Synthesis of Biotin-Tagged Chitosan Oligosaccharides and Assessment of Their Immunomodulatory Activity

Chitin, a polymer of β-(1→4)-linked N-acetyl-d-glucosamine, is one of the main polysaccharide components of the fungal cell wall. Its N-deacetylated form, chitosan, is enzymatically produced in the cell wall by chitin deacetylases. It exerts immunomodulative, anti-inflammatory, anti-cancer, anti-bacterial, and anti-fungal activities with various medical applications. To study the immunobiological properties of chitosan oligosaccharides, we synthesized a series of β-(1→4)-linked N-acetyl-d-glucosamine oligomers comprising 3, 5, and 7 monosaccharide units equipped with biotin tags. The key synthetic intermediate employed for oligosaccharide chain elongation, a disaccharide thioglycoside, was prepared by orthogonal glycosylation of a 4-OH thioglycoside acceptor with a glycosyl trichloroacetimidate bearing the temporary 4-O-tert-butyldimethylsilyl group. The use of silyl protection suppressed aglycon transfer and provided a high yield for the target disaccharide donor. Using synthesized chitosan oligomers, as well as previously obtained chitin counterparts, the immunobiological relationship between these synthetic oligosaccharides and RAW 264.7 cells was studied in vitro. Evaluation of cell proliferation, phagocytosis, respiratory burst, and Th1, Th2, Th17, and Treg polarized cytokine expression demonstrated effective immune responsiveness and immunomodulation in RAW 264.7 cells exposed to chitin- and chitosan-derived oligosaccharides. Macrophage reactivity was accompanied by significant inductive dose- and structure-dependent protective Th1 and Th17 polarization, which was greater with exposure to chitosan- rather than chitin-derived oligosaccharides. Moreover, no antiproliferative or cytotoxic effects were observed, even following prolonged 48 h exposure. The obtained results demonstrate the potent immunobiological activity of these synthetically prepared chito-oligosaccharides.


INTRODUCTION
The fungal cell wall protects the cell from environmental stresses and is essential for cell morphogenesis and pathogenicity (Bowman and Free, 2006;Gow and Hube, 2012;Erwig and Gow, 2016;Gow et al., 2017). It comprises mostly polysaccharides of different types (Latgé, 2010) that account for ∼90% of the cell wall, each carrying out a specific biological function. Chitin, a homopolymer of β-(1→4)-linked N-acetyl-D-glucosamine, is an important constituent of the innermost layer of the fungal cell wall and forms a covalent complex with β-(1→3)-glucan, which is responsible for the mechanical strength and integrity of the cell wall (Free, 2013). Chitin can undergo enzymatic N-deacetylation in the fungal cell wall by chitin deacetylases to form chitosan, a polymer of β-(1→4)-D-glucosamine or its copolymer with N-acetyl-D-glucosamine. The biological role of chitosan in the cell wall is not completely clear; however, research suggests that chitosan is important for morphogenesis (Geoghegan and Gurr, 2016;Upadhya et al., 2018), cell integrity (Baker et al., 2007), and virulence (Baker et al., 2011;Geoghegan and Gurr, 2016;Upadhya et al., 2018). Chitosan can be either prepared by chemical deacetylation of chitin or isolated from the cell walls of Zygomycota or other fungi, such as Basidiomycota, e.g., Cryptococcus neoformans. Cell wall polysaccharides are also engaged in the pathogen-associated molecular patterns associated with interactive crosstalk between immunocytes and immune sensing of the host organism, triggering various immune responses-innate and/or adaptive. FIBCD1, NKR-P1, and RegIIIc have been identified as chitin-binding pattern recognition receptors in mammals. Other receptors that participate in the mediation of immune responses to chitin are Toll-like receptor (TLR) 2, dectin-1, and mannose receptor CD 206 (Bueter et al., 2013). Recently, Wagener et al. (2014) identified NOD2 and TLR9 as essential fungal chitin-recognition receptors engaged in chitin-induced selective secretion of IL-10. Studies by Da Silva and Kogiso (Da Silva et al., 2009;Kogiso et al., 2011) suggested the importance of chitin particle size on the character of the elicited immune response; large particles (>40 µm) reportedly induced a classical Th2 response, whereas small particles (1-10 µm) induced both protective Th1, and anti-inflammatory responses.
Generally, immune sensing and recognition of fungi is cooperating with the interactive system of microbial-, pathogen-, and danger-associated molecular patterns (MAMPs, PAMPs, and DAMPs) engaged in interactions with soluble-and cellassociated pattern recognition receptors (PRRs). The synthetic glycans and oligosaccharides with well-defined composition are appropriate structures to characterize the minimal acquired immunobiologically active structures-referred as antigenic determinants or epitopes engaged in immune responses. Several synthetic immunogenic oligosaccharides partially mimicking the structure of fungal cell wall PAMP molecules, sensed by germline encoded PRRs and engaged in innate immune system cells signaling, have been designed and constructed (Xin et al., 2008;Costello and Bundle, 2012;Dang et al., 2012;Cartmell et al., 2015;Colombo et al., 2018 etc.). The immunobiological effectivity of the synthetically prepared oligosaccharides mimicking the natural fungal cell wall glycans demonstrated in vitro and in vivo the induction of effective cell proliferation, cell phagocytosis, Tand B-cell responses, macrophage secretion of Th1, Th2, Th17, and Treg inflammatory and anti-inflammatory interleukins, and growth factors (Paulovičová et al., 2013(Paulovičová et al., , 2014(Paulovičová et al., , 2015(Paulovičová et al., , 2017Karelin et al., 2016).
Difficulties associated with isolation and purification, poor solubility, and possible irregularity of fungal polysaccharides themselves contribute to the inconvenience of studying their functions, immunological properties, and biological activities. Chemically synthesized regular polysaccharides might provide useful substitutes; however, polysaccharide synthesis is rather complex and time-consuming (Kochetkov et al., 1987). For this reason, synthetic oligosaccharides that are structurally related to fungal polysaccharides take on special significance for investigation of their immunological and other biological activities.
In particular, over the past few years, we have synthesized an array of biotinylated synthetic oligosaccharides representing fragments of fungal cell wall polysaccharides on streptavidincoated surfaces (Krylov and Nifantiev, 2020). Such arrays have been applied to assess carbohydrate specificity of monoclonal antibodies (Matveev et al., 2018(Matveev et al., , 2019Krylov et al., 2019;Schubert et al., 2019;Kazakova et al., 2020) and polyclonal antibodies in model sera induced by immunization of animals with cell wall preparations of various fungal species (Komarova et al., 2015;Paulovičová et al., 2016;Krylov et al., 2018a,b), as well as in sera obtained from patients with fungal infections (Komarova et al., 2018;Kazakova et al., 2020;Wong et al., 2020).
Here, we report the efficient synthesis of biotin-tagged chitosan oligomers composed of 3, 5, and 7 glucosamine units. Using synthesized chitosan oligomers, as well as previously prepared chitin counterparts (Yudina et al., 2016), we demonstrate the immunomodulative activities of synthetically prepared chitin-and chitosan-derived oligosaccharide formulas of various lengths and evaluate the structure-immunomodulation relationship.

Chemistry
General Chemicals were purchased from Acros Organics (Geel, Belgium) and Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. All moisture-sensitive reactions were carried out using dry solvents under dry argon. Solutions were concentrated under reduced pressure using a rotatory evaporator at 40 • C (bath temperature).
NMR spectra were obtained using a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA). Protected oligosaccharides were measured in chloroform-d (CDCl 3 ), and 1 H NMR chemical shifts were referenced to the solvent residual signal (δ H 7.27). 13 C chemical shifts were referenced to the central resonance of CDCl 3 (δ C 77.0). Free oligosaccharides were measured in deuterium oxide (D 2 O) using acetone (δ H 2.225, δ C 31.45) as an internal standard. Signal assignment was made using COZY, TOCSY, and HSQC experiments. Unit A refers to the reducing end monosaccharide in the description of NMR data. NMR spectra of synthesized compounds are presented in the Supplementary Material.
Optical rotations were measured using a JASCO DIP-360 polarimeter at 18-22 • C in the specified solvents.

Preparation of Chitin-and Chitosan-Derived Oligosaccharides
Stock solutions and partial dilutions of chitosan (25-27, Scheme 3) and chitin (28-30, Scheme 3) oligosaccharide formulas were prepared aseptically using pre-sterilized disposable plastic wares (Eppendorf, Hamburg, Germany) and sterile water for injection (Fresenius Kabi Italia S.r.l., Verona, Italy). Solutions were sterilized using a 0.2-µm filter (Q-Max R ; Frisenette, Knebel, Denmark) in a laminar flow hood. The laminar flow cabinet was pre-sterilized with 70% ethanol and UV irradiation for 30 min prior to each experiment. Stock solutions were assayed with the EndoLISA R endotoxin determination kit (Hyglos, Bernried, Germany) to ascertain endotoxin-free content.

Cell Maintenance, Culture, and Cell Culture Exposure
Murine macrophage cell line RAW 264.7 cells (European Collection of Authenticated Cell Cultures, Salisbury, UK) were cultured in complete Dulbecco's Modified Eagle Medium and a high-glucose medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10,000 U/ml penicillin, and 10 mg/ml streptomycin (Sigma-Aldrich) at 37 • C in a humidified atmosphere with 5% carbon dioxide until cells reached ∼80% confluence. Cell exposure experiments were performed within 24-48 h. Cell viability was assessed using Trypan blue dye exclusion assay (TC20 TM automated cell counter (Bio-Rad Laboratories). The starting inoculum of 1.33 × 10 6 cells/ml/well (92% viable cells) was cultured in a 24-well cell culture plate (Sigma-Aldrich), and cells were exposed to 10 and 100 µg/well of oligosaccharides 25-27 and 28-30 for 24 and 48 h, respectively. Cell mitogens concanavalin A (Con A, 10 µg/ml), phytohemagglutinin (PHA, 10 µg/ml), lipopolysaccharide (LPS, 1 µg/ml), and poke weed mitogen (PWM, 1 µg/ml) (all Sigma-Aldrich) were used as positive controls. Cell morphology and viability were assayed prior to cell immunophenotyping and evaluation of phagocytosis and cytotoxicity. The cell culture media were separated and frozen at −20 • C until further use.

Determination of Cell Proliferation and Cytotoxicity
The influence of oligosaccharides 25-27 and 28-30 on cytotoxicity and proliferation of RAW 264.7 cells was evaluated using the ViaLight TM Plus bioassay kit (Lonza, Walkersville, MD, USA) according to the manufacturer's recommendations. The impact of oligosaccharide exposure on cellular adenosine triphosphate (ATP) was determined by luciferase-based luminescence quantification. Briefly, the intensity of emitted light was measured using the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc., Winooski, VT, USA). Light emission was recorded continuously for 1 s, and peak values were evaluated and expressed as relative light units (RLU). Values of unexposed cells were considered the control baseline. The proliferation index was calculated as the ratio of stimulated cell proliferation, i.e., cells treated with oligosaccharides 25-27 and 28-30, to that of unexposed cells. The proliferation index of the negative control, i.e., baseline unexposed cells, was equal to one.

In vitro Quantification of Interleukins and Growth Factors
Cytokine levels in cell culture supernatants induced by exposure to oligosaccharides were assayed using Platinum ELISA R kits: Mouse IL-12 p70 [Minimum Detectable Dose [MDD] 4 pg/ml], Mouse GM-CSF (MDD 2 pg/ml), Mouse IL-17 (MDD 1.6 pg/ml), Mouse IL-6 (MDD 6.5 pg/ml), and Mouse IL-2 (MDD 5.3 pg/ml) and Instant ELISA R kits: Mouse IFNγ (MDD 4 pg/ml), Mouse tumor necrosis factor (TNF)-α (MDD 4 pg/ml), Mouse IL-10 (MDD 5.28 pg/ml), Mouse IL-1β (MDD 3 pg/ml), and Mouse IL-4 (MDD 0.6 pg/ml), all from Thermo-Fisher Scientific (Waltham, MA, USA) according to the manufacturer's instructions. The mean percentage of phagocytic cells was represented by the percentage of cells that ingested at least one SPA-FITC particle. The mean percentage of respiratory burst was represented by the percentage of cells tagged by ethidium. The mean percentage of metabolic activity was represented by the percentage of cells that ingested at least one SPA-FITC and was tagged by ethidium.

Fluorescence Quenching Cytometric Assay
Extracellular fluorescein isothiocyanate (FITC) fluorescence was quenched by 0.4% Trypan blue dye (Sigma-Aldrich). Immunocytometric analysis of Trypan blue-treated RAW 264.7 cells was performed following cell incubation for 15 min in the dark at 37 • C. The amount of membrane-attached FITCconjugated SPA was expressed as the difference between wholecell phagocytosis, i.e., cells with cell-bound and internalized SPA-FITC, and the Trypan blue-quenched cell population. Metabolic activity was expressed as the percentage of cells simultaneously undergoing phagocytosis and oxidative burst.

Macrophage Immunophenotyping
For immunocytometric assays, RAW 264.7 cells were stained directly with fluorescein FITC-conjugated rat anti-mouse monoclonal antibodies: F4/80, CD11b, and CD14 (Thermo Fisher Scientific). The appropriate antibody isotype-negative controls were used for setting gates. The FITC-conjugated monoclonal antibodies (5 µl) and RAW 264.7 cells (50 µl) were added to the 96-well microtiter plate and incubated for 30 min in the dark at 4 • C. Then, the samples were evaluated by immunoflow cytometry (CytoFLEX, Beckman Coulter Life Sciences, Inc.).

Chitin and Chitosan
Preparation Approximately 60 ml of wet Candida albicans CCY 29-3-100 cells was extracted according to Ferreira et al. (2006) to obtain a pellet with a sufficient amount of insoluble β-1,3-glucans (and chitin). The obtained mass of 3.80 g was extracted with 5 mass% potassium hydroxide (80 ml) for 30 min at 90 • C. The sediment was washed 3× with water. Next, 30 mass% peroxide (40 ml) and acetic acid (40 ml) were added. The suspension was stirred with heating for 20 min at 100 • C and then washed 3× with ultrapure water. The obtained crude chitin was washed 3× with 5 mass% potassium hydroxide at 100 • C for 30 min, with three subsequent water washing steps. Finally, the sediment was suspended in 1.0 M hydrochloric acid and stirred for 30 min at RT, followed by washing 5× with ultrapure water. After lyophilization, 0.213 g of chitin was obtained (5.6% of the glucan mass).
Chitin (0.113 g) and 50 mass% sodium hydroxide (1.50 ml) were incubated at 120 • C for 6 h with occasional shaking. The suspension was cooled at 5 • C overnight. The next day, the suspension was centrifuged (2,000×g, 10 min, Hettich R Universal 320R centrifuge), the sediment was dissolved in water, and 1 M hydrochloric acid was added. The insoluble fraction was dialyzed against ultrapure water and then lyophilized. Then, 45 mg of chitosan was obtained (40% yield).

FTIR
Fourier-transform infrared (FTIR) spectra were measured using the Nicolet iS50 FT-IR spectrometer (Thermo Fisher Scientific) equipped with a deuterated-triglycine sulfate detector and the Omnic 9.0 software (Thermo Fisher Scientific). For each sample, 64 scans were averaged with a spectral resolution of 4 cm −1 in the middle region from 4,000 to 400 cm −1 . A diamond attenuated total reflectance sampling accessory was employed for solidstate measurements. The FTIR spectra of chitin and chitosan are shown in Supplementary Figure 1.

NMR
Prior to NMR spectroscopy, protons in the samples were exchanged with heavy water. 1 H NMR spectra were acquired in deuterium oxide (99.97% D) plus acetic acid (1 µl) on a Bruker AVANCE III HD 400 MHz spectrometer (Bruker, Germany) equipped with a 5-mm broad band BB-(H-F)-D-05-Z liquid N 2 Prodigy probe with an automatic chemical shift calibration and processed using the MestReNova 14.0.1 software. The 1 H signal of acetic acid (1.950 ppm) was used as a reference for chemical shifts. The 1 H NMR spectrum of chitosan is shown in Supplementary Figure 2.

Determination of the Degree of Acetylation
Degree of acetylation (DA) was measured as previously reported by Czechowska-Biskup et al. (2012).

Statistical Analysis
Results were expressed as mean ± SD. Normality of data distribution was determined according to the Shapiro-Wilk test at the 0.05 level of significance. Statistical evaluation was performed via one-way ANOVA and post-hoc Bonferroni test. Results were considered significant when differences equaled or exceeded the 95% confidence level (P < 0.05). Statistical analysis was performed using the ORIGIN 2018 software (OriginLab Corporation, Northampton, MA, USA). Pearson's correlation coefficients were used to compare the strength of the relationship between immunobiological variables.

Chemistry
We recently described the synthesis of 2-aminoethyl glycosides of chito-oligomers containing 3, 5, and 7 N-acetyl-D-glucosamine residues (Yudina et al., 2015) and their biotinylated derivatives 28-30 (Scheme 3) (Yudina et al., 2016). Azide and phthaloyl groups were employed as a precursor of the aglycon amino group and for N-protection of glucosamine, respectively. Elongation of the oligosaccharide chain was accomplished using a disaccharide donor containing acetyl or chloroacetyl groups for temporary protection of 4-OH (Yudina et al., 2015). An attempt to prepare donor 3 directly by orthogonal glycosylation of thioglycoside 2 with imidate 1 was not overly successful (Scheme 1) due to predominant transfer of ethylthio aglycon from acceptor 2 onto donor 1 (Yudina et al., 2015). For this reason, we employed an indirect approach for the disaccharide donor, consisting of initial glycosylation of an acceptor with p-methoxyphenyl anomeric protection, followed by two-step transformation of p-methoxyphenyl glycoside into the corresponding trichloroacetimidate (Yudina et al., 2015).
The same protecting group pattern was used in the present work, but we improved considerably the preparation of the key disaccharide donor. Glycosylation of acceptor 2 with various types of donors (glycosyl bromides, sulfoxides, and trichloroacetimidates) bearing different protecting groups at O-4 (acyl, silyl) was examined. The results of this examination, together with published data (Barry et al., 2013), demonstrated that electron-withdrawing acyl protection at O-4 facilitated aglycon transfer and should thus be excluded.
The optimal result was achieved with 4-O-TBDMS imidate 6, which was efficiently prepared from 2 by silylation of 4-OH (→4), hydrolysis of the anomeric SEt-group (Lay et al., 1998) (→5), and trichloroacetimidate formation, for a total yield of 81% in three steps (Scheme 1). As 4-O-silylated donor 6 must possess higher reactivity than 4-O-acylated counterparts (Tanaka et al., 2017), one could anticipate a decrease of the proportion of aglycon transfer in its reaction with 2 Gildersleeve, 2006, 2007). Indeed, TMSOTf-promoted glycosylation of acceptor 2 with imidate 6 at low temperature provided the requisite disaccharide donor 7 in high yield and was practically unaccompanied by aglycon transfer. Thus, thioglycoside 7 was synthesized in four steps from single precursor 2 with an overall yield of 71%.
N-iodosuccinimide-TfOH-promoted glycosylation of 2azidoethyl glycoside 8 (Yudina et al., 2015) with thioglycoside 7 to smoothly produce trisaccharide 9; from which removal of the silyl group with aqueous hydrofluoric acid in acetonitrile resulted in new glycosyl acceptor 10 (Scheme 2). Desilylation with hydrofluoric acid proceeded slowly (48-72 h) but smoothly, with minimal formation of side products. Desilylation with tetrabutylammonium fluoride in tetrahydrofuran was much faster (2-3 h), but yields of 10 were noticeably lower, apparently due to insufficient stability of N-phthaloyl groups under basic conditions. Glycosylation with 7 and desilylation of the glycosylation product was reiterated twice to give derivatives of chitopentaose (11 and 12) and chitoheptaose (13 and 14). Further chain elongation was not performed, as it was previously demonstrated that pentamers and hexamers possess the highest affinity for antichitosan polyclonal antibodies (Kim et al., 2000).
N-phthaloyl groups in protected oligomers 10, 12, and 14 were removed by treatment with hydrazine hydrate, and free amino groups were protected again by trifluoroacetylation to give derivatives 15-17 (Scheme 3). These were subjected to simultaneous debenzylation and azide reduction by SCHEME 1 | Synthesis of disaccharide donor 7. SCHEME 2 | Assembly of protected chito-oligosaccharides.
hydrogenolysis over Pd (OH) 2 /C in aqueous methanol in the presence of acetic acid (Yudina et al., 2016). 1 H and 13 C NMR data of obtained N-trifluoroacetylated 2-aminoethyl glycosides 18-20 unequivocally demonstrated that all glucosamine residues exhibited β-configuration and were connected by (1→4)linkage. Thus, signals for H-1 in the 1 H NMR spectra appeared as doublets with the coupling constant value J 1,2 ∼8 Hz. The signal location for C-1 at δ ∼101.5 ppm in the 13 C NMR spectra also confirmed β-configuration. A downfield location of signals for C-4 (δ ∼80 ppm) of the internal glucosamine residues, compared with that of terminal unsubstituted glucosamine (δ ∼71 ppm), confirmed the position of the glycoside bonds.
N-acylation of the spacer amino group in 18-20 with active ester of biotin 21 (Tsvetkov et al., 2012) containing the hydrophilic hexa (ethylene glycol) linker resulted in the formation of biotinylated products 22-24. Removal of the N-trifluoroacetyl groups by mild alkaline hydrolysis produced the requisite biotin-tagged chitosan oligomers 25-27. The presence of signals corresponding to oligosaccharide, biotin, and linker moieties in the NMR spectra of 25-27 confirmed their structure.

Effect of Chitin-and Chitosan-Derived Oligosaccharides on RAW 264.7 Proliferation
The capability of synthetically prepared chitin or chitosan oligosaccharides to affect proliferation of RAW 264.7 cells was monitored by ATP bioluminescence as a cell viability marker (Figure 1) compared with that of natural chitin isolated from C. albicans serotype A or prepared chitosan. Obtained results after 24 h treatment revealed higher capability of chitosan oligosaccharides 25, 26, and 27 to increase RAW 264.7 cell SCHEME 3 | Transformation of the protected chito-oligomers in biotin-tagged chitosan oligosaccharides 25-27. Structures of previously synthesized chito-oligosaccharides 28-30 used in this work. proliferation than that of chitin oligosaccharides 28, 29, and 30. Additionally, stimulatory activity of prepared chitosan was higher than that of natural chitin. Stimulation with longer chitosan oligosaccharides (27, 10 µg/ml: P = 2.5 × 10 8 , 100 µg/ml: P = 0.001) for 24 h induced significantly more pronounced RAW 264.7 cell proliferation than that with shorter chitosan oligosaccharides (26: 10 µg/ml: P = 0.12, 100 µg/ml: P = 0.0012, 25: 10 µg/ml: P = 0.061, 100 µg/ml: P = 0.043), which was even greater than that of prepared chitosan (10 µg/ml: P = 0.0012, 100 µg/ml: P = 0.0026). Chitosan heptasaccharide 27 was the most effective inducer of RAW 264.7 cell proliferation (10 µg/ml: 1.32fold and 100 µg/ml: 1.36-fold increase compared with the control). After 48 h treatment, no significant difference was observed between prepared chitin or chitosan oligosaccharides in their capability to increase proliferation of RAW 264.7 macrophages. The increase in macrophage proliferation induced by chitin or chitosan oligosaccharides was higher than (Con A, PHA, and LPS) or comparable with (PWM) the proliferation induced by mitogens.

Effect of Chitin-and Chitosan-Derived
Oligosaccharides on RAW 264.7 Phagocytic Activity RAW 264.7 macrophage cells exposed to chitin-and chitosanderived oligosaccharides 28-30 and 25-27, as well as natural Candida chitin and chitosan, were subjected to bacterial phagocytosis and respiratory burst analysis (Tables 1, 2). In parallel, phagocytosis of cells exposed to cell mitogens was used as positive control ( Table 3). The influence of applied concentrations of oligosaccharides on phagocytic activity favored mainly the higher (100 µg/ml) concentration. After 24 h exposure, the highest phagocytic activity was observed for heptasaccharides 30 and 27 (1.7-fold increases compared with the control). After 48 h exposure, heptasaccharide 30 remained the most effective inducer of phagocytic activity of the chitinderived oligosaccharides; however, the higher concentration of all tested chitosan-derived oligosaccharides showed comparable inductions of increased phagocytic activity.
Although increased phagocytic activity was induced by longer oligosaccharides (30−24 h and 48 h, 27−24 h) and higher concentration (100 µg/ml), SPA-FITC particles remained mostly attached to the cell membrane. Higher internalization was induced by shorter oligosaccharides and/or lower concentration (10 µg/ml). Exposure of RAW 264.7 cells to chitosan-derived oligosaccharides for 48 h increased internalization of SPA-FITC particles for all tested oligosaccharides (25, 26, and 27) and for both concentrations. Exposure to all tested chitin-and chitosan-derived oligosaccharides enhanced the respiratory burst of RAW 264.7 macrophages after 24 h (from 1.2-to 1.8-fold increases). The 48 h treatment demonstrated that the higher concentration of chitin-derived oligosaccharides more effectively induced the respiratory burst of RAW 264.7 macrophages than the lower concentration.  (27), and chitosan. Untreated RAW 264.7 cells were used as the negative control. Concanavaline A (Con A, 10 µg/ml), phytohemagglutinin (PHA, 10 µg/ml), pokeweed mitogen (PWM, 1 µg/ml), and lipopolysaccharide (LPS, 10 µg/ml) were used as positive controls. Horizontal dashed line represents baseline. Results are expressed as the mean stimulation indexes (average relative light units in the presence of antigens/average relative light units obtained without antigen). All data are presented as mean stimulation indexes ± SD. Tests were carried out in triplicate. Statistical significance of differences between untreated and stimulated cells using one-way ANOVA and post-hoc Bonferroni test is expressed as: ***P < 0.001, ** 0.001 < P < 0.01, * 0.01 < P < 0.05.
Similar to IL-2 production, stimulation with chitosan-derived oligosaccharides 25, 26, and 27 significantly increased IL-17 production, induced markedly increased IL-12 production, Phagocytic activity, oxidative burst, and metabolic activity were expressed as percentage of cells actually undergoing these processes in a population of 10,000 cells. All data are presented as mean ± SD. Tests were carried out in triplicate. Statistical significance of differences between untreated and stimulated cells using one-way ANOVA and post-hoc Bonferroni tests is expressed as: ***P < 0.001, ** 0.001 < P < 0.01, * 0.01 < P < 0.05.  Table 4).

DISCUSSION
In this study, an array of synthetically prepared chitinand chitosan-derived oligosaccharides of different lengths was examined to elucidate their influence on the behavior of macrophage RAW 264.7 cells. To evaluate the relationship between the chito-oligosaccharide structure and immunobiological activity, functional tests based on the induction of various immune responses, including proliferation, cytotoxicity, immunological inflammation, and polarization of T-helper responses (Th1, Th2, Th17, or Treg), were applied following RAW 264.7 cell exposure to natural Candida chitin and chitosan and synthetic oligosaccharide fragments 28-30 and 25-27. Proliferation results after exposure for 24 and 48 h demonstrated good biocompatibility without cytotoxic effects for all tested formulas, even at the higher concentration (Figure 1). Chitosan-derived structures more effectively promoted and accelerated macrophage cell proliferation compared with chitin derivatives (Figure 1), which concurred with results from other studies. Yang et al. (2019) revealed good cytocompatibility of chito-oligosaccharides after pretreatment of RAW 264.7 cells for 24 h, even at 100 µg/ml concentration. Active stimulation and enhancement of cell proliferation was reported with chitosan and chitosan-derived structures in different cell systems, such as human skin fibroblasts and keratinocytes (Howling et al., 2001), neuron-like PC12 cells (Alhosseini et al., 2012), Saos-2 cells (Isikli et al., 2012), and L929 fibroblasts (Tangsadthakun et al., 2007). Chang et al. (2019) revealed significantly increased mitogen-induced proliferation of splenocytes and Peyer's patch lymphocytes after in vivo administration of chitosan hydrolytic products in mice. Hoseini et al. (2016) determined that chitin and chitosan microparticles (<40 µm) subcutaneously injected into Balb/c mice induced cell proliferation.
Chitin, chitosan, and their derivatives may be potentially employed in immunomodulating adjuvant formulas based on their ability to affect macrophage polarization to proinflammatory M1 phenotype induced by inflammatory cytokines, such as TNFα and IFNγ, polarization to antiinflammatory M2 phenotype induced by anti-inflammatory  cytokines, such as IL-10, IL-4, and IL-13, as well as the release of relevant cytokine patterns. Research by Da Silva et al. (2010) demonstrated that chitin is a potent adjuvant that augments Th2, Th1, and Th17 responses in vivo and in vitro. Mori et al. (2012) revealed that a combination of chitosan and TLR9 agonist CpG as an adjuvant activated the NLRP3 inflammasome and enhanced secretion of IL-12 and the other key Th1 and Th17 cell-polarizing cytokines. Further, Jesus et al. (2018) characterized adjuvant activity of poly-ε-caprolactone/chitosan nanoparticles and mastocyte activation accompanied by IFN-γ and IL-17 release. Particularly of interest is the inhibition of LPS-induced inflammatory cytokines TNF-α, IL-1, and IL-6 in RAW 264.7 macrophage cells by either chitosan nanoparticles (Ma et al., 2016) or chitosan oligosaccharides (Yoon et al., 2007). Chang et al. (2019) demonstrated in RAW 264.7 macrophage cells that 156 and 72 kDa chitosans significantly inhibited the production of TNFα and IL-6, whereas 7.1 kDa chitosan and chito-oligosaccharides significantly induced their production. Evidently, chitosan particle size played an important role, with small particles eliciting the greatest activity. This concurs with a similar pattern regarding size-dependent production of macrophage TNF and IL-10 observed by Da Silva et al. (2009). Their study revealed that large chitin fragments were inert, whereas intermediate-sized chitin (40-70 µm) and small chitin (<40 µm, largely 2-10 µm) stimulated TNF expansion. In contrast, only small chitin induced IL-10 expansion. Shibata et al. (1997) observed the induction of IL-12, TNFα, and IFNγ in mouse splenocytes with phagocytosable-size chitin particles (1-10 µm), whereas the release of these cytokines was not observed with larger particles (50-100 µm). Co-formulation of chitosan with IL-12 has been suggested as an effective antitumor immunotherapy (Heffernan et al., 2011;Smith et al., 2015). Additionally, antitumor efficacy has been enhanced by co-delivery of doxorubicin and IL-2 using chitosan-based nanoparticles (Wu et al., 2017).

CONCLUSIONS
Biotinylated chitosan oligomers comprising 3, 5, and 7 glucosamine residues were efficiently synthesized. Oligosaccharide chain elongation was accomplished using a disaccharide thioglycoside donor bearing a temporary tertbutyldimethylsilyl group at O-4 ′ . The disaccharide donor was prepared in a straightforward manner by orthogonal glycosylation of a thioglycoside acceptor with 4-O-TBDMS trichloroacetimidate. In contrast to glycosylation with 4-O-acyldonors that was accompanied by intensive aglycon transfer, application of the silyl-protected donor almost completely obviated this side reaction. In vitro immunobiological evaluation of chitin-and chitosan-derived oligosaccharides (28-30 and 25-27, respectively) in RAW 264.7 cells demonstrated effective immunomodulation with respect to induction of cytokine release, cell proliferation, phagocytosis, and respiratory burst. Macrophage reactivity was accompanied by significant inductive concentration-and structure-dependent Th1 and Th17 polarization, including increased Th1 cytokine production for IL-2, IL-12 (p70), TNFα, GM-CSF, and Th17 cytokine IL-17, which was greater with exposure to chitosan-rather than chitin-derived oligosaccharides. Tested oligomers triggered significant cell release of anti-inflammatory IL-10 and were efficient inducers of internalization and phagocytosis of S. aureus particles by RAW 264.7 cells. Moreover, the absence of antiproliferative/cytotoxic effects, even following prolonged 48 h exposure, is a promising result for further studies with these oligosaccharides. Synthetically prepared chitin-and chitosan-derived oligosaccharides are suitable for use in vitro and prospectively in vivo for further immunobiological and immunotoxicological studies, as potential antigens for in vitro diagnostics of candidosis, and for anti-fungal therapy monitoring.

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, analyzed the data, acquired funding, and prepared the original draft. PF performed the modification and characterization of chitin and chitosan. YT performed the chemical syntheses, analyzed the data, and prepared the original draft. All authors contributed to manuscript revision and read and approved the submitted version.