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MyD88 pathway mediated activation of macrophages
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A homogalacturonan from Hippophae rhamnoides L. Berries enhance immunomodulatory activity through TLR4/MyD88 pathway mediated activation of macrophages Hailiang Wang a , Hongtao Bi b , Tingting Gao b , Bin Zhao a , Weihua Ni c,∗ , Jun Liu a,∗∗ a
Department of Neurosurgery, The Second Hospital of Jilin University, Changchun 130021, China Qinghai Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China c Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130033, China b
a r t i c l e
i n f o
Article history: Received 16 June 2017 Received in revised form 11 September 2017 Accepted 20 September 2017 Available online xxx Keywords: Homogalacturonan Hippophae rhamnoides L Polysaccharide Macrophage Toll-like receptor 4 TLR4
a b s t r a c t Our previous study isolated a natural high-methoxyl homogalacturonan (HRWP-A) from Hippophae rhamnoides and showed antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of HRWP-A were further investigated. Results showed that HRWP-A could recover the body condition and activated macrophage in Cyclophosphamide (CTX)-induced immunosuppressed mice. Further, we investigated the possible mechanism underlying the effects of HRWP-A on mouse peritoneal macrophages. qPCR and western blot revealed that HRWP-A upregulated the expression of TLR4 mRNA in vitro. This process was accompanied by a clear increase in MyD88 expression and p-IB-␣, but these effects were largely abrogated by pretreatment with anti-TLR4 antibodies. The effects of HRWPA on macrophage NO, IL-1 and IL-6 production were also inhibited by anti-TLR4 antibodies and were greatly influenced by the NF-B inhibitor PDTC. Moreover, HRWP-A failed to induce the production of NO, IL-1 and IL-6 in peritoneal macrophages prepared from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, suggesting the involvement of the TLR4 molecule in HRWP-A-mediated macrophage activation. These results may have important implications for our understanding of the structure-activity relationship of immunopotentiating polysaccharides from medicinal herbs. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Many natural polysaccharides isolated from medicinal plants or mushrooms have been found to exhibit diverse activities [1–5]. The activity of polysaccharides might be caused and influenced by their glycosidic linkages, chain length, branch-point number, molecular size and tertiary structure [2]. Previous studies of polysaccharide structure-activity relationships have revealed that many polysaccharides, such as 1,3--glucans, amylopectin-like polysaccharides (␣-1,4-/1,6-glucans) [6–8] and pectins [1,4,9–11] show promising potential as immunological adjuvants or antitumor agents because of their interactions with immunological cell surface receptors.
∗ Corresponding author at: Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130021, China. ∗∗ Corresponding author at: Department of Neurosurgery, The Second Hospital of Jilin University, Changchun, 130033, China. E-mail addresses: [email protected] (W. Ni), [email protected] (J. Liu).
The stimulation of macrophages is an important method for enhancing immunological activity [3]. Many polysaccharides activate macrophages by binding to receptors on the surface of immune cells. Polysaccharides interact with macrophages via dectin-1, Tolllike receptor 4 (TLR4), CD14, Scavenger receptor (SR), complement receptor 3 (CR3) and the mannose receptor (MR) [2,3,6]. Activation of these receptors leads to intracellular signaling cascades, resulting in transcriptional activation and the production of proinflammatory cytokines. The Hippophae rhamnoides L. berry has been a traditional medicinal food of the Tibetan plateau for thousands of years [12,13]. Recently, our research group purified a water-soluble polysaccharide (HRWP-A) from H. rhamnoides berry that was identified as a high-methoxyl homogalacturonan (with repeating units of (1 → 4)--d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups). HRWP-A exhibits anti-fatigue and anti-tumor activity [12,13]. Notably, it augmented macrophage phagocytic activity, increased macrophage secretion of NO and TNF-␣ in tumor-bearing mice.
http://dx.doi.org/10.1016/j.ijbiomac.2017.09.083 0141-8130/© 2017 Elsevier B.V. All rights reserved.
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In this study, to further reveal its mechanism of immunomodulatory activity, we investigate the immunomodulatory activities of HRWP-A in cyclophosphamide (CTX)-induced immunosuppressed mice. The effect of HRWP-A on TLR4 pathway signal in peritoneal macrophages was also analyzed to further elucidate the mechanism of macrophage activation. The results may provide insights into underlying mechanisms of activity and valuable information for desigening of more effective adjuvant for cancer therapy. 2. Materials and methods 2.1. Mice Female BALB/c, C3H/HeN and C3H/HeJ mice (6–8 weeks old, weighing 20.0 ± 2.0 g) were purchased from the Pharmacology Experimental Center of Jilin University (Changchun, China). The mice were housed on a 12/12-h light–dark cycle at room temperature and allowed free access to standard rodent food and water during the experiments. Animal handling procedures were conducted under National Institutes of Health animal care and use guidelines. All efforts were made to minimize the animals’ suffering and to reduce the number of animals used. 2.2. Reagents TRIzol reagent and primers were purchased from Invitrogen (Carlsbad, CA, USA). MTS, M-MLV reverse transcriptase and oligodT primers from Promega (Madison, WI, USA). qPCR TaqMix from TaKaRa Bio (Sakado-shi, Saitama, Japan). Mouse TNF-␣, IL-12p70, IL-1 and IL-6 enzyme-linked immunosorbent assay kits and FITCF4/80 were purchased from eBioscience (San Diego, CA, USA). The NO kit and Annexin V-fuorescein isothiocyanate (FITC) apoptotic detection kit was purchased from Beyotime (Nantong, Jiangsu, China). Fluorescence conjugated antibodies specific for MHCII, CD80 and CD86, TLR4/MD-2 were obtained from BioLegend (San Diego, CA, USA). Antibodies specific for MyD88 and GAPDH were purchased from Abcam (Cambridge, UK), while antibodies specific for IB-␣ and the corresponding phospho-antibodies were all purchased from Cell Signaling Technology (Beverly, MA, USA). Pyrrolidine dithiocarbamate (PDTC), mouse lymphocyte separation medium, Lipopolysaccharide (LPS; E. coli 055:B5) and polymyxin B (PMB) were purchased from Sigma–Aldrich (St. Louis, MO, USA). MTS was from Promega (Madison, WI, USA). Cyclophosphamide (CTX) was from Jiangsu Hengrui (Lianyungang, Jiangsu, China). 2.3. Preparation of polysaccharide Polysaccharide from Hippophae rhamnoides L. berries was prepared as described previously [13]. Briefly, crude polysaccharide was obtained from fresh berries by hot water extraction and 80% ethanol precipitation. Then, the extract was successively purified through DEAE-Cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and DEAE-Sepharose Fast Flow ion-exchange chromatography, finally resulting in a homogeneous polysaccharide fraction named HRWP-A. HRWPA was found to be a high-methoxyl homogalacturonan with a molecular weight of 4992 Da through HPGPC, FT-IR and NMR analysis; this polysaccharide contains repeating units of (1 → 4)-d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups. Endotoxin contamination was analyzed with a gel-clot Limulus amebocyte lysate assay. The endotoxin level in each polysaccharide solution was less than 0.005 EU (endotoxin units)/mg.
2.4. Immunomodulatory activity in CTX-induced immunosuppressed mice 2.4.1. Animal treatment and experimental design Cyclophosphamide (CTX) (25 mg/kg) were administered intraperitoneally to establish the immunosuppressive animal model (5 mice/group). One day later, HRWP-A (50 mg/kg) were administered intragastrically each day for 10 consecutive days. The CTX group was treated only with CTX, and the P.S. group was given physiologic saline as normal control. The dose volume was 0.2 ml. On the day 11, the mice were sacrificed. The spleens and thymus were weighed and their weights relative to the final body weights (organ index) were calculated. 2.4.2. Preparation of splenic mononuclear cells and peritoneal macrophages The spleen from each mouse was harvested and a mononuclear cell suspension was prepared. The splenic mononuclear cells were isolated by density gradient centrifugation on mouse lymphocyte separation medium. The splenic mononuclear cells were collected for analyzing the expression of CD80, MHCII or CD86. Cells from the peritoneal exudate (PEC) were collected from the immuno-suppressed mouse by peritoneal lavage with cold PBS. PECs were washed twice and resuspended in RPMI-1640 medium. Peritoneal macrophages were further isolated by incubating the PECs (1 × 107 /well) in a 6-well plate at 37 ◦ C in a humidified atmosphere with 5% CO2 for 3 h to allow the peritoneal macrophages to adhere. After removing the non-adherent cells with cold PBS, the macrophage monolayer was collected. The adherent cell population was > 95% macrophages, as determined by flow cytometric (FACSCalibur, San Diego, CA, USA) analysis of FITC-F4/80 staining. The macrophages (4 × 105 cells/well) were added into 96-well microwell plates and cultured for 48 h, and then culture supernatants were harvested to detect cytokine secretion by ELISA. 2.4.3. Flow cytometry assay The splenic mononuclear cells (1 × 106 cells) were prepared as described above and fixed with paraformaldehyde for 1 h and washed twice with FACS solution. The cells were incubated with either FITC-labeled anti-mouse MHCII, CD80 and CD86 antibody at 4 ◦ C for 30 min in the dark, and then washed twice with FACS solution. Subsequently, the stained cells were analyzed with a flow cytometer. The FITC-labeled mouse IgG isotype was used as a control. 2.4.4. MTS assay Peritoneal macrophages were prepared as described. The cell viabilities of peritoneal macrophages were determined by MTS assay. The macrophages (4 × 105 cells/ml, 200 l/well) were seeded in a 96-well plate and cultured at 37 ◦ C, 5% CO2 for 20 and 44 h. MTS (5 mg/ml, 20 l) was added to each well and incubated for an additional 4 h. The absorbance at 490 nm was measured using a microplate reader (Bio-Rad, West Berkeley, CA, USA). Relative cell viability was calculated as A490 nm (HRWP-A-treated group)/A490 nm (control group) × 100%. 2.4.5. Apoptosis assay Peritoneal macrophages were placed in a 6-well culture plate at a density of 1 × 106 cells/well for 24 h or 48 h. Apoptosis was measured by staining with Annexin V-FITC/propidium iodide assay kit. Briefly, cells were washed with PBS and resuspended in 500 l of binding buffer. Then, 1 l of AnnexinV-FITC was added to the samples at 4 ◦ C in the dark. After incubation for 5 min, 1 l of propidium iodide was added, and the mixture was incubated for 5 min. Sub-
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Fig. 1. Effect of HRWP-A on immune organ indices in mice. Organ indices = weight of organ (mg)/body weight (g). P.S. group = mice untreated with CTX and treated with saline, CTX group = mice pretreated with CTX. Each value represents the mean ± SD (n = 5). Significant differences from the Model group were evaluated using Student’s t test: a P < 0.05 vs. P.S. group; b P < 0.05 vs. CTX group.
sequently, flow cytometry analysis was performed. Results were expressed as% of apoptotic cells. 2.5. HRWP-A-treated peritoneal macrophages in vitro Peritoneal macrophages were obtained from either BALB/c, C3H/HeN or C3H/HeJ mouse peritoneal as described above. For qPCR and western blot, macrophages were treated with various concentrations of HRWP-A (2.5 − 20 g/ml) or LPS (5 g/ml) in the presence or absence of polymyxin B (5 or 10 g/ml) prior to being seeded in 24-well plates at 4 × 105 cells/well for 6 h. For NO production and cytokine secretion, macrophages were incubated in complete medium in the presence or absence of LPS (5 g/ml) or HRWP-A (10 g/ml) for 24 h. 2.6. Detection of nitric oxide The NO levels in the culture supernatant of mouse peritoneal macrophages were determined with an NO kit. Each data point is an average of the values obtained from 6 mice. The absorbance at 540 nm in each well was measured with an automated microtiter plate reader. 2.7. Cytokine secretion assay The culture supernatants were assayed for IL-1, IL-6, TNF-␣ and IL-12p70 with a commercial ELISA kit according to the manufacturer’s protocol. The absorbance at 450 nm in each well was measured with an automated microtiter plate reader.
2.9. Western blot TLR4 and MyD88 expression and IB phosphorylation were detected using western blot. Macrophage lysates were prepared with RIPA lysis buffer in the presence of the protease inhibitor leupeptin and a phosphatase inhibitor. The protein concentration was determined using the bicinchoninic acid protein detection system. Proteins were separated by SDS-PAGE and subjected to western blot. The following antibodies were used: anti-GAPDH, anti-TLR2/9, anti-MyD88, anti-IB, anti-p-IB. 2.10. Blocking experiments For TLR4 or PDTC blocking experiments, macrophages were cultured in 24-well plates and pretreated with anti-TLR4 (20 g/ml) or a mouse IgG2a (20 g/ml) isotype control antibody for 60 min. In some experiments, the NF-B inhibitor PDTC (20 M) was added to the cell cultures 1 h prior to HRWP-A (10 g/ml) treatment and left in the culture media throughout the experiment. LPS (5 g/ml) was added as a control. Then, NO, IL-1 and IL-6 secretion was analyzed. 2.11. Statistical analysis Statistical significance was determined by one-way ANOVA followed by Student’s t-test or Dunnett’s post hoc comparisons (SPSS statistical software package, version 16.0, SPSS, Chicago, IL, USA) when appropriate. All values were expressed as mean ± S.D. A Pvalue of < 0.05 was considered to be significant.
2.8. Real-time PCR
3. Results and discussion
Total RNA was isolated with TRIzol reagent according to the manufacturer’s protocol. Total RNA was converted to cDNA using M-MLV reverse transcriptase and oligo-dT primers according to the manufacturer’s instructions. TLR4 levels were analyzed with realtime PCR using qPCR TaqMix. The primers used to amplify cDNA were as follows: TLR4 5 -TCA GTG TGC TTG TGG TAG CC-3 and 5 -TCG TTT CTC ACC CAG TCC TC-3 . The real-time PCR reactions were performed on an ABI PRISM@ 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). These values were expressed as the ratios of the relative abundance of target genes to GAPDH.
3.1. Immunomodulatory activity of HRWP-A in CTX-induced immunosuppressed mice 3.1.1. Effects of polysaccharides on organ weight Body weight was recorded after the experiment (10 days, final). Spleen and thymus as immune function-related organs were evaluated (Fig. 1). As expected, CTX obviously decreased the relative thymus and spleen weight as compared to the normal group. However, HRWP-A significantly increased the relative spleen and thymus weights as compared to the model groups (CTX-treated group). These findings suggested that HRWP-A partially recov-
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Fig. 2. The effects of HRWP-A on macrophages from immuno-suppressive mice. (A) The splenic mononuclear cells were stained with either FITC-labeled anti-mouse MHCII, CD80 or CD86 antibody. The stained cells were analyzed by flow cytometry. (B) Effects of HRWP-A on peritoneal macrophage viability. Cell viability was determined by MTS assay, absorbance was measured at 490 nm and values were expressed relative to the viability of cells incubated in medium. (C) Effect of HRWP-A on inhibition of macrophage apoptosis. The apoptotic ratio of peritoneal macrophage was assessed by Annexin V-FITC/PI binding and measured using fllow cytometry. (D) Effects of HRWP-A on macrophage NO (as nitrite), IL-1, TNF-␣, IL-6 and IL-12p70 production of peritoneal macrophage from immuno-suppressive mice. The culture supernatants were collected for NO and cytokine (IL-1, TNF-␣, IL-6 and IL-12p70) analyses with Griess reagent and an ELISA kit. The reported values are the means ± SDs (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc comparisons: a P < 0.05 vs. P.S. group; b P < 0.05 vs. CTX group.
ered the immunosuppressive accompanied with an improvement of immune organs.
3.1.2. HRWP-A activated and enhanced viability of peritoneal macrophages from CTX-induced immunosuppressed mice Macrophages are considered the pivotal immunocytes of host defense, and they provide an important bridge between innate and adaptive immunity. MHCII, CD80 and CD86 is highly expressed on the surface of activated macrophages. To investigate the effect of HRWP-A on macrophages, the expression of MHCII, CD80 and CD86 was analyzed using flow cytometry. As shown in Fig. 2A, the expression of MHCII, CD80 and CD86 in the HRWP-A groups was significantly up-regulated compared with the Model group (P < 0.05). These data indicate that HRWP-A increases the percentage of activated macrophages. Meanwhile, although CTX decrease the macrophage viability and increase the macrophage apoptosis, but HRWP-A can prolong the cell survive for it can enhance macrophage viability detecting by MTS assay (Fig. 2B) and inhibit the macrophage apoptosis detecting by Annexin V-FITC/PI apoptosis assay (Fig. 2C). The analysis of cytokine secretion showed that CTX decreased the level of NO, IL-1, IL-6, TNF-␣ and IL-12p70 of macrophages (Fig. 2C). IL1 has a wide range of immunomodulatory effects and may be directly involved in the inflammatory process [14]. IL-6 is a cytokine with multiple immunomodulatory functions, and NO are important mediators in the destruction of tumor cells [15–17]. However,
HRWP-A significantly increased the level of NO, IL-1, TNF-␣ and IL-6. Meanwhile, HRWP-A partially recovered the level of NO, IL1 and IL-6 inhibiteed by CTX. HRWP-A showed little effect on the IL-12 secretion, indicated that HRWP-A might have less effect on macrophage polarization in the CTX-induced immunosuppressed mice. Altogether, these results indicated that HRWP-A might counteract the damage of CTX to macrophage to some extent.
3.2. HRWP-A upregulates TLR4 expression and activates the MyD88 pathway via TLR4 Subsequently, we explored the possible mechanism underlying macrophage activation by HRWP-A. Several studies have reported that botanical polysaccharides interact with macrophages via TLR2 or TLR4. Thus, we first detected the expression of TLR2/4 on macrophages treated with HRWP-A. QPCR showed that the TLR2 mRNA level in macrophages did not change after stimulation with HRWP-A (data not shown), while HRWP-A significantly upregulated TLR4 expression in a concentration-dependent manner (2.5–20 g/ml) (Fig. 3A), and this effect was not abrogated by treatment with polymyxin B at 5 g/ml, which eliminated the possibility of LPS contamination. As a positive control, LPS (5 g/ml) also upregulated TLR4 expression, but this effect was reversed after preincubating the LPS with polymyxin B. These results indicated that TLR4 may relevant to the interaction between HRWP-A and macrophages.
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Fig. 3. HRWP-A activates MyD88/NF-B via TLR4. (A) The effect of HRWP-A on TLR4 expression patterns in macrophages was detected using real-time PCR. Peritoneal macrophage cells were treated with various concentrations of HRWP-A (2.5 − 20 g/ml) or LPS (5 g/ml) in the presence or absence of polymyxin B (5 or 10 g/ml) prior to being seeded in 24-well plates at 4 × 105 cells/well for 6 h. (B) The effect of HRWP-A on MyD88/NF-B expression was detected with western blot. Macrophages were pretreated with an anti-mouse TLR4 (20 g/ml) antibody or 5 g/ml polymyxin B (PMB) prior to the addition of HRWP-A (10 g/ml) or LPS (5 g/ml) for 6 h; MyD88 and IB-␣ activation was then determined. The data are expressed as the means ± SDs of three independent experiments. Statistical significance was determined by Student’s t-test: *P < 0.01 vs. the medium-treated group, # P < 0.01 vs. the HRWP-A or LPS (alone)-treated group, respectively.
Subsequently, to confirm that HRWP-A may be considered a ligand for TLR4, the receptor that plays an important role in the activation, polarization and function of macrophages, we further studied the downstream signaling pathways. As shown in Fig. 3B, HRWP-A (10 g/ml) increased MyD88 expression and IB phosphorylation, and the addition of polymyxin B did not reverse the effect of HRWP-A, suggesting that HRWP-A was responsible for activating the MyD88/NF-B pathway in these experiments. In contrast, although LPS (5 g/ml) also upregulated MyD88 and pIB expression, the LPS-induced activation of IB-␣ was reversed after preincubation of LPS with PMB. Furthermore, our results showed that pretreating macrophages with an anti-TLR4 antibody (20 g/ml) almost eliminated MyD88 expression and IB-␣ phosphorylation; these results strongly suggests that HRWP-A induces the activation of the MyD88/NF-B signaling pathway via TLR4 in macrophages, presumably leading to proinflammatory cytokine secretion and macrophage activation. Although it remains unclear how HRWP-A works with TLR4, HRWP-A treatment results in the recruitment of MyD88 to TLR4, which initiates the signaling cascade that leads to the activation of NF-B.
3.3. Blocking TLR4/NF-B inhibits the HRWP-A-induced increase in NO, IL-1ˇ and IL-6 levels To further confirm the involvement of TLR4 in the interaction of HRWP-A and macrophages, an anti-TLR4 antibody and the NF-B inhibitor PDTC were used in vitro to pre-treat the macrophage, after which NO, IL-1 and IL-6 secretion was examined. Results showed that HRWP-A (10 g/ml) increased the level of NO, IL-1 and IL-6 produced by macrophages (Fig. 4A–F). Treatment with anti-TLR4 (20 g/ml) completely abrogated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels (Fig. 4A-C), and PDTC (20 M) partially attenuated (Fig. 4D-F) the HRWP-A-induced increase in NO, IL-1 and IL-6. These results support the evidence that TLR4 is involved in the HRWP-A-mediated stimulation of macrophages and that the MyD88/NF-B pathway is associated with macrophage activation. In addition, we compared normal mice (C3H/HeN) and mutant TLR4 mice (C3H/HeJ) to further verify this phenomenon. The results show that macrophages from C3H/HeN mice responded to HRWPA stimulation by producing higher levels of NO, IL-1 and IL-6 (Fig. 3G-H); however, macrophages from C3H/HeJ mice did not respond to HRWP-A (10 g/ml) stimulation in vitro, suggesting TLR4 as one of the candidate HRWP-A receptors.
TLR4 triggers immune responses mainly through 2 signaling pathways: the MyD88-dependent pathway and the TRIFdependent pathway. MyD88 is utilized by TLR4 and activates downstream ERK, NF-B or p38 MAPK signaling, and MyD88 recruits TRAF6, leading to the activation of NF-B. TRIF recruits TRAF3 and TRAF6, which then activate NF-B and IRF3, respectively [18]. In this paper, we clearly demonstrate that HRWP-A stimulates TLR4 and induces NF-B activation in peritoneal macrophages. This result is consistent with several reports that polysaccharides from several other medicinal herbs are able to activate the MyD88/NFB signaling pathway via TLR4 in macrophages [5,18–22]. These plant-derived polysaccharides might be important in the initiation of some forms of chronic intestinal inflammation. An anti-TLR4 antibody (20 g/ml) completely abrogated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels, suggesting that the interaction between HRWP-A and macrophages relies on TLR4. The NF-B inhibitor PDTC partially attenuated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels, suggesting that in addition to NF-B, HRWP-A may also be involved in other pathways downstream of MyD88, such as p38 MAPK or TRAF. Further study of these intracellular signal transduction mechanisms is underway in our laboratory. Previous studies have revealed that many glucans with ␣(1,3),(1,4), ␣-(1,4),(1,6) or -(1–3)(1–6) linkages may interact with TLR4 [2,5]. Pectin, structurally and functionally the most complex polysaccharide in plant cell walls, is a family of galacturonic acid-rich polysaccharides including homogalacturonan (HG), ramified rhamnogalacturonan (RG) I and II. Arabinogalactan structural motifs of RG-I are usually important for the immunomodulating activity [2,4]. Several studies have reported that pectic polysaccharides also interact with macrophages via TLR4 [5,18–22]. Our study showed that the high-methoxyl homogalacturonan pectin (with repeating units of (1 → 4)--d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups) HRWP-A from the H. rhamnoides berry and TLR4 interact in the HRWP-A-mediated stimulation of macrophages and that the MyD88/NF-B pathway is associated with macrophage activation, leading to downstream signal transmission and inducing the production of proinflammatory cytokines. In contrast, some research has reported that pectic polysaccharide, such as arabinogalactan with a back-bone of (1 → 3)-linked -d-galactopyranosyl residues which were mainly substituted at the O-6 position by the branches composed of arabinosyl, galactosyl, and rhamnosyl residues [21], attenuates inflammation through inhibiting the TLR4/NF-B signaling path-
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Fig. 4. Effects of HRWP-A on macrophage NO (as nitrite), IL-1 and IL-6 production in the presence of TLR4 blockade. (A–C) For the TLR4 blocking experiments, peritoneal macrophages were cultured in 24-well plates and pretreated with an anti-TLR4 antibody (20 g/ml) or a mouse IgG2a (20 g/ml) antibody for 60 min. (D-F) For the PDTC blocking experiments, the NF-B inhibitor PDTC (20 M) was added to the cell cultures 1 h prior to HRWP-A (10 g/ml) treatment and remained in the culture media throughout the experiment. (G-H) Comparison of the pro-inflammatory factors in wild-type C3H/HeN and C3H/HeJ mice. The culture supernatants were collected for NO and cytokine (IL-1, IL-6) analyses with Griess reagent and an ELISA kit. The reported values are the means ± SDs (n = 6). Statistical significance was determined by Student’s t-test: *P < 0.01 vs. the medium-treated group, # P < 0.01 vs. the HRWP-A or LPS (alone)-treated group, respectively.
way. The differences in these data may result from the glycan structures, specifically the molecular weight, chain length and tertiary structure [2–5]. In summary, our study provides evidence that HRWP-A, which has hitherto been considered a potent proinflammatory stimulus, activates macrophages via TLR4 and the activation of the NF-B pathway. HRWP-A may have intrinsic adjuvant-like properties that enhance the immune response. Research on this structure may contribute to understanding the structure-activity relationship (SAR) of polysaccharides. Acknowledgments This work was funded by the Department of Science and Technology of Jilin Province (No. 20160101018JC), the National Natural Science Foundation of China (No. 31300291), the Bethune plan of Jilin University (No. 2015434), and “The Western Light” Personnel Training Project and Applying Basic Research Program of Qinghai province (No. 2014-ZJ-751 and 2015-ZJ-727).
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A homogalacturonan from Hippophae rhamnoides L. Berries enhance immunomodulatory activity through TLR4/MyD88 pathway mediated activation of macrophages Hailiang Wang a , Hongtao Bi b , Tingting Gao b , Bin Zhao a , Weihua Ni c,∗ , Jun Liu a,∗∗ a
Department of Neurosurgery, The Second Hospital of Jilin University, Changchun 130021, China Qinghai Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China c Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130033, China b
a r t i c l e
i n f o
Article history: Received 16 June 2017 Received in revised form 11 September 2017 Accepted 20 September 2017 Available online xxx Keywords: Homogalacturonan Hippophae rhamnoides L Polysaccharide Macrophage Toll-like receptor 4 TLR4
a b s t r a c t Our previous study isolated a natural high-methoxyl homogalacturonan (HRWP-A) from Hippophae rhamnoides and showed antitumor activity in vivo. In this study, the immunomodulatory activity and mechanisms of action of HRWP-A were further investigated. Results showed that HRWP-A could recover the body condition and activated macrophage in Cyclophosphamide (CTX)-induced immunosuppressed mice. Further, we investigated the possible mechanism underlying the effects of HRWP-A on mouse peritoneal macrophages. qPCR and western blot revealed that HRWP-A upregulated the expression of TLR4 mRNA in vitro. This process was accompanied by a clear increase in MyD88 expression and p-IB-␣, but these effects were largely abrogated by pretreatment with anti-TLR4 antibodies. The effects of HRWPA on macrophage NO, IL-1 and IL-6 production were also inhibited by anti-TLR4 antibodies and were greatly influenced by the NF-B inhibitor PDTC. Moreover, HRWP-A failed to induce the production of NO, IL-1 and IL-6 in peritoneal macrophages prepared from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, suggesting the involvement of the TLR4 molecule in HRWP-A-mediated macrophage activation. These results may have important implications for our understanding of the structure-activity relationship of immunopotentiating polysaccharides from medicinal herbs. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Many natural polysaccharides isolated from medicinal plants or mushrooms have been found to exhibit diverse activities [1–5]. The activity of polysaccharides might be caused and influenced by their glycosidic linkages, chain length, branch-point number, molecular size and tertiary structure [2]. Previous studies of polysaccharide structure-activity relationships have revealed that many polysaccharides, such as 1,3--glucans, amylopectin-like polysaccharides (␣-1,4-/1,6-glucans) [6–8] and pectins [1,4,9–11] show promising potential as immunological adjuvants or antitumor agents because of their interactions with immunological cell surface receptors.
∗ Corresponding author at: Department of Immunology, College of Basic Medical Science, Jilin University, Changchun 130021, China. ∗∗ Corresponding author at: Department of Neurosurgery, The Second Hospital of Jilin University, Changchun, 130033, China. E-mail addresses: [email protected] (W. Ni), [email protected] (J. Liu).
The stimulation of macrophages is an important method for enhancing immunological activity [3]. Many polysaccharides activate macrophages by binding to receptors on the surface of immune cells. Polysaccharides interact with macrophages via dectin-1, Tolllike receptor 4 (TLR4), CD14, Scavenger receptor (SR), complement receptor 3 (CR3) and the mannose receptor (MR) [2,3,6]. Activation of these receptors leads to intracellular signaling cascades, resulting in transcriptional activation and the production of proinflammatory cytokines. The Hippophae rhamnoides L. berry has been a traditional medicinal food of the Tibetan plateau for thousands of years [12,13]. Recently, our research group purified a water-soluble polysaccharide (HRWP-A) from H. rhamnoides berry that was identified as a high-methoxyl homogalacturonan (with repeating units of (1 → 4)--d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups). HRWP-A exhibits anti-fatigue and anti-tumor activity [12,13]. Notably, it augmented macrophage phagocytic activity, increased macrophage secretion of NO and TNF-␣ in tumor-bearing mice.
http://dx.doi.org/10.1016/j.ijbiomac.2017.09.083 0141-8130/© 2017 Elsevier B.V. All rights reserved.
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In this study, to further reveal its mechanism of immunomodulatory activity, we investigate the immunomodulatory activities of HRWP-A in cyclophosphamide (CTX)-induced immunosuppressed mice. The effect of HRWP-A on TLR4 pathway signal in peritoneal macrophages was also analyzed to further elucidate the mechanism of macrophage activation. The results may provide insights into underlying mechanisms of activity and valuable information for desigening of more effective adjuvant for cancer therapy. 2. Materials and methods 2.1. Mice Female BALB/c, C3H/HeN and C3H/HeJ mice (6–8 weeks old, weighing 20.0 ± 2.0 g) were purchased from the Pharmacology Experimental Center of Jilin University (Changchun, China). The mice were housed on a 12/12-h light–dark cycle at room temperature and allowed free access to standard rodent food and water during the experiments. Animal handling procedures were conducted under National Institutes of Health animal care and use guidelines. All efforts were made to minimize the animals’ suffering and to reduce the number of animals used. 2.2. Reagents TRIzol reagent and primers were purchased from Invitrogen (Carlsbad, CA, USA). MTS, M-MLV reverse transcriptase and oligodT primers from Promega (Madison, WI, USA). qPCR TaqMix from TaKaRa Bio (Sakado-shi, Saitama, Japan). Mouse TNF-␣, IL-12p70, IL-1 and IL-6 enzyme-linked immunosorbent assay kits and FITCF4/80 were purchased from eBioscience (San Diego, CA, USA). The NO kit and Annexin V-fuorescein isothiocyanate (FITC) apoptotic detection kit was purchased from Beyotime (Nantong, Jiangsu, China). Fluorescence conjugated antibodies specific for MHCII, CD80 and CD86, TLR4/MD-2 were obtained from BioLegend (San Diego, CA, USA). Antibodies specific for MyD88 and GAPDH were purchased from Abcam (Cambridge, UK), while antibodies specific for IB-␣ and the corresponding phospho-antibodies were all purchased from Cell Signaling Technology (Beverly, MA, USA). Pyrrolidine dithiocarbamate (PDTC), mouse lymphocyte separation medium, Lipopolysaccharide (LPS; E. coli 055:B5) and polymyxin B (PMB) were purchased from Sigma–Aldrich (St. Louis, MO, USA). MTS was from Promega (Madison, WI, USA). Cyclophosphamide (CTX) was from Jiangsu Hengrui (Lianyungang, Jiangsu, China). 2.3. Preparation of polysaccharide Polysaccharide from Hippophae rhamnoides L. berries was prepared as described previously [13]. Briefly, crude polysaccharide was obtained from fresh berries by hot water extraction and 80% ethanol precipitation. Then, the extract was successively purified through DEAE-Cellulose ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and DEAE-Sepharose Fast Flow ion-exchange chromatography, finally resulting in a homogeneous polysaccharide fraction named HRWP-A. HRWPA was found to be a high-methoxyl homogalacturonan with a molecular weight of 4992 Da through HPGPC, FT-IR and NMR analysis; this polysaccharide contains repeating units of (1 → 4)-d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups. Endotoxin contamination was analyzed with a gel-clot Limulus amebocyte lysate assay. The endotoxin level in each polysaccharide solution was less than 0.005 EU (endotoxin units)/mg.
2.4. Immunomodulatory activity in CTX-induced immunosuppressed mice 2.4.1. Animal treatment and experimental design Cyclophosphamide (CTX) (25 mg/kg) were administered intraperitoneally to establish the immunosuppressive animal model (5 mice/group). One day later, HRWP-A (50 mg/kg) were administered intragastrically each day for 10 consecutive days. The CTX group was treated only with CTX, and the P.S. group was given physiologic saline as normal control. The dose volume was 0.2 ml. On the day 11, the mice were sacrificed. The spleens and thymus were weighed and their weights relative to the final body weights (organ index) were calculated. 2.4.2. Preparation of splenic mononuclear cells and peritoneal macrophages The spleen from each mouse was harvested and a mononuclear cell suspension was prepared. The splenic mononuclear cells were isolated by density gradient centrifugation on mouse lymphocyte separation medium. The splenic mononuclear cells were collected for analyzing the expression of CD80, MHCII or CD86. Cells from the peritoneal exudate (PEC) were collected from the immuno-suppressed mouse by peritoneal lavage with cold PBS. PECs were washed twice and resuspended in RPMI-1640 medium. Peritoneal macrophages were further isolated by incubating the PECs (1 × 107 /well) in a 6-well plate at 37 ◦ C in a humidified atmosphere with 5% CO2 for 3 h to allow the peritoneal macrophages to adhere. After removing the non-adherent cells with cold PBS, the macrophage monolayer was collected. The adherent cell population was > 95% macrophages, as determined by flow cytometric (FACSCalibur, San Diego, CA, USA) analysis of FITC-F4/80 staining. The macrophages (4 × 105 cells/well) were added into 96-well microwell plates and cultured for 48 h, and then culture supernatants were harvested to detect cytokine secretion by ELISA. 2.4.3. Flow cytometry assay The splenic mononuclear cells (1 × 106 cells) were prepared as described above and fixed with paraformaldehyde for 1 h and washed twice with FACS solution. The cells were incubated with either FITC-labeled anti-mouse MHCII, CD80 and CD86 antibody at 4 ◦ C for 30 min in the dark, and then washed twice with FACS solution. Subsequently, the stained cells were analyzed with a flow cytometer. The FITC-labeled mouse IgG isotype was used as a control. 2.4.4. MTS assay Peritoneal macrophages were prepared as described. The cell viabilities of peritoneal macrophages were determined by MTS assay. The macrophages (4 × 105 cells/ml, 200 l/well) were seeded in a 96-well plate and cultured at 37 ◦ C, 5% CO2 for 20 and 44 h. MTS (5 mg/ml, 20 l) was added to each well and incubated for an additional 4 h. The absorbance at 490 nm was measured using a microplate reader (Bio-Rad, West Berkeley, CA, USA). Relative cell viability was calculated as A490 nm (HRWP-A-treated group)/A490 nm (control group) × 100%. 2.4.5. Apoptosis assay Peritoneal macrophages were placed in a 6-well culture plate at a density of 1 × 106 cells/well for 24 h or 48 h. Apoptosis was measured by staining with Annexin V-FITC/propidium iodide assay kit. Briefly, cells were washed with PBS and resuspended in 500 l of binding buffer. Then, 1 l of AnnexinV-FITC was added to the samples at 4 ◦ C in the dark. After incubation for 5 min, 1 l of propidium iodide was added, and the mixture was incubated for 5 min. Sub-
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Fig. 1. Effect of HRWP-A on immune organ indices in mice. Organ indices = weight of organ (mg)/body weight (g). P.S. group = mice untreated with CTX and treated with saline, CTX group = mice pretreated with CTX. Each value represents the mean ± SD (n = 5). Significant differences from the Model group were evaluated using Student’s t test: a P < 0.05 vs. P.S. group; b P < 0.05 vs. CTX group.
sequently, flow cytometry analysis was performed. Results were expressed as% of apoptotic cells. 2.5. HRWP-A-treated peritoneal macrophages in vitro Peritoneal macrophages were obtained from either BALB/c, C3H/HeN or C3H/HeJ mouse peritoneal as described above. For qPCR and western blot, macrophages were treated with various concentrations of HRWP-A (2.5 − 20 g/ml) or LPS (5 g/ml) in the presence or absence of polymyxin B (5 or 10 g/ml) prior to being seeded in 24-well plates at 4 × 105 cells/well for 6 h. For NO production and cytokine secretion, macrophages were incubated in complete medium in the presence or absence of LPS (5 g/ml) or HRWP-A (10 g/ml) for 24 h. 2.6. Detection of nitric oxide The NO levels in the culture supernatant of mouse peritoneal macrophages were determined with an NO kit. Each data point is an average of the values obtained from 6 mice. The absorbance at 540 nm in each well was measured with an automated microtiter plate reader. 2.7. Cytokine secretion assay The culture supernatants were assayed for IL-1, IL-6, TNF-␣ and IL-12p70 with a commercial ELISA kit according to the manufacturer’s protocol. The absorbance at 450 nm in each well was measured with an automated microtiter plate reader.
2.9. Western blot TLR4 and MyD88 expression and IB phosphorylation were detected using western blot. Macrophage lysates were prepared with RIPA lysis buffer in the presence of the protease inhibitor leupeptin and a phosphatase inhibitor. The protein concentration was determined using the bicinchoninic acid protein detection system. Proteins were separated by SDS-PAGE and subjected to western blot. The following antibodies were used: anti-GAPDH, anti-TLR2/9, anti-MyD88, anti-IB, anti-p-IB. 2.10. Blocking experiments For TLR4 or PDTC blocking experiments, macrophages were cultured in 24-well plates and pretreated with anti-TLR4 (20 g/ml) or a mouse IgG2a (20 g/ml) isotype control antibody for 60 min. In some experiments, the NF-B inhibitor PDTC (20 M) was added to the cell cultures 1 h prior to HRWP-A (10 g/ml) treatment and left in the culture media throughout the experiment. LPS (5 g/ml) was added as a control. Then, NO, IL-1 and IL-6 secretion was analyzed. 2.11. Statistical analysis Statistical significance was determined by one-way ANOVA followed by Student’s t-test or Dunnett’s post hoc comparisons (SPSS statistical software package, version 16.0, SPSS, Chicago, IL, USA) when appropriate. All values were expressed as mean ± S.D. A Pvalue of < 0.05 was considered to be significant.
2.8. Real-time PCR
3. Results and discussion
Total RNA was isolated with TRIzol reagent according to the manufacturer’s protocol. Total RNA was converted to cDNA using M-MLV reverse transcriptase and oligo-dT primers according to the manufacturer’s instructions. TLR4 levels were analyzed with realtime PCR using qPCR TaqMix. The primers used to amplify cDNA were as follows: TLR4 5 -TCA GTG TGC TTG TGG TAG CC-3 and 5 -TCG TTT CTC ACC CAG TCC TC-3 . The real-time PCR reactions were performed on an ABI PRISM@ 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). These values were expressed as the ratios of the relative abundance of target genes to GAPDH.
3.1. Immunomodulatory activity of HRWP-A in CTX-induced immunosuppressed mice 3.1.1. Effects of polysaccharides on organ weight Body weight was recorded after the experiment (10 days, final). Spleen and thymus as immune function-related organs were evaluated (Fig. 1). As expected, CTX obviously decreased the relative thymus and spleen weight as compared to the normal group. However, HRWP-A significantly increased the relative spleen and thymus weights as compared to the model groups (CTX-treated group). These findings suggested that HRWP-A partially recov-
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Fig. 2. The effects of HRWP-A on macrophages from immuno-suppressive mice. (A) The splenic mononuclear cells were stained with either FITC-labeled anti-mouse MHCII, CD80 or CD86 antibody. The stained cells were analyzed by flow cytometry. (B) Effects of HRWP-A on peritoneal macrophage viability. Cell viability was determined by MTS assay, absorbance was measured at 490 nm and values were expressed relative to the viability of cells incubated in medium. (C) Effect of HRWP-A on inhibition of macrophage apoptosis. The apoptotic ratio of peritoneal macrophage was assessed by Annexin V-FITC/PI binding and measured using fllow cytometry. (D) Effects of HRWP-A on macrophage NO (as nitrite), IL-1, TNF-␣, IL-6 and IL-12p70 production of peritoneal macrophage from immuno-suppressive mice. The culture supernatants were collected for NO and cytokine (IL-1, TNF-␣, IL-6 and IL-12p70) analyses with Griess reagent and an ELISA kit. The reported values are the means ± SDs (n = 5). Statistical significance was determined by one-way ANOVA followed by Dunnett’s post hoc comparisons: a P < 0.05 vs. P.S. group; b P < 0.05 vs. CTX group.
ered the immunosuppressive accompanied with an improvement of immune organs.
3.1.2. HRWP-A activated and enhanced viability of peritoneal macrophages from CTX-induced immunosuppressed mice Macrophages are considered the pivotal immunocytes of host defense, and they provide an important bridge between innate and adaptive immunity. MHCII, CD80 and CD86 is highly expressed on the surface of activated macrophages. To investigate the effect of HRWP-A on macrophages, the expression of MHCII, CD80 and CD86 was analyzed using flow cytometry. As shown in Fig. 2A, the expression of MHCII, CD80 and CD86 in the HRWP-A groups was significantly up-regulated compared with the Model group (P < 0.05). These data indicate that HRWP-A increases the percentage of activated macrophages. Meanwhile, although CTX decrease the macrophage viability and increase the macrophage apoptosis, but HRWP-A can prolong the cell survive for it can enhance macrophage viability detecting by MTS assay (Fig. 2B) and inhibit the macrophage apoptosis detecting by Annexin V-FITC/PI apoptosis assay (Fig. 2C). The analysis of cytokine secretion showed that CTX decreased the level of NO, IL-1, IL-6, TNF-␣ and IL-12p70 of macrophages (Fig. 2C). IL1 has a wide range of immunomodulatory effects and may be directly involved in the inflammatory process [14]. IL-6 is a cytokine with multiple immunomodulatory functions, and NO are important mediators in the destruction of tumor cells [15–17]. However,
HRWP-A significantly increased the level of NO, IL-1, TNF-␣ and IL-6. Meanwhile, HRWP-A partially recovered the level of NO, IL1 and IL-6 inhibiteed by CTX. HRWP-A showed little effect on the IL-12 secretion, indicated that HRWP-A might have less effect on macrophage polarization in the CTX-induced immunosuppressed mice. Altogether, these results indicated that HRWP-A might counteract the damage of CTX to macrophage to some extent.
3.2. HRWP-A upregulates TLR4 expression and activates the MyD88 pathway via TLR4 Subsequently, we explored the possible mechanism underlying macrophage activation by HRWP-A. Several studies have reported that botanical polysaccharides interact with macrophages via TLR2 or TLR4. Thus, we first detected the expression of TLR2/4 on macrophages treated with HRWP-A. QPCR showed that the TLR2 mRNA level in macrophages did not change after stimulation with HRWP-A (data not shown), while HRWP-A significantly upregulated TLR4 expression in a concentration-dependent manner (2.5–20 g/ml) (Fig. 3A), and this effect was not abrogated by treatment with polymyxin B at 5 g/ml, which eliminated the possibility of LPS contamination. As a positive control, LPS (5 g/ml) also upregulated TLR4 expression, but this effect was reversed after preincubating the LPS with polymyxin B. These results indicated that TLR4 may relevant to the interaction between HRWP-A and macrophages.
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Fig. 3. HRWP-A activates MyD88/NF-B via TLR4. (A) The effect of HRWP-A on TLR4 expression patterns in macrophages was detected using real-time PCR. Peritoneal macrophage cells were treated with various concentrations of HRWP-A (2.5 − 20 g/ml) or LPS (5 g/ml) in the presence or absence of polymyxin B (5 or 10 g/ml) prior to being seeded in 24-well plates at 4 × 105 cells/well for 6 h. (B) The effect of HRWP-A on MyD88/NF-B expression was detected with western blot. Macrophages were pretreated with an anti-mouse TLR4 (20 g/ml) antibody or 5 g/ml polymyxin B (PMB) prior to the addition of HRWP-A (10 g/ml) or LPS (5 g/ml) for 6 h; MyD88 and IB-␣ activation was then determined. The data are expressed as the means ± SDs of three independent experiments. Statistical significance was determined by Student’s t-test: *P < 0.01 vs. the medium-treated group, # P < 0.01 vs. the HRWP-A or LPS (alone)-treated group, respectively.
Subsequently, to confirm that HRWP-A may be considered a ligand for TLR4, the receptor that plays an important role in the activation, polarization and function of macrophages, we further studied the downstream signaling pathways. As shown in Fig. 3B, HRWP-A (10 g/ml) increased MyD88 expression and IB phosphorylation, and the addition of polymyxin B did not reverse the effect of HRWP-A, suggesting that HRWP-A was responsible for activating the MyD88/NF-B pathway in these experiments. In contrast, although LPS (5 g/ml) also upregulated MyD88 and pIB expression, the LPS-induced activation of IB-␣ was reversed after preincubation of LPS with PMB. Furthermore, our results showed that pretreating macrophages with an anti-TLR4 antibody (20 g/ml) almost eliminated MyD88 expression and IB-␣ phosphorylation; these results strongly suggests that HRWP-A induces the activation of the MyD88/NF-B signaling pathway via TLR4 in macrophages, presumably leading to proinflammatory cytokine secretion and macrophage activation. Although it remains unclear how HRWP-A works with TLR4, HRWP-A treatment results in the recruitment of MyD88 to TLR4, which initiates the signaling cascade that leads to the activation of NF-B.
3.3. Blocking TLR4/NF-B inhibits the HRWP-A-induced increase in NO, IL-1ˇ and IL-6 levels To further confirm the involvement of TLR4 in the interaction of HRWP-A and macrophages, an anti-TLR4 antibody and the NF-B inhibitor PDTC were used in vitro to pre-treat the macrophage, after which NO, IL-1 and IL-6 secretion was examined. Results showed that HRWP-A (10 g/ml) increased the level of NO, IL-1 and IL-6 produced by macrophages (Fig. 4A–F). Treatment with anti-TLR4 (20 g/ml) completely abrogated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels (Fig. 4A-C), and PDTC (20 M) partially attenuated (Fig. 4D-F) the HRWP-A-induced increase in NO, IL-1 and IL-6. These results support the evidence that TLR4 is involved in the HRWP-A-mediated stimulation of macrophages and that the MyD88/NF-B pathway is associated with macrophage activation. In addition, we compared normal mice (C3H/HeN) and mutant TLR4 mice (C3H/HeJ) to further verify this phenomenon. The results show that macrophages from C3H/HeN mice responded to HRWPA stimulation by producing higher levels of NO, IL-1 and IL-6 (Fig. 3G-H); however, macrophages from C3H/HeJ mice did not respond to HRWP-A (10 g/ml) stimulation in vitro, suggesting TLR4 as one of the candidate HRWP-A receptors.
TLR4 triggers immune responses mainly through 2 signaling pathways: the MyD88-dependent pathway and the TRIFdependent pathway. MyD88 is utilized by TLR4 and activates downstream ERK, NF-B or p38 MAPK signaling, and MyD88 recruits TRAF6, leading to the activation of NF-B. TRIF recruits TRAF3 and TRAF6, which then activate NF-B and IRF3, respectively [18]. In this paper, we clearly demonstrate that HRWP-A stimulates TLR4 and induces NF-B activation in peritoneal macrophages. This result is consistent with several reports that polysaccharides from several other medicinal herbs are able to activate the MyD88/NFB signaling pathway via TLR4 in macrophages [5,18–22]. These plant-derived polysaccharides might be important in the initiation of some forms of chronic intestinal inflammation. An anti-TLR4 antibody (20 g/ml) completely abrogated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels, suggesting that the interaction between HRWP-A and macrophages relies on TLR4. The NF-B inhibitor PDTC partially attenuated the HRWP-A-induced increase in NO, IL-1 and IL-6 levels, suggesting that in addition to NF-B, HRWP-A may also be involved in other pathways downstream of MyD88, such as p38 MAPK or TRAF. Further study of these intracellular signal transduction mechanisms is underway in our laboratory. Previous studies have revealed that many glucans with ␣(1,3),(1,4), ␣-(1,4),(1,6) or -(1–3)(1–6) linkages may interact with TLR4 [2,5]. Pectin, structurally and functionally the most complex polysaccharide in plant cell walls, is a family of galacturonic acid-rich polysaccharides including homogalacturonan (HG), ramified rhamnogalacturonan (RG) I and II. Arabinogalactan structural motifs of RG-I are usually important for the immunomodulating activity [2,4]. Several studies have reported that pectic polysaccharides also interact with macrophages via TLR4 [5,18–22]. Our study showed that the high-methoxyl homogalacturonan pectin (with repeating units of (1 → 4)--d-galactopyranosyluronic residues, 85.16% of which were esterified with methyl groups) HRWP-A from the H. rhamnoides berry and TLR4 interact in the HRWP-A-mediated stimulation of macrophages and that the MyD88/NF-B pathway is associated with macrophage activation, leading to downstream signal transmission and inducing the production of proinflammatory cytokines. In contrast, some research has reported that pectic polysaccharide, such as arabinogalactan with a back-bone of (1 → 3)-linked -d-galactopyranosyl residues which were mainly substituted at the O-6 position by the branches composed of arabinosyl, galactosyl, and rhamnosyl residues [21], attenuates inflammation through inhibiting the TLR4/NF-B signaling path-
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Fig. 4. Effects of HRWP-A on macrophage NO (as nitrite), IL-1 and IL-6 production in the presence of TLR4 blockade. (A–C) For the TLR4 blocking experiments, peritoneal macrophages were cultured in 24-well plates and pretreated with an anti-TLR4 antibody (20 g/ml) or a mouse IgG2a (20 g/ml) antibody for 60 min. (D-F) For the PDTC blocking experiments, the NF-B inhibitor PDTC (20 M) was added to the cell cultures 1 h prior to HRWP-A (10 g/ml) treatment and remained in the culture media throughout the experiment. (G-H) Comparison of the pro-inflammatory factors in wild-type C3H/HeN and C3H/HeJ mice. The culture supernatants were collected for NO and cytokine (IL-1, IL-6) analyses with Griess reagent and an ELISA kit. The reported values are the means ± SDs (n = 6). Statistical significance was determined by Student’s t-test: *P < 0.01 vs. the medium-treated group, # P < 0.01 vs. the HRWP-A or LPS (alone)-treated group, respectively.
way. The differences in these data may result from the glycan structures, specifically the molecular weight, chain length and tertiary structure [2–5]. In summary, our study provides evidence that HRWP-A, which has hitherto been considered a potent proinflammatory stimulus, activates macrophages via TLR4 and the activation of the NF-B pathway. HRWP-A may have intrinsic adjuvant-like properties that enhance the immune response. Research on this structure may contribute to understanding the structure-activity relationship (SAR) of polysaccharides. Acknowledgments This work was funded by the Department of Science and Technology of Jilin Province (No. 20160101018JC), the National Natural Science Foundation of China (No. 31300291), the Bethune plan of Jilin University (No. 2015434), and “The Western Light” Personnel Training Project and Applying Basic Research Program of Qinghai province (No. 2014-ZJ-751 and 2015-ZJ-727).
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