Immune-enhancing effects of anionic macromolecules extracted from Codium fragile on cyclophosphamide-treated mice

Immune-regulation and homeostasis are critical in cancer therapy and immunomodulatory biomaterials have been used to decrease side effects of immunosuppressant drugs. Anionic macromolecules (CFAMs) were isolated from the seaweed Codium fragile, and its immune-enhancing biological activities were examined in CY-induced immunosuppressed mice. CFAMs improved the splenic lymphocyte proliferation, NK cell activity, and spleen index. The expression of immune-associated genes was highly upregulated in splenic lymphocytes, and gene expression was differently regulated according to mitogens such as T-cell (Con A) and B-cell (LPS) mitogens. Additionally, CFAMs boosted the proliferation, NO production, and phagocytosis of peritoneal macrophages. CFAMs also considerably stimulated immune-associated gene expression in peritoneal macrophages. Moreover, our results showed CFAMs mediated its immune-enhancing effects via the MAPK pathway. These suggested CFAMs can be used as a potent immunomodulatory material under immune-suppressive condition. Furthermore, CFAMs may also be used as a bio-functional and pharmaceutical material for improving human health and immunity.


Introduction
Immunomodulation, consisting of immune-stimulation and immunosuppression, is a sophisticated mechanism regulating the pathophysiology and etiology of various diseases affecting the immune system [1]. Immuno-regulation and homeostasis are crucial factors affecting cellular processes [2]. An immunomodulatory material can be used as an immune-stimulator to decrease the side effects of immunosuppressant drugs [3]. From ancient times, natural products with immune-regulatory properties have been considered to be important. They also act as important nutrients for improving human health and preventing chronic diseases and physiological problems, such as allergies, infections, and autoimmune diseases. Beta-glucan, a polysaccharide, is useful for treating respiratory tract infections [4]. Hemicellulose is known to possess immune-regulatory, antibacterial, and antitumor biological activities [5]. Chitin also enhances innate immunity and modulates inflammation in occupational allergies in humans PLOS

Isolation of the polysaccharides
Extraction and purification of crude anionic macromolecules from C. fragile (CFAMs) were performed as did previously [16] and these CFAMs were used in this study. Briefly, CFAMs were extracted from the milled sample of C. fragile using EtOH and distilled water, following centrifugation, filtration, and evaporation after removal of proteins [19].

Induction of immunosuppression in mice
Mice were randomly divided into seven groups (n = 5), after acclimatizing them for one week. One group was designated as the control group (normal group) and was administrated saline orally. The other groups were orally administrated saline (saline group) supplemented with varying concentrations of CFAMs (50,100,250, and 500 mg/kg BW) or with 100 mg/kg BW of commercial ginseng syrup (ginseng group). All groups received the respective treatment once per day for 10 consecutive days. At day 4-6 post-administration, mice (except those in the normal group) were injected intraperitoneally once a day with CY (80 mg/kg BW; Sigma-Aldrich, USA), and all the mice were sacrificed 24 h after completion of the treatment regimen.

Preparation of peritoneal macrophages and splenocytes
Peritoneal macrophages were prepared using the Ray and Dittel method [20]. Five milliliters of ice-cold phosphate buffered saline (PBS, supplemented with 3% FCS) was injected into the peritoneal cavity of each mouse, and subsequently the macrophages were collected. After collection, the cell suspension was centrifuged and cell pellet were resuspended in the RPMI-1640 medium or PBS for cell counting. Splenocytes were isolated from the spleen of BALB/c mice. The spleen was weighed and collected in ice-cold PBS. After treating the spleen with 1 × RBC Lysis Buffer (eBioscience, USA), the lysate cells were centrifuged at 400 × g for 10 min and washed using PBS. The splenocytes were resuspended in RPMI-1640 growth medium supplemented with 10% fetal bovine serum, streptomycin (100 μg/mL), and penicillin (100 IU/mL). Spleen index was evaluated using the following formula: Spleen index ¼ spleen weight ðmgÞ mouse body weight ðgÞ

Determination of peritoneal macrophage proliferation and nitric oxide production
In order to determine the proliferation of macrophages, the EZ-Cytox Cell Viability Assay kit (Daeil Labservice, Korea) was used, as described previously [21]. Macrophage proliferation ratio (%) was analyzed using the following formula: Macrophage proliferation ratio % ð Þ ¼ Absorbance of the test group Absorbance of the control group x 100 The immune-enhancing activity was measured based on nitric oxide (NO) production by macrophages. The cells were cultured with or without LPS, and NO concentration was measured using the Griess reagent (Sigma-Aldrich, USA) [22].

Phagocytosis of peritoneal macrophages
Phagocytosis of peritoneal macrophages was determined using the neutral red uptake method, as described previously [23]. Macrophages were seeded in triplicates in microplates and grown overnight in complete RPMI-1640 medium at 37˚C in a humidified atmosphere of 5% CO 2 . Phagocytosis of macrophages was measured using a microplate reader [absorbance (A), 540 nm]. Phagocytosis ratio was calculated using the following formula

Proliferation of splenic lymphocytes
Isolated splenic lymphocytes were induced using a T-cell (Con A, 5 μg/mL) or B-cell (LPS, 10 μg/mL) mitogen. Cells in the negative control group were incubated in RPMI at 37˚C in a humidified atmosphere of 5% CO 2 . After culturing for 48 h, cell proliferation was evaluated using the EZ-Cytox Cell Viability Assay kit.

Cytotoxicity assays for measuring the natural killer cell activity of splenocytes
Natural killer cell activity of splenocytes (effector cells) was analyzed using YAC-1 cells (target cells) [24] and lactate dehydrogenase (LDH) solution (Promega Co., USA) [25,26]. Splenocyte suspensions were added to the YAC-1 cells to obtain an effector-to-target cell ratio of 50:1. After 4 h incubation, the culture supernatants were mixed with a LDH solution. Absorbance was measured at 490 nm and the percentage of NK cell cytotoxicity was evaluated using the following formula: Cytotoxicity (%) = [(Experimental release-Spontaneous release)/(Maximum release-Spontaneous release)] × 100.

RNA isolation and reverse transcription
Total RNA was prepared from the LPS-stimulated peritoneal macrophages and mitogen-stimulated (Con A and LPS) splenic lymphocytes using the Tri reagent 1 (Molecular Research Center, Inc., USA). After isolation, RNA was dissolved in nuclease-free water and its concentration was analyzed using a nanophotometer (Implen, Germany). cDNA was synthesized from the extracted RNA using the High Capacity cDNA Reverse Transcription kit (Applied biosystems, USA), according to the manufacturer's instructions.

Analysis of immune gene expression using real-time PCR
Quantification of immune gene expression in peritoneal macrophages and splenocytes was performed using the QuantStudio™ 7 FlexReal-Time PCR System (ThermoFisher scientific, USA) and SYBR1 Premix Ex Taq™ II (Takara Bio Inc., Japan). Relative gene expression was calculated using the 2 -ΔΔC T method [27] and β-Actin as the reference gene (Table 1).

Statistical analysis
Statistical analysis was performed using the Statistix 8.1 software. Data were analyzed using one-way ANOVA followed by comparison with the saline or ginseng group. Differences between groups were considered to be statistically significant at P < 0.05 and P < 0.01. Compared to the normal group, the CY-treated group showed a marked reduction in the spleen index and spleen size. Compared to the CY group, spleen index of the group treated with 250 or 500 mg/kg BW CFAMs was significantly different; however, no significant differences in the body weight were observed between these groups (S1 Fig). Further, significant differences were not observed in the spleen size and spleen index of the group treated with 50 or 100 mg/kg BW CFAMs and the CY group.

Effect of CFAMs treatment on splenic lymphocyte proliferation
Splenocytes from each group were incubated with two mitogens, namely Con A and LPS. Con A and LPS stimulate the T and B cells in splenocytes, respectively. Fig 1C shows that compared to the normal group, the proliferative response of splenic lymphocytes to both the T-and Bcell mitogens decreased markedly in the CY-treated group. However, compared to the CYtreated group, the CFAMs-treated group showed enhanced T and B cell proliferation in a dose-dependent manner. Further, the proliferation in the CFAMs-treated group was similar to that observed in the red ginseng-treated group, and significantly higher than that observed in the normal group.

Effect of CFAMs on NK cell activity of splenocytes
In order to evaluate the NK cell activity of splenocytes, the splenocytes were co-cultured with YAC-1 cells. Cytotoxicity was determined using the LDH assay. Fig 1D shows that CY treatment suppressed splenic NK cell activity. However, CFAMs treatment gradually stimulated the NK cell activity of splenocytes. Notably, compared to the red ginseng-treated and normal groups, higher splenic NK cell activity was observed in the group treated with a high dose of CFAMs.

Effect of CFAM on immune-associated gene expression in splenic lymphocytes
By analyzing the expression of immune-associated genes (including those for cytokines) using qRT-PCR, the immuno-enhancing effects of CFAMs were elucidated. Fig 2 shows that compared to the normal group, the CY-treated group showed reduced expression of immune-associated genes in the spleen lymphocytes. However, treatment with varying concentrations of CFAMs enhanced immune-associated gene expression in splenic lymphocytes, which responded to the T-and B-cell mitogens. After CFAMs treatment, genes such as IL-4, IL-10, TNF-α, IFN-γ, TLR-4, and COX-2 in splenic lymphocytes were highly upregulated and responded more to the B cell mitogen (LPS) than to the T cell mitogen (Con A). In contrast, the expression of IL-1β, IL-2, and IL-6 was considerably higher in the Con A-stimulated cells than in the LPS-stimulated cells.

Effect of CFAMs treatment on peritoneal macrophage proliferation and nitric oxide production
In order to investigate the effect of CFAMs on macrophage proliferation and nitric oxide production, peritoneal macrophages were isolated from each group of mice and stimulated using LPS. As shown in Fig 3A and 3B, compared to the CY-treated mice, the CFAMs-treated mice displayed a significant and dose-dependent increase in peritoneal macrophage proliferation and nitric oxide (NO) production. At high doses (250-500 mg/kg BW), CFAMs restored peritoneal macrophage proliferation and nitric oxide production. These restored levels were similar to those observed for the non-treated mice (normal group), and similar or higher than those observed for the ginseng-treated group.

Effect of CFAMs on peritoneal macrophage phagocytosis
The neutral red uptake method has been used for the evaluation of phagocytosis by mouse peritoneal macrophages. Fig 3C shows that peritoneal macrophage phagocytosis was significantly lower in the CY group than in the normal group. Compared to the CY-treated group, peritoneal macrophage phagocytosis rates were remarkably and dose-dependently higher in the CFAMs-treated group. High doses of CFAMs also induced the recovery of peritoneal macrophage phagocytosis.

Effect of CFAMs treatment on immune-associated gene expression in peritoneal macrophages
Immune-associated gene expression in peritoneal macrophages, in addition to that observed in splenic lymphocytes, was analyzed in order to investigate the effect of CFAMs treatment on the activation of peritoneal macrophages from the CY-treated mice. Fig 4 shows that the expression of iNOS, IL-6, TNF-α, COX-2, and IFN-γ was remarkably downregulated in the CY-treated mice. Compared to the CY-treated group, the CFP-treated group showed a dosedependent and significantly higher expression of these immune-associated genes. Furthermore, peritoneal macrophages treated with 100, 250, and 500 mg/kg BW CFAMs showed similar or higher gene expression than the red ginseng-treated group (positive control).

Fig 1. Effects of various concentrations of Codium fragile extracts.
The spleen was collected from each group of mice (A). The spleen weight was divided with body weight for calculating the spleen index (B). The splenocytes were isolated and were stimulated with mitogens for splenic lymphocyte proliferation assay (C). The splenocytes were co-cultured with YAC-1 cells for splenic NK cells cytotoxic activity (D). Data were observed to be significantly different compared to the saline group ( � , P < 0.01) and ginseng group (#, P < 0.05).

Effect of CFAMs treatment on NF-κB and MAPK signaling pathways
To analyze the mechanism by which CFAMs regulated immune responses in physiological systems, RAW264.7 cells were grown in the presence of varying concentrations of CFAMs. As shown in Fig 5, compared to the negative control (RPMI), CFAMs treatment increased phospholyration of IκBα and NF-κB-p65 in a dose-dependent manner. In addition, phospholyration of ERK, JNK, and p38, which are the key biomarkers for the mitogen-activated protein kinase (MAPK) pathway, was also investigated. It was observed that CFAMs increased the phosphorylation of these proteins in a dose-dependent manner.

Effect of CFAMs treatment on NF-κB and MAPK activation inhibited-RAW264.7 cells
In order to investigate the effects of CFAMs on cellular signaling for immune-regulation, TNF-α expression was examined in NF-κB and MAPK activation inhibited-RAW264.7 cells (Fig 6). Compared to the RPMI group (negative control), the CFAMs-treated group showed increased expression of TNF-α in the NF-κB and MAPK activation inhibited-macrophages in Peritoneal macrophages were isolated from each group of mice in the sacrifice day. The cells were stimulated with or without 1 μg/mL of LPS. LPS was used to investigate the immune response of macrophages isolated from each group. After 24h stimulated, the stimulated-cells were used for the peritoneal macrophage proliferation assay (A), and the cultured medium was used for the nitric oxide production assay (B). Phagocytosis of peritoneal macrophages was determined using the neutral red uptake (C). Significant differences were observed at P < 0.01 compared with the saline group ( � ) and at P < 0.05 compared with the ginseng group (#). https://doi.org/10.1371/journal.pone.0211570.g003 Immune-enhancing effects of Codium fragile polysaccharides on immune-suppressed mice a dose-dependent manner. Notably, in the NF-κB and ERK inhibited-RAW264.7 cells treated with high doses of CFAMs (for NF-κB inhibited cells, CFAMs = 7.5 and 15 μg/mL; for ERK inhibited cells, CFAMs = 15 μg/mL), TNF-α expression was restored and similar to or higher than that observed for the RPMI group (negative control). Immune-enhancing effects of Codium fragile polysaccharides on immune-suppressed mice

Discussions
In humans, immune-regulation is important for protection against several diseases, including tumorigenesis [30]. CY, a well-known chemotherapeutic drug used for tumor treatment, induces adverse effects on the immune system and causes immunosuppression, which can be life threatening [31]. Therefore, many natural compounds having immune-modulation activities have been reported recently. These compounds alleviate the deleterious effects of chemotherapeutic drugs [1,[30][31][32]. Particularly, polysaccharides with low toxic side-effects have been used as adjuvant medications or immunomodulators for strengthening host defense responses [33,34]. In the present study, we determined the efficacy of polysaccharides isolated from C. fragile in enhancing immunological functions in immunosuppressed BALB/c mice. The spleen is an important lymphoid organ consisting of immune cells, such as monocytes, macrophages, B and T lymphocytes, for mediating immune responses [35]. Spleen-associated factors, such as spleen index and NK cell activities, reflect the immune status of an individual [36]. Especially, lymphocyte proliferation is the most direct indicator of the state of host immunity [37]. T and B lymphocytes are known to react with stimulation activated by antigens or mitogens, which is a common type of non-specific immune regulation [12]. Proliferation of T and B lymphocytes is induced by Con A and LPS, respectively [38]. Fig 1 shows that CFAM accelerated the recovery of spleen size and spleen index in the CY-induced immunosuppressed mice. CFAM also promoted T-and B-cell proliferation and NK cell activities in splenocytes in a dose-dependent manner. Previous studies reported that CY could inhibit both humoral and cellular immune responses [39,40]. Notably, CFAM-treated mice did not show any mortality. Additionally, compared to the normal group, their body weights also did not change significantly (S1 Fig) [30,41,42]. These results indicated that under immunosuppressive conditions, CFAMs treatment establishes immune homeostasis in physiological systems.
Macrophages are immune cells which play important roles in combating infection and removing tumor cells in hosts. They are also closely associated with immune-modulation in cancer therapy [43]. Phagocytes, including macrophages, are important for the regulation of innate immune responses. Phagocytosis is the mechanism by which the phagocytes ingest and kill microorganisms or cancer cells [32]. Increased production of NO by macrophages protects host cells from infection [44]. Macrophages also play an important role as antigen-presenting cells and coordinate with T lymphocytes for regulation of adaptive immune responses [32]. Fig 3 shows that in immunosuppressed mice, CY impaired peritoneal macrophage proliferation and NO production; however, CFAMs enhanced proliferation and NO production of peritoneal macrophages in a dose-dependent manner. Furthermore, CFAMs considerably increased phagocytosis by peritoneal macrophages in a dose-dependent manner, suggesting that CFAMs could enhance non-specific immune functions in CY-treated immunosuppressed mice to improve immune homeostasis.
Several subtypes of T cells, including T helper (Th) cells, cytotoxic T cells, suppressor T cells, effector T cells, are present. Particularly, the type 1 (Th1) and type 2 (Th2) T helper cells play different roles under different immune conditions. Th1 cells are activated during cellmediated immune responses; however, Th2 cells are stimulated during humoral or allergic responses [45]. Th1 and Th2 cells produce specific cytokines to directly and indirectly control specific immune responses. IL-2, IFN-γ, and TNF-α are regulated by Th1 cells, whereas IL-4, IL-6, and IL-10 are modulated by Th2 cells [46]. Our results showed that cytokine expression in the CFAMs-treated group was higher than that in the CY-treated group. This suggested that CFAMs, unlike in CY-treated group, stimulated the secretion of Th1 and Th2 cytokines to restore immunosuppression and control the dynamic balance between the Th1 and Th2 cells in mice [47].
To regulate immune responses, intermediates of the NF-κB signaling pathway coordinate with inflammatory intermediates, including iNOS, COX-2, and the pro-inflammatory cytokines [48]. In addition, the MAPK signaling pathway involving ERK1/2, JNK, and p38 regulates cellular differentiation and growth by modulating immune responses during stress [49]. MAPK, along with NF-κB, regulates inflammatory cytokine production and related processes [50]. Our results showed that CFAMs increased the phosphorylation of IκBα, NF-κB-p65, ERK, JNK, and p38. Among these, phosphorylation of JNK was the highest. Moreover, it was shown that TNF-α expression, widely used as an experimental model for studying immunomodulatory activities of polysaccharides [36], was affected in the NF-κB and MAPK inhibited cells. In these cells, the expression of TNF-α was observed to be considerably low. These data suggested that CFAMs activated macrophages via the MAPK pathway and involved the phosphorylation of JNK.

Conclusions
The present study demonstrated that CFAMs, anionic macromolecules from C. fragile, enhanced the immune responses in CY-treated immunosuppressed mice via peritoneal macrophage proliferation, NO production and phagocytosis by peritoneal macrophages, enhancing macrophage gene expression, increasing splenic lymphocyte proliferation and NK cell activity, spleen index, and splenic lymphocyte gene expression. Additionally, it was shown using macrophages and NF-κB-and MAPK-inhibited cells that CFAMs stimulated macrophages via the MAPK pathway. Consequently, our results might help in elucidating the immune-enhancing mechanisms of CFAMs under immune-suppressive conditions in cancer treatment. In conclusion, CFAMs may be used as a potent biofunctional and pharmaceutical material for enhancing immunity in humans.