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Candida albicans is a pleiomorphic microorganism. The yeast-to-hypha transition has been shown to be one of several virulence attributes that enable C. albicans to invade human tissues (1). Therefore, studies aimed at determining whether candidal morphology impacts host defense mechanisms are prudent.

Previously, we showed that the most pathogenic C. albicans relative to Candida krusei has the ability to create an environment rich in interleukin-10 (IL-10) and poor in IL-12 and gamma interferon (12). Furthermore, the fact that C. krusei and heat-killed C. albicans lack the ability to germinate raised the question of whether the interaction of monocytes (MN) with germinating C. albicans may differentially induce IL-12 production, leading to initiation of different types of immune responses.

In the present study, isogenic strains of C. albicans that differ only in the ability to produce hyphae were used to explore the role of hyphae versus that of the yeast form in IL-12 production by MN. SC 5314 is a wild-type strain of C. albicans (5), and isogenic strain HLC54 (with deletions of the EFG1 and CPH1 genes, which are involved in C. albicans germination) was derived from SC 5314 (6), while HLC84 is a revertant strain of HLC54 to which the EFG1 gene was reintroduced, thereby restoring the ability to germinate (10). HLC54 and HLC84 were generously provided by J. Kohler (The Whitehead Institute). Live organisms and heat-killed C. albicans were prepared as previously described (12).

MN were prepared from fresh normal peripheral blood with a MACS separation column (Miltenyi Biotec, Auburn, Calif.), and the purity of the MN was >90%. MN (2 × 10⁶ cells) or MN with heat-killed C. albicans at a C. albicans-to-MN ratio of 1:2 in the plates were incubated at 37°C in a 5% CO₂ incubator for 2 h. Live C. albicans was added to activated MN at different ratios and incubated further for 20 h, and then supernatants were collected and stored at 70°C until use. In all experiments, the level of endotoxin was less than 0.06 U/ml, as determined by an E-TOXATE kit (Sigma). For the assay of the viability of MN cocultured with C. albicans, a LIVE/DEAD Viability/Cytotoxicity kit (Molecular Probes, Inc., Eugene, Oreg.) was employed by following the manufacturer's instructions.

The levels of IL-12 p70 in culture supernatants were determined in duplicate by enzyme-linked immunosorbent assay using a pair of mouse anti-human IL-12 p70 monoclonal antibodies (Endogen, Cambridge, Mass.). The total RNA of MN was extracted with an RNeasy total RNA kit (Qiagen, Chatsworth, Calif.), and reverse transcription (RT)-PCR was performed as previously described (4).

We first compared C. albicans and Saccharomyces cerevisiae since they represent pathogenic and nonpathogenic yeasts, respectively. More importantly, C. albicans appears as both the yeast form and hyphae while S. cerevisiae maintains yeast morphology under experimental conditions. After 20 h of incubation with MN, SC5314 failed to induce IL-12 p70 production (23 ± 7 pg/ml, n = 7) while S. cerevisiae strongly induced IL-12 production (162 ± 41 pg/ml, n = 3). These results confirm our previous observation (12) that the yeast form of fungi induced monocytic IL-12 p70 production but live, filament-forming C. albicans did not.

We further exposed MN to heat-killed C. albicans for 2 h to induce IL-12 production and also exposed MN to different concentrations of live C. albicans. As shown in Fig. 1, heat-killed C. albicans, at the optimal 1:2 ratio of C. albicans to MN (12), induced high levels of IL-12 (222 ± 41 pg/ml, n = 10). Adding live C. albicans, at a 3:10 ratio of C. albicans to MN, to the activated MN resulted in statistically significant inhibition of IL-12 p70 production (P < 0.01). These results showed that live C. albicans suppressed induced IL-12 p70 production by MN and did so in a dose-dependent manner.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Live C. albicans (L-CA) inhibits heat-killed (HK) C. albicans-induced IL-12 production by MN. IL-12 p70 levels are expressed as means ± the standard errors of the means. The ratios 1:10 and 3:10 represent the C. albicans-to-MN ratios used. n = 4 to 10.

To rule out the killing of MN by C. albicans during coculturing, the viability of MN exposed to live C. albicans was determined (Fig. 2). Our data indicate that the inhibition of IL-12 p70 production by C. albicans at a ratio of 3:10 was due to C. albicans-associated factors and not to killing of MN by C. albicans.

View larger version (29K): [in this window] [in a new window]   FIG. 2.   Viability of monocytes cocultured with different ratios of C. albicans (CA). Fluorescence intensity represents MN viability as determined by cytofluorometer. The ratios 1:10, 3:10, and 1:2 represent the C. albicans-to-MN ratios used. n = 2.

We next used a set of isogenic C. albicans strains, as mentioned above, to compare their abilities to induce IL-12 production. As shown in Fig. 3A, SC5314 inhibited (20 ± 8 pg/ml) while HLC54 strongly induced IL-12 p70 production (216 ± 40 pg/ml). The differences in the levels of IL-12 between SC5314 and HLC54 were statistically significant (P < 0.01). RT-PCR was performed to determine whether IL-12 p70 protein production is mirrored at the mRNA level. As shown in Fig. 3B, the pattern of IL-12 p40 mRNA expression paralleled IL-12 p70 protein production following stimulation with wild-type and agerminating mutant C. albicans. Although there is a trend toward a lower level of IL-12 protein production by HLC54, there is no statistically significant difference. Thus, there might be a weak case for translational inhibition. We further used heat-killed C. albicans to induce IL-12 production by MN and then added SC5314 and HLC54 to study the IL-12-inhibitory effect of C. albicans. As shown in Fig. 4, SC5314 but not HLC54, at a 3:10 ratio of C. albicans to MN, significantly inhibited the induction of IL-12 p70 (84 ± 22 and 331 ± 36 pg/ml for SC5314 and HLC54, respectively; P < 0.01). These results indicated that the germinating parental strain (SC5314) significantly inhibited IL-12 production by MN while the hypha-deficient strain (HLC54) did not. To further confirm that germination is important for IL-12 inhibition, we performed experiments with revertant strain HLC84. Our results showed that IL-12 p70 was significantly reinhibited by HLC84 (223 ± 16 pg/ml) relative to HLC54 (331 ± 36 pg/ml) (P < 0.05). Similarly, IL-12 p40 mRNA was associated with IL-12 protein production by MN stimulated with C. albicans (Fig. 4B). These results are consistent with our contention that germination of C. albicans plays a critical role in inhibition of IL-12 production by MN.

View larger version (33K): [in this window] [in a new window]   FIG. 3.   C. albicans (CA) hyphae and blastospores differentially induce IL-12 production by MN. (A) IL-12 p70 levels in supernatants were measured by enzyme-linked immunosorbent assay. n = 2 to 6. (B) RT-PCR for IL-12 p40 mRNA expression by MN. The data shown represent three experiments with similar results. Refer to the text for a description of the Candida strains used. HK-CA, heat-killed C. albicans.

View larger version (37K): [in this window] [in a new window]   FIG. 4.   Inhibition of induced IL-12 production by C. albicans. (A) IL-12 p70 levels are expressed as means ± the standard errors of the means. n = 3 to 9. (B) RT-PCR for IL-12 p40 mRNA expression by MN. HK-CA, heat-killed C. albicans.

The mechanism underlying C. albicans regulation of IL-12 production by MN is not known. Recent findings showed that C. albicans hyphae as a ligand can bind to the integrin CR3 (CD11b/CD18) on MN (3). Similarly, our findings and observations by others indicated that iC3b and Histoplasma capsulatum as natural ligands binding to CR3 can specifically downregulate IL-12 secretion by MN in vitro (7, 8, 13). Thus, similar mechanisms may be operational in IL-12 inhibition by C. albicans. Another possibility is the presence of soluble factors released by C. albicans. For example, mannoproteins, a water-soluble component of the C. albicans cell wall, have been reported to exert significant immunosuppressive activity both in vivo and in vitro (2, 11). In this regard, the culture supernatant of C. albicans inhibited nitric oxide production by activated macrophages and nitric oxide is known to induce IL-12 gene expression (9). This supports our findings that IL-12 production by MN is suppressed by C. albicans. An understanding of how C. albicans hyphae are involved in altering host immune responses may stimulate the development of novel immunorelated therapies for the treatment of candidiasis.