We have demonstrated that live C. albicans
suppresses ROS production by phagocytes in vitro. This suppression did not occur with S. cerevisiae
, which is infrequently pathogenic. Suppression of ROS production was not dependent on phagocytosis and overrode the stimulatory signals of the cell wall, including 1,3-β-glucan. Many recent studies of phagocyte interactions with Candida
have focused on the cell wall and phagocyte pathogen recognition receptors, such as dectin, mannose receptor, and Toll-like receptors (22
). Given the topology of Candida
-phagocyte interactions, phagocyte recognition of the cell wall is clearly important in modulating the host response to yeast. However, our data demonstrate that live Candida
suppressed production of ROS in a manner that appeared to override the stimulatory effects of the cell wall and thus may represent a novel immune evasion strategy for Candida
One possible mechanism through which live C. albicans could decrease ROS in these assays is by scavenging of ROS by SOD, catalase, or other redox-reactive molecules, such as thiols. To investigate this, we compared ROS scavenging by HK and live C. albicans, as well as by S. cerevisiae, which does not suppress ROS production. We found that both types of yeast were more effective at ROS scavenging when alive compared to HK. However, both live C. albicans and live S. cerevisiae scavenged ROS at similar levels. Thus, while scavenging of ROS may be active in these assays, there appears to be an additional mechanism through which C. albicans actively suppresses ROS production by the phagocytes.
ROS production in BMDM from BALB/c mice was not suppressed by live C. albicans
, whereas ROS production was suppressed in BMDM from C57BL/6 mice, primary murine PMN, and human phagocytes. The differences observed with BMDM from the two mouse strains raise interesting questions. Responses in BALB/c and C57BL/6 mice have often been compared because of their preponderance to Th2 or Th1 responses, respectively, as demonstrated by the classic experiments in which C57BL/6 mice are more resistant to leishmaniasis than are BALB/c mice (26
). This is thought to be due, in part, to the ability of macrophages from C57BL/6 mice to more effectively stimulate a Th1 response (28
). Thus, it was not wholly unexpected that the BMDM from these strains would respond differently to C. albicans
. In fact, it has been demonstrated that BALB/c and C57BL/6 mice, while both susceptible to gastric candidiasis, produce different cytokine profiles after Candida
). Despite these differences, both strains of mice are considered to be similarly resistant to systemic challenge with Candida
These considerations highlight a limitation of these studies: in analyzing these interactions in vitro, we do not know how well our findings model host-pathogen interactions in vivo. It is possible that, in an immunocompetent host, other components of the immune system can activate phagocytes and allow them to overcome suppression of ROS production, or that ROS is not necessary for the clearance of C. albicans. Nevertheless, the overall finding that suppression occurs in hPBMC and hPMN suggests this process may be important for Candida in some fashion, perhaps by providing an immune evasion mechanism that is important in maintenance of its commensal state.
Several studies have investigated phagocyte ROS production in response to C. albicans
. Smail et al. described the release of a soluble inhibitory product from hyphae treated with UV light (31
). This product, later identified as adenosine (30
), suppressed production of ROS in PMN stimulated by the chemoattractant peptide fMLP, but not by PMA. In contrast, we found that suppression of ROS production occurred for both PMA-stimulated and unstimulated phagocytes. Thus, it seems unlikely that adenosine mediates suppression of ROS production in our system.
Donini et al. investigated production of ROS in dendritic cells (DC) exposed to C. albicans
); consistent with our results, they found that live C. albicans
suppressed PMA-stimulated production of ROS by DC. They also found that treatment of DC with dectin-1 agonists stimulated production of ROS, whereas treatment with agonists for both dectin-1 and the mannose receptor (CD206) resulted in lower production of ROS. Thus, they postulated that activation of the mannose receptor inhibited dectin-1-dependent ROS production.
Our finding that caspofungin treatment of yeast suppresses ROS production is consistent with the postulated inhibition of dectin-1 by mannose receptor signals, because caspofungin-treated Candida have both mannans and 1,3-β-glucan exposed on the surface. However, we found that UV-inactivated Candida, with an intact surface mannoprotein layer, only partially suppressed ROS production. If ROS suppression were solely due to mannose receptor activation, we would expect UV-inactivated yeast to fully suppress ROS production. Furthermore, we found that cell wall ghosts prepared from live Candida stimulated ROS production. The cell wall ghosts were prepared under nondenaturing conditions designed to preserve the native cell wall structure. Thus, it is unlikely that the suppression of ROS production we observed is due to stimulation of the mannose receptor, as it did not occur in the presence of cell wall ghosts, which should have an intact mannoprotein layer. It is possible that the physical disruption used to produce the cell wall ghosts resulted in the loss of some key mannoprotein constituent that might be responsible for suppression of ROS production. However, the bulk mannose/mannoprotein state of the cell wall ghosts should resemble that of live, intact cells. Thus, our data are more consistent with the possibility that suppression of ROS production in phagocytes is mediated by a pathway unrelated to the mannose receptor.
Given the intermediate suppression of ROS production seen with UV-inactivated Candida, which was paralleled by a similar decrease in metabolic activity, it is possible that the mechanism of ROS suppression requires metabolic activity, such as the production of a small metabolite that could function as a phagocyte toxin. Thus, we investigated whether a secreted product might be responsible. We did not find ROS-suppressive activity in spent culture medium or in medium harvested from phagocytes exposed to live Candida. If a secreted product is involved, it is either not stable or is present at a low concentration in the supernatant. The production of a secreted compound that would only function at relatively high concentrations is an interesting possibility: such a compound might be expected to be in high concentration at the site of Candida-phagocyte interactions but be diluted below the active concentration after diffusion into the environment. In this way, Candida could inhibit ROS production in closely associated phagocytes, in a paracrine-like fashion.
Phagocyte-generated ROS are well-known to kill many microbial pathogens; suppression of ROS production might thus represent an important mechanism for Candida
to evade phagocytic killing. However, data to suggest that ROS also carry out important signaling functions are accumulating. For example, in some cell lines, activation of the proinflammatory transcription factor NF-κB is dependent on ROS (29
). ROS may also be important in regulation of tyrosine phosphorylation as well as activation of mitogen-activated protein kinases, protein kinase C, and phospholipase A2
in leukocytes (10
). As these signaling pathways are important in activation of host immune responses, suppression of ROS production by Candida
may result in significant modulation of anti-Candida
An immunomodulatory role for ROS is supported by studies in which mice deficient in production of both ROS and reactive nitrogen intermediates were inoculated with Candida
via the gastrointestinal tract (3
). Although all of these mice died after inoculation, the cause of death appeared to be an exaggerated immune response rather than overwhelming fungal infection. Furthermore, phagocytes from normal mice and ROS/reactive nitrogen intermediate-deficient mice were equally able to kill C. albicans
in vitro. These data suggest that production of ROS in response to C. albicans
may not be important for direct killing of Candida
. This is consistent with our finding that Candida
suppresses ROS production in phagocytes. Thus, ROS production in response to Candida
infection may be more important for regulation of inflammatory responses than for direct anti-Candida
In summary, we have demonstrated that live Candida suppresses production of ROS by phagocytes. Suppression of ROS production does not occur with S. cerevisiae, suggesting that it may be important in the pathogenesis of Candida disease. Suppression of ROS production appears to override stimulatory signals from the cell wall, including 1,3-β-glucan. Thus, the ability of Candida to control host production of ROS may represent an important factor in host-Candida interactions.