loaded and separated by 10% SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Immunoblotting was performed with specific Abs and visualized by using the ECL Western blotting detection kit (Amersham Biosciences).

RT-PCR for detection of IL-12 p35 mRNA

DCs were left unstimulated or stimulated with LPS (1 µg/ml), Pneumocystis beta-glucan (2.5 x 106 particles/ml), or Saccharomyces beta-glucan (2.5 x 106 particles/ml) for a period of 4 h. Reverse transcription was performed as follows. Total RNA was isolated with an RNeasy mini kit (Qiagen). During isolation, all samples were digested with DNase I to remove possible contaminating genomic DNA. A 20-µl first-strand cDNA synthesis reaction was performed using SuperScript III (Invitrogen Life Technologies), and 2.0 µl of cDNA was used in a 50 µl PCR. The following primers were used in amplification: human IL-12 p35 sense primer, GAT GAG CTG ATG CAG GCC; antisense primer, AAG CAT TCA GAT AGC TCA TCA. As a loading control, the housekeeping gene hypoxanthine phosphoribosyltransferase was also amplified, as described previously (14). For both cDNA amplifications, conditions consisted of an initial 2-min hot start at 94°C, followed by 30 cycles at 94°C for 60 s, 55°C for 60 s, and 72°C for 60 s, and a final 10-min extension at 72°C. PCR amplicons were run on a 3% agarose gel and photographed.

Statistical analysis

All data are expressed as mean ± SD, unless otherwise noted. Differences between experimental groups were determined using two-tailed Student’s t tests for normally distributed variables, and nonparametric data were analyzed using the Mann-Whitney U test using the StatView statistical software (SAS Institute). Statistical differences between groups were considered significant if p was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Pneumocystis cell wall beta-glucans induce costimulatory molecule expression in human DCs

We first investigated the ability of fungal beta-glucans derived from both Pneumocystis and Saccharomyces to induce costimulatory molecule expression in human primary DCs. DC costimulatory molecule expression was compared with that occurring under the influence of the bacterial cell wall component, LPS, a well-known stimulant of DC costimulatory molecules. Peripheral blood monocytes were isolated and cultured in the presence of IL-4 and GM-CSF for a period of 7 days before challenge with the microbial cell wall components. Phenotypic identification and costimulatory molecule expression status of the DCs were determined by flow cytometry based on the expression of specific HLA class II and CD11c surface markers. We observed that the majority of stimulated DCs exhibited abundant surface expression for HLA class II and CD11c (Fig. 1A). We further studied the expression of costimulatory molecules, including CD40, CD80, and CD86 in beta-glucan or LPS-challenged DC. Expression of CD80, CD86, and CD40 increased upon stimulation with either Pneumocystis or Saccharomyces beta-glucans and with LPS when compared with nontreated cells (Fig. 1A). Hence, fungal cell wall beta-glucans induce DC costimulatory molecule expression in a manner parallel to that induced by the bacterial cell wall component LPS.


Figure 1
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  		FIGURE 1. Human DCs stimulated with Pneumocystis beta-glucan or LPS express HLA-II, costimulatory molecules, and chemokine receptors on their surfaces. Primary human DCs were either left untreated (NT) or treated for 18 h with either Pneumocystis beta-glucan (PC beta-glucan), Saccharomyces beta-glucan (SC beta-glucan), or LPS. A, Surface expression of HLA-II, CD11c, CD80, CD86, and CD40 was measured by flow cytometry. B, Human DCs were also analyzed for the lymph node homing chemokine receptor CCR7 after stimulation with beta-glucan or LPS, as described above. The percentage of positive expressing cells is displayed in the upper left corner of the grid. Results are representative of four independent experiments.

 
An essential aspect of DC activation program involves changes in the migratory capacity of these cells. Maturing DC migrate from inflamed tissues to draining lymph nodes, where they interact with naive T cells. The mechanisms responsible for this change in migratory capacity involve a complete switch in the expression of chemokine receptors, particularly CCR7. Therefore, we measured the expression of CCR7 as an additional marker of DC activation after Pneumocystis or Saccharomyces beta-glucan exposure. As shown in Fig. 1B, beta-glucan-stimulated cells, as well as LPS-matured DCs exhibited up-regulated CCR7 expression when compared with nonstimulated cells. Thus, both fungal beta-glucans and LPS-challenged DCs increased the expression of chemokine receptors needed for lymph node migration during the DC maturation process.

Pneumocystis beta-glucan-challenged DCs secrete a unique cytokine profile compared with LPS

To further characterize the functional activation of DC cultured in the presence of Pneumocystis beta-glucan, we next investigated the cytokine secretion induced by this fungal cell wall component and compared it with LPS. We first analyzed the secretion of IL-12, a proinflammatory cytokine frequently released by DCs and phagocytes in response to pathogens (5, 15, 16). Interestingly, despite our observations that both Pneumocystis and Saccharomyces beta-glucan induced costimulatory molecule expression in a manner highly similar to LPS, the production of total IL-12 (IL-12 p70) by DCs after exposure to these fungal cell wall preparations was not stimulated by the fungal isolates (Fig. 2A). In contrast, LPS was observed to markedly induce secretion of IL-12 p70. To exclude the possibility of delayed DC response to Pneumocystis and Saccharomyces beta-glucan, we exposed the cells for up to 72 h to those agents, but still did not observe any discernable IL-12 p70 secretion (Fig. 2B).


Figure 2
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  		FIGURE 2. DCs activated with fungal beta-glucans do not produce IL-12 compared with LPS-stimulated DCs. A, DC IL-12 production was measured by ELISA after 18-h incubation with either Pneumocystis beta-glucan (PC beta-glucan), Saccharomyces beta-glucan (SC beta-glucan), or LPS at the indicated concentrations. B, Time course of total IL-12 release following stimulation with the microbial cell wall components. Total IL-12 (IL-12 p70) was determined by ELISA from the culture supernatant of DCs after exposure to either PC beta-glucan (10 x 106 particles/ml), Saccharomyces beta-glucan (10 x 106 particles/ml), or LPS (1 µg/ml) stimulation over the indicated period of time. C, Next, the IL-12 p40 subunit was measured by ELISA from the same supernatants described above. The data are expressed as the mean ± SD of a representative experiment typical of four separate experimental runs conducted with different DC preparations. D, RNA was isolated from either nonstimulated or DCs stimulated with LPS, or either Pneumocystis or Saccharomyces beta-glucans for 4 h. Total RNA was subjected to RT-PCR to measure IL-12 p35 mRNA expression. Hypoxanthine phosphoribosyltransferase mRNA was also analyzed as a loading control.

 
IL-12 is a heterodimer consisting of an L chain of ~35 kDa (p35) and an H chain of 40 kDa (p40) (17, 18). Although the p35 subunit is retained in cells and secreted only when associated with the p40 chain, p40 may be secreted independently in significant quantities (17, 18). To further assess whether the defect in IL-12 p70 production was due to a lack of one of these subunits, we analyzed the secretion of p40 in response to Pneumocystis and Saccharomyces beta-glucans (Fig. 2C). Of note, both Pneumocystis and Saccharomyces beta-glucan-treated DCs secreted substantial amounts of p40 in a fashion parallel to LPS stimulation. Based on these observations, we postulated that the lack of IL-12 secretion from DCs challenged with fungal beta-glucans was most likely due to defective expression of the p35 subunit. To confirm this, IL-12 p35 mRNA expression was analyzed in LPS and beta-glucan-stimulated DCs. As demonstrated in Fig. 2D, both Pneumocystis and Saccharomyces beta-glucans fail to induce IL-12 p35 mRNA when compared with LPS. We, therefore, conclude that the absence of total IL-12 secretion seen in beta-glucan-activated DCs was related to impaired expression of the p35 subunit.

We further analyzed whether Pneumocystis beta-glucan-challenged DCs were impaired in the production of other cytokines following activation. Specifically, TNF-{alpha}, IL-10, and IL-1beta were also measured in the medium following stimulation with these various microbial cell wall components (Fig. 3). In contrast to IL-12, abundant secretion of TNF-{alpha} and IL-10 was detected in DCs challenged with either Pneumocystis or Saccharomyces beta-glucans, in a manner parallel to LPS-treated cells. However, we observed that Pneumocystis and Saccharomyces beta-glucan-challenged DC induced greater amounts of IL-1beta compared with LPS-stimulated cells (Fig. 4). Taken together, these data indicate that Pneumocystis and Saccharomyces beta-glucan and LPS induced contrasting patterns of cytokine expression by DCs. Although TNF-{alpha} and IL-10 secretion levels were similar, the bacterial and fungal cell wall components differed in their ability to promote secretion of other cytokines, particularly IL-12 and IL-1beta. Although LPS-challenged DCs secreted substantial amounts of IL-12, Pneumocystis and Saccharomyces glucan-stimulated DCs produced greater amounts of IL-1beta.


Figure 3
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  		FIGURE 3. Pneumocystis beta-glucan or LPS stimulates DCs to produce substantial quantities of TNF-{alpha} and IL-10. A, TNF-{alpha} and IL-10 production was measured by ELISA after 18-h incubation with either Pneumocystis beta-glucan (PC beta-glucan), Saccharomyces beta-glucan (SC beta-glucan), or LPS at the indicated concentrations. B, We further determined the kinetics of cytokine release in response to these microbial cell wall components. TNF-{alpha} and IL-10 were evaluated by ELISA from the culture supernatant of DCs after Pneumocystis beta-glucan (10 x 106 particles/ml), Saccharomyces beta-glucan (10 x 106 particles/ml), or LPS (1 µg/ml) stimulation over the indicated time periods. The data are shown as the mean ± SD of a representative experiment typical of four separate experimental runs conducted with different DC preparations.

 

Figure 4
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  		FIGURE 4. Fungal beta-glucans, but not LPS-stimulated DCs produce abundant IL-1beta. Primary human DCs were left untreated or treated with either Pneumocystis beta-glucan (PC beta-glucan) (10 x 106 particles/ml), Saccharomyces beta-glucan (SC beta-glucan) (10 x 106 particles/ml), or LPS (1 µg/ml) for the indicated periods of time. IL-1beta secretion from the DCs was evaluated by ELISA. The data are shown as the mean ± SD of a representative experiment typical of four separate experimental runs conducted with different DC preparations.

 
We also observed that even in the absence of IL-12, T cells secrete large amounts of IFN-{gamma} when cocultured with beta-glucan-primed DCs. Additionally, beta-glucan-activated DCs were observed to secrete large amounts of IL-1beta. We, therefore, sought to investigate whether IL-1beta secretion would be responsible for the induction of IFN-{gamma} observed in T cells cocultured with beta-glucan-primed DCs. To address this, neutralizing anti-IL-1beta was added to T cells before culture with beta-glucan-primed DCs in an attempt to block IL-1beta and subsequent IFN-{gamma} production by T cells. As expected, T cells that were pretreated with anti-IL-1beta resulted in decreased IFN-{gamma} secretion when compared with nontreated cells (Fig. 5). Therefore, we conclude that in the absence of IL-12, secretion of IL-1beta is at least partially responsible for IFN-{gamma} secretion and the related Th1 response.


Figure 5
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  		FIGURE 5. IL-1beta partially mediates IFN-{gamma} secretion by T cells cocultured with beta-glucan-stimulated DCs. Primary CD3+ cells were preincubated with or without anti-IL-1beta Ab (20 µg/ml) for 1 h before coculture with DCs. DCs were either left untreated or treated with either Pneumocystis beta-glucan (PC beta-glucan) or Saccharomyces beta-glucan (SC beta-glucan) for 18 h before coculture with T cells. IFN-{gamma} secretion was measured by ELISA. (*, Denotes p < 0.05 comparing IFN-{gamma} secretion in the presence or absence of anti-IL-1beta Ab.)

 
Pneumocystis beta-glucans stimulate cytokine generation in part through ligation of dectin-1 receptors

We next sought to determine receptor mechanisms used by DCs to respond to Pneumocystis beta-glucans. Dectin-1 receptors are expressed on human DCs, acting as major beta-glucan receptors on these cells (19). Our group has recently shown that dectin-1 is necessary for both efficient internalization of beta-glucan particles by macrophages, as well as cytokine release in response to this fungal cell wall component (20). We, therefore, studied the role of dectin-1 receptors in cytokine secretion by DCs after stimulation with Pneumocystis beta-glucan. DCs were incubated with a human anti-dectin-1 Ab or nonimmune isotype control Ig before challenge with the fungal cell wall isolate (Fig. 6). The presence of anti-dectin-1 Ab, but not the isotype control, significantly impaired TNF-{alpha} secretion from DCs stimulated with Pneumocystis beta-glucan. These data indicate that dectin-1 receptors mediate, at least partially, beta-glucan-induced cytokine secretion from DCs. We also conclude that beta-glucan rather than other components present in these cell wall preparations is responsible for cytokine secretion by DCs.


Figure 6
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  		FIGURE 6. Inhibition of dectin-1 receptors results in impaired TNF-{alpha} secretion by DCs. DCs were left untreated (NT) or preincubated with different concentrations of human anti-dectin-1 Ab or isotype control Ig for 1 h before stimulation with Pneumocystis beta-glucan (10 x 106 particles/ml). TNF-{alpha} was measured by ELISA in the supernatant of the cells after 18 h of subsequent incubation. (*, Denotes p < 0.05 comparing TNF-{alpha} secretion in the presence or absence of anti-dectin-1 Ab.)

 
Pneumocystis beta-glucan-stimulated DCs induce proliferation of T cells and IFN-{gamma} production in the absence of IL-12

Effective T cell responses are necessary for the elimination of Pneumocystis pneumonia (21). Mature DCs migrate from peripheral tissues to draining lymph nodes, where they interact with naive T cells, inducing T cell proliferation and differentiation. Mature DCs are primed by pathogens to provide T cells with specific signals required to promote protective T cell responses. For instance, LPS-challenged DCs produce an environment rich of IL-12 that induces an efficient Th1 response with increased IFN-{gamma} production by T cells. In contrast, lack of IL-12 production generally heralds a switch to a prototypic Th2 response with associated T cell production of IL-4 (22). To further understand the functional impact of Pneumocystis beta-glucans on mature DC activation of T cells, we next studied proliferation and polarization of T cells primed with Pneumocystis and Saccharomyces beta-glucan-stimulated DCs. Cell proliferation was analyzed by BrdU incorporation and IL-2 production. Our results demonstrated that beta-glucan-stimulated DCs induced T cell proliferation and a moderate increase in IL-2 secretion, when compared with controls (Fig. 7), indicating that T cells were activated by beta-glucan-primed DCs.


Figure 7
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  		FIGURE 7. DCs challenged with Pneumocystis beta-glucans induce proliferation of T cells. A, T cell proliferative responses were measured in the presence or absence of DCs stimulated with either Pneumocystis beta-glucan (PC beta-glucan) (10 x 106 particles/ml) or Saccharomyces beta-glucan (SC beta-glucan) (10 x 106 particles/ml). Naive T cells were incubated with DCs at the indicated ratios, which had been primed with the beta-glucans for 18 h before the cells were cocultured. T cell proliferation was measured by BrdU incorporation after 5 days. (*, Denotes p < 0.05 comparing T lymphocyte proliferation in the presence of beta-glucan-matured DCs with those cultured with nontreated DCs (NT-DCs).) B, IL-2 levels were further measured in the culture supernatants by ELISA following 24 h of these mixed lymphocyte cultures. (*, Denotes p < 0.05.) The data are shown as the mean ± SD of a representative experiment typical of three separate experimental runs conducted with different DC preparations.

 
To gain further insight into the type of T cell responses generated by beta-glucan-primed DCs, IFN-{gamma} and IL-4 were also measured in the supernatant of the cells after cognate DC-T cell interactions. T cell responses to beta-glucan-primed DCs were compared with those of LPS-primed DCs. Surprisingly, in our model, beta-glucan-polarized T cells did not secrete detectable levels of IL-4, even in an environment free of IL-12 (Fig. 8A). In contrast, they produced large amounts of IFN-{gamma}, comparable to that produced by LPS-polarized T cells (Fig. 8B). Naive T cells were also directly exposed to beta-glucan particles in the absence of DC to rule out the possibility of direct beta-glucan T cell activation and were shown not to induce IL-4 (Fig. 8A) or IFN-{gamma} production in the absence of DCs (Fig. 8B).


Figure 8
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  		FIGURE 8. Human DCs challenged with Pneumocystis beta-glucan polarize T cells toward a Th1 response. Human DCs were stimulated with Pneumocystis beta-glucan (PC beta-glucan) (10 x 106 particles/ml), Saccharomyces beta-glucan (SC beta-glucan) (10 x 106 particles/ml), or LPS (1 µg/ml) for 18 h, and subsequently cultured with naive T cells at a ratio of 1:5 over the indicated time periods. IL-4 (A) and IFN-{gamma} (B) were measured in the culture medium by ELISA. As an additional control, Saccharomyces beta-glucan was also added to naive T cell cultures in the absence of DCs. The data are shown as the mean ± SD of a representative experiment typical of three separate experimental runs conducted with different DC preparations.

 
Pneumocystis beta-glucans promote DC expression of Fas and related molecules

Although Fas expression induces apoptotic signals in many cell types, there is recent evidence that Fas-induced signaling leads to proliferation and differentiation of DCs. This is due to coordinate expression of FLIP (23). In addition, it has been further recognized that Fas-FasL interactions also partially regulate production of IL-1beta by DCs. Because IL-1beta represents one of the major cytokines secreted by DCs after challenge with Pneumocystis beta-glucans, we further studied the potential involvement of Fas-FasL interactions in the activation of DCs. To address this, the surface expression of Fas (CD95) was evaluated in DCs exposed to fungal beta-glucans (Fig. 9A). Even though DCs constitutively express Fas, incubation with either Pneumocystis or Saccharomyces beta-glucan for 48 h induced enhanced surface expression of this receptor. DCs were also found to be inherently resistant to Fas-induced apoptosis due to expression of FLIP. DC viability was not altered in fungal beta-glucan-treated cells over the course of these cultures (data not shown). Consistent with that observation, we further demonstrated that after 24 h of incubation with fungal beta-glucans, FLIP expression was coordinately increased compared with nontreated cells (Fig. 9B). Thus, fungal beta-glucans induce expression of Fas and the Fas-related protein FLIP in DCs.


Figure 9
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  		FIGURE 9. Pneumocystis beta-glucans induce the expression of Fas and the anti-apoptotic factor FLIP by human DCs. A, Human DCs were left untreated or pulsed with either Pneumocystis (PC beta-glucan) or Saccharomyces beta-glucan (SC beta-glucan), as indicated in the figure, over various time periods. Fas surface expression was evaluated by flow cytometry. The percentage of positive expressing cells is displayed in the upper left corner of the grid. Results are representative of three independent experiments. B, Whole cell extracts were isolated from DCs either left untreated or incubated with Pneumocystis-derived beta-glucan (10 x 106 particles/ml) or with FasL (500 ng/ml) for 24 h. FLIP expression was analyzed by immunoblot.

 
Inhibition of Fas-FasL binding suppresses IL-1beta and TNF-{alpha} production during DC-T cell interactions

Having observed enhanced Fas expression on DCs challenged with either Pneumocystis or Saccharomyces beta-glucans, we next investigated the cytokine secretion patterns produced during DC-T cell interactions in the presence of soluble FasL inhibitor. DCs were challenged with either Pneumocystis or Saccharomyces beta-glucans and subsequently cultured with T cells over various time periods. The T cells were preincubated with FasL inhibitor before addition to DC cultures. IL-1beta production as well as TNF-{alpha} secretion by DCs were significantly, though partially, inhibited after 72 h of DC-T cell interaction in the presence of the FasL inhibitor (Fig. 10, A and B). To exclude the possibility that the impairment of cytokine production was due to DC death from inhibitor toxicity, cell viability was measured during DC-T cell interaction in the presence or absence of FasL inhibitor. Cell viability was not impaired by the FasL inhibitor (data not shown).


Figure 10
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  		FIGURE 10. Inhibition of Fas-FasL suppresses secretion of IL-1beta, TNF-{alpha}, and IFN-{gamma} during DC-T cell interactions. Human DCs were stimulated with Pneumocystis beta-glucan (PC beta-glucan) (10 x 106 particles/ml) or Saccharomyces beta-glucan (SC beta-glucan) (10 x 106 particles/ml) for 18 h before being cultured with naive T cells at a ratio of 1:5 for the indicated periods of time. T cells were left untreated or treated with the indicated concentrations of FasL inhibitor for 1 h before and throughout the subsequent coculture with the primed DCs. IL-1beta (A), TNF-{alpha} (B), and IFN-{gamma} (C) were measured by ELISA in the culture medium. (*, Denotes p < 0.05 comparing the presence or absence of inhibitor at the indicated concentrations.) The data are shown as the mean ± SD of a representative experiment typical of three separate experimental runs.

 
We further investigated whether disruption of Fas-FasL interactions would also alter IFN-{gamma} production by T cells. Cocultures of T cells with Pneumocystis or Saccharomyces beta-glucan-activated DC were further analyzed for IFN-{gamma} secretion in the presence or absence of FasL inhibitor (Fig. 10C). Significant reductions of this cytokine were also observed in the presence of the Fas-FasL inhibitor when compared with nontreated cells, suggesting that Fas-FasL interactions mediate this response. In addition, we observed that stimulating DCs directly with FasL also resulted in inflammatory activation of DCs. DCs stimulated with 500 ng/ml FasL release 269 ± 42 pg/ml TNF-{alpha} compared with unstimulated DCs, which released 35 ± 12 pg/ml (p = 0.0007). Taken together, these studies suggest that Fas-FasL interactions in part regulate the secretion of DC-derived cytokines, including TNF-{alpha} and IL-1beta during DC-T cell interactions in the presence of fungal beta-glucans. In addition, IFN-{gamma} generation is also modulated by Fas-FasL mechanisms during challenge with Pneumocystis beta-glucans, suggesting this also is an important mechanism in the control of T cell-derived cytokines during this infection.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Pneumocystis and related fungal pathogens have emerged as significant threats to the ever-growing number of individuals with impaired lymphocytic immunity, including HIV and malignancies (4). Relatively little is known about innate and acquired immunity to these fungal species. In contrast, a great deal has been discovered about the bacterial cell wall component LPS and its ability to stimulate inflammation and induce the maturation of DCs. In the current study, we analyzed the ability of beta-glucans, carbohydrate polymeric components of fungal cell walls, to induce costimulatory molecule expression and activation of human DCs. Our study demonstrates that, like LPS, fungal beta-glucans derived from P. carinii and S. cerevisiae induce costimulatory molecule expression on DCs, with polarized T lymphocyte activation. DC costimulatory molecule expression is also associated with the expression of the CCR7 lymphocyte homing receptor on the surface of DCs. However, unlike LPS, the fungal cell wall beta-glucans appear to polarize T cell activation toward a Th1 patterned response, in the absence of IL-12. Our studies further indicate that DC activation by beta-glucan occurs through dectin-1 receptors and is partially regulated by interactions of Fas-FasL.

DC activation is associated with cytokine generation and expression of defined surface markers, with acquisition of the ability of DCs to stimulate effective T cell responses. In the case of fungal beta-glucans, the resulting cytokine profile appears to polarize generally toward a Th1 pattern. However, IL-12 was conspicuously lacking, due to defective generation of the p35 subunit of this cytokine. This observation is parallel to prior studies demonstrating that impaired production of IL-12 p70 in response to Mycobacterium tuberculosis is related to a defective expression of p35 rather than the p40 component of this cytokine (24). Interestingly, earlier studies of cytokine responses to yeast zymosan, a cell wall fraction containing both beta-glucan and mannan components, have reported IL-12 secretion by DCs. In contrast to that earlier study, both the purified fungal beta-glucans in our current study and bacterial peptidoglycan in that earlier report failed to generate this Th1-associated cytokine (25). Thus, microbial surface components vary in their ability to elicit specific key cytokines, which will impact host responses to these various microbial pathogens.

Our study also demonstrates for the first time that beta-glucan, a major component of the Pneumocystis cell wall, stimulates Fas expression by DCs. This induction of Fas is accompanied by coordinate expression of FLIP, rendering the DCs resistant to FAS-induced apoptosis (23). Furthermore, we observed that FasL inhibitor significantly blunted both TNF-{alpha} and IL-1beta expression in response to beta-glucans. These observations are consistent with prior studies indicating that Fas engagement induces DC maturation and expression of IL-1beta during interaction of DC and T lymphocytes in the absence of IL-12. Thus, Fas expression by DCs in response to the Pneumocystis cell wall may represent an additional mechanism that promotes and prolongs lung inflammation in response to this organism.

One might initially speculate that the up-regulation of Fas may present a mechanism of immune evasion for Pneumocystis, as its induction by Pneumocystis components may render DCs susceptible to apoptosis upon interaction with activated T cells expressing FasL. However, we observed that induction of Fas is accompanied by coordinate expression of FLIP, and beta-glucans further did not appear to alter the viability of DCs in our culture systems. Alternatively, and more provocatively, one may hypothesize that Fas-FasL is actually important for clearance of Pneumocystis. Parallel to our study, a recent investigation demonstrates that infection of macrophages with Leishmania donovani up-regulates Fas expression on infected cells and enhanced the levels of soluble FasL in supernatants from infected cultures (26). An additional study demonstrates that the clearance of Leishmania major infection requires both FasL and IFN-{gamma} (27). These investigators illustrate that Fas pathways are essential for organism elimination. They further demonstrate that FasL exerts a direct effect by reducing organism viability within macrophages (27). Additional investigation will be needed to determine whether Fas-FasL interactions are also active in the clearance of Pneumocystis infection.

DC costimulatory molecule expression and cytokine activation in response to Pneumocystis most likely represent an auxiliary component of the innate inflammatory response to this pathogen, as well as an essential mechanism of Ag presentation for the eventual generation of effective acquired lymphocytic responses to this organism. As lymphocytic immunity wanes during the course of HIV infection or other immunosuppressive conditions, the DCs may also provide an alternate means to stimulate effective immunity against this organism. Intriguing studies by Zheng et al. (28) have shown that murine bone marrow-derived DCs that express the murine CD40L can be stimulated ex vivo with Pneumocystis Ags. Such cells, when transferred to lymphocyte-depleted mice, confer protection against subsequent Pneumocystis pneumonia (28). Thus, increased understanding of DC activation by this organism and related fungi holds promise for novel means to prevent and treat these devastating infections. One may postulate that activation of DCs by Pneumocystis glucan through ligation of innate receptors provides an additional signal that prepares the DC in the context of Pneumocystis Ag presentation to T cells and stimulation of a robust adaptive immune response. In the context of vaccine development, one can argue that in combination with Pneumocystis protein Ags, poorly immunogenic, but well-tolerated compounds such as glucans may serve as potent adjuvants to enhance and promote specific host DC functions against Pneumocystis.

The mechanisms through which Pneumocystis and other fungal beta-glucans initiate activation of DCs are not fully understood. Emerging evidence has supported a number of distinct receptors in mediating host cell responses to this fungal cell wall component (6, 12, 29, 30, 31, 32). Our current data support earlier studies that dectin-1 serves as a major receptor for fungal beta-glucans found on DCs and macrophages (29, 33, 34). Additional recent studies from our laboratory further document the beta-glucans stimulate macrophages through MyD88-dependent mechanisms (6). Other investigations have demonstrated that dectin-1 may act synergistically with TLR2 to mediate cytokine generation by DCs and macrophages in response to zymosan, a glucan-rich cell wall fraction of yeast (35). However, other investigations indicate that under other conditions, cells of the lower respiratory tract can respond to fungal beta-glucans in the absence of dectin-1 (12, 32). In this light, other receptors including lactosylceramide can also independently mediate host inflammatory signaling to Pneumocystis and other fungal cell wall beta-glucans (12, 32, 36, 37). The precise interactions of these various beta-glucan receptors in mediating DC recognition of the fungal cell wall will require additional investigation.

Accumulating evidence suggests that distinct strains or species of Pneumocystis specifically infect various mammalian hosts. We are currently only able to isolate <1 mg of purified beta-glucan from 40 heavily infected rat lungs (5). Unfortunately, it is not feasible at this time to isolate significant amounts of beta-glucans from human-derived Pneumocystis. Nonetheless, ultrastructural analysis and other studies support the presence of similar beta-glucan structures in human-derived Pneumocystis organisms (38, 39, 40). beta-Glucans are structural carbohydrates that most likely lack the fine structural diversity observed with surface protein epitopes between species. Indeed, in our studies, we have observed highly similar responses with beta-glucan, derived from the nonpathogenic S. cerevisiae, to that derived from Pneumocystis, thus making it a useful model to evaluate cell responses to fungi (20). We further propose that these responses to beta-glucan may be generalized responses to fungi, and are predictive of the types of responses occurring in patients with Pneumocystis infection.

In summary, we have demonstrated that the cell wall beta-glucan components of the Pneumocystis and other fungal cell wall can induce costimulatory molecule expression by DCs and subsequent stimulation of T cells to express a Th1 pattern cytokine response. This occurs in the absence of IL-12 secretion. In addition, DC activation in response to Pneumocystis beta-glucan is at least partially mediated by dectin-1 receptors and interactions of Fas-FasL. Understanding innate and adaptive immune response to Pneumocystis should provide additional insights for more effective therapies for this important infection of immune compromised patients.


   Acknowledgments
 
We thank Dr. Stephen Murphy and Kathy Streich for the preparation of the manuscript. We also thank the members of the Mayo Clinic Thoracic Diseases Research Unit for their many helpful discussions.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by National Institutes of Health Grants R01 HL55934 and HL62150 (to A.H.L.). Back

2 Address correspondence and reprint requests to Dr. Andrew H. Limper, 8-24 Stabile Building, Mayo Clinic, Rochester, MN 55905. E-mail address: limper.andrew{at}mayo.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; FasL, Fas ligand. Back

Received for publication April 21, 2005. Accepted for publication April 14, 2006.


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