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Keywords:

  • CpG-ODN;
  • Cryptococcus neoformans;
  • macrophages;
  • nitric oxide

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-γ. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-γ. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-κB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-γ-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.

List of Abbreviations: 
BAL

bronchoalveolar lavage

C. neoformans

Cryptococcus neoformans

CpG-ODN

CpG motif-containing oligodeoxynucleotide

dCap67

disrupted Cap67

GXM

glucuronoxylomannan

HPRT

hypoxanthine phosphoribosyl transferase

iNOS

inducible nitric oxide synthase

LPS

lipopolysaccharide

NF-κB

nuclear factor kappa B

NO

nitric oxide

PDA

potato dextrose agar

rIFN-γ

recombinant γ-interferon

Cryptococcus neoformans, an opportunistic fungal pathogen, infects via an airborne route. The infection is usually controlled within the primary infection site in healthy individuals, whereas this fungal microorganism hematogenously disseminates to the central nervous system and causes a life-threatening meningoencephalitis in patients with severely compromised immune responses, like AIDS (1). Meningoencephalitis under such conditions is often refractory to chemotherapy with anti-fungal agents, and the mortality rate is still high in the era when highly active anti-retroviral therapy is widely available (2).

The host defense to C. neoformans is largely mediated by cellular immune responses (3), in which type-1 helper T (Th1) cells play a critical role by producing IFN-γ (4). Mice with a genetic disruption of Th1-related cytokines, such as IFN-γ, interleukin (IL)-12 and IL-18, are highly susceptible to cryptococcal infection (5–9). C. neoformans grows within macrophages by resistance to their killing mechanism and persists in the primary infected tissues for long periods of time (10). IFN-γ leads to the expression of iNOS by macrophages, which catalyzes NO synthesis from l-arginine (11). NO is a central mediator in eradicating C. neoformans by armed macrophages and plays an important role in the host defense to this fungal pathogen (12–14).

In previous investigations, C. neoformans has been reported to interfere with the immune responses to survive the host defense mechanisms (10, 15–18). The production of pro-inflammatory cytokines such as IL-1β and tumor necrosis factor (TNF)-α by activated macrophages is inhibited by GXM, a major capsular polysaccharide of this fungal pathogen (17). Similar findings are also reported in the expression of costimulatory molecules on dendritic cells that are important for efficient antigen presentation to T cells (15, 18). In earlier studies, we indicated that C. neoformans inhibited the synthesis of IL-12 and NO by macrophages stimulated with LPS (19, 20).

A decade ago, Krieg and coworkers discovered that bacterial DNA with an unmethylated CpG motif had an immunostimulatory activity on B cells (21, 22). The synthetic oligo-DNA (ODN) containing this motif activates dendritic cells to produce IL-12 and expression of costimulatory molecules such as CD40, which leads to the development of Th1-type immune responses (22–24). The receptor recognizing this DNA motif has been identified as Toll-like receptor (TLR) 9, which delivers the signals through activation of NF-κB (22, 25). Many investigations have addressed the therapeutic application of CpG-ODN in infections, malignancies and allergic diseases on the basis of its immune stimulatory activity (22). Recently, we and other investigators demonstrated that CpG-ODN treatment protected mice from infection with C. neoformans through altering the Th1-Th2 cytokine balance toward Th1-biased immune responses (26, 27).

In the present study, we addressed the question of whether C. neoformans affects the production of NO by macrophages activated via a TLR9-mediated pathway, which was triggered by CpG-ODN. We found that C. neoformans strongly inhibited these responses in a fashion independent of capsular polysaccharides. This effect was not necessarily associated with the suppression of TLR9-mediated NF-κB activation in a luciferase reporter assay with HEK293T cells.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Culture medium and reagents

RPMI-1640 medium was purchased from Nipro (Osaka, Japan) and fetal calf serum (FCS) from Cansera (Rexdale, Ontario, Canada). LPS from Escherichia coli serotype O111:B4, peptidoglycan (PG) was purchased from Sigma Chemical Co. (St Louis, MO, USA). Murine recombinant (r)IFN-γ (specific activity >2 × 107 units/mg) was purchased from (PeproTech EC, London, UK).

Macrophage cultures

A SV40-transformed mouse alveolar macrophage cell line, designated as MH-S, was obtained from American Type Culture Collection (Manassas, VA, USA). These cells were cultured at 5 × 105/mL in RPMI-1640 medium supplemented with 10% FCS, 100 U/mL penicillin G, 100 μg/mL streptomycin and 50 μM 2-mercaptoethanol in a 5% CO2 incubator. The cells were stimulated with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the presence or absence of C. neoformans or its derivatives for 24 hr. In some experiments, the culture was set in a 24-well double chamber separated by a 0.45-μm-pore membrane (Intercell, Kurabo Industries, Tokyo, Japan). The culture supernatants were harvested and kept at −70 °C until measurement of NO. In some experiments, alveolar macrophages from bronchoalveolar lavage (BAL) fluids of C57BL/6 mice were used in place of MH-S. These mice were kept under specific pathogen-free conditions at the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine, and the experiments were conducted according to the institutional guidelines and were approved by the institutional ethics committees.

CpG motif-containing oligodeoxynucleotide

The sequence of CpG-ODN was TCC ATG ACG TTC CTG ACG TT, which was phosphorothioated and purified by high-performance liquid chromatography (HPLC) at Hokkaido System Science (Sapporo, Japan). The endotoxin content measured by Limulus amoebocyte lysate assay was less than 10 pg/mL. CpG-ODN-induced NO synthesis by MH-S did not receive any influence by the addition of polymyxin B (10 μg/mL), although that caused by LPS (1 μg/mL) was completely abolished by the same treatment.

Cryptococcus neoformans

A serotype A-encapsulated strain of C. neoformans, designated as YC-11, was established from a patient with pulmonary cryptococcosis. The strain had a thick capsule when examined shortly after harvesting from infected mouse lungs (28). In some experiments, a serotype D-acapsular strain of C. neoformans, designated as Cap67, and its parent strain, designated as B3501, (kind gifts of Dr Stuart M. Levitz, Boston University, Boston, MA, USA and Dr Kwon-Chung, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA, respectively) were used. The yeast cells were cultured at 30 °C on PDA (Eiken, Tokyo, Japan) plates. After 2 to 3 days of culture, the cells were collected, washed three times in normal saline, and counted using a hemocytometer.

Preparation of C. neoformans culture supernatants

The YC-11 strain of C. neoformans was cultured in RPMI-1640 medium supplemented with 10% FCS at 37 °C in a CO2 incubator for 5 days. The culture supernatants were centrifuged, passed through 0.2 μm milipore filter (Millipore, Billerica, MA, USA) and kept at −70 °C before use. The level of capsular GXM in the culture supernatants was measured using the latex agglutination test (Eiken).

Disruption of C. neoformans

Cap67 collected from PDA cultures were washed three times and suspended at 2 × 09/mL in normal saline. The yeast cells were disrupted with the same volume of 0.5 mm glass beads in a Multi-beads shocker (MB400U; Yasui kikai, Osaka, Japan) at a setting of 2700 r.p.m., 60 s on and off each time with five cycles per operation, and this operation was repeated five times with 20 to 30 min intervals. The cell suspension was continuously kept at 4 °C during disruption. This procedure enabled us to make a complete disruption of the yeast cells, as confirmed by microscopic observation and loss of their growth on PDA plates.

Nitric oxide assay

Nitric oxide produced in the macrophage cultures is converted to nitrite and nitrate within a short period of time. Therefore, we evaluated the level of NO production by measuring the amount of nitrite accumulating in the cultures using the method described by Stuehr and Nathan (29). Briefly, 100 μL culture supernatant was mixed with the same volume of Griess reagent and absorbance was read at 550 nm using an automated microplate reader. The concentration of nitrite was calculated from a NaNO2 standard curve.

Extraction of RNA and RT-PCR

Total cellular RNA was extracted from macrophages stimulated with CpG-ODN and rIFN-γ in the presence or absence of C. neoformans using ISOGEN (Wako, Osaka, Japan), followed by reverse transcription (30). The cDNA obtained was then amplified in an automatic DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT, USA) using specific primers 5′-AGG TAC TCA GCG TGC TCC AC-3′ (sense) and 5′-GCA CCG AAG ATA TCT TCA TG-3′ (antisense) for iNOS, and 5′-CTC ATG ACC ACA GTC CAT GC-3′ (sense) and 5′-CAC ATT GGG GGT AGG AAC AC-3′ (antisense) for HPRT. We added 1.0 μL of the sample cDNA solution to 49 μL of the reaction mixture, which contained the following concentrations: 10 mM Tris-HCl (pH = 8.3), 50 mM KCl, 1.5 mM MgCl2, 10 μg/mL gelatin, dNTP (each at a concentration of 200 μM), 1.0 μM sense and antisense primer, and 1.25 U of AmpliTaq DNA polymerase (Perkin-Elmer Cetus). The mixture was incubated for 1 min at 94 °C, 1 min at 55 °C and 1 min 30 s at 72 °C for iNOS and HPRT. The number of cycles was determined for samples not reaching the amplification plateau (32 cycles for iNOS and 28 cycles for HPRT. The PCR products were electrophoresed on 2% agarose gels, stained with 0.5 μg/mL ethidium bromide and observed with a UV transilluminator.

Preparation of lipids from C. neoformans

Lipids of C. neoformans were extracted from disrupted Cap67 with chloroform-methanol 2:1 and 1:2 (vol/vol) (31). The crude lipid extract was partitioned according to the method of Folch et al. (32). The lipids recovered from Folch's lower phase were fractionated on a silica gel column eluted with chloroform, acetone, and methanol. The lipids recovered from Folch's lower phase were subjected to fractionation by a using silica gel column, and neutral lipid and phospholipid were then eluted with chloroform and methanol, respectively. The neutral lipid or phospholipid obtained was analyzed by thin-layer chromatography (TLC) as well as liquid chromatography combined with mass spectrometry, and subjected to cell-culture study.

TLR9 cloning and NF-κB reporter assay

The full-length TLR9 was polymerase chain reaction (PCR)-amplified from a cDNA of mouse macrophage cell line RAW264.7 cells (RIKEN Cell Bank, Tsukuba, Japan). Specific primers for amplification of TLR9 were designed based on the GeneBank accession number AF314224 (33). The sequences of primers were 5′-CAC CAT GGT TCT CCG TCG AAG G-3′ (forward) and 5′-CTA TTC TGC TGT AGG TCC CCG GC-3′ (reverse). The amplified full-length cDNA was cloned into pcDNA6.2/TOPO (Invitrogen Japan, Tokyo, Japan) and sequenced. Endothelial leukocyte adhesion molecule 1 (ELAM1) luciferase reporter plasmids and mouse TLR4, MD2 and CD14 cDNA were kindly provided by Dr Masao Mitsuyama (Kyoto University, Kyoto, Japan). The coding region for each gene was inserted into the mammalian expression vector p3xFLAG CMV-1 (Sigma). For NF-κB luciferase reporter assay, HEK293T cells were transiently transfected with reporter plasmid, together with expression plasmids of TLR9 or TLR4/MD2/CD14 or empty control plasmid. At 24 hr after transfection, the cells were treated with CpG-ODN or LPS in the presence or absence of disrupted Cap67 for 6 hr. Luciferase activity in the total cell lysates was measured with the Dual-luciferase reporter assay system (Promega, Tokyo, Japan). The Renilla luciferase reporter gene was simultaneously transfected as an internal control. Relative luciferase activities were calculated as folds of induction compared with unstimulated vector control.

Statistical analysis

Data were analyzed using Statview II software on a Macintosh computer. Data are expressed as mean ± standard deviation (SD). Differences between groups were examined for statistical significance using one-way analysis of variance (anova) with a post-hoc analysis (Fisher PLSD test). A P value less than 0.05 was considered significant.

Results

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Inhibition of macrophage NO production by C. neoformans

Initially, we examined the effect of C. neoformans (YC-11) on the production of NO by an alveolar macrophage cell line, MH-S, stimulated with CpG-ODN and IFN-γ. In our preliminary experiments, MH-S produced only a small amount of NO upon stimulation with CpG-ODN alone and the addition of IFN-γ strikingly enhanced this production, although IFN-γ alone did not show such activity. Thus, we stimulated MH-S in combination with both agents in further experiments. As shown in Figure 1a, YC-11 strongly suppressed the NO synthesis by MH-S in a dose-dependent fashion. Similar results were obtained in alveolar macrophages from BAL fluids (data not shown). Because YC-11 is coated by a thick capsule, a major virulent factor, and secretes a large amount of GXM, which has been reported to make profound influences on various immune responses (15–18), we tested the effect of YC-11 culture supernatants prepared in RPMI-1640 medium supplemented with 10% FCS that contained 3.2 mg/mL GXM. As shown in Figure 1b, addition of the culture supernatants did not affect the NO synthesis by MH-S. These results suggested that capsular polysaccharides did not contribute much to this suppression.

imageimage

Figure 1. Suppression of NO synthesis by C. neoformans. MH-S were cultured with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the presence or absence of indicated doses of (a) YC-11 or (b) YC-11 culture supernatants for 24 hr, and then nitrite content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures. CpG, CpG-ODN; YC11-sup, culture supernatants of YC-11.

Role of capsules in the inhibition of NO synthesis by C. neoformans

To further elucidate the involvement of capsular polysaccharides, we compared an acapsular strain of C. neoformans, Cap67, and its parent strain, B3501, in their effect on the synthesis of NO by MH-S stimulated with CpG-ODN and IFN-γ. As shown in Figure 2, both strains inhibited NO production to an equivalent level in a similar dose–response curve. Similar results were obtained in alveolar macrophages from BAL fluids (data not shown). These results indicated that capsule was not required for this suppression. Thus, in further experiments, Cap67 was used to approach the mechanism of the inhibitory effect of C. neoformans on macrophage NO synthesis.

image

Figure 2. Suppression of NO synthesis by an acapsular strain of C. neoformans. MH-S were cultured with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the presence or absence of indicated doses of Cap67 or B3501 for 24 hr, and then nitrite content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures.

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Physical contact-dependent inhibition of macrophage NO production by C. neoformans

To investigate the mechanism of NO inhibition by C. neoformans, the cultures were set in two chambers separated by a membranous septum, which acted as a barrier for cell movement between the upper and lower chambers but allowed the diffusion of macromolecules as large as 0.45 μm. As shown in Figure 3, NO production was strongly inhibited when CpG-ODN and IFN-γ-stimulated MH-S were cultured with Cap67 yeast cells in the same chamber, whereas such inhibition was not observed when these cells were cultured separately in the lower and upper chambers. These results suggested that direct physical contact or close proximity of C. neoformans to macrophages was required for the inhibition of NO synthesis.

image

Figure 3. Direct contact-dependent suppression of NO synthesis. The culture was set in a 24-well double chamber separated by a membrane of 0.45 μm pores. MH-S were cultured with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the lower or upper chamber with or without Cap67 (1 × 107/mL) in a total volume of 1.0 mL for 24 hr, and then nitrite content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures. Lower, lower chamber; Upper, upper chamber.

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Inhibition of macrophage NO production by disrupted C. neoformans

In further studies, we tested whether disrupted C. neoformans showed a similar effect on the synthesis of NO by macrophages. For this purpose, Cap67 was disrupted by glass beads under a condition continuously kept at 4 °C. By this treatment, any yeast cells were not found under microscopic observation, and any live colonies were not grown on PDA plates. As shown in Figure 4a, the disrupted (d)Cap67 suppressed NO production in a dose-dependent manner with a highest activity at 10%, which was an equivalent level to that detected by untreated Cap67. Compatible with this result, the expression of iNOS mRNA by CpG-ODN and IFN-γ-stimulated MH-S was inhibited by the addition of dCap67 in a reverse transcription–polymerase chain reaction (RT-PCR) analysis (Fig. 4b).

imageimage

Figure 4. Suppression of NO and iNOS synthesis by dCap67. MH-S were cultured with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the presence or absence of Cap67 (1 × 107/mL) or indicated doses of dCap67 for 24 hr. (a) NO content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures. (b) Expression of iNOS or HPRT mRNA was examined by reverse transcription–polymerase chain reaction. dCap67, disrupted Cap67.

Previously, we demonstrated that NO was essential for killing of C. neoformans by macrophages (14). Indeed, activation of MH-S by CpG-ODN and IFN-γ caused not only NO production but also reduction in the growth of YC-11, which were completely abrogated by addition of NG-monomethyl-l-arginine (l-NMMA), but not by NG-monomethyl-d-arginine (D-NMMA; data not shown). These results indicated the contribution of NO to the growth inhibition of C. neoformans by activated MH-S, which promoted us to test the effect of dCap67 on the growth of this fungal pathogen. As shown in Figure 5, MH-S significantly reduced the number of live colonies upon stimulation with CpG-ODN and IFN-γ, and the addition of dCap67 interfered with the growth inhibition of this fungus by the activated MH-S.

image

Figure 5. Effect of dCap67 on the MH-S fungicidal activity. YC-11 were cultured at 1 × 105/mL with or without MH-S in the presence or absence of CpG-ODN (1 μg/mL), rIFN-γ (10 ng/mL) or dCap67 (10%) for 24 hr, and then the number of live colonies of YC-11 was counted. dCap67, disrupted Cap67. *P < 0.05.

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Characterization of NO inhibition by C. neoformans

Next, we tested the effect of heat treatment by boiling the whole Cap67 or dCap67 for 10 or 30 min. As shown in Figure 6a, this treatment completely abolished the suppression of MH-S NO synthesis by the whole yeast cells, whereas such effect was not detected in the case of dCap67. These results may reduce the possibility that a certain proteinous molecule acts as an inhibitor in dCap67, although this possibility cannot be excluded in undisrupted yeast cells. Considered collectively with our data showing the potent inhibition of NO synthesis by a mutant strain of C. neoformans lacking capsular polysaccharides, some lipid could be a candidate molecule for such inhibition. Thus, we examined the influence of neutral and phospholipids derived from Cap67 on the production of NO by activated MH-S. As shown in Figure 6b, these lipids did not show any inhibitory effect.

imageimage

Figure 6. Effect of heat-treated Cap67 and their lipid fractions on NO synthesis. MH-S were cultured with CpG-ODN (1 μg/mL) and rIFN-γ (10 ng/mL) in the (a) presence or absence of boiled or untreated Cap67 (1 × 107/mL) or dCap67 (10%) or (b) indicated doses of lipid fractions of Cap67 for 24 hr, and then nitrite content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures. dCap67, disrupted Cap67; boil −10 min or boil −30 min, boiled for 10 min or 30 min, respectively; NL, neutral lipid; PL, phospholipid.

Effect of C. neoformans on the signals via TLR

Finally, we questioned which signals triggered by CpG-ODN or IFN-γ were affected by C. neoformans. For this purpose, these signals were separated using two-step cultures, in which MH-S were transiently incubated with either CpG-ODN or IFN-γ for 1 hr and washed out three times to delete residual stimulants, followed by re-stimulation with the counterpart reagent for 23 hr. To test the effect of this fungal pathogen, intact or disrupted Cap67 was added to the second cultures. As shown in Figure 7, the first stimulation for 1 hr was not sufficient to induce the synthesis of NO, because MHS did not produce NO when stimulated with both reagents just for 1 hr. Addition of either intact or disrupted Cap67 to the second culture containing CpG-ODN or IFN-γ resulted in the suppression of NO synthesis by MH-S prestimulated with the counterpart reagent in the first culture. These results suggested that C. neoformans may affect both signals caused by CpG-ODN and IFN-γ.

image

Figure 7. Two-step activation of MH-S by CpG-ODN and IFN-γ. MH-S were cultured with CpG-ODN (1 μg/mL) and/or rIFN-γ (10 ng/mL) for 1 hr (first culture). The cultures were washed three times by culture medium and then cultured with CpG-ODN and/or rIFN-γ in the presence or absence of dCap67 (10%) for 23 hr (second culture). Nitrite content in the culture supernatants was measured. Data are the mean ± SD of triplicate cultures. dCap67, disrupted Cap67.

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In further experiments, to directly test the effect of C. neoformans on the TLR9-mediated signaling, we performed a luciferase reporter assay using HEK293T cells transfected with the gene for TLR9 and the luciferase gene linked to the promoter sequence containing an NF-κB-binding site. As shown in Figure 8, CpG-ODN induced the luciferase activity in TLR9-expressing HEK293T cells, and addition of dCap67 did not result in reduction, but rather caused augmentation in this activity. In addition, we tested the effect of dCap67 in the same assay using LPS-stimulated HEK293T cells transfected with genes for TLR4, MD2 and CD14. LPS caused the activation of NF-κB and addition of dCap67 led to the reduction in this activity.

image

Figure 8. Effect of dCap67 on the TLR-mediated NF-κB activation. HEK293T cells transfected with TLR9 or TLR4/MD2/CD14 gene or control vector were treated with CpG-ODN (1 μM) or LPS (1 μg/mL), respectively, in the presence or absence of dCap67 (3%). The luciferase activity in each sample was determined. Data are expressed as the mean ± SD of relative values to those of control vector in triplicate cultures. dCap67, disrupted Cap67.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The findings of the present study are as follows: (i) CpG-ODN induced the production of NO by alveolar macrophages in combination with IFN-γ; (ii) both encapsulated and acapsular strains of C. neoformans (B3501 and Cap67, respectively) inhibited this response to an equivalent level; (iii) the culture supernatants of C. neoformans containing capsular polysaccharides did not show such effect; (iv) physical or closer contact between macrophages and Cap67 was required for this inhibition; (v) dCap67 also showed the inhibitory activity that was resistant to heat treatment; (vi) neither neutral nor phospholipids from Cap67 suppressed the NO production; and (vii) dCap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-κB in a luciferase reporter assay with HEK293T cells.

Previous investigations have reported the immunosuppressive effects of capsular polysaccharides including GXM (e.g. production of inflammatory cytokines and expression of co-stimulatory molecules on macrophages and dendritic cells (15–18)). In contrast to these previous observations, in our study, capsular polysaccharides were not likely to be essential for the suppression of NO synthesis by alveolar macrophages, because the culture supernatants containing a large amount of capsular polysaccharides did not show the inhibitory activity and there was no significant difference in this activity between the acapsular mutant strain of C. neoformans and its encapsulated parent strain. In addition, physical or closer contact with macrophages was required for this fungal pathogen to exert the inhibitory activity, as shown by the data in the trans-well experiments. The major activity in dCap67 was ascribed to precipitates, but not to supernatants, obtained by their regular centrifugation (data not shown). Although the precise mechanism remains to be understood, certain cell wall, plasma membrane or cytosolic compartments might be involved in this phenomenon.

The present study indicated that heat treatment by boiling for 10 and even 30 min did not affect the inhibitory activity of dCap67, whereas the same treatment completely abolished that of undisrupted Cap67. Therefore, there could be two different mechanisms in the NO inhibition by C. neoformans: one is heat labile and another is heat resistant. The latter feature suggests that the molecule responsible may be something other than proteinous molecules, but similar to polysaccharides or lipids. As mentioned above, capsular polysaccharides were not likely in this case, although the possible contribution of cell wall polysaccharides was not examined here. Therefore, in further experiments, to address the involvement of lipids, we tested the effect of neutral and phospholipids derived from dCap67, which accounted for most of their lipids (data not shown), on the synthesis of NO by MH-S, but the results were negative. Thus, in the current study, characteristics of active molecules in the dCap67-induced NO suppression remain to be substantiated.

In our earlier studies (19, 20), we demonstrated that C. neoformans inhibited the synthesis of NO and IL-12 by peritoneal macrophages stimulated with LPS, a TLR4 ligand (34), in combination with IFN-γ. The present study showed similar results in the activation of an alveolar macrophage cell line caused by CpG-ODN, a TLR9 ligand (22, 25), and IFN-γ. These findings raised a possibility that C. neoformans may affect the intracellular signaling mediated by TLR. In fact, our study showed that dCap67 suppressed the LPS-induced activation of NF-κB in TLR4/MD2/CD14-transfected HEK293T cells in a luciferase reporter assay. However, such inhibition was not detected using TLR9-transfected HEK293T cells upon stimulation with CpG-ODN. Whereas LPS is sensed by TLR4 expressed on the surface of macrophages (34), CpG-ODN is translocated into the endosomal compartments and interacts with TLR9 at this site (35, 36). The distinct effect of dCap67 on the NF-κB activation in this assay might be related to such difference. Because it is not clear whether HEK293T cells respond to dCap67 similarly as MH-S, the current study did not clarify the answer to the question as to why C. neoformans suppressed NO synthesis by MH-S stimulated with CpG-ODN and IFN-γ. To address this issue, effects of this fungal pathogen on the intracellular signaling mediated by TLR9 in MH-S need to be examined in our future investigations.

In conclusion, in the present study, we demonstrated that C. neoformans suppressed the production of NO by macrophages activated via TLR9 and IFN-γ signaling in a fashion independent of capsular polysaccharides and suggested that this activity could be a mechanism for them to survive the NO-mediated killing by armed macrophages. The present findings may provide a better understanding of the pathogenic mechanisms of intractable infectious diseases caused by C. neoformans.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The authors thank Dr Masao Mitsuyama for kind gifts of luciferase reporter plasmids, HEK293T cells and mouse TLR4, MD2 and CD14 cDNA. This work was supported in part by a Grant-in-Aid for Science Research (C) (KAKENHI12670261 and 18590413) from the Ministry of Education, Culture, Sports, Science and Technology, by Tohoku University 21st COE program ‘CRESCEND’ and by Japan-China Medical Association.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. Results
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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