1 INTRODUCTION

Numerous studies have reported that some specific polysaccharides have immunomodulatory, antitumor, antibacterial, and antiviral activities (Ramberg et al., 2010). The sources of these polysaccharides are diverse, involving higher plants, animals, fungi, and bacteria. In addition, polysaccharides from marine animals and plants, such as fucoidan extracted from brown algae and marine invertebrates, have also been found to exhibit immune regulation and antiviral activity (Senni et al., 2011). Because surface seawater contains large amounts of bacteria (~10⁶ bacterial cells/mL; Hagström et al., 2002), marine bacteria have attracted increasing attention as a source of bioactive compounds, such as secondary metabolites from marine cyanobacteria (Tan, 2007; Nunnery et al., 2010).

The polar environment contains a large number of microorganisms. The unique conditions of high salinity and low temperature in this environment have resulted in the development of unusual microbial populations. Exopolysaccharides (EPSs) produced by Antarctic bacteria are abundant in the Antarctic marine environment, such as in sea ice and ocean particles, where they might assist microbial communities to endure extreme temperatures, salinities, and nutrient limitations (Nichols et al., 2005). However, in recent years there have been only a few reports regarding immune activities by Antarctic bacterial EPSs, such as a study by Bai et al. (2012).

Macrophages are widely distributed in human body tissues and play important roles in both innate and adaptive immunities. They have diverse functions, including phagocytosis, antigen presentation, and secretion of cytokines, enzymes, and complement components. Macrophages are central to many disease states and have emerged as important therapeutic targets in many diseases (Wynn et al., 2013). A large number of polysaccharides from fungi and higher plants have been found to activate macrophages and regulate their immune responses (Wasser, 2002; Schepetkin and Quinn, 2006; Li et al., 2007). Polysaccharides from bacteria have also been found to affect macrophage activity (Kitazawa et al., 2000; Bai et al., 2012). Moreover, macrophages express many polysaccharide receptors, such as toll-like receptors (TLRs), scavenger receptors (SRs), complement receptor 3 (CR3), β-glucan receptors, and mannose receptor (Leung et al., 2006; Schepetkin and Quinn, 2006). The macrophage cell line, RAW264.7, is considered to be a good model for studying macrophage activation in vitro. The present study investigated RAW264.7 cell activation by a B-3 EPS isolated from an Antarctic psychrophilic bacterium B-3.

2 MATERIAL AND METHOD 2.1 Bacteria source

The Antarctic Psychrobacter sp. B-3 was isolated from collected ice samples at Uruguay Station (62°11′50.52″S and 58°55′50.4″W) during the 24th Chinese Antarctic scientific expedition by screening and isolation in Zobell 2216E medium(containing 0.5% peptone, 0.1% yeast extract and 2.0% agar). The isolated bacterial strain was confirmed to be a Psychrobacter sp. through 16S rDNA identification.

2.2 Isolation, purification, and compositional analysis of B-3 EPS

After recovery of Antarctic Psychrobacter sp. B-3 from -80℃ refrigerator, it was inoculated into 2216E liquid medium at a concentration of 1% (by vol) and cultured at 10℃ and 150 r/min for 72 h. Subsequently, the fermentation broth was centrifuged (6 000×g, 10 min) and the supernatant mixed with three volumes of 95% ethanol and incubated for ethanol precipitation overnight at 4℃. After centrifugation at 7 000×g’s, the precipitate was collected, redissolved in distilled water, and crude polysaccharides obtained by removing protein using Sevage reagent (chloroform/ butanol, 5/1, v/v; Fang, 1984). The crude EPS preparation was then purified using a DEAESephadex A-50 ion exchange column (50 cm H× 2.6 cm D; Sigma-Aldrich, Inc., St. Louis, MO, USA) with phosphate buffer eluent (0.02 mol/L, pH 7.4) at a flow rate of 24 mL/h. Fractions (4 mL each) were tested quantitatively for carbohydrates (Li et al., 1982), and positive fractions were pooled, freezedried, and further purified by Sephadex G-100 gel chromatography (100 cm H×2 cm D; Sigma-Aldrich, Inc.) using ammonium acetate eluent (0.05 mol/L) at a flow rate of 8 mL/h. EPS carbohydrate content was measured using the phenol-sulfuric acid method (Fang, 1984). The molecular weight of the purified sample was determined by high-performance gel permeation chromatography (HP-GPC). Hydrolyzed monosaccharides from EPS were conventionally converted into alditol acetates and analyzed by gas chromatography (GC) with column OV-225 at temperature of 240℃, and the monosaccharide composition were determined (Li et al., 1982).

2.3 Measurement of cell viability

RAW264.7, a mouse macrophage cell line, was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China) and cultured in Gibco DMEM medium (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Cells were grown at 37℃ in a humidified incubator with 5% CO₂ and cell viability were measured using an MTT assay (Yoshimura et al., 1993). Subsequently, RAW264.7 cells were cultured in 96-well plates to a density of 2×10⁴ cells/well with or without varying concentrations of B-3 EPS (0.02–100 μg/mL) in a total volume of 200 μL/well for 24 or 48 h. After culturing, 20 μL of MTT (5 mg/mL in PBS; SigmaAldrich, Inc.) were added to each well and incubated for 4 h. Then, the supernatant was discarded and 100 μL of DMSO (AppliChem Inc., St. Louis, USA) added to each well. Finally, the plate was incubated in a thermostatic oscillation incubator for 15 min to dissolve the crystals, and the A₄₉₃ was measured using a microplate reader (Multiskan MK3, Thermo Fisher Scientific Inc.).

2.4 Determination of phagocytic ability

The phagocytic ability of RAW264.7 cells was measured by FITC-dextran (Sigma-Aldrich, Inc.) internalization using a flow cytometer. Following culturing with B-3 EPS or LPS (from E. coli 055:B5; purity, >97%; Sigma-Aldrich, Inc.) for 24 h, FITCdextran then was added to the wells to a final concentration of 1 mg/mL and incubated at 37℃ for 45 min, except in a well without B-3 EPS for a control. Phagocytosis was stopped by discarding the supernatants and washing with cold PBS. Subsequently, these cells were washed thrice, harvested, and resuspended in PBS, and phagocytic activity were analyzed using a flow cytometer (BD Accuri^TM C6; Becton, Dickinson & Co., Franklin Lakes, NJ, USA).

2.5 Measurement of nitric oxide

Nitric oxide (NO) production in the culture supernatant was determined using the Griess reagent assay. A standard NaNO₂ gradient was diluted to 100, 60, 40, 20, 10, and 0 μmol/mL. After treating cells with varying B-3 EPS concentrations for 48 h, culture supernatants and a volume of a NaNO₂ solution, for a total volume of 50 μL/well, were moved to 96-well plates and 50 μL of Griess reagent (Beyotime Biotechnology Inc., Jiangsu, China) added to each well. The A₅₄₀ was determined and sample NO concentrations were obtained using a standard NaNO₂ curve.

2.6 Determination of TNFα secretion

Cellular TNFα secretion was evaluated using an ELISA kit (Mouse TNF-alpha ELISA Kit 70-EEK2823, MultiSciences Biotechnology Co., Ltd. Hangzhou, China). After culturing cells with B-3 EPS for 48 h, the culture supernatants were diluted twofold. Meanwhile, standard mouse TNFα was diluted to 400, 160, 64, 25.6, 10.24, 4.10, and 0 pg/mL, and the diluted samples and standard TNFα solutions, for a total volume of 50 μL/well, were added to the wells of an ELISA plate. The specific determination step was performed according to the manufacturer’s instructions.

2.7 Inhibition of signaling transduction by TLR4

The potential signaling pathway involved in the activation of RAW264.7 cells by B-3 EPS was explored by investigating the effects of B-3 EPS on TLR4-mediated immune response in these cells. The TLR4 inhibitor TAK-242 (Kawamoto et al., 2008) was used to test TLR4’s effect on NO production by RAW264.7 cells after being stimulated by B-3 EPS. The cells were cultured in 12-well plates for 24 h and, subsequently, TAK-242 (InvivoGen, San Diego, CA, USA) was added to some of these wells to a final 1 μg/mL and incubated for 1 h. Next, all of the wells, with and without TAK-242, were incubated with B-3 EPS (20 μg/mL) or LPS (1 μg/mL) for 24 h. NO production in culture supernatants was determined using the Griess reagent method, as mentioned above.

2.8 Western blot analysis

Total protein was extracted using an efficient RIPA tissue and cell lysate (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and protein concentrations were determined using a bicinchoninic acid assay kit (Beijing Solarbio Science & Technology Co.). A 5-μg protein sample from each well was transferred to an individual lane, the protein components separated by 10% SDS-PAGE, and then the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% albumin bovine (Beijing Solarbio Science & Technology Co.) in 1× Tris buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) at room temperature for 1 h. Subsequently, membranes were incubated with primary antibodies, antiphospho-p65, anti-Iκβα (Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-β actin, at 4℃ overnight. The membranes were then incubated with horseradish peroxidase conjugated anti-rabbit antibody at room temperature for 1 h and rinsed with TBS-T. Protein bands were detected using a Pro-light Horseradish Peroxidase Chemiluminescent Kit (Tiangen Biotechnology Co., Ltd., Beijing, China) and Molecular Imager^® Gel Doc^TM XR+ and ChemiDoc^TM XRS+ Systems (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.9 Statistical analysis

Data were expressed as mean±SD. SPSS 11.0 (IBM Corp, Chicago, IL, USA) was used for statistical analyses and significant differences between means were analyzed using Student’s t-test. Values of P < 0.05 were regarded as statistically significant.

3 RESULT 3.1 Characteristics of B-3 EPS

HP-GPC revealed only a single symmetrical peak, indicating B-3 EPS purity and homogeneity and its suitability for structural analysis (Fig. 1a). B-3 EPS’s molecular weight was determined to be 5 400 Da. GC determination of the sugar composition showed that B-3 EPS was composed of two monosaccharides, including mannose and glucose (Fig. 1b).

3.2 Effect of B-3 EPS on RAW264.7 cell viability

The effects of varying B-3 EPS concentrations on RAW264.7 cell viability were analyzed using the MTT method. The results showed that B-3 EPS at < 20 μg/mL had no cytotoxic effect on these cells after stimulation for 24 or 48 h. However, the B-3 EPS significantly improved these cells’ viability, as shown in Fig. 2.

3.3 Effects of B-3 EPS on phagocytosis

Macrophages are strategically located throughout the body tissues, where they ingest and process foreign materials, dead cells, and debris as well as recruit additional macrophages in response to inflammatory signals (Murray and Wynn, 2011). Phagocytosis is critical for macrophage defense against invading pathogens and for metabolic waste clearance. Therefore, the ability of B-3 EPS to affect RAW264.7 cell phagocytic capability was studied by analyzing the uptake of FITC-dextran by these cells using flow cytometry. Phagocytic uptake of FITCdextran by cells incubated with B-3 EPS increased in a dose-dependent manner, suggesting that these cells’ phagocytic ability was enhanced by B-3 EPS (Fig. 3).

3.4 Effects of B-3 EPS on NO production

NO is one of the inflammatory mediators secreted by macrophages for mediating the inflammatory response. Both NO as well as TNFα have the ability to destroy pathogenic microorganisms and cancer cells. The effects of B-3 EPS on RAW264.7 cell NO production were determined using the Griess reagent assay. At concentrations of 2, 20, and 100 μg/mL, B-3 EPS significantly increased NO production in cell culture supernatants, indicating that, under these conditions, B-3 EPS induced macrophages to produce more NO (Fig. 4).

3.5 Effect of B-3 EPS on TNFα concentration

At the concentrations of 2, 20, and 100 μg/mL, B-3 EPS was able to increase TNFα concentrations in RAW264.7 cell culture supernatants, especially at 20 and 100 μg/mL (P < 0.05, Fig. 5).

3.6 Effects of TAK-242 on NO production by RAW264.7 cells incubated with B-3 EPS

TLR4 is one of the TLRs identified as a signaling receptor for LPS (Akashi et al., 2000). LPS delivered by CD14 to TLR4/MD-2 initiates a signaling cascade, which eventually leads to NF-κB activation, resulting in production of proinflammatory mediators, such as NO and TNFα (Akira and Takeda, 2004). In addition, other polysaccharides and complexes have been found to be capable of activating NF-κB via TLR4, resulting in NO and TNFα production (Ando et al., 2002; Kawai and Akira, 2011). The signaling pathway involved in RAW264.7 cell activation by B-3 EPS was explored by employing a TLR4 inhibitor, TAK-242, to test whether it affected NO production induced by B-3 EPS. The results showed that TAK-242 significantly reduced NO production induced by B-3 EPS (Fig. 6, P < 0.05), suggesting that B-3 EPS might primarily have triggered downstream signaling in these cells by acting on TLR4.

3.7 NF-κB-signaling pathways in RAW264.7 cell activation by B-3 EPS

NF-κB is a transcription factor that plays a critical role in many biological processes, including the immune system (Hayden and Ghosh, 2004; Sun, 2012), and usually participates in acute inflammatory macrophage activation. Therefore, in the present study, the activation of NF-κB-signaling pathway in RAW264.7 cells treated with B-3 EPS was investigated and the results showed phosphorylation of p65, a subunit of NF-κB, and degradation of IκBα. Furthermore, when compared with controls, p65 phosphorylation was clearly increased by both B-3 EPS and LPS stimulation. Meanwhile, IκBα was completely degraded after B-3 EPS or LPS treatment at certain time points (Fig. 7). These findings indicated that NF-κB was activated in RAW264.7 cells incubated with B-3 EPS.

4 DISCUSSION

Extreme environments offer novel microbial biodiversity, resulting in the production of varied and promising EPSs (Nichols et al., 2005). In fact, a large number of EPSs produced by microorganisms are known to play an important role in maintaining ecosystem stability and are beneficial to other organisms. Recently, increasing attention has been paid to low-temperature adaptation in EPSs and potential applications for their unique properties. In the present study, a new immunoactive EPS, EPS B-3, was isolated and purified from an Antarctic bacterium, and the results obtained provide some evidence for potential biotechnological applications of this Antarctic resource.

Macrophages and other lymphocytes proliferate in response to polysaccharide stimulation, which is mediated through polysaccharide binding to their corresponding receptors (Leung et al., 2006). The results obtained here showed that B-3 EPS, at concentrations of 0.02, 0.2, and 2 μg/mL, significantly promoted RAW264.7 cell viability. This implied that this EPS might have bound to a receptor on RAW264.7 cells and induced downstream signals that altered cell viability. Activated macrophages can release many active substances, including enzymes, cytokines, and inflammatory mediators. Among them, TNFα and NO have a role in destroying tumor cells. The present findings demonstrated that B-3 EPS, at concentrations of 2, 20, and 100 μg/mL, improved TNFα and NO production by RAW264.7 cells, implying that this EPS increased macrophage ability to destroy tumor cells. Moreover, it must be noted that at low concentrations, B-3 EPS promoted cell viability without inducing increased TNFα and NO release, whereas, at high concentrations, this EPS significantly induced increased TNFα and NO production. Therefore, it was concluded that B-3 EPS activated these cells to different levels depending on its concentration. Macrophages have strong phagocytosis and digestive functions, which can ingest large antigen particles, such as pathogens, and consume and clear metabolic waste. Thus, phagocytosis is critical for macrophages in executing their functions. These results demonstrated that B-3 EPS enhanced the phagocytic ability of these cells, indicating that it might have helped initiate and strengthen the macrophage immune response against pathogens and tumor cells.

TLR stimulation is known to upregulate the expression of hundreds of genes in macrophages. These genes are potentially involved in antimicrobial defense, metabolic changes, and tissue repair; and, more importantly, some gene products are associated with the positive and negative regulation of inflammatory responses by controlling TLR-signaling pathways (Takeuchi and Akira, 2010). The results obtained in the present study showed that a TLR4 inhibitor significantly reduced NO production in cells already activated by B-3 EPS, which suggested that TLR4 might have involved in macrophages activation by B-3 EPS.

The NF-κB-signaling pathway plays an important role in immune regulation, particularly in the early stages of immune and inflammatory responses. NF-κB plays an essential role in early events of innate immune responses through TLR-signaling pathways. In various stages of the inflammatory response, NF-κB regulates many molecules, including proinflammatory factors, anti-inflammatory factors, chemokines, adhesion molecules, and colony stimulating factors. In addition, NF-κB can broadly influence gene expression events that affect cell survival, differentiation, and proliferation beyond the confines of the immune response (Hayden and Ghosh, 2008). In cytoplasm of unstimulated cells, NF-κB exists in an inactive form bound to IκBα. After receiving an activation signal from upstream, IκBα is phosphorylated, ubiquitinated, and then degraded. Subsequently, some NF-κB subunits are phosphorylated at certain sites. Among them, p65 phosphorylation is the first event recognized after IκBα degradation (Naumann and Scheidereit, 1994; Neumann et al., 1995). Later, NF-κB is transferred to the nucleus and bound to DNA to activate transcription. Therefore, in the present study, IκBα degradation and p65 phosphorylation were measured and the results demonstrated that B-3 EPS activated the NF-κBsignaling pathway in these cells. This indicated that this EPS might have affected macrophage immune responses as well as other aspects of cell activity by activating transcription factor NF-κB.

5 CONCLUSION

In conclusion, B-3 EPS extracted from an Antarctic psychrophilic bacterium was found to activate RAW264.7 cells and regulate their immune response. Stimulation by B-3 EPS over a certain concentration range enhanced cell viability, phagocytic ability, and TNFα and NO secretion in these cells. Furthermore, cellular NO production following treatment with B-3 EPS was inhibited by a TLR4 inhibitor. After stimulation by B-3 EPS, the NF-κB-signaling pathway in these cells was activated. In conclusion, B-3 EPS activated RAW264.7 cells by acting on TLR4 and inducing NF-κB activation.

6 ACKNOWLEDGMENT

We thank B. S. ZHANG for language rhetoric guidance.