Soluble Branched β-(1,4)Glucans from Acetobacter Species Show Strong Activities to Induce Interleukin-12 in Vitro and Inhibit T-helper 2 Cellular Response with Immunoglobulin E Production in Vivo
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m304948200
ISSN1083-351X
AutoresKimika Saito, Toshiki Yajima, Hitoshi Nishimura, Keiko Aiba, Ryotaro Ishimitsu, Tetsuya Matsuguchi, Takashi Fushimi, Yoshifumi Ohshima, Yoshinori Tsukamoto, Yasunobu Yoshikai,
Tópico(s)Microbial metabolism and enzyme function
ResumoAn Extracellular Polysaccharide, Ac-1, Produced By Acetobacter Polysaccharogenes Is Composed Of β-(1,4)GluCan With Branches Of Glucosyl Residues. We Found That Ac-1 Showed A Strong Activity To Induce Production Of Interleukin-12 P40 And Tumor Necrosis Factor-α By MacroPhage Cell Lines In Vitro. Cellulase Treatment Completely Abolished The Activity Of Ac-1 To Induce Tumor Necrosis Factor-α Production By Macrophages, Whereas Treatment Of Ac-1 With Polymyxin B Or Proteinase Did Not Affect The Activity. Results Of Experiments Using Toll-Like Receptor (Tlr) 4-Deficient Mice And Tlr4-Transfected Human Cell Line Indicated That Tlr4 Is Involved In Pattern RecogniTion Of Ac-1. In Vivo Administration Of Ac-1 Significantly Reduced The Serum Levels Of Ovalbumin (Ova)-Specific Ige And Interleukin-4 Production By T Cells In Response To Ova In Mice Immunized With Ova. Ac-1, A Soluble Branched β-(1,4)Glucan May Be Useful In Prevention And Treatment Of Allergic Disorders With Ige Production. An Extracellular Polysaccharide, Ac-1, Produced By Acetobacter Polysaccharogenes Is Composed Of β-(1,4)GluCan With Branches Of Glucosyl Residues. We Found That Ac-1 Showed A Strong Activity To Induce Production Of Interleukin-12 P40 And Tumor Necrosis Factor-α By MacroPhage Cell Lines In Vitro. Cellulase Treatment Completely Abolished The Activity Of Ac-1 To Induce Tumor Necrosis Factor-α Production By Macrophages, Whereas Treatment Of Ac-1 With Polymyxin B Or Proteinase Did Not Affect The Activity. Results Of Experiments Using Toll-Like Receptor (Tlr) 4-Deficient Mice And Tlr4-Transfected Human Cell Line Indicated That Tlr4 Is Involved In Pattern RecogniTion Of Ac-1. In Vivo Administration Of Ac-1 Significantly Reduced The Serum Levels Of Ovalbumin (Ova)-Specific Ige And Interleukin-4 Production By T Cells In Response To Ova In Mice Immunized With Ova. Ac-1, A Soluble Branched β-(1,4)Glucan May Be Useful In Prevention And Treatment Of Allergic Disorders With Ige Production. 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In the present study, we found that a bacteria cellulose AC-1 derived from Acetobacter species was a potent inducer of IL-12 p40 and TNF-α production by macrophages in vitro. AC-1, a β-glucan composed of β-(1,4)glucan with branches of glucosyl residues, has a molecular mass of 1 × 106 daltons. Polymyxin B- and proteinase-treated AC-1 stimulated spleen-adherent cells to produce TNF-α, whereas such cytokine production was not detected in cellulase-treated AC-1. Results of experiments using TLR4-deficient mice and TLR4-transfected human cell line indicated that TLR4 is involved in pattern recognition of AC-1. When oral administration of AC-1 was begun immediately after ovalbumin (OVA) immunization, significant decreases in the serum levels of OVA-specific IgE and IgG1 accompanied by augmented IFN-γ production occurred. These results suggest that AC-1, a potent IL-12 and TNF-α inducer, suppresses allergic inflammation with IgE production, thus offering an approach for the treatment of allergic disorders. Animals—C3H/HeN mice, C3H/HeJ mice and BALB/c mice (SLC, Shizuoka, Japan) were used in the experiments at 6–10 weeks of age. Mutant mice (F2 interbred from 129/Ola x C57BL/6) with a deficiency in TLR2 were generated by gene targeting and kindly provided by S. Akira (Osaka University, Japan) (4Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2785) Google Scholar). Age- and sex-matched groups of TLR2-deficient (TLR2–/–) mice and their littermate (TLR2+/–) mice were used for the experiments. These mice were bred in our Institute under specific pathogen-free conditions. Purification of AC—The polysaccharide AC series of the Acetobacter species used in this study were prepared and purified by the method reported in a previous paper (30Minakami H. Entani E. Tayama K. Fujiyama S. Masai H. Agric. Biol. Chem. 1984; 48: 2405-2414Google Scholar). Briefly, five strains of polysaccharide-producing Acetobacter species were cultivated in a shaking flask at 30 °C for 5 days, and the cells were removed by both centrifugation (10,000xg) of diluted broth and filtration with celite. The cell-free polysaccharides were precipitated by the addition of isopropyl alcohol, and the precipitate was dissolved in water. Then 5% aqueous cetyltrimethylammonium bromide (CTAB) solution was added until no more precipitate was formed. The insoluble acidic polysaccharide-cetyltrimethylammonium bromide complex was collected by centrifugation and redissolved in 20% sodium chloride solution. After dialysis against running water, the polysaccharide was precipitated with ethanol and dissolved in water. The acidic polysaccharide thus obtained was dialyzed against distilled water and lyophilized. We named these polysaccharides AC series as follows: AC-1, Acetobacter polysaccharogenes MT-11-2; AC-2, A. polysaccharogenes 1007; AC-3, A. polysaccharogenes 1011; AC-4, Acetobacter xylinum MH-1597; AC-5, A. xylinum 1053). The endotoxin content of AC-1 (100 μg/ml) was estimated by using an endotoxin-specific chromogenic Limulus test (Wako) to be 32 pg/ml. This level of endotoxin does not affect TNF-α production in a culture supernatant of peritoneal adherent cells of C3H/HeN mice. In some experiments, AC-1 (100 μg/ml) was treated with 10 μg/ml of polymyxin B (Wako) at 37 °C for 2 h for deactivation of LPS activity or treated with actinases E (Kakenshouyaku) 37 °C for 24 h and then DEAE anion chromatography for removal of protein. AC-1 (5 mg) was incubated with 1 mg of β-(1,4)glucanase (Wako) in 0.05 m acetate buffer (2.5 ml) at pH 5.0 and 37 °C for 24 h. LPS was treated in the same manner. Analysis with High-performance Anion-exchange Chromatography Coupled with a Pulsed Amperometric Detection—The polysaccharides from Acetobacter species were subjected to acid hydrolysis using trifluoroacetic acid and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. The 10-mg sample was mixed with a final concentration 2 n trifluoroacetic acid containing 10 g/ml fucose as an internal standard in a glass tube sealed with a screw cap. After 18 h at 100 °C, solutions were dried under a stream of nitrogen; the residues were then redissolved in water and the hydrolyzate solutions transferred to autosampler vials. The hydrolyzates were stable in water at –20 °C for at least 1 month. Samples were analyzed by high-performance anion-exchange chromatography on a Dionex 500 system (Dionex Corp.) supplied with a pulsed amperometric detector. The system was equipped with a CarboPac column (packed silica appropriate for mono-, di-, tri-, and oligosaccharide analysis). Sodium hydroxide solution (NaOH, 250 mm in water) was used as the eluant. Analyses were performed in the isocratic mode (% water/% NaOH = 48:52). Flow rate was set to 0.6 ml/min. To minimize carbonate formation in the system, which leads to a dramatic reduction of the retention times, a small amount of Ba(CH3COO)2 (4 mm) was added to the alkaline eluant. Data were collected and analyzed on computers equipped with the Dionex PeakNet software. Cell Preparation—All cell lines were grown in tissue culture flasks at 37 °C in 5% CO2, 95% air and passaged every 2 or 3 days to maintain logarithmic growth. Two mouse macrophage cell lines, J744.1 and RAW264.7, and a human embryonic kidney cell line, HEK 293 (human embryonic kidney 293), were obtained from the Institute of Physical and Chemical Research Cell Bank (Tsukuba, Japan) and maintained in Dulbecco's minimum essential medium with 10% fetal bovine serum (JRH Bioscience). Adherent cells from the peritoneal cavity of C3H/HeN, C3H/HeJ, TLR2+/–, or TLR2–/– mice were used as a source of mouse macrophages. Briefly, peritoneal exudate cells (PEC) were suspended in RPMI 1640 containing 10% fetal bovine serum were cultured on plastic plates for 2 h at 37 °C. After nonadherent cells had been removed, fresh complete medium was added to the adherent cells with or without AC-1 or control reagents in the presence of IFN-γ (30 units/ml). Isolation of the Full-length cDNA Clone Encoding Mouse TLRs— Messenger RNA was isolated from RAW264.7 cell using a Quick Prep Micro mRNA Purification kit (Amersham Biosciences). mRNA (1 μg) was treated with DNase prior to reverse transcription-PCR using a Superscript 2 preamplification system. The resulting templates were subjected to PCR reactions with murine each TLR-specific primers. The primers used were as follows: TLR1 sense, 5′-TGCGGCCGCACCATGACTAAACCAAATTCC-3′; TLR1antisense, 5′-CGGGATCCTTGCTGTGTGTAACACGTTCCT-3′; TLR2 sense, 5′-TGCTGGAGCCCATTGAGAGGA-3′; TLR2 antisense, 5′-GGACTTTATTGCAGTTCTCAG-3′; TLR4 sense, 5′-TTGCGGCCGCTGCCAGGATGATGCCTCCCT-3′; TLR4 antisense, 5′-CGGGATCCGGTCCAAGTTGCCGTTTCTTGT-3′; TLR6 sense, 5′-TGCGGCCGCACCATGGTAAAGTCCCTCTGG-3′; TLR6 antisense, 5′-CGGGATCCAGTTTTCACATCCTCATTGACT-3′; TLR9 sense, 5′-AGCTTCCTGCTGGCTCAGC-3′; TLR9 antisense, 5′-GGACGCAGGATCACCAACAC-3′. Each PCR product was electrophoresed and transferred to and hybridized with murine TLR cDNA as a probe. DNA sequence analysis was performed on these plasmid clones using a DNA sequencer (model 373A) and a Thermo Sequenase cycle sequencing kit (PE Biosystems). Plasmids—Murine TLR tagged with p3xFLAG at the carboxyl terminus was generated by PCR and ligated into the expression plasmid CMV14 (Sigma). All TLR plasmids used in transfections were purified using an Endo-free plasmid kit (Qiagen). A mouse CD14 expression plasmid, pcDNA3.1(+)-mCD14, and a NF-κB-responsive reporter, pGL3-NFκB-Luc, have been described previously (39Matsuguchi T. Takagi K. Musikacharoen T. Yoshikai Y. Blood. 2000; 95: 1378-1385Crossref PubMed Google Scholar). A carboxyl-terminal FLAG-tagged each TLRs expression plasmid and mouse MD2 expression plasmid were generated by inserting the whole mTLR and MD-2 coding region cDNA into the p3xFLAG-CMV14 vector. TLR Expression by Western Blotting—Cells were lysed in lysis buffer (50 mm HEPES (pH 7.0), 150 mm NaCl, 10% glycerol, 1% Triton X-100, 1.5 mm MgCl2, 1 mm EGTA, 100 mm NaF, 10 mm NaPPi, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) at 1 × 108 cells/ml. The lysates were separated in SDS-polyacrylamide gels and then electrotransferred to Immobilon polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked for 1 h in 5% nonfat milk-TBST (20 mm Tris-HCl pH 7.6, 0.15 m sodium chloride, 0.1% Tween 20), incubated with anti-FLAG monoclonal antibody M2 (Sigma) in TBST for 1 h, washed three times with TBST, and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-rabbit Ig (Amersham Biosciences) diluted 1:5000 in 5% nonfat milk-TBST. After three washes in TBST, the blot was developed using an enhanced chemiluminescence system (Amersham Biosciences) and analyzed using a lumino-image analyzer (LAS-1000plus, Fujifilm). Luciferase Assay—HEK 293 cells were seeded in 6-well plates at a density of 1 × 106 cells/well 1 day before transfection. HEK 293 cells were transiently transfected with 0.1 μg of pGL3-NF-κB/Luc (a luciferase reporter construct containing the consensus NF-κB binding sequence), 0.4 μg of pSV-β-galactosidase as an internal control (Promega, Madison, WI), and either 1.0 μg of mTLRs/FLAG using LipofectAMINE™ (Invitrogen). At 48 h after transfection, the HEK 293 cells were stimulated with AC-1 for 8 h. The cells were harvested, washed, and lysed in 200 μl of lysis buffer, and the luciferase activity was measured using a luminometer (Wallac 1420, PerkinElmer Life Sciences) with a Dual-Luciferase Reporter Assay System (Toyo Ink, Tokyo, Japan) or Steady-Glo Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Background luciferase activity was subtracted. Cytokine Measurement—J744.1 or RAW264.7 cells (5 × 105 cells/ml) were stimulated with the AC series, and the levels of IL-12 p40 and TNF-α production were determined by using an enzyme-linked immunosorbent assay (ELISA) 24 h after incubation. Adherent cells of PEC from C3H/HeN and C3H/HeJ or TLR2+/– and TLR2–/– mice were incubated for 24 h with AC-1, LPS (Escherichia coli O55, Sigma), synthetic lipoprotein (palmitoyl-Cys(R,S)-2,3-di(plamitoyloxy)-propyl-Ala-Gly-OH, Bachem AG, Bubendorf, Switzerland), or synthetic lipid A (Ono-4007, Osaka Japan) in the presence or absence of IFN-γ, and the culture supernatant was then collected. IL-12 p40 and TNF-α levels in the culture supernatant were determined by ELISA. ELISAs for IL-12 p40 and TNF-α were performed using commercially available kits from Genzyme (Cambridge, MA). Immunization and Challenge—Mice were immunized intraperitoneally with 50 μg of chicken OVA (Gradee, Sigma) absorbed in 100 μl of the alum on day 0. Twelve days later, the mice were injected intraperitoneally with 50 μg of OVA in 100 μl of alum. The mice were challenged intragastrically with a suspension of 100 μl of AC-1 (10 mg/ml) or PBS on days 1, 4, 9, and 14. Spleens and sera were obtained on day 29 and stored at –20 °C until analysis. Measurement of Cytokine Production of Spleen Cells—Spleen cells were incubated on a nylon wool column at 37 °C in 5% CO2 for 60 min. The cell population eluted from the column contained >90% T cells as determined by flow cytometric analysis with anti-CD3ϵ monoclonal antibody. T cells (5 × 105) and mitomycin C-treated naive spleen cells (5 × 105) were cultured in 96-well cell culture plates (Falcon, BD Biosciences) with 200 μg of OVA. After 48 h of culture, the cultured supernatants were collected, and the amounts of secreted IL-4 and IFN-γ in the supernatants were determined by ELISA. Commercial ELISA kits were used to measure the levels of IL-4 and IFN-γ (Genzyme Diagnostics) according to the manufacturer's instructions. Measurement of OVA-specific IgE, IgG1, and IgG2a—Levels of OVA-specific IgE, IgG1, and IgG2a were determined by ELISA. Sample wells of an ELISA plate were coated with OVA and left overnight and then blocked with 1% bovine serum albumin in borate-buffered saline (0.05 m borate, 0.15 m NaCl, pH 8.6, 100 μl/well) at 37 °C for 30 min. Diluted samples (100 μl/well) were incubated for 90 min at room temperature (Samples for IgE, IgG1, and IgG2a were diluted 1:1000, 1:10000, and 1:10000, respectively). The plates were washed with borate-buffered saline with 0.05% Tween 20 and incubated with peroxidase-conjugated anti-mouse IgE (GAM/IgE(Fc) PO, Nordic), IgG1 (peroxidase rabbit anti-mouse IgG1, γ1-specific, Zymed Laboratories Inc.), or IgG2a (peroxidase rabbit anti-mouse IgG2a, γ2-specific, Zymed Laboratories Inc.) for 90 min at room temperature. After further washing, plates were incubated for 20 min at room temperature with 100 μl/well of o-phenylendiamine solution (1 μg/ml with 3% H2O2), and the OD was read at 492 nm. Statistical Analysis—The statistical significance of the data was determined by Student's t test. A p value of less than 0.05 was taken as significant. Structure of the Polysaccharide AC Series—Five strains of Acetobacter species that produce a new type of extracellular soluble polysaccharide were isolated, and the composition of their carbohydrate components was analyzed using high-performance anion-exchange chromatography with a pulsed amperometric detector. A representative polysaccharide, AC-1, isolated from the culture filtrate of A. polysaccharogenes MT-11-2(1005) is composed of d-glucose, d-galactose, d-mannose, and d-glucuronic acid in the molar ratio of 3.0:1.0:1.1:1.5. The composition of the carbohydrate components in each sample is shown in Table I. NMR spectroscopy indicated that the dominant d-glycosidic linkages must be in β-configuration (30Minakami H. Entani E. Tayama K. Fujiyama S. Masai H. Agric. Biol. Chem. 1984; 48: 2405-2414Google Scholar). To obtain information on the mode of glycosidic linkages, both native and carboxyl-reduced polysaccharides were methylated, and the partially methylated sugars in the acid hydrolysate were analyzed by gas liquid chromatography. The identities and proportions of the cleavage fragments of the native and carboxyl-reduced polysaccharides indicate that polysaccharide AC-1 has a highly branched structure with a repeating unit of 11 sugar residues. It contains a backbone chain of β-(1,4)-linked d-glucose, residues and two of the four d-glucose residues are branched at the O-3 position. There are two kinds of side chains; one is terminated with d-glucose residues and the other with d-glucuronic acid residues, as indicated by the increase in approximately 1 mol of tetra-O-methyl-d-glucose in the methylated, carboxyl-reduced polysaccharide. In addition, the polysaccharide contains (1,6)-linked d-glucose, (1,6)-linked d-galactose, and (1,2)-linked d-mannose residues. They are most probably located in the side chains, as revealed by the fragmentation analysis (31Tayama K. Minakami H. Entani E. Fujiyama S. Masai H. Agric. Biol. Chem. 1985; 49: 959-966Google Scholar). Similar analysis was performed on AC-4 derived from A. xylinum MH-1597. The structural feature of polysaccharide AC-4 resembles that of polysaccharide AC-1 except for the sugar arrangement in the side chains. Thus, AC is a β-glucan composed of β-(1,4)glucan with branches of glucosyl residues (Fig. 1).Table IMolar ratio of carbohydrate components of polysaccharides AC seriesStrainGlcGalFucManRhaGlcAManAGlcNAcGalNAcnmolAC-1A. polysaccharogenes MT-11-2 (1005)3.01.0-1.1-1.50.1-+AC-2A. polysaccharogenes 10073.00.5-0.5-0.3-+-AC-3A. polysaccharogenes 10113.01.1-1.9-0.70.1--AC-5A. xylinum 10533.0--2.20.80.60.1-- Open table in a new tab Bacterial Cellulose Derived from Acetobacter Species Stimulated Mouse Macrophages to Produce IL-12 p40 and TNF-α—We screened soluble β-(1,4)glucans derived from Acetobacter species by measuring the levels of cytokine production in the mouse macrophage cell lines J774.1 and RAW264.7. Among the various preparations, AC-1, -2, and -3, all of which are derived from A. polysaccharogenes, stimulated J774.1 to produce IL-12 p40 (Fig. 2A). AC-1, derived from Acetobacter polysaccharogenes MT-11-2, at a final concentration of 100 μg/ml induced the maximal level of TNF-α production in both mouse macrophage cell lines among the preparations (Fig. 2, B and C). RAW264.7 cells did not produce IL-12 p40 in response to AC-1 or LPS (data not shown). AC-1 was used in the following experiments. Next, an experiment was carried out to determine whether AC-1 induces production of IL-12 p40 and TNF-α by primary culture of macrophages from naive mice. As shown in Fig. 3A and B, AC-1 induced significantly high levels of IL-12 p40 and TNF-α production by peritoneal adherent cells from BALB/c mice. To further purify the polysaccharides from AC-1, we treated AC-1 with protease and subjected them to ion exchange chromatography. Protease-treated products of AC-1 exhibited almost the same level of activity to induce TNF-α production as that of nontreated AC-1 (Fig. 3, A and B). Thus, the protein moiety of AC-1 is not required for the activity of AC-1. Because Acetobacter species are Gram-negative bacteria containing LPS, we examined the effect of treatment of AC-1 with polymyxin B, which neutralizes LPS activities, on the production of TNF-α by macrophages. As shown in Fig. 3C, polymyxin B inhibited the activity of LPS (100 ng/ml) but not the activity of AC-1. Thus, the possibility that induction of the activity of AC-1 is due to contamination by LPS is ruled out. Backbone Chain of β-(1,4)Glucan Is Important for AC-1 Activity—As shown in Fig. 1, AC-1 has a branched structure containing a backbone chain of β-(1,4)-linked d-glucose, two of every four glucose residues being substituted at the O-3 positions to form two kinds of branches. To determine the involvement of β-(1,4)glucose linkage of AC-1 in stimulation of macrophages, AC-1
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