Importance of Candida Antigenic Factors: Structure-Driven Immunomodulation Properties of Synthetically Prepared Mannooligosaccharides in RAW264.7 Macrophages

The incidence and prevalence of serious fungal infections is rising, especially in immunosuppressed individuals. Moreover, co-administration of antibiotics and immunosuppressants has driven the emergence of new multidrug-resistant pathogens. The significant increase of multidrug-resistant pathogens, together with their ability to form biofilms, is associated with morbidity and mortality. Research on novel synthetically prepared immunomodulators as potential antifungal immunotherapeutics is of serious interest. Our study demonstrated the immunobiological activity of synthetically prepared biotinylated mannooligosaccharides mimicking Candida antigenic factors using RAW264.7 macrophages. Macrophage exposure to the set of eight structurally different mannooligosaccharides induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The observed immune responses were tightly associated with structure, dose, exposure time, and selected signature cytokines. The viability/cytotoxicity of the mannooligosaccharide formulas was assessed based on cell proliferation. The structure-based immunomodulatory activity of the formulas was evaluated with respect to the length, branching and conformation of the various formulas. Glycoconjugate formulas with terminal β-mannosyl-units tended to be more potent in terms of Candida relevant cytokines IL-12 p70, IL-17, GM-CSF, IL-6, and TNFα induction and cell proliferation, and this tendency was associated with structural differences between the studied glycoconjugate formulas. The eight tested mannooligosaccharide conjugates can be considered potential in vitro immunomodulative agents suitable for in vitro Candida diagnostics or prospectively for subcellular anti-Candida vaccine design.


INTRODUCTION
Most Candida species, including the facultative pathogenic strains, belong to the normal commensal mycobiota of immunocompetent individuals. The factors affecting the candidosis are diverse, including the prolonged antifungal treatment in long-term care, immunosuppression associated with anticancer therapy and transplantation of solid organ or bone marrow, immunosuppressive states as diabetes mellitus and HIV, use of vascular devices and hospitalization at intensive care units (Richter et al., 2005;Angiolella et al., 2008;Adiguzel et al., 2010;Cortés and Corrales, 2018). Next, immunocompromised persons with genetic immune system defects are at high risk for mucocutaneous and invasive fungal infections (Vinh, 2011;Cunha and Carvalho, 2012;Pichard et al., 2015;Beenhouwer, 2018). Approximately 17 different Candida species are known etiological agents of human infections; more than 90% of systemic infections are caused by Candida albicans (C. albicans), Candida glabrata (C. glabrata), Candida parapsilosis (C. parapsilosis), Candida tropicalis (C. tropicalis), and Candida krusei (C. krusei) (Pfaller et al., 2002). The new multidrugresistant species Candida auris (C. auris) was recently isolated (Sears and Schwartz, 2017;Forsberg et al., 2019). CD4 + -derived T-cell subpopulations Th1, Th2, and Th17 contribute to anti-Candida cellular immune protection. The protective anticandidal Th1 response requires the activity of various cytokines, such as interferon gamma (IFN-γ), transforming growth factor beta (TGF-β), interleukin 6 (IL-6), tumor necrosis factor alpha (TNFα), and IL-12. The induction of the protective antifungal Th1 immune response is inhibited by Th2 cytokines, such as IL-4 and IL-10 (Ito, 2011;Netea et al., 2015;Richardson and Moyes, 2015;Gow et al., 2017). In early infection, neutralization of Th1 cytokines, mainly IFN-γ and IL-12, leads predominately to the onset of Th2 rather than Th1 responses. Th2-type responses are frequently associated with susceptibility to recurrent or persistent infection and fungal allergy. TNFα, IL-1β, IL-6, IL-8, and colonystimulating factors (CSFs) are among the major proinflammatory cytokines associated with the interaction of immune-competent cells with Candida cells. TNFα is thought to be essential in the primary control of disseminated infection caused by C. albicans. Although IL-1 shares common properties with TNFα, both IL-1β and IL-6, acting mainly through recruitment of polymorphonuclear neutrophils (PMNs), presumably are not as essential as TNFα in the innate antifungal response. IL-12 is recognized as essential to induce the protective Th1 response to the fungus, simultaneously blocking the Th2 response. The crucial role of the Th17 subset has been associated with anti-Candida effectiveness, especially the mucosal immune response (Romani, 2003;Rizzetto et al., 2010;van de Veerdonk and Netea, 2010). Proinflammatory cytokines, such as IL-12, IL-15, and TNFα, have been studied as candidate adjuvants in preclinical trials based on their ability to upregulate the antifungal Th1 response (Ashman and Papadimitriou, 1995;Romani, 2011;Pikman and Ben-Ami, 2012;Naglik, 2014).
Mannan has also been studied as a promising bioactive material for drug nanocarrier systems and vaccine adjuvant formulations (Tang et al., 2009). Moreover, nanoliposomes with orthogonally bound mannan represent a platform for the development of targeted drug delivery systems and self-adjuvanted carriers for construction of recombinant vaccines (Bartheldyova et al., 2019). Concerning the design of anti-fungal vaccination therapy, apart from Candida cell wall moieties, potential new anti-Candida drugs have targeted the growth and virulence factors of C. albicans, including core signaling components of the high-osmolarity glycerol (HOG) and target of rapamycin (TOR) signaling pathways (Li et al., 2015), as well as various immunomodulators, e.g., colony-stimulating factors and proinflammatory cytokines (Pikman and Ben-Ami, 2012).
Natural Candida mannan is a complex polysaccharide structure containing linear and branched fragments composed of α-and β-mannose units, as seen in Figure 1 (Klis et al., 2001), with the carbohydrate sequences represented according to symbol carbohydrate nomenclature (Varki et al., 1999). Thus, the use of such a heterogenic structure is problematic for the assessment of the biological role of its distinct fragments. However, the application of synthetic mannooligosaccharide derivatives, related to the structures of selected antigenic factors of Candida mannan, creates the opportunity to assess the biological roles of each antigenic factor.
Our work investigated the immunomodulation properties of antigenically active distinct parts of C. albicans mannan by using a series of structurally related synthetic mannooligosaccharides.

Isolation of Natural Cellular Mannan and Preparation of FITC-Labeled Mannan
The yeast strain Candida albicans CCY 29-3-100 (serotype A) (CCY Culture Collection of Yeasts, Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia) was used to isolate and purify cellular mannan from fresh biomass. Mannan was extracted by autoclaving in 0.2 mol/l NaCl (120 • C, 700 kPa) for 10 min and purified using precipitation with Fehling reagent according to a previously described method (Peat et al., 1961).
For the preparation of FITC-labeled mannan, C. albicans CCY 29-3-100 mannan (54 mg) was suspended in 1.00 mL of dimethyl sulfoxide and 2.0 µL of pyridine was added. The suspension was heated in a thermoblock at 95 • C until the mannan dissolved (3 h). Then, 20 mg of isothiocyanatofluorescein (FITC) was added and heated for another 2 h at 95 • C. The reaction was quenched by addition of 10 mL of water, and the result was dialyzed using cellulose membrane tube (cut-off = 14,000, Sigma) against 0.05 % NaHCO3 (1 × 0.9 L, 4 h stirred) and deionized water [8 × 0.9 L, 4 h on stirrer or 12 h in the refrigerator (5 • C)] and then lyophilized (FreeZone 18 Liter Console Freeze Dry System, Labconco Corporation, Kansas City, USA).

Preparation of Stock Solutions of Natural Cellular Mannan and Synthetically Prepared Mannooligosaccharides
Stock solutions and different dilutions of natural cellular mannan and glycoconjugate formulas 1-8 were prepared aseptically using pre-sterilized disposable plastic wares and sterile, apyrogenic aqua pro injectione (Fresenius Kabi Italia S.r.l., Verona, Italy). All solutions were prepared in a laminar flow hood and sterilized using a 0.2-µm filter (Q-Max R Syringe filter, Frisenette ApS, Knebel, Denmark) before exposure. The laminar flow cabinet was sterilized with 70% ethanol p.a. and UV for 30 min prior to each experiment. The stock solutions were assayed with EndoLISA R ELISA-based Endotoxin Detection Assay (Hyglos, Bernried am Starnberger See, Germany) and evaluated using the Cytation 5 Imager Multi-Mode Reader (BioTek, Winooski, USA) to ascertain endotoxin-free exposure conditions.

Cell Maintenance and Culture, Cell Exposure
The murine macrophage-like RAW 264.7 cell line was selected in the present study because this cell model has been frequently used in in vitro studies on phagocytosis, cytokine production, and to evaluate potential bioactive substances to predict their effect in vivo.

Cell Proliferation and Cytotoxicity
The influence of glycoconjugates on RAW 264.7 cell proliferation and cytotoxicity was evaluated using the cell proliferation assay ViaLight TM plus kit (Lonza, Rockland, ME, USA) according to the manufacturer's recommendations. Cellular ATP was determined with luciferase-based luminescence quantification. The intensity of emitted light was measured using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc.). Light emission was recorded continuously for 1 s and peak values were evaluated and expressed as relative light units (RLU). The values of unexposed cells were considered the baseline. The proliferation index was calculated as the ratio between the stimulated cells (glycoconjugate formula-treated cells) and the baseline proliferation of unexposed cells. Thus, the proliferation index of the negative control, i.e., unexposed cells, was equal to one.
To compare the effect of different glycoconjugates on RAW 264.7 macrophage interleukins and growth factors, analyses were performed on raw cytokine concentration data and cytokine concentration data normalized to viable cell counts of untreated control RAW 264.7 cells. The raw concentrations of cytokines determined by ELISA were divided by the RLU [ATP detection systems to quantify viable cells, ViaLightTM plus kit (Lonza, USA)] of living cells in a corresponding sample and multiplied by the RLU of untreated control RAW 264.7 cells.

Statistical Analysis
The experimental results were expressed as mean values ± SD. Normality of data distribution was established according to the Shapiro-Wilk test at the 0.05 level of significance. Statistical comparisons were performed by one-way ANOVA and post-hoc Bonferroni tests. Pearson's correlation coefficient was used to compare the strength of the relationship between immunobiological variables. Results were significant when the differences equaled or exceeded the 95% confidence level (P < 0.05). Statistics were performed using ORIGIN 7.5 PRO software (OriginLab Corporation, Northampton, USA).
As natural Candida mannan (section Isolation of natural cellular mannan and preparation of FITC-labeled mannan) was utilized in all experiments as a comparative substance, evaluation of its interaction with RAW 264.7 cells was essential. Fluorescently labeled natural mannan was used to visualize the cell interaction and endocytotosis of Candida mannan by the murine macrophage RAW 264.7 cells. Evaluation of the interaction was performed with light and fluorescence microscopy ( Figure 3A) and 3D Z-stack imaging ( Figure 3B). The patterns documented the ingestion of mannan and its inclusion into subcellular compartments.

Interactions of Natural Mannan and Glycoconjugate Formulas 1-8 With Murine
Macrophage Cell Line RAW 264.7, and Influence on Cell Proliferation The effect of glycoconjugate formulas 1-8 on macrophage cell line RAW 264.7 proliferation was monitored by adenosine triphosphate (ATP) bioluminescence as a marker of cell viability (Figure 4). The lower concentration of glycoconjugate formulas 1-4 (10 µg/mL, Figure 4A) slightly decreased the proliferation of RAW 264.7 macrophages. Improved proliferation was observed for formula 1, which is comprised of three α-1,2-Man units (24 h treatment). The higher concentration of formulas 1-4 (100 µg/mL, Figure 4A), which are comprised exclusively of α-linkages between Man residues, significantly decreased the proliferation of RAW 264.7 macrophages (between 94 and 98% reduction). As opposed to the α-mannooligosaccharides, treatment of RAW 264.7 macrophages with formulas 5-8, which also contain β-1,2-linked Man units, slightly increased proliferation after 24 h, and the increase was more significant after 48 h stimulation ( Figure 4B). The highest proliferations were observed for the 10 µg/mL concentration of tetramer formula 5, which contains one terminal β-1,2-linked Man unit (2.1 times higher than control), and hexamer formula 8, which contains a tetrameric block of β-1,2-linked Man units (2.2 times higher than control). The proliferation of RAW 264.7 macrophages treated by glycoconjugates for 48 h was significantly lower (formulas 1-4: p < 0.01, formulas 5-8: p < 0.01) compared with natural C. albicans mannan (M, Figure 4C).

Cytokine Responses of RAW 264.7
Macrophages in vitro to Glycoconjugate Formulas 1-8 The in vitro stimulatory effect of glycoconjugate formulas 1-8 on RAW 264.7 macrophage cytokine production was determined by the levels of pro-inflammatory cytokines TNFα, IL-6, IL-17, IL-12, anti-inflammatory cytokine IL-10, and haemopoietic growth factor GM-CSF in supernatants obtained from cultures of RAW264.7 macrophages after 24 or 48 h treatments [not normalized raw cytokine concentrations (Supplementary Figures 1, 2) and cytokine concentrations normalized to viable cell counts of untreated control RAW 264.7 cells (Figures 5, 6)].
Non-normalized raw cytokine concentrations data showed that stimulation of RAW 264.7 cells with the lower concentration of glycoconjugate formulas 1-4 (10 µg/mL), which contain linked Man residues, resulted in a slight increase of TNFα production; maximal effect was observed for formula 1 (24 h treatment: 1.37-fold increase and 48 h treatment: 1.48fold increase, Supplementary Figure 1). The stimulation of RAW 264.7 macrophages with the higher concentration of glycoconjugate formulas (100 µg/mL) significantly decreased TNFα production (more than 70% decrease compared to the control). However, IL-6 and GM-CSF production showed different concentration dependencies. The higher concentration of glycoconjugate formulas 1-4 (100 µg/mL) induced comparable or higher IL-6 and GM-CSF secretion than the lower concentration (10 µg/mL) (Supplementary Figure 1). The highest IL-6 and GM-CSF release was observed for glycoconjugate formula 3 (IL-6: 3.2-fold increase, GM-CSF: 1.9-fold increase).
Due to the tested glycoconjugates having a significant effect on RAW 264.7 macrophage proliferation, especially for glycoconjugate formulas 1-4 that contain α-linked Man residues, the raw data of cytokine concentrations in the culture supernatants were normalized to the viable cell counts of untreated control RAW 264.7 cells for each experiment. We observed that the normalization of cytokine concentration data showed no significant trend change for stimulation of RAW 264.7 macrophages with the β-mannooligosaccharide glycoconjugates (formulas 5-8) (Figure 6). Out of all tested β-mannooligosaccharide glycoconjugates, the most effective cytokine inducers were glycoconjugate formulas 6 and 8. The highest TNFα (24 h: 23.2-fold increase), IL-6 (24 h, 38.5-fold increase), IL-12 (24 h: 15.6-fold increase), and IL-10 (24 h: 15.3-fold increase) secretion was induced by glycoconjugate formula 6. The GM-CSF (24 h: 14.5-fold increase) and IL-17 (24 h: 3.1-fold increase) was most effectively induced by βmannooligosaccharide glycoconjugate formula 8. Normalization of the cytokine concentration data after stimulation with α-mannooligosaccharide glycoconjugates (formulas 1-4) (Figure 5) accentuated the release of cytokines induced by the higher concentration of glycoconjugates (100 µg/mL). We observed significant capability to induce TNFα, IL-6, GM-CSF, IL-17, and IL-12 production accompanied by an increase of IL-10 after stimulation with all α-mannooligosaccharide glycoconjugates, induced especially with higher 100 µg/mL concentration, that strongly reduced the proliferation of RAW 264.7 cells. The highest production of TNFα, IL-6, GM-CSF, and IL-10 was observed after the shorter exposure time (24 h) with glycoconjugate formula 3.
The influence of glycoconjugate formulas 1-8 on Th1 and Th2 polarization was revealed based on the TNFα (Th1) to IL-10 (Th2) and IL-6 (Th2) to IL-10 (Th2) ratios (Figure 7). Th1 dominance was represented by a higher ratio, while a lower ratio expressed a Th2 dominated environment. Concerning the ratios following 24 and 48 h exposures with 100 and 10 µg/mL of glycoconjugate formulas 1-4, Th1 dominance based on the TNFα/IL-10 ratio was revealed for conjugate formulas 1 and 4, while conjugate formulas 2 and 3 exerted Th1 dominance with higher TNFα/IL-10 ratios over IL-6/IL-10 ratios only at the lower concentration (10 µg/mL) after 48 h exposure. For conjugate formulas 5-8, the values of the TNFα/IL-10 ratios overcame the values of the IL-6/IL-10 ratios following 24 and 48 h exposures with both concentrations for all conjugates, reflecting Th1 dominance.
Moreover, the sera reactivity and determination of antigenspecific isotypic antibodies against synthetically prepared mannooligosaccharides were evaluated in a cohort of patients with vulvovaginal candidosis Paulovicova et al., 2016Paulovicova et al., , 2017. Postvaccination antisynthetic heptamannoside polyclonal sera inhibited growth of the azole-resistant clinical strain C. albicans CCY 29-3-164 and reduced the number of colony-forming units throughout an experimental mucosal infection . Next, sera cytokine patterns of Th1/Th2/Th17 polarization of immune responses FIGURE 7 | Influence of glycoconjugate formulas 1-8 on Th1 and Th2 polarization based on TNFα to IL-10 and IL-6 to IL-10 ratios.
Here, the proliferating and cytokine-inducing activities of a series of synthetically prepared nanopolymers mimicking native C. albicans cell wall immunogenic moieties were studied using RAW264.7 cell exposure (Figure 4). The cell proliferation results revealed almost immunoinhibitory activity of α-mannoside formulas 1-4 (trisaccharide through hexasaccharide), with more pronounced activity with increasing concentration (p < 0.001), in contrast with native C. albicans cell wall mannan (Figure 4). These findings concur with previously published studies (Podzorski et al., 1989(Podzorski et al., , 1990) that reported the immunoinhibitory influence of members of a family of mannose oligosaccharides (disaccharide through hexasaccharide) derived from cetyltrimethylammonium bromide (CTAB) mannan (native C. albicans mannan prepared by complexation with CTAB). CTAB mannan was a potent stimulator of lymphoproliferative when added to human peripheral blood mononuclear cells (PMBCs) from donors responsive to Candida; it had no inhibitory influence on lymphoproliferation induced by Candida or other antigens. Two major oligomannosyl components, mannobiose and mannotriose, of CTAB mannan with an inhibitory effect on cell proliferation were demonstrated to be bound mainly through α(1,2) linkages (Hayette et al., 1992). In contrast, synthetically prepared mannooligosaccharide formulas 5-8 with terminal β-mannosyl units (Figure 3) exerted a stimulatory effect on RAW264.7 cell proliferation (p < 0.001). Thus, the immunobiological properties of the studied mannooligosaccharides are dose-and structuredependent. Cell release of interleukins and growth factors associated with inflammation and proliferation was induced by mannooligomers to different extents depending on the oligomer structures (normalized data: Figures 5, 6, not normalized data: Supplementary Figures 1, 2).
Upregulation of cytokines such as TNFα, IL-6, IL-12, GM-CSF was more evident with mannooligosaccharides with terminal β-mannosyl units. Acceleration of secretion of anti-inflammatory cytokine IL-10 with Th1-inhibiting properties was also revealed with β-mannooligosaccharides (Supplementary Figure 2). Association between pro-and anti-inflammatory cytokines, in addition to Th1, Th2 and Th17 polarization, is an important prerequisite for the assessment of immunogenic substance behavior. The influence of glycoconjugate formulas 1-8 on Th1 and Th2 polarization, based on TNFα to IL-10 and IL-6 to IL-10 ratios (Figure 7), resulted in a predominant Th1 immune response. Th1 dominance, represented by higher TNFα/IL-10 and IL-6/IL-10 ratios with higher dose and following prolonged treatment, was revealed with tested 1-8 formulas, and was more evident for formulas 5-8 with β-terminal mannosyls; the Th2 dominated environment was not determined. The observed immune response was tightly associated with dose, exposure time, and selected signature cytokines. The TNFα/IL-10 ratio was more descriptive than the IL-6/IL-10 ratio, presumably due to dual IL-6 roles, i.e., anti-inflammatory activities of IL-6 are mediated by classic signaling, whereas proinflammatory responses of IL-6 are mediated by trans-signaling (Scheller et al., 2011). Saijo et al. reported induced release of cytokines, such as IL-12p40, IL-6, TNFα, and IL-10, from wild-type bone marrow-derived dendritic cells (BMDCs) by treatment with C. albicans water soluble fraction (CAWs) and C. albicans mannans (Saijo et al., 2010). Moreover, they also observed the secretion of yeast-and hyphae-specific cytokines following cell exposure with both C. albicans morphoforms. C. albicans mannan, glucomannoprotein and phospholipomannan, containing β-1,2 oligomannosides, induced TNFα in association with degree of polymerization (DP). Jouault et al. noted that TNFα-release occurred most in the presence of relatively long chains of β-oligomannosides (i.e., an oligomannoside comprised of eight mannose units was superior to shorter chains, and oligomannosides of less than four mannose units were not active) (Jouault et al., 1995). Evidently a minimal DP of 4 was necessary to induce production of cytokine (Poulain et al., 1997). With synthetically prepared α-and β-oligomannosides, effective TNFα release was triggered by trimannosides. The ability of β-oligomannosides to induce release of TNFα was also demonstrated by Cutler (2001).

CONCLUSIONS
Our data suggest an immunobiological role for synthesized mannooligosaccharides that closely resemble Candida cell wall mannooligomers. The observed Th1/Th2/Th17 immune responses were tightly associated with structure, dose, exposure time, and selected signature cytokines. Glycoconjugate formulas 5-8, with terminal β-mannosyl-units, tended to be more potent than glycoconjugate formulas 1-4 in terms of Candida relevant cytokines IL-12 p70, IL-17, GM-CSF, IL-6, and TNFα induction and cell proliferation, and this tendency was associated with structural differences between the studied glycoconjugate formulas. Obtained results warrant further systematic investigation of the immunological properties of carbohydrate antigens of the Candida cell wall toward the selection of efficient structures suitable for application as immunomodulative agents either for in vitro Candida diagnostics or prospectively for subcellular anti-Candida vaccine design.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
EP, LP, and NN contributed to the conception and design of the study, performed the immunobiological research and analyzed data, acquired funding, and prepared the original draft. PF performed the modification and characterization of mannan. AK, YT, and VK performed the chemical syntheses and analyzed data. All authors contributed to manuscript revision, read, and approved the submitted version.

FUNDING
The synthetic part of this work was supported by the Russian Science Foundation (grant 19-73-30017), while the immunological part of the work was supported by the Scientific Grant Agency of the Slovak Republic VEGA (project 2/0098/17) and the Slovak Research and Development Agency (contract N • APVV-15-0161).