1,4-β-d-Glucomannan from Dendrobium officinale Activates NF-кB via TLR4 to Regulate the Immune Response

2,3-O-acetylated-1,4-β-d-glucomannan (DOP-1-1) is a polysaccharide isolated from the stem of Dendrobium officinale. DOP-1-1 has been demonstrated to have remarkable immunomodulatory properties, but little is known about the influence of its structural diversity on bioactivity (and even less about the exact mechanism underlying its immune responses). First, DOP-1-1 was stabilized at different temperatures and pH conditions based on differential scanning calorimetry and size exclusion-chromatography–high-performance liquid chromatography. Then, a detailed study on the effects of DOP-1-1 on a human leukemia monocytic cell line (THP-1) under normal conditions was undertaken. DOP-1-1 promoted the translocation of nuclear factor-kappa B (NF-κB) and degradation of IκB proteins. The expression of genes and proteins closely associated with the immune, survival and apoptotic functions of NF-κB were analyzed by quantitative real-time RT-PCR. Furthermore, CCL4 and IP10 were confirmed to be the novel targets of the immune response stimulated by DOP-1-1. The phosphorylation of NF-кB was inhibited by treatment with a toll-like receptor 4 (TLR4) antagonist (TAK-242) and myeloid differentiation factor 88 (MyD88) inhibitor (ST2825). These data suggested: (i) the O-acetylated glucomannan DOP-1-1 is present in the steady state in low-pH solutions; (ii) DOP-1-1 can induce an immune response through NF-кB mediated by a TLR4 signaling pathway; and (iii) CCL4 and IP10 could be the novel targets of the immune response stimulated by O-acetylated glucomannan.

In addition, polysaccharides, as the major active constituent of Dendrobium species, recently have been reported to have immunomodulatory, antioxidant, and antitumor effects, to inhibit apoptosis, In addition, polysaccharides, as the major active constituent of Dendrobium species, recently have been reported to have immunomodulatory, antioxidant, and antitumor effects, to inhibit apoptosis, and to prevent liver injury and fibrosis [5]. Moreover, the conformational changes of biopolymers, such as proteins, lipids, and polysaccharides have pivotal roles in biologic activities [6][7][8][9][10]. However, the study about the relationship between the conformational dynamics of polysaccharides and biological processes are scarce.
Polysaccharides are not only basic nutrients but also play a key part in herbal medicine [11]. Additionally, it is commonly believed that polysaccharides have immunoregulatory activities [12][13][14]. However, the immune responses of various polysaccharides refer to different signaling pathways [15,16].
The regulation of immunity plays an important part in life processes and the treatment of diseases [17,18]. The immune system of an organism provides an extraordinary defense against infections, where innate immunity is the first line of defense in mediating the initial protection against foreign attacks. Once it recognizes matter as "non-self", it activates multiple chemical and physiologic processes to control and eliminate the pathogen. Thus, the immune response is associated with driving signaling pathways such as nuclear factor-kappa B (NF-κB) [19] and janus kinase/signal transducers and activators of transcription (JAK/STAT) [20,21].
The immunomodulatory mechanism of action of the polysaccharide O-acetylated glucomannan from D. officinale has not been studied even though its immunomodulatory activities have been noted widely [1,22,23]. In our previous study, O-acetylated glucomannan from D. officinale showed remarkable immune-enhancing properties [22]. Additionally, the thermal stability of polysaccharides from D. officinale under a thermal transition temperature with changing pH has not been reported although its structural characterization has been fully elucidated [24,25]. In fact, O-acetylated glucomannan was shown to have a molecular weight of 1.78 × 10 5 Da and a backbone composed of →4)-Manp-(1→ and →4)-Glcp-(1→ residues (Figure 1).  [22,24,25].
Taking the thermal stability of O-acetylated glucomannan into consideration, the immunomodulatory mechanism of action of O-acetylated glucomannan was further investigated under normal conditions. This study was carried out because O-acetylated glucomannan from D. officinale is widely used in ethnopharmacology and the functional-food industry.

Carbohydrate and Protein Contents of Crude and Pure O-Acetylated Glucomannan
In our previous study, the DOP-1-1 (a type of O-acetylated glucomannan) present in D. officinale was isolated by HPLC [22]. To further confirm the homogeneity of O-acetylated glucomannan isolated from D. officinale, carbohydrate and protein contents were measured.
The total sugar content of D. officinale increased from 76% (w/w, DOP, after dialysis) to 99% (w/w, DOP-1-1) after purification by HPLC [22], which explains the decrease in the percentage content of contaminating proteins to zero after purification (Table 1). Taking the thermal stability of O-acetylated glucomannan into consideration, the immunomodulatory mechanism of action of O-acetylated glucomannan was further investigated under normal conditions. This study was carried out because O-acetylated glucomannan from D. officinale is widely used in ethnopharmacology and the functional-food industry.

Carbohydrate and Protein Contents of Crude and Pure O-Acetylated Glucomannan
In our previous study, the DOP-1-1 (a type of O-acetylated glucomannan) present in D. officinale was isolated by HPLC [22]. To further confirm the homogeneity of O-acetylated glucomannan isolated from D. officinale, carbohydrate and protein contents were measured.
The total sugar content of D. officinale increased from 76% (w/w, DOP, after dialysis) to 99% (w/w, DOP-1-1) after purification by HPLC [22], which explains the decrease in the percentage content of contaminating proteins to zero after purification (Table 1).

Stability of DOP-1-1 under Different pH Conditions
Biopolymers are induced by intramolecular non-covalent forces to fold into well-defined tertiary structures [26]. Therefore, DSC was used to ascertain the thermal stability of DOP-1-1 under different pH conditions. Little change in DOP-1-1 was observed in different pH solutions ( Figure 2b) compared with buffer, which may have been due to unfolding or a monomeric state of DOP-1-1. Thus, the DSC profiles of different concentrations of DOP-1-1 were further analyzed. Similarly, little change was observed among these samples (Figure 2a,b). These results suggested that DOP-1-1 might be extremely stable under different pH conditions (Figure 2b). To further confirm this conclusion, the stability of DOP-1-1 under different pH conditions was examined with HPLC. HPLC data also demonstrated that DOP-1-1 was insensitive to pH ( Figure 2c). There are many challenges to overcome when developing manufacturing processes for polysaccharides, such as temperature and pH.

Stability of DOP-1-1 under Different pH Conditions
Biopolymers are induced by intramolecular non-covalent forces to fold into well-defined tertiary structures [26]. Therefore, DSC was used to ascertain the thermal stability of DOP-1-1 under different pH conditions. Little change in DOP-1-1 was observed in different pH solutions ( Figure 2b) compared with buffer, which may have been due to unfolding or a monomeric state of DOP-1-1. Thus, the DSC profiles of different concentrations of DOP-1-1 were further analyzed. Similarly, little change was observed among these samples (Figure 2a,b). These results suggested that DOP-1-1 might be extremely stable under different pH conditions (Figure 2b). To further confirm this conclusion, the stability of DOP-1-1 under different pH conditions was examined with HPLC. HPLC data also demonstrated that DOP-1-1 was insensitive to pH ( Figure 2c). There are many challenges to overcome when developing manufacturing processes for polysaccharides, such as temperature and pH.

Toxicity of DOP-1-1 to THP-1 Monocytes
THP-1 is a human leukemia monocytic cell line that has become a common model to estimate the modulation of monocyte and macrophage activities. As THP-1 monocytes are a type of terminally differentiated cells [27], the toxicity of DOP-1-1 to mature macrophages was examined. Results suggested that DOP-1-1 had absolutely no effect on macrophages derived from a human leukemia monocytic cell line (Figure 3a-

Toxicity of DOP-1-1 to THP-1 Monocytes
THP-1 is a human leukemia monocytic cell line that has become a common model to estimate the modulation of monocyte and macrophage activities. As THP-1 monocytes are a type of terminally differentiated cells [27], the toxicity of DOP-1-1 to mature macrophages was examined. Results suggested that DOP-1-1 had absolutely no effect on macrophages derived from a human leukemia monocytic cell line (Figure 3a-d).

Effects of DOP-1-1 on NF-κB Signaling
Our previous study showed that DOP-1-1 has immunomodulatory properties [22]. To further study the mechanism of DOP-1-1-induced immunoregulation, the proteins associated with immune responses were examined. The cellular response to different concentrations of DOP-1-1 resulted in a phosphorylation increase of NF-κB in a dose-dependent manner and degradation of IκBα (Figure 4a). Other proteins were not detected. NF-κB pathway can be activated via at least three distinct routes: Canonical, atypical and noncanonical pathways. Our results suggested that DOP-1-1 induced activation of NF-κB signaling via Iκκ-independent means (Iκκ-β and Iκκ-α were not detected). The activation of NF-κB can induce its nuclear translocation. In order to ascertain whether DOP-1-1 translocated NF-κB into the nucleus, western blotting for cytoplasmic and nuclear fractions was carried out using a Nuclear and Cytoplasmic Protein Extraction Kit. The expression of NF-κB (p65) in the cytoplasmic fractions of THP-1 cells decreased, and that of NF-κB (p65) in nuclear extracts increased, in a dose-dependent manner (Figure 4b). In order to confirm these results, NF-κB expression was determined by an immunofluorescence method using a secondary antibody (Alexa Fluor 488). Green fluorescent staining was stronger in the nucleus than in plasma (Figure 4c), a result that was consistent with the data of using a Nuclear and Cytoplasmic Protein Extraction Kit. Taken together, these results demonstrated that DOP-1-1 induced an immune response via the NF-κB pathway.

Effects of DOP-1-1 on NF-κB Signaling
Our previous study showed that DOP-1-1 has immunomodulatory properties [22]. To further study the mechanism of DOP-1-1-induced immunoregulation, the proteins associated with immune responses were examined. The cellular response to different concentrations of DOP-1-1 resulted in a phosphorylation increase of NF-κB in a dose-dependent manner and degradation of IκBα ( Figure 4a). Other proteins were not detected. NF-κB pathway can be activated via at least three distinct routes: Canonical, atypical and noncanonical pathways. Our results suggested that DOP-1-1 induced activation of NF-κB signaling via Iκκ-independent means (Iκκ-β and Iκκ-α were not detected). The activation of NF-κB can induce its nuclear translocation. In order to ascertain whether DOP-1-1 translocated NF-κB into the nucleus, western blotting for cytoplasmic and nuclear fractions was carried out using a Nuclear and Cytoplasmic Protein Extraction Kit. The expression of NF-κB (p65) in the cytoplasmic fractions of THP-1 cells decreased, and that of NF-κB (p65) in nuclear extracts increased, in a dose-dependent manner ( Figure 4b). In order to confirm these results, NF-κB expression was determined by an immunofluorescence method using a secondary antibody (Alexa Fluor 488). Green fluorescent staining was stronger in the nucleus than in plasma (Figure 4c), a result that was consistent with the data of using a Nuclear and Cytoplasmic Protein Extraction Kit. Taken together, these results demonstrated that DOP-1-1 induced an immune response via the NF-κB pathway.

TLR4 Is a Pattern-Recongnition Receptor for DOP-1-1
In order to detect the specificity and affinity of DOP-1-1 to the surface receptors on THP-1 cells, the latter were grown on COP-1 chips (compatible polystyrene coated sensor surface). A large shift in the frequency was observed and most of the cells were washed away during the analysis ( Figure  5a). This observation suggested that cells did not adhere strongly to the surface of THP-1 cells. Hence, the protocol was changed and COP-1 chips were coated with poly-L-lysine. Most cells remained on the sensor surface by fixation, whereas without fixation the THP-1 cells were washed away by the flow (Figure 5b). However, a similar low-frequency shift was detected in fixed and unfixed cells ( Figure 5c). Combining these results, we could conclude that DOP-1-1 had very little interaction with the receptors on THP-1cells.
TLR4 has a central role in the enhancement of the innate immune response and the production of cytokines induced by polysaccharides. Therefore, TLR4 expression was analyzed by western blotting. The expression of TLR4 protein increased in a dose-dependent manner (Figure 5d). In order to ascertain whether TLR4 is a pattern-recognition receptor for DOP-1-1, blockade of TLR4 protein using anti-mouse TLR4 monoclonal antibodies was employed to interfere in the interaction between DOP-1-1 and TLR4. Upon addition of anti-mouse TLR4 monoclonal antibodies, the DOP-1-1-induced NF-κB activation in THP-1 cells was strongly inhibited (Figure 5e), which suggested that DOP-1-1 stimulated the immune response via TLR4.

TLR4 Is a Pattern-Recongnition Receptor for DOP-1-1
In order to detect the specificity and affinity of DOP-1-1 to the surface receptors on THP-1 cells, the latter were grown on COP-1 chips (compatible polystyrene coated sensor surface). A large shift in the frequency was observed and most of the cells were washed away during the analysis (Figure 5a). This observation suggested that cells did not adhere strongly to the surface of THP-1 cells. Hence, the protocol was changed and COP-1 chips were coated with poly-L-lysine. Most cells remained on the sensor surface by fixation, whereas without fixation the THP-1 cells were washed away by the flow (Figure 5b). However, a similar low-frequency shift was detected in fixed and unfixed cells (Figure 5c). Combining these results, we could conclude that DOP-1-1 had very little interaction with the receptors on THP-1cells.
TLR4 has a central role in the enhancement of the innate immune response and the production of cytokines induced by polysaccharides. Therefore, TLR4 expression was analyzed by western blotting. The expression of TLR4 protein increased in a dose-dependent manner (Figure 5d). In order to ascertain whether TLR4 is a pattern-recognition receptor for DOP-1-1, blockade of TLR4 protein using anti-mouse TLR4 monoclonal antibodies was employed to interfere in the interaction between DOP-1-1 and TLR4. Upon addition of anti-mouse TLR4 monoclonal antibodies, the DOP-1-1-induced NF-κB activation in THP-1 cells was strongly inhibited (Figure 5e), which suggested that DOP-1-1 stimulated the immune response via TLR4.

Expression of the Genes Associated with Nf-Κb Signaling
To further test the mechanism of NF-κB activation and targets of the immune response induced by DOP-1-1, the expression of some of the genes involved in the immune response was measured by treatment with PDTC and Bay 11-7082. PDTC (80 μmol/L) and Bay 11-7082 (20 μmol/L) inhibited the phosphorylation of NF-κB ( Figure  6a). RT-PCR analyses suggested that DOP-1-1 gave rise to changes in the expression of almost all of the genes associated with NF-κB (including those associated with inflammation, survival, and apoptosis) compared with control (Figure 6b-f) but pretreatment with PDTC (80 μmol/L) or Bay 11-7082 (20 μmol/L) resulted in reduced expression of many of these genes. These results provided further evidence that DOP-1-1 induced an immune response through the NF-κB pathway. Besides, expression of CCL4 and IP10 showed the greatest changes among these genes ( Figure 6 and Table 2, 4023-and 2954-fold higher, respectively). Further, the up-regulation of the expression of CCL4 and IP10 could be suppressed after treatment with the NF-κB inhibitors PDTC and Bay 11-7082, which confirmed that CCL4 and IP10 had a specific response to the NF-κB activation induced by DOP-1-1. Therefore, CCL4 and IP10 could be considered to have molecular signatures in the immunoregulation by DOP-1-1.

Expression of the Genes Associated with Nf-Kb Signaling
To further test the mechanism of NF-κB activation and targets of the immune response induced by DOP-1-1, the expression of some of the genes involved in the immune response was measured by treatment with PDTC and Bay 11-7082. PDTC (80 µmol/L) and Bay 11-7082 (20 µmol/L) inhibited the phosphorylation of NF-κB (Figure 6a). RT-PCR analyses suggested that DOP-1-1 gave rise to changes in the expression of almost all of the genes associated with NF-κB (including those associated with inflammation, survival, and apoptosis) compared with control (Figure 6b-f) but pretreatment with PDTC (80 µmol/L) or Bay 11-7082 (20 µmol/L) resulted in reduced expression of many of these genes. These results provided further evidence that DOP-1-1 induced an immune response through the NF-κB pathway. Besides, expression of CCL4 and IP10 showed the greatest changes among these genes ( Figure 6 and Table 2, 4023-and 2954-fold higher, respectively). Further, the up-regulation of the expression of CCL4 and IP10 could be suppressed after treatment with the NF-κB inhibitors PDTC and Bay 11-7082, which confirmed that CCL4 and IP10 had a specific response to the NF-κB activation induced by DOP-1-1. Therefore, CCL4 and IP10 could be considered to have molecular signatures in the immunoregulation by DOP-1-1.

Discussion
The water extracts of D. officinale can be infused in tea or wine, or consumed as a decoction, by sick individuals. Polysaccharides, as the major active component of D. officinale, encompass many physicochemical, and physiologic effects that could stimulate the immune system.
In the present study, the conformational change of DOP-1-1 was analyzed. Our results showed that DOP-1-1 had good stability under pH ranged from 2 to 8 (Figure 2a-c). So, in order to fully extracted the polysaccharide from D. officinale, a small amount of an acid or base may be added in solvent. This observation is in contrast with researchers who have often assumed that

Discussion
The water extracts of D. officinale can be infused in tea or wine, or consumed as a decoction, by sick individuals. Polysaccharides, as the major active component of D. officinale, encompass many physicochemical, and physiologic effects that could stimulate the immune system.
In the present study, the conformational change of DOP-1-1 was analyzed. Our results showed that DOP-1-1 had good stability under pH ranged from 2 to 8 (Figure 2a-c). So, in order to fully extracted the polysaccharide from D. officinale, a small amount of an acid or base may be added in solvent. This observation is in contrast with researchers who have often assumed that polysaccharides in acidic solutions can be readily degraded. This assumption may be due to the DOP-1-1 linkage type, which is composed of →4)-Manp-(1→ and →4)-Glcp-(1→ residues (Figure 1) [22,24,25].
In jawed vertebrates, the immune response is composed of innate immunity and adaptive immunity. The former is carried mainly by macrophages and neutrophils, which are the first line of defense against infections. The innate response is usually triggered when microbes are identified by pattern-recognition receptors (PRRs). But, as polysaccharides cannot enter cells directly because of their large molecular mass, the first step for polysaccharides to exert their functions is the recognition by PRRs [28,29].
This study revealed that DOP-1-1 was weakly bound to the surface of THP-1 cells (Figure 5a-c). There are two explanations for this result. First, very few receptors may be expressed on the surface of THP-1 cells. Second, the number of receptors may be sufficient, but there might be weak interactions between DOP-1-1 and the proteins on the surface of THP-1 cells [30]. In addition, different polysaccharides can be recognized by different PRRs, such as TLR2-4, CR-3, and NOD-like receptor protein (NLRP)1-7 [12,31]. Among these receptors, TLR4 has been reported to be the most common PRR for the recognition of natural polysaccharides [32]. In order to ascertain whether TLR4 is a PRR of DOP-1-1 when it binds to macrophages, the changes of TLR4 expression was analyzed by treatment with DOP-1-1 (Figure 5d). Further, previous treatment with an anti-TLR4 antibody inhibited NF-κB activation (Figure 5d), which suggested that DOP-1-1 activates macrophages via TLR4-mediated signaling pathways.
It would be useful to understand the recognition pattern of O-acetylated glucomannan. Hence, the pathways associated with the immune response were systematically investigated. DOP-1-1 promoted the degradation of IκB complexes and activated NF-κB phosphorylation (Figure 4a). Once activated, the NF-κB dimer translocates to the nucleus, where it binds to specific DNA sequences [33]. Therefore, the changes in levels of NF-κB proteins in the cytoplasm and nuclei were also tested. Finally, the expression of proteins and genes associated with the NF-κB signaling pathway were tested by RT-PCR in this study. When inhibitors of NF-κB phosphorylation were added, the gene-expression levels were similar to those of control. Taken together, our results showed that O-acetylated glucomannan induced an immune response through NF-κB mediated by a TLR4 signaling pathway. Furthermore, CCL4 and IP10 were confirmed to be the novel targets of the immune response stimulated by O-acetylated glucomannan.
CCL4 and IP10 are chemotactic cytokines that have a vital role in the regulation of migration of various body cells, especially immune cells such as monocytes, macrophages, neutrophils, natural killer cells and T cells. The importance of chemokines has been shown in recent years. It has become recognized that they are key players in many disease processes, including inflammation, autoimmune disease, infectious diseases, and cancer [34,35]. Our data suggested that DOP-1-1 upregulated expression of CCL4 and IP10, which could provide new targets for the development of immunologic intervention strategies. Our results also suggest that DOP-1-1 could have an important role in the treatment of autoimmune disease, infectious diseases, wound healing, and cancer.
The immunoregulatory mechanisms of polysaccharides vary depending on the type of in polysaccharide [12][13][14]36]. A hypothesis for the immunoregulatory mechanism of DOP-1-1 is shown in Figure 7.

Carbohydrate and Protein Contents of Crude and Pure O-Acetylated Glucomannan
The carbohydrate contents of crude and pure O-acetylated glucomannan were measured using the anthrone-sulfuric acid reaction [37]. Glucose was used as the standard and the concentration range was 0-72 μg/mL with a coefficient value of 0.994. The concentrations of crude polysaccharide and purified O-acetylated glucomannan were 46.1 μg/mL and 40 μg/mL, respectively. The anthronesulfuric acid reagent was prepared immediately before use. The protein contents of crude and purified O-acetylated glucomannan were measured by the bicinchoninic acid method [38]. The concentrations range was 0-500 ng/mL with a coefficient value of 0.999. The concentrations of crude polysaccharide and purified O-acetylated glucomannan were 2 and 2.35 mg/mL, respectively.

Carbohydrate and Protein Contents of Crude and Pure O-Acetylated Glucomannan
The carbohydrate contents of crude and pure O-acetylated glucomannan were measured using the anthrone-sulfuric acid reaction [37]. Glucose was used as the standard and the concentration range was 0-72 µg/mL with a coefficient value of 0.994. The concentrations of crude polysaccharide and purified O-acetylated glucomannan were 46.1 µg/mL and 40 µg/mL, respectively. The anthrone-sulfuric acid reagent was prepared immediately before use. The protein contents of crude and purified O-acetylated glucomannan were measured by the bicinchoninic acid method [38]. The concentrations range was 0-500 ng/mL with a coefficient value of 0.999. The concentrations of crude polysaccharide and purified O-acetylated glucomannan were 2 and 2.35 mg/mL, respectively.

Thermal Stability Analysis of O-Acetylated Glucomannan
The thermal stability of O-acetylated glucomannan was determined by differential scanning calorimetry (DSC) and size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC). Samples solutions were prepared at the pH indicated unless specified otherwise ( Figure 2). Two sets of identical samples of each polysaccharide were prepared: One set for DSC analysis and the other set for HPLC analysis. The storage temperature of polysaccharides for HPLC analysis was 65 • C. A MicroCal VP-DSC system (Malvern Instruments, Malvern, UK) was used at 25-90 • C with a scan rate of 1 • C/min. Analyses of thermograms were done with Origin™ software (OriginLab, Northampton, MA, USA). All experiments were carried out at a polysaccharide concentration of 2 mg/mL for DSC and HPLC analyses. For SEC-HPLC, 0.1 M NaCl was used as the eluent at a flow rate of 0.5 mL/min and the injection volume was 20 µL; a PL aquagel-OH column (Agilent Technologies, Santa Clara, CA, USA) was employed at 30 • C.

MTT Assay
The toxicity of DOP-1-1 against THP-1 cells was tested using a modification of the method described by Carmichael et al. [39]. THP-1 cells (3 × 10 4 /well) were plated with improved complete RPMI 1640 (HyClone Laboratories) supplemented with 0.2 ng/mL PMA into a 96-well plate and incubated for 48 h. DOP-1-1 (25, 50, 100, 150, 300 µg/mL) was added and cells were incubated at 37 • C for 12, 24, 36 and 48 h, respectively. Then, cells were washed twice with phosphate-buffered saline (PBS) and MTT solution (0.5 mg/mL) was added to induce a reaction for 4 h. The purple MTT-formazan crystals were dissolved with 150 µL of dimethyl sulfoxide. Spectrophotometric absorbance at 492 nm was determined immediately using a microplate reader. Percent survival was calculated according to the following formula: where A is the rate of cell growth; A E is the optical density of the experimental group; and A C is the optical density of control group.

Kinetic Evaluation of the Interactions between DOP-1-1 and THP-1 Cells
The surface of a COP-1 chip (compatible polystyrene coated sensor surface) was incubated with 100 µL poly-l-lysine solution (50 µg/mL) for 20 min. Then, the surface was washed with tissue culture-grade water and dried under a sterile hood ≥2 h before the introduction of THP-1 cells. A total of 100,000 THP-1 cells were seeded on the COP-1 surface in 700 µL of medium (improved RPMI 1640 supplemented with 10% FBS, 1% penicillin/streptomycin and 20 ng/mL PMA), and cultured for 48 h at 37 • C under an atmosphere with 5% CO 2 /95% air. Subsequently, the medium was removed, and 700 µL of improved RPMI 1640 without FBS or penicillin/streptomycin was added. After 20 h, one chip was fixed with 3.7% formaldehyde (or not) at 4 • C for 10 min (the unfixed surface was not treated with formaldehyde). The cell coverage on the sensor surface was evaluated by staining the nuclei with Hoechst 33342 and visualized under a fluorescence microscope (Olympus, Tokyo, Japan). Then, the chip was docked into an A200 QCM instrument (Attana, Stockholm, Sweden) and a biosensor experiment measured the interactions between DOP-1-1 and cells.

Western Blotting
THP-1 cells (1 × 10 7 /well) were induced into a mature macrophage-like state as noted above (Section 4.5). After pretreatment with DOP-1-1 (25, 50, 75, 100, 150 µg/mL), the samples of extracted protein were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes. Cytosol and nuclear proteins were prepared using a Cytosol and Nuclear Extracts kit (Beyotime Biotechnology, ShangHai, China). After blockade with 5% bovine serum albumin (BSA), PVDF membranes were incubated overnight with primary antibodies. After washing three times, a secondary antibody was added to PVDF membranes along with 5% bovine serum albumin for 1 h. β-tubulin was used as an internal control.
For visualization of the protein bands, a detection system of enhanced chemiluminescent, colorimetric and UV fluorescent gels and blots (FluorChem E system; ProteinSimple, Santa Clara, CA, USA) was applied.

Immunofluorescent Staining
THP-1 cells (1 × 10 6 ) were plated onto a six-well plate in a total volume of 2 mL culture medium containing 0.2 ng/mL PMA. After attachment, cells were cultured in the medium with 5% FBS for 20 h. Then, cells were fixed in paraformaldehyde for 20 min at room temperature and washed gently thrice with PBS. Afterwards, cells were blocked with 1% BSA for 30 min and incubated with anti-p-NF-κB/p65 Ser536 antibody in 0.1% BSA at 4 • C overnight. Subsequently, cells were rinsed three times with PBS at room temperature and labelled with secondary antibodies with Alexa Fluor 488 (donkey anti-rabbit, Life Technologies, Carlsbad, CA, USA) for 1 h in the dark. The expression of NF-κB was photographed using a confocal microscope.

Statistical Analysis
The results are expressed as the means ± standard deviations (SD) of data obtained from triplicate experiments. Statistical analyses were performed using a one-way Analysis of Variance (ANOVA) with Prism6 software (GraphPad, San Diego, CA, USA). p < 0.05, p < 0.01, or p < 0.001 were considered as statistically significant (* p < 0.05, ** p < 0.01, or *** p < 0.001).

Conclusions
The O-acetylated glucomannan DOP-1-1 is present in the steady state in low-pH solutions. DOP-1-1 may induce an immune response through NF-κB mediated by a TLR4 signaling pathway. CCL4 and IP10 may be the novel targets of the immune response stimulated by O-acetylated glucomannan.