Previous studies have demonstrated that C.albicans influences immune response (Th1/Th2 balance) patterns during infection [15, 16]. Since IL-12 plays a central role in linking the innate and acquired immune systems [5], suppression of this cytokine could be a key to survival of any potential pathogen. IL-12 produced by monocytes/macrophages has been demonstrated to play a central role in the production of IFN-γ by NK and T cells, acting in concert with IL-18, IL-1β, TNF-α and IL-2. Moreover, IL-12 stimulates both NK and T cells to produce cytokines evoking an IFN-γ response. IFN-γ production results in increased cytotoxic activity and enhanced pathogen killing. Conversely, IL-12 suppression may lead to a predominant Th2 type response to Candida infections. Our previous studies indicate that the factor(s) responsible for inhibition of HKCA stimulated IL-12 associated with virulent strains of C. albicans at serum culture condition, is released into the media as a soluble secretory IL-12 inhibitory factor (CA-SIIF) [8]. In this study, to exclude the possible effect of serum and HKCA on further characterization and purification of this protein, we collected CA-SIIF at serum-free condition and induced IL-12 by IFN-γ priming and LPS stimulation, and compared different doses with media control and another supernatant obtained from the non-pathogenic control, Saccharomyces cerevisiae. Our results suggested CA-SIIF is serum-independent but CA-specific, and 200 μg CA-SIIF from serum-free conditions is enough to show a significant inhibitory effect on IL-12 production by 1 × 106 human monocytes. This finding was used as background for performing further investigations on CA-SIIF with human monocytes.
Macrophages, in many cases, are localized monocytes entering damaged tissue through blood vessel epithelium with a series of changes in morphology and biological functions. We speculate that since human monocytes are suppressed in their ability to produce IL-12 following exposure to CA-SIIF, that this same phenotype can be predicted for murine macrophages as well. Therefore, we established a mouse model to investigate the impact of CA-SIIF on IL-12 production by murine macrophages in vitro and in vivo. Treatment with LPS and IFN-γ resulted in stimulation of IL-12 production, while in vitro addition or in vivo injection of CA-SIIF repressed IL-12 production. In this regard, IL-12 production in TG-elicited murine peripheral macrophages was decreased by 32% and 45% in vitro and in vivo respectively, following CA-SIIF treatment. This demonstrates the same trend as human Monocytes. Human monocytes stimulated by LPS and IFN-γ were inhibited approximately 67% by CA-SIIF, as shown above in Figure 2. The difference in the levels of IL-12 inhibition between murine macrophages and human monocytes might be related to the state of cell differentiation (monocytes versus macrophages). Significantly, these results showed the ability of murine-derived cells to respond to CA-SIIF in a fashion similar to that observed for human immune cells, demonstrating the clinical relevance of our results.
It also has been reported that, when treated with C. albicans, peripheral blood mononuclear cells produce MCP-1, RANTES and IL-8 [17, 18], and elevations of MCP-1 level in human alveolar macrophages are also induced [19]. Provided IL-12 is inhibited by CA-SIIF, it had been our interest to explore whether CA-SIIF has any inhibitory effect on other pro-inflammatory cytokines and chemokines. Our data shows that addition of CA-SIIF repressed various pro-inflammatory cytokines compared to IFN-γ/LPS-stimulated monocytes alone. Pro-inflammatory cytokines or chemokines such as IL-8, IL-12, MCP-2, MIP-1Δ, RANTES and two generally implicated in allergic responses, IL-13 and Eotaxin-2, were down-regulated by monocytes exposure to CA-SIIF. Also, three cancer-associated proteins, GRO, LIF, TIMP-2 were also down-regulated, along with Leptin, an energy intake and expenditure regulator. It is also worth mentioning that levels of the anti-inflammatory cytokine IL-10 were also reduced significantly, which indicates a complex interaction of cytokines and chemokines in response to fungal infections. Interestingly, a newly reported role for IL-17A in host defense against C. albicans infection has also been published [20]. These authors demonstrate that IL-17A plays a critical role in host defense. It is possible, although we did not examine it directly in these studies, that CA-SIIF may also suppress other anti-Candida cytokines such as IL-17.
In our study, levels of MCP-4, MIP-3α and especially MIF, a chemokine that inhibits macrophage migration, were up-regulated. Overall, large scale suppression of pro-inflammatory cytokines or chemokines and up-regulation of specific anti-inflammatory factor like MIF suggests C. albicans' ability to use CA-SIIF to suppress inflammatory effects of immune cells. Such ability may contribute to possible refractory Candida infections in patients. However, whether some of the cytokines or chemokines' differential expression were the result of a larger scale cytokine/chemokine cross-talk remains unknown.
Dendritic cells, which are differentiated from precursor monocytes, express Toll-like receptors and other surface receptors interacting with pathogens, which play an active role in host protection against Candida infections, especially in the aspect of antigen presentation [21]. Since ERK and p38 MAPK are involved in CA-SIIF's inhibitory effect [8] and reciprocally regulate the differentiation of monocyte-derived dendritic cells [13], we suspected that the derivation from monocytes to dendritic cells might also be inhibited by CA-SIIF. By measuring cell surface markers specific to monocytes or derived dendritic cells through two color immunofluorescence staining flow cytometry, we found CA-SIIF significantly decreased CD1a expression on monocyte-derived dendritic cells induced by GM-CSF and IL-4. This provides us an increased understanding of other aspects of CA-SIIF inhibitory effect on monocytes, in terms of preventing them from becoming more specific and mature dendritic cells, which are responsible for local antigen presentation and establishment of (Th1) protective immune responses against Candida infection. Nevertheless, a recent report discovered that, even though subpopulations of moncoyte-derived dendritic cells (MoDCs) are phenotypically related to CD34 positive stem cell-derived dendritic cells (CD34DCs), MoDCs express a specific integrin VLA-6 but CD34DCs does not. Additionally, the adhesion and binding to components of cutaneous extracellular matrix between the two also differ, which suggests more investigations need to be performed before we draw a simple conclusion of CA-SIIF's effect on other dendritic cells subpopulations [22].
Many biologically functional proteins are glycosylated. Recent study of C. albicans secreted proteinaceous materials by proteomic analysis suggests a large portion of glycosylated proteins, of which many are also similar to the components present in cell wall/surface fractions and were generally not considered within the classical Candida secretome [23]. In our previous study, we hypothesized and performed preliminary biochemical analysis to show that IL-12 inhibitory activity of CA-SIIF might be due to the presence of a carbohydrate(s) [8]. Thus, in this study, to determine whether CA-SIIF is glycoprotein in nature, we performed one-step purification of CA-SIIF by passing the crude CA-SIIF preparation through commercially-made ConA lectin column. Next, we evaluated the inhibitory effects of fractionated CA-SIIF on monocyte IL-12 production. Our results showed that the ConA-bound glycoprotein fraction had a higher inhibitory efficiency (fold decrease/mg sample), compared to the non-glycoprotein fraction and the crude CA-SIIF preparation. These results clearly demonstrated that the inhibitory activity of CA-SIIF is mediated by glycoprotein(s) rich in high mannose-type and hybrid-type oligosaccharides [14]. This purified CA-SIIF fraction allows us to perform more detailed biochemical and molecular analyses regarding the mechanism of action of this factor in the future.
Several studies have reported β-glucans isolated from Candida cell walls to exhibit immunomodulatory activities [24–26]. However, isolation of glucans from Candida cell walls involves stringent extraction steps, including treatment with NaClO and dimethylsulfoxide [26], and hot acid and alkali treatments [25, 27]. While presence of serum also induces secretion of glucans by C. albicans [27], our studies revealed that the IL-12 inhibitory activity of CA-SIIF was retained even in the absence of serum. Furthermore, we found that IL-12 inhibitory activity of CA-SIIF is abrogated by proteinase treatment, indicating this activity is retained in the protein fraction of CA-SIIF and is not due to glucan.
It has been previously shown that, after phagocytosis of C. albicans yeast forms, monocytes failed to differentiate to dendritic cells and their IL-12 production was also inhibited. In contrast, while phagocytosis of germ tube forms of C. albicans leads to inhibition of IL-12, the maturation from monocytes to dendritic cells remains unaffected [28]. Another study showed that hyphal-form of C. albicans can suppress IL-12 production even in the absence of phagocytosis [29]. Results described in the current study, and our previous study showed that IL-12 inhibition can also be achieved by soluble factors secreted by C. albicans [8]. The growth medium (RPMI-1640) used in these studies is known to induce hyphal formation and we did see more hyphae when collecting CA-SIIF, suggesting CA-SIIF is possibly secreted more by hyphal-form of C. albicans. Since hyphal forms of C. albicans are generally associated with increased virulence, it is possible that CA-SIIF could have a larger role in disease, contributing to the pathogenicity of this organism. However, further studies need to be done to confirm this hypothesis. Release of C. albicans proteins or molecules that regulate cytokine production is in agreement with other studies. For example, the C. albicans Water-Soluble Mannoprotein-β-glucan Complex (CAWS), which resembles the free β-1,3-D-glucan in patient blood, is known to modulate the growth and cytokine production of murine macrophage cell line [30]. A recent study has also found that farnesol pretreatment reduced both IFN-γ and IL-12, but not TNF-α and exhibited IL-5 increase [15].
Taken together, our studies suggest another novel way that C. albicans may suppress the immune response, namely by secreting CA-SIIF, which can modulate the Th1 protective immune responses, immune cell differentiation, and inflammatory responses.