In Vivo and In Vitro Study of Immunostimulation by Leuconostoc lactis-Produced Gluco-Oligosaccharides

Glycosyltransferase-producing Leuconostoc lactis CCK940 produces CCK- oligosaccharides, gluco-oligosaccharide molecules, using sucrose and maltose as donor and acceptor molecules, respectively. In this study, the immunostimulatory activities of CCK-oligosaccharides on RAW264.7 macrophages and BALB/c mice were evaluated. CCK-oligosaccharides induced the expression of phosphorylated-p38, extracellular-signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) and upregulation of phagocytic activity in RAW264.7 macrophages, suggesting their involvement in mitogen-activated protein kinase (MAPK) signaling pathway and phagocytosis. When CCK-oligosaccharides were administered to mice intraperitoneally injected with cyclophosphamide (CY), spleen indices and expressions of interleukin (IL)-6, IL–10, and tumor necrosis factor-α increased, compared with those in only CY-treated group. These findings suggest that CCK-oligosaccharides can be used as an effective immunostimulating agent.

Cyclophosphamide (CY) is a well-known alkylating agent [17], and is an important chemotherapeutic drug used in tumor treatment [18]. It is also used in organ transplantation, and to treat autoimmune diseases and cancer patients. CY causes an immunosuppressive effect by damaging DNA of normal treat autoimmune diseases and cancer patients. CY causes an immunosuppressive effect by damaging DNA of normal cells [19] and is one of the major immunosuppressive medicines used in clinical trials [20]. Therefore, it is unfavorable for the immune system of organisms because immunosuppression can be fatal [18].
In the present study, the mechanism of immnunostimulatory effects of CCK-oligosaccharides was determined by Western blot and real-time polymerase chain reaction (PCR) in RAW264.7 cells in vitro. Furthermore, the effects of CCK-oligosaccharides on spleen weight and mRNA expression levels of various cytokines (IL-6, IL-10, and TNF-α) in CY-treated mice were evaluated in vivo.

Effect of CCK-Oligosaccharides on MAPK Signaling Pathway of RAW264.7 Cells
MAPKs include ERK, JNK, and P38, which control various immune responses, such as cell proliferation and differentiation, and play a particularly important role in macrophage activation [21,22]. Furthermore, CCK-oligosaccharides increase nitric oxide (NO) production in a dosedependent manner [6]. When RAW264.7 macrophage cells were treated with CCK-oligosaccharides, the expression of P38, JNK, and ERK1/2 MAPK was determined ( Figure 1). The expression of phosphorylated P38, ERK1/2, and JNK in 1 mg/mL CCK-oligosaccharide-treated RAW264.7 cells was higher than those in control-treated cells. Particularly, the expression of phosphorylated JNK increased in CCK-oligosaccharide-treated RAW264.7 cells in a dose-dependent manner.  (a) Western blot images. Adherent RAW264.7 cells were incubated in medium with 0.1, 1, and 10 mg/mL CCK-oligosaccharides. The whole-cell lysates were prepared from the treated cells and the levels of both phosphorylated and non-phosphorylated MAPKs were analyzed by Western blot. Each blot is representative of three independent experiments and the numbers below blots indicate the relative expression level of MAPKs. (b) Quantification of Western blots. One-way ANOVA was used for comparison of group mean values, followed by Duncan's multiple range test for significance of individual comparisons (p < 0.05). Different alphabet letters among groups represent statistically significant difference.

Effect of CCK-Oligosaccharides on Phagocytic Activity of RAW264.7 Cells
Phagocytosis is very important for innate and adaptive immunity as it stimulates defense against pathogens, tissue repair promoting, and chronic inflammation [25,26]. Activation of Fcγ receptors on the surface of the monocytes, macrophages, and neutrophils leads to broad-spectrum antimicrobial activities, including phagocytosis, secretion, cytokine synthesis, Ab-dependent cellular cytotoxicity, and generation of reactive oxidant species [27][28][29]. Effective phagocytosis by macrophages is caused by opsonization of pathogens, by Ig or complement proteins [18]. Phagocytosis of activated RAW264.7 macrophages has been determined by fluorescent IgG-coated latex beads [30]. To determine the effect of CCK-oligosaccharides on the phagocytic activity of RAW264.7 macrophages, FITC-conjugated IgG-coated latex beads were incubated with the cells after treatment ( Figure 2). ANOVA was used for comparison of group mean values, followed by Duncan's multiple range test for significance of individual comparisons (p < 0.05). Different alphabet letters among groups represent statistically significant difference.

Effect of CCK-Oligosaccharides on Phagocytic Activity of RAW264.7 Cells
Phagocytosis is very important for innate and adaptive immunity as it stimulates defense against pathogens, tissue repair promoting, and chronic inflammation [25,26]. Activation of Fcγ receptors on the surface of the monocytes, macrophages, and neutrophils leads to broad-spectrum antimicrobial activities, including phagocytosis, secretion, cytokine synthesis, Ab-dependent cellular cytotoxicity, and generation of reactive oxidant species [27][28][29]. Effective phagocytosis by macrophages is caused by opsonization of pathogens, by Ig or complement proteins [18]. Phagocytosis of activated RAW264.7 macrophages has been determined by fluorescent IgG-coated latex beads [30]. To determine the effect of CCK-oligosaccharides on the phagocytic activity of RAW264.7 macrophages, FITC-conjugated IgG-coated latex beads were incubated with the cells after treatment ( Figure 2). Quantification of rabbit IgG and FITC-coated latex bead uptake by RAW264.7 macrophages. Oneway ANOVA was used for comparison of group mean values, followed by Duncan's multiple range test for significance of individual comparisons (p < 0.05). Different alphabet letters among groups The phagocytic index increased in a dose-dependent manner in CCK-oligosaccharide-treated RAW264.7 macrophages. The phagocytic indices of LPS-treated and 10 mg/mL CCK-oligosaccharidetreated cells were calculated to be 1.26 and 1.2, respectively. These results indicate that both LPS-and CCK-oligosaccharide-induction increased phagocytic activity, although CCK-oligosaccharide has lower phagocytic activity than LPS. The polysaccharide fraction of Solanum nigrum induces phagocytosis activity [30] and the purified polysaccharide isolated from Caulerpa lentillifera increases NO production, expression of P38 in MAPK signaling pathways, and phagocytosis activity, in a dose-dependent manner [31]. As the primary purpose of studying oligosaccharide produced by lactic acid bacteria is to examine the prebiotic activity of oligosaccharide, few studies have focused on the phagocytic activity of oligosaccharides, and this study is important as it shows that the oligosaccharides produced from L. lactis CCK 940 have a good phagocytic activity.

Effect of CCK-Oligosaccharides on Spleen Indices of CY-Induced Mice
The spleen is an important organ involved in nonspecific and specific immunity and spleen index reflects immune function and prognosis. Immunostimulation can increase the weight of the spleen [20,32]. Figure 3 shows that the spleen indices of the CY group mice was lower than those of the control and CY + CCK group mice, and spleen index had increased in the CY + CCK group mice. This result suggests that immunity decreases upon treating mice with CY. Treating mice with CCK-oligosaccharide increased the spleen indices. These findings indicate that CCK-oligosaccharides enhance immunity in immunosuppressed model mice. In other studies, the spleen indices of mice increased when CY-treated mice were exposed to polysaccharides of Sophora subprosrate, water-soluble polysaccharides of Sargassum fusiforme, and chito-oligosaccharides [20,33,34]. Many studies have shown that polysaccharides exert immunostimulating effects in CY-induced mice; however, only limited studies have focused on the immunostimulating effects of oligosaccharides obtained from lactic acid bacteria. The phagocytic index increased in a dose-dependent manner in CCK-oligosaccharide-treated RAW264.7 macrophages. The phagocytic indices of LPS-treated and 10 mg/mL CCK-oligosaccharidetreated cells were calculated to be 1.26 and 1.2, respectively. These results indicate that both LPS-and CCK-oligosaccharide-induction increased phagocytic activity, although CCK-oligosaccharide has lower phagocytic activity than LPS. The polysaccharide fraction of Solanum nigrum induces phagocytosis activity [30] and the purified polysaccharide isolated from Caulerpa lentillifera increases NO production, expression of P38 in MAPK signaling pathways, and phagocytosis activity, in a dosedependent manner [31]. As the primary purpose of studying oligosaccharide produced by lactic acid bacteria is to examine the prebiotic activity of oligosaccharide, few studies have focused on the phagocytic activity of oligosaccharides, and this study is important as it shows that the oligosaccharides produced from L. lactis CCK 940 have a good phagocytic activity.

Effect of CCK-Oligosaccharides on Spleen Indices of CY-Induced Mice
The spleen is an important organ involved in nonspecific and specific immunity and spleen index reflects immune function and prognosis. Immunostimulation can increase the weight of the spleen [20,32]. Figure 3 shows that the spleen indices of the CY group mice was lower than those of the control and CY + CCK group mice, and spleen index had increased in the CY + CCK group mice. This result suggests that immunity decreases upon treating mice with CY. Treating mice with CCKoligosaccharide increased the spleen indices. These findings indicate that CCK-oligosaccharides enhance immunity in immunosuppressed model mice. In other studies, the spleen indices of mice increased when CY-treated mice were exposed to polysaccharides of Sophora subprosrate, watersoluble polysaccharides of Sargassum fusiforme, and chito-oligosaccharides [20,33,34]. Many studies have shown that polysaccharides exert immunostimulating effects in CY-induced mice; however, only limited studies have focused on the immunostimulating effects of oligosaccharides obtained from lactic acid bacteria.

Effect of CCK-Oligosaccharides on mRNA Expression of Peritoneal Macrophages
Activated macrophages stimulate the production of various immunomodulatory cytokines, such as IL-6, IL-10, and TNF-α [35]. IL-6 is an important inflammatory and immune mediator that increases complement production and phagocytosis; it also regulates diverse cell functions as a growth factor, to enhance the proliferation and differentiation of neuronal cells, endothelial cells, Tand B-lymphocytes, and keratinocytes [36,37]. IL-10, an anti-inflammatory cytokine, is a Th2-type

Effect of CCK-Oligosaccharides on mRNA Expression of Peritoneal Macrophages
Activated macrophages stimulate the production of various immunomodulatory cytokines, such as IL-6, IL-10, and TNF-α [35]. IL-6 is an important inflammatory and immune mediator that increases complement production and phagocytosis; it also regulates diverse cell functions as a growth factor, to enhance the proliferation and differentiation of neuronal cells, endothelial cells, T-and Blymphocytes, and keratinocytes [36,37]. IL-10, an anti-inflammatory cytokine, is a Th2-type cytokine and tends to inhibit a broad range of inflammatory responses [38][39][40]. IL-10 is released under various conditions of immune activation by T cells, B cells, macrophages, and monocytes, and is known to be an important factor for supporting homeostasis of general immune responses [39,41]. It also exerts immunostimulatory effects, and it is involved in the activation of mast, T, B, and NK cells [42]. TNF-α is a powerful pro-inflammatory multifunctional cytokine, which is involved in inflammation, cell differentiation, proliferation, apoptosis, and promotion of immune cell functions [43][44][45].
To determine the effects of CCK-oligosaccharides on CY-induced immunosuppression, peritoneal macrophages of BALB/c mice were isolated and mRNA expression was evaluated. In peritoneal macrophages, the intraperitoneal treatment of CY inhibited the expression of cytokines. The administration of CCK-oligosaccharides increased the expression of IL-6, IL-10, and TNF-α compared with only those in the CY-treated mice (Figure 4a-c).
Molecules 2019, 24, x FOR PEER REVIEW 5 of 11 cytokine and tends to inhibit a broad range of inflammatory responses [38][39][40]. IL-10 is released under various conditions of immune activation by T cells, B cells, macrophages, and monocytes, and is known to be an important factor for supporting homeostasis of general immune responses [39,41]. It also exerts immunostimulatory effects, and it is involved in the activation of mast, T, B, and NK cells [42]. TNF-α is a powerful pro-inflammatory multifunctional cytokine, which is involved in inflammation, cell differentiation, proliferation, apoptosis, and promotion of immune cell functions [43][44][45].
To determine the effects of CCK-oligosaccharides on CY-induced immunosuppression, peritoneal macrophages of BALB/c mice were isolated and mRNA expression was evaluated. In peritoneal macrophages, the intraperitoneal treatment of CY inhibited the expression of cytokines. The administration of CCK-oligosaccharides increased the expression of IL-6, IL-10, and TNF-α compared with only those in the CY-treated mice (Figure 4a-c). When mice were administered with CCK-oligosaccharides at a concentration of 200 mg/kg, mRNA expression levels of IL-6, IL-10, and TNF-α were 10 times higher than those in mice only administered with CY. These results show that CCK-oligosaccharide administration led to the enhancement of immune functions in an immunosuppressed mice model. Curdlan oligosaccharides from Alcaligenes faecalis var. myxogenes activate MAPK and NF-κB pathways and improve immunostimulatory activity in CY-induced imuunosuppressed mice [46]. Polysaccharides of Lonicera japonica, a typical Chinese herbal medicine, and wheat bran-derived polysaccharides markedly promote the serum cytokine levels [47,48], and polysaccharides derived from Stichopus japonicus (sea cucumber), up-regulate the expression of cytokines, such as IL-1β, IL-4, IL-6, IL-10, TNF-α, and IFNγ, in the CY-induced mice [49]. As CCK-oligosaccharides have an immunostimulating activity, it can be used as an ingredient for functional foods to regulate immune responses. Even though glucooligosaccharides are considered safe, the structural changes by the intestinal microflora might result in undesirable effects. Correspondingly, the safety issue of oligosaccharides would be considered in the development of functional foods.

Preparation of CCK-Oligosaccharides
CCK-oligosaccharides were purified by the protocol described in our previous study [6]. Briefly, L. lactis CCK940 was cultured in Lactobacilli MRS broth (BD, Franklin Lakes, NJ, USA) at 30 °C for 20 h. The culture was inoculated to LM media [50], supplemented with 9.6% (w/v) sucrose and 7.4% (w/v) maltose, and incubated at 30 °C for 9 h. The culture supernatants were concentrated and separated by gel permeation chromatography (GPC; Bio-gel P2; Bio-Rad, Hercules, CA, USA). The When mice were administered with CCK-oligosaccharides at a concentration of 200 mg/kg, mRNA expression levels of IL-6, IL-10, and TNF-α were 10 times higher than those in mice only administered with CY. These results show that CCK-oligosaccharide administration led to the enhancement of immune functions in an immunosuppressed mice model. Curdlan oligosaccharides from Alcaligenes faecalis var. myxogenes activate MAPK and NF-κB pathways and improve immunostimulatory activity in CY-induced imuunosuppressed mice [46]. Polysaccharides of Lonicera japonica, a typical Chinese herbal medicine, and wheat bran-derived polysaccharides markedly promote the serum cytokine levels [47,48], and polysaccharides derived from Stichopus japonicus (sea cucumber), up-regulate the expression of cytokines, such as IL-1β, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, in the CY-induced mice [49]. As CCK-oligosaccharides have an immunostimulating activity, it can be used as an ingredient for functional foods to regulate immune responses. Even though gluco-oligosaccharides are considered safe, the structural changes by the intestinal microflora might result in undesirable effects. Correspondingly, the safety issue of oligosaccharides would be considered in the development of functional foods.

Preparation of CCK-Oligosaccharides
CCK-oligosaccharides were purified by the protocol described in our previous study [6]. Briefly, L. lactis CCK940 was cultured in Lactobacilli MRS broth (BD, Franklin Lakes, NJ, USA) at 30 • C for 20 h. The culture was inoculated to LM media [50], supplemented with 9.6% (w/v) sucrose and 7.4% (w/v) maltose, and incubated at 30 • C for 9 h. The culture supernatants were concentrated and separated by gel permeation chromatography (GPC; Bio-gel P2; Bio-Rad, Hercules, CA, USA). The oligosaccharide fractions were collected and lyophilized (SunilEyela, Seongnam, Korea) to determine their immunological effects.

Phagocytosis Assay
RAW264.7 cells were cultured in 24-well plates (2 × 10 5 cells/well) for 24 h with 5% CO 2 at 37 • C Cells were treated with 0.1, 1.0, and 10.0 mg/mL CCK-oligosaccharides, or 1 µg/mL lipopolysaccharide (LPS) as a control, and incubated at 37 • C in a 5% CO 2 incubator for 24 h. Each group was treated with specific FITC-conjugated rabbit IgG-coated latex beads (Cayman Chemical, Ann Arbor, MI, USA) to determine whether CCK-oligosaccharides affected the phagocytic activity of fluorescence particles in RAW264.7 cells. Random sites were photographed, and Live Imaging software was used to acquire real-time microscope (Nikon ECLIPSE Ti, Nikon Instruments Inc., Tokyo, Japan) images over a period of 24 h. Imaging software files were exported and analyzed in MetaMorph software version 7.8.9.0 (Molecular device, Sunnyvale, CA, USA).

Animals
The study was performed on 6-week old male BALB/c mice, with medium weight 23 ± 2 g, obtained from Orient Bio Inc. (Suwon, Korea). The mice were housed with sterile bedding under 12/12 h light/dark schedule in a temperature-and humidity-controlled room, with ad-libitum access to food and water. Animal experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. injected with 80 mg/kg CY on days 1-3 and fed 200 mg/kg CCK-oligosaccharide on days 4-10. On day 11, the mice were euthanized by carbon dioxide exposure.

Calculation of Spleen Indices
Mice were sacrificed on day 11 and the spleen of each mouse was aseptically removed and weighed. The spleen index was calculated using the following formula: Spleen index = Spleen weight (mg)/Body weight (g) (1)

Isolation of Mouse Peritoneal Macrophages
For isolation of peritoneal macrophages, mice were intraperitoneally injected with 10 mL cold phosphate buffered saline solution (PBS) after scarification. After a soft abdominal massage, the peritoneal PBS was collected to sterilize tubes. The collected PBS was centrifuged at 200× g for 10 min and peritoneal macrophages were isolated using RBC lysis buffer (Sigma Aldrich, St. Louise, MO, USA). Peritoneal macrophages were cultured in RPMI 1640 media (HyClone, GE Healthcare, Chicago, IL, USA) at 37 • C, in a 5% CO 2 incubator.

RNA Extraction and cDNA Synthesis
The expression levels of cytokines were measured to confirm the immunostimulating effect of the CCK-oligosaccharides on mouse peritoneal macrophages, and cells were cultured in a 12-well plate at 37 • C in a 5% CO 2 incubator. RNA was isolated using easy-BLUETM Total RNA Extraction kit (iNtRON Biotechnology, Inc., Seongnam, Korea) from the cultured cells. Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) was used to synthesize cDNA from the isolated RNA, and the synthesized cDNA was used for real-time PCR (LightCycler96, Roche, Basel, Switzerland), using the FastStart Essential DNA Green Master Kit (Roche, Basel, Switzerland).
3.5.6. Real-Time PCR IL-6, IL-10, and TNF-α cytokine genes were amplified. GAPDH was used as an endogenous control gene and the expression levels of cytokine mRNA relative to those of the GAPDH mRNA were analyzed using the 2 -ddCT method. The sequences of the primers used in this study are listed in Table 1.

Statistical Analysis
Data are expressed as mean ± standard deviation (SD) of values from triplicate experiments. Statistical analyses were performed using SPSS 23 (SPSS Inc., Chicago, IL, USA). Statistical significance between groups was determined by a paired t-test for repeated measures. Data with p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant. One-way ANOVA was used for comparison of group means, followed by Duncan's multiple range test for significance of individual comparisons (p < 0.05).

Conclusions
This study demonstrated that CCK-oligosaccharides have a potent immunomodulating activity, which predominantly results from CCK-oligosaccharide-induced upregulation of phagocytic activity and MAPK signaling pathways in RAW264.7 macrophages. These activities were due to the activation of major signaling proteins, such as P38, JNK, and ERK, as well as TNF-α. In vivo study also showed that CCK-oligosaccharides can promote immune function in CY-induced immunosuppressed mice, presumably due to the activation of the spleen. To the best of our knowledge, this is the first study to report the immunostimulatory activity of gluco-oligosaccharides produced from Leuconostoc sp. and demonstrate that CCK-oligosaccharides may be used as an agent developed from functional foods to regulate immune responses.