Enhanced Cytokine Secretion from Primary Macrophages due to

Dec 2, 2010 - Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu,. Fukuoka 808-0135, Japa...
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Bioconjugate Chem. 2011, 22, 9–15

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Enhanced Cytokine Secretion from Primary Macrophages due to Dectin-1 Mediated Uptake of CpG DNA/β-1,3-Glucan Complex Jusaku Minari,† Shinichi Mochizuki,† Tsubasa Matsuzaki,† Yoshiyuki Adachi,‡ Naohito Ohno,‡ and Kazuo Sakurai*,†,§ Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan, Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan. Received March 4, 2010; Revised Manuscript Received September 21, 2010

Unmethylated CpG sequences (CpG DNA) can induce Th1 response and thus become a potential immunotherapeutic agent. A key step in the treatment is to transport CpG DNA to its receptor TLR9 located in the endocytosis pathway of target immune cells. For the effective transport, we prepared a novel complex from a β-1,3-glucan schizophyllan (SPG) and CpG DNA, and administered the complex to murine peritoneal macrophages that had been previously activated by thioglycollate and expressed a major β-1,3-glucan receptor Dectin-1 on the cellular surface. Flow cytometric analysis and microscopic observation showed that the complex was taken up by the macrophage through Dectin-1 mediated pathway. Indeed, ELISA demonstrated that IL-12 production was increased sigmoidally with increasing SPG/CpG DNA ratio in the complexation, and reached the maximum at the SPGrich composition. In the present work, we describe a new approach to deliver CpG DNA to immune cells by use of a β-1,3-glucan/DNA complex.

INTRODUCTION The innate immune system has the remarkable ability to distinguish subtle differences between pathogenic and host molecules with the aid of Toll-like receptors (TLRs) (1, 2). In particular, TLR9 is located in the endocytosis compartments of immunocytes, and lowering the pH at the late endosome can lead TLR9 to bind to CpG DNA (3, 4). A recent study has shown that dimerization of TLR9 is critical to its activation by CpG DNA (5). After the activation, NF-κB is recruited through a MyD88-dependent pathway, and eventually, a Th1 response is initiated, following the production of various cytokines including IL-12. The optimal CpG motifs for murine TLR9 are defined as an unmethylated CpG dinucleotide flanked by two 5′ purines and two 3′ pyrimidines, i.e., Pu-Pu-C-G-Py-Py (6). Methylation of the cytosine or sequence inversion from CpG to GpC does not activate TLR9 at all, indicating that this recognition is highly sensitive to molecular structure (7). In the past 10 years, the molecular mechanism of CpG DNA/TLR9 interactions has gradually become understood. In accordance with this, clinical trials of CpG DNA have been conducted for allergies, asthma, cancer, and certain infectious disease (8). CpG DNA itself does not have cellular selectivity, and thus, targetability to immunocytes is of great importance in developing a CpG DNA carrier. In many studies including our preceding work, cationic functional groups or cell-fusion peptides were attached to carriers to enhance cellular uptake (9, 10). Although these chemical modifications enhanced the cytokine secretion dramatically, they could not have targetability at all. Especially * Corresponding author. Name: Prof. Kazuo Sakurai. Mailing address: Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka, 808-0135, Japan. Tel: +81-93-695-3294, Fax: +81-93-695-3368. † The University of Kitakyushu. ‡ Tokyo University of Pharmacy and Life Science. § Japan Science and Technology Agency.

in vivo, cationic compounds are rapidly eliminated from blood and thus may create problems for practical application. Schizophyllan (SPG) is a member of the β-1,3-glucan family and known to exist as a right-handed triple helix in neutral water and as a single chain in alkaline solution (>0.25 N NaOHaq) (11). When the alkaline solution of SPG is neutralized to pH ) 7-8, the single chain retrieves its original triple helix owing to the hydrophobic and hydrogen bonding interactions. When a certain homopolynucleotide such as poly(C) or poly(dA) with enough chain length (ca., bp >100) coexists in this neutralization reaction, a novel complex is formed between SPG and the polynucleotide. As represented in Figure 1, two glucoses of SPG main chains and one base of the polynucleotide bind with each other in the complex, and accordingly, two SPG chains and one polynucleotide chain form a triple helix (12, 13). Contrary to these long homo sequences, short hetero sequences such as antisense and CpG DNA do not bind to SPG, and thus to induce the binding, we attached an oligo-dA tail in the range of 30-60 bases to the hetero sequences (14) and applied SPG to the delivering vehicle (10). In addition, phosphorothioation of the dA tails can induce more stable complex formation, and therefore, in this study, we use CpG DNA with a phosphorothioated dA tail denoted as CpG-dA and demonstrate delivering performance for the CpG-dA/SPG complex. Recently, Gordon et al. found a major β-1,3-glucan receptor called Dectin-1, expressed on the surface of immune cells such as murine peritoneal macrophages and dendric cells (15, 16). We presume that the CpG-dA/SPG complex can be recognized by Dectin-1 and induce the uptake of the bound CpG-dA. If this hypothesis is correct, it can be possible to specifically deliver CpG DNA into the immune cells with Dectin-1. In fact, our preceding papers have represented an encouraging in vivo result that the complexed CpG-dA can induce larger amounts of IL12 production in the blood after intraperitoneal administration, compared with the uncomplexed one (17). Despite these positive results, there is no direct evidence to support our hypothesis.

10.1021/bc1001196  2011 American Chemical Society Published on Web 12/02/2010

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Figure 1. Repeating unit of SPG and a structural model for the complex consisting of CpG-dA and SPG. The dA tail is incorporated in the complex forming hydrogen bonding between the two SPG main chain glucoses and one dA base, while the CpG heterosequenced moiety is not binding with SPG.

In the present study, we demonstrate that a β-1,3-glucan SPG can provide immune cell-specific delivery of CpG DNA, using murine peritoneal macrophages highly expressing Dectin-1.

Scheme 1. Synthesis of Fluorescein-Labeled SPG (F-SPG)a

EXPERIMENTAL PROCEDURES Materials and Complex Formation. SPG (Mw ) 1.5 × 105, Mw/Mn ) 1.47) was supplied by Mitsui Sugar Co. Ltd. (Tokyo, Japan), where Mw and Mn are the weight- and number-average molecular weights, determined with gel permeation chromatography. The sequence for CpG DNA was 5′-TCC ATG ACG TTC CTG ATG CT-A60-3′, denoted by CpG-dA60 (6, 9). This sequence had a poly(dA)60 tail attached at the 3′ end in order to form a stable complex with SPG. CpG-dA60 was synthesized by FASMAC Co., Ltd. (Kanagawa, Japan), and purified with high-performance liquid chromatography. We used phosphorothioate types for CpG-dA60 owing to the strong binding ability with SPG and good cellular uptake. 5′-Fluorescein-labeled CpGdA60 (F-CpG) and Rhodamine red-X(RRX)-labeled CpG (RCpG) were used for binding and uptake to cells, and fluorescence microscopic observation, respectively. For sample preparation, a 3.5 mg/mL solution of CpG-dA60, an appropriate amount of SPG solution (50 mg/mL as a stock solution, 0.25 N NaOHaq), and a phosphate buffer solution (pH ) 4.0, 330 mM NaH2PO4) were mixed. The mixture (the CpG-dA60 concentration, 5 µM; pH ) 7.4) was stored at 4 °C for 1 day to lead to the complexation. By changing the mixing ratio, [mG]/[dA] was controlled to 0.5-30. Synthesis of Biotin-Labeled and Fluorescein-Labeled SPG. Selective 1,2-diol oxidation of the SPG side chain, subsequent reaction between 2-aminoethanol and the SPG formyl terminate, and synthesis of biotin-labeled SPG (B-SPG) were carried out with the methods described in the previous paper (18–20). For synthesis of fluorescein-labeled SPG (FSPG, Scheme 1), amine-induced SPG (100 mg) and fluorescein4-isothiocyanate (FITC-I, 100 mg, Dojindo Lab., Kumamoto, Japan) were dissolved in DMSO (100 mL). After 3 days with stirring under N2, the product was dialyzed in a NaHCO3-NaOH buffer solution (pH ) 9.0, 200 mM) with a dialysis membrane (a cutoff molecular weight of (1.2-1.4) × 104, Sanko Junyaku, Tokyo, Japan). Freeze-drying treatment gave F-SPG as a yellow powder (95 mg). 1H NMR (500 MHz, 10 mg/mL, DMSO-d6, 25 °C): δ ) 10.25 (br, 1H), 8.15 (br, 1H), 8.05 (br, 1H, ArH), 7.25 (br, 1H, ArH), 7.19 (br, 1H, ArH), 6.71 (s, 2H, ArH), 6.60 (m, 4H, ArH). The modification rate was 20 mol %, determined with an F-4500 fluorescence spectrometer (Hitachi, Tokyo, Japan), and F-SPG maintained its ability of complex formation (Supporting Information).

a (i) NaIO4, H2O, 4 °C, 3 days; (ii) 2-aminoethanol, DMSO, rt, 2 days, then NaBH4, DMSO, rt, 1 day; (iii) fluorescein-4-isothiocyanate (FITC), DMSO, rt, 3 days.

Cell Culture. HEK 293 cells and the stable Dectin-1 transfectants (d-HEK) were prepared by N. Ohno and Y. Adachi (21) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Wako, Osaka, Japan) supplemented with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel) and 1 wt % penicillin and streptomycin mixture (Invitrogen, Carlsbad, CA) and 1 wt % Geneticin (for d-HEK, G418, Promega, Madison, WI). The cells were incubated at 37 °C in humidified air containing 5% of CO2. All animal experiments were performed according to the guidelines of Kyushu Institute of Technology. Male C57BL/6 mice (7 weeks of age) were purchased from Kyudou Co. Ltd. (Kumamoto, Japan). The mice (7-11 weeks of age) received an intraperitoneal (i.p.) injection with 3 mL of thioglycollate medium (3%, Nissui Pharmaceutical, Tokyo, Japan), according to the standard procedure (22). After 3 days, the elicited peritoneal macrophages were harvested by peritoneal lavage using 6 mL of RPMI-1640 medium (containing 10% FBS and 1 wt % penicillin and streptomycin mixture). The lavage fluids were pooled and centrifuged at 1000 rpm for 5 min. The cell pellet was washed three times with 10 mL of the medium, and the cells were seeded into plates and allowed to adhere for 5 h. To remove nonadherent cells, the cells were washed and then were incubated for 24 h. Circular Dichroism Spectroscopy (CD) and Gel Shift Assay. The circular dichroism (CD) in 185-305 nm regions was measured on a Jasco J-820 spectropolarimeter (JASCO, Tokyo, Japan) at 5 °C using a 1 cm quartz cell with a water jacket. Gel electrophoresis was carried out on a 12% polyacrylamide gel in a TBE buffer solution (Nippon Gene, Toyama,

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Figure 2. Circular dichroism (CD) spectral changes with increase of the SPG molar ratio in the complexes ([mG]/[dA] ) 0, 0.5, 1.0, 2.0, 10, 30) (A), and composition dependence of the CD intensity at 218 nm (B).

Japan) for 60 min at 100 V, and DNA was visualized by UV illumination after SYBR Gold staining (Invitrogen, Carlsbad, CA). Dectin-1 Detection. For the detection of Dectin-1 in d-HEK, the cells were blocked at 4 °C with 1.5 µg/mL of 2.4G2 (antiFcγRII and III mAb; BD Pharmingen, San Diego, CA) before the addition of primary antibodies. Dectin-1 on the cell surface was detected using 2A11 mAb (rat IgG2b anti-βGR; Serotec, Oxford, UK). Alexa 488-labeled goat antirat IgG (Invitrogen) was used as secondary Abs to detect primary Abs. The stained cells were analyzed using an EPICS XL Flow Cytometer (Beckman Coulter, Fullerton, CA). Binding and Uptake of the Complex. In the binding assay for HEK and d-HEK, each cell (5 × 105 cells in 100 µL of DMEM)wasincubatedwithB-SPG(stainedwithstreptavidin-Alexa 488 conjugates later) or F-CpG for 0.5 h at 4 °C and then washed twice with PBS. The fluorescence intensity from the cells with the streptavidin-Alexa 488 or F-CpG was measured with an EPICS XL Flow Cytometer (Beckman Coulter). For the binding and uptake in peritoneal macrophages, the cells were seeded in a 96-well plate at a cell density of 1 × 105 cells/well and incubated for 24 h at 37 °C, and then, F-CpG was added at 0.5 µM to each well. After the cells were incubated for 0.5 h at 4 °C (for binding) or 37 °C (for uptake) and then washed twice with PBS, the fluorescence intensity from the cell lysate was detected using a microplate reader (Wallac 1420 ARVO MX, Perkin-Elmer, Waltham, MA, USA). Fluorescence Microscopy and Enzyme-Linked Immunosorbent Assay (ELISA). For microscopic observation, peritoneal macrophages were seeded on a 35 mm/glass base dish (Iwaki, Chiba, Japan) at a cell density of 1 × 105 cells/ dish and incubated for 48 h at 37 °C. R-CpG was added at 0.5 µM, and then the cells were incubated for 0.5 h and fixed with 5% HCHO (PBS) in the presence of DAPI (Dojindo Lab., Kumamoto, Japan) at 4 °C for 20 min. The fluorescent images were captured using a BZ-9000 digital fluorescence microscope (Keyence, Osaka, Japan). For ELISA, peritoneal macrophages were cultured in a 96well plate at a cell density of 1 × 105 cells/well and incubated for 24 h at 37 °C, and CpG-dA60 were added at 0.5 µM to each well for 24 h. After supernatants were collected, IL-12 production was measured by ELISA using commercially available kits (mouse total IL-12, Thermo Fisher Scientific Inc., Rockford, IL).

RESULTS AND DISCUSSION Complex Formation between CpG and SPG. Figure 2A shows the CD spectral changes of CpG-dA60 when SPG was added in the range of [mG]/[dA] ) 0-30 at a fixed CpG concentration ([CpG-dA60] ) 0.83 µM or [dA] ) 50 µM). Here,

Figure 3. Complexation of CpG-dA60 and SPG confirmed by 12% polyacrylamide gel with SYBR Gold stain. [CpG-dA60] indicates the number of CpG-dA60 molecules included in one complex that is calculated from [mG]/[dA]. The upper illustrations show how the SPG and CpG-dA60 molecules exist in the solutions; at [mG]/[dA] < 2, there is free CpG-dA60 and the complex is fully loaded with CpG-dA60, while at [mG]/[dA] > 2, there is uncomplexed SPG and the complex has fewer CpG-dA60.

[mG] and [dA] mean the molar concentration of the main-chain glucose of SPG and the dA base of CpG-dA60, respectively. With increasing [mG]/[dA], the positive band at 218 nm increased and the negative band at 250 nm disappeared. These spectral changes can be ascribed to the conformational change of the SPG-bound dA tail (14). Figure 2B plots the CD intensity at 218 nm against [mG]/[dA], where the increment seems to be saturated at [mG]/[dA] ) 2.0. This value agrees with the stoichiometric proportion previously reported for SPG/poly(dA) complex (12, 13). On the basis of this fact, we can conclude that only the dA moiety of CpG-dA60 binds to SPG, and the CpG moiety does not bind to SPG and might be hanging around from the complex, as represented in Figure 1. Figure 3 compares polyacrylamide gel electrophoresis patterns between CpG-dA60 and its mixtures with SPG. With increase of [mG]/[dA], the naked CpG band faded away and the band of the complex appeared. At [mG]/[dA] ) 2.0, the naked band almost became invisible, and at [mG]/[dA] ) 5.0, it disappeared completely. At the higher molar ratio (in lanes 6-8), the complex bands become weaker than those of lanes 4-5. The ratio of complexation determined by luminance contrast was 56% in lane 2, 78% in lane 3, 96% in lane 4, and 100% in lanes 5-8, respectively, confirming the results of the CD measurements. With GPC-MALS (multiangle light scattering coupled to size exclusion chromatography), we determined the weight-average molecular weight (Mw) of the complex to be 2.5 × 105 (Supporting Information). Since Mw of SPG as the single chain is 1.5 × 105, one complex contains approximately 11 CpGdA60 molecules at [mG]/[dA] ) 2.0 (the stoichiometric propor-

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Figure 4. Binding ability of SPG and its complex for d-HEK. B-SPG (0, 3, 30, and 300 µg/mL) was administrated to d-HEK (A) and HEK (B) at 4 °C. After incubation for 0.5 h, the cells were stained with streptavidin-Alexa 488. (C) Dectin-1 binding of F-CpG and its complex (CpG; 0.5 µM, [mG]/[dA] ) 5) in d-HEK after incubation for 0.5 h at 4 °C.

tion). Above this composition, with increase of [mG]/[dA], the number of CpG-dA60 in a single complex should decrease. Binding with Dectin-1-Transduced Cells (d-HEK). The interaction between SPG and Dectin-1 was investigated by flow cytometric analysis in HEK 293 cells (HEK) and the Dectin-1 transfectants (d-HEK) at 4 °C. The fluorescence intensity in d-HEK drastically increased with increase of the B-SPG concentration (Figure 4A). Here, since ATP-dependent uptake should not occur at 4 °C, the added B-SPG was presumably absorbed by the receptor and remained on the cellular surface. Contrary to d-HEK, HEK did not show any binding of B-SPG even at 300 µg/mL. In addition, we carried out a competitive binding assay with unmodified SPG or other polysaccharides

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such as amylase, dextran, and pullulan and found that only unmodified SPG suppressed the binding of B-SPG to Dectin-1 (date not shown), consistent with previous results (15). These results indicated that SPG specifically binds to Dectin-1 in a dose-dependent manner. Figure 4C shows flow cytometry profiles of d-HEK with naked F-CpG and its complex ([mG]/ [dA] ) 5). This result shows that the complex has more affinity to d-HEK than the naked CpG-dA60, and this difference should be ascribed to binding of the complex to Dectin-1. These results lead to the conclusion that the SPG/CpG-dA60 complex can be recognized by Dectin-1. Binding and Cellular Uptake with Murine Peritoneal Macrophages. Peritoneal injection of thioglycollate induces a dramatic increase in the total number of peritoneal macrophages. Unlike resident macrophages, these macrophages are activated and thus expected to express Dectin-1 (22). Figure 5A shows the flow cytometric analysis, confirming that most of macrophages are expressing Dectin-1. For these macrophages, we applied F-CpG in various [mG]/[dA] compositions at the same CpG-dA60 concentration. After cell incubation for 0.5 h at 4 and 37 °C, the fluorescence intensity from the cells was measured (Figure 5B). Since receptor-mediated uptake is considerably suppressed at 4 °C, only the ligand-receptor binding at the cellular surface should be observed; namely, the gray bars in the figure can represent the amount of F-CpG bound on the surface. On the other hand, receptor-mediated uptake is active at 37 °C. Therefore, the difference between the black and gray bars can be related to the net amount of introduced CpG-dA60. At [mG]/[dA] ) 0, meaning the naked CpG-dA60, some amount of CpG-dA60 was taken up due to the potential of phosphorothioate DNA (23). With increasing [mG]/[dA], the fluorescence intensity at 37 °C increased and reached the highest level around [mG]/[dA] ) 2-5. The intensity was almost 2-fold higher than that of naked CpG-dA60. This result can be interpreted by the complexed CpG-dA60 being taken up through Dectin-1. In addition, in the range [mG]/[dA] > 2 (SPG rich compositions), the intensity slightly decreases. This can be explained by there being free SPG molecules in the composition and these free SPG competing in the binding with the complex. Fluorescence Microscopic Observation of the Complex. Figure 6 shows the fluorescence microscopic images of the peritoneal macrophages when the complex ([mG]/[dA] ) 5.0) prepared from R-CpG and F-SPG was applied. CpG-dA60 and SPG were distributed heterogeneously within the cytosol in a spotty fashion, which indicate localization of CpG-dA60 and SPG

Figure 5. Dectin-1 expression and binding and uptake of F-CpG for murine peritoneal macrophages. (A) The macrophages were unstained (upper) and stained (bottom) with anti-Dectin-1 antibody. (B) The F-CpG/SPG complex with different composition (CpG; 0.5 µM) was administered to the macrophages at 4 °C (gray bars) and at 37 °C (black bars) for 0.5 h (n ) 3).

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Figure 6. Fluorescence microscopic images for the murine macrophages after R-CpG/F-SPG complex (CpG; 0.5 µM, [mG]/[dA] ) 5) was applied for 0.5 h and the cells stained with DAPI. The scale bar shows 15 um.

Figure 7. The complex composition dependence (CpG; 0.5 µM) of IL-12 production from the murine peritoneal macrophages after incubation for 24 h (n ) 3). The abbreviation (ND) means “not detectable”.

in vesicles, presumably in endosome and lysosome. In addition, the yellow color (red/green merge) evidence that CpG-dA60 and SPG located in the same vesicles. This co-localization phenomenon was not observed in the uncomplexed mixture of R-CpG and F-SPG (Supporting Information). These results indicate that the complex was transported into the endosomal compartments. IL-12 Production Due to Administration of the Complex. Peritoneal macrophages express TLR9 to induce CpG DNA-mediated IL-12 (24). Accordingly, the complexes with various compositions were applied to the activated peritoneal macrophages, and the amount of secreted IL-12 was measured with ELISA (Figure 7). Here, [CpG-dA60] was fixed at 0.5 µM and we confirmed that IL-12 production from the cell did not

reach the maximum (Supporting Information). The naked CpGdA60 produced low levels of IL-12 (0.8 ng/mL) in macrophages, and this value was consistent with the other group’s results (24). In the range of [mG]/[dA] ) 0-1.0, the production slightly increased with increase of the amount of SPG, and dramatically enhanced at [mG]/[dA] ) 2. At [mG]/[dA] ) 5, the complex induced about 7-fold (5.2 ng/mL) higher production than that of naked CpG-dA60, and the production decreased with further increase of amount of SPG. Interestingly, between [mG]/[dA] ) 1 and 5, there is a significant difference in the IL-12 production from cells, while the amount of CpG-dA60 taken up by the cells differed only slightly among them, as represented in Figure 5. In addition, this drastic increase did not occur when the mixture of CpG-dA60 and SPG, but not the complex, was treated (Supporting Information). When multiple CpG DNAs are conjugated on one substrate and this substrate is taken up by macrophages, the cytokine production is dramatically (or allosterically) induced (25–28). This is explained by the observation that the multiple DNAs increase local concentrations of CpG sequences in endolysosomes, and subsequently, TLR9 recognition of CpG DNA and further signals are enhanced. The allostericity is probably related to the fact that dimerization of TLR9 is necessary for its activation (5). In addition, our recent study has shown that the flexibility of the polymer containing CpG DNA is a key to induce the large amount of IL-12 (29). Once one CpG branch binds to one TLR9 dimer, the polymer is locked adjacently to the vesicle surface where other TLR9 dimers are present. When the structure is flexible enough to turn, the second binding can easily occur, and the adjacent second binding seems to be critical

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for the allosteric effect. At [mG]/[dA] < 2 in Figure 7, the complex is considered to have the maximum number of CpGdA60 due to the DNA-rich composition. On the other hand, at [mG]/[dA] > 2, most of the complexes have fewer CpG-dA60 than that at [mG]/[dA] < 2 due to the SPG-rich composition. With increasing [mG]/[dA], the flexibility of the complex is considered to increase. The complex with rigid structure could be difficult to access the adjacent TLR9 dimers, while the complex with flexible structure could access them easily. Therefore, the cytokine secretion drastically increased at [mG]/ [dA] > 2 because of the easy accessibility of the complex to TLR9 dimers. In conclusion, we characterized the molecular architecture of SPG/CpG-dA with CD and gel electrophoresis, and showed that the dA tail binds to SPG and the CpG moieties are hanging around from the complex. Dectin-1 expressed on the activated peritoneal macrophages can recognize the complex and induce the uptake. The Dectin-1 recognition reached the maximum at the stoichiometric composition ([mG]/[dA] ) 2), while IL-12 production reached the maximum at an SPG-rich composition ([mG]/[dA] ) 5). These results suggested the existence of a particular higher-order structure to activate TLR9 more efficiently. The present work shows a novel strategy to deliver CpG DNA to immune cells by use of β-1,3-glucan/DNA complex.

ACKNOWLEDGMENT This work was supported by Grant-in-Aid for JSPS Fellows (19 · 11715) and the fund (07-16) from the National Institute of Biomedical Innovation, Japan. Supporting Information Available: The electrophoresis pattern to examine the complexation ability of F-SPG, the weight-average molecular weight (Mw) of the complex determined by GPC, fluorescence microscopic observation for the uncomplexed mixture of R-CpG and F-SPG, dose dependence of CpG-dA60 and the complex in IL-12 production, and the comparison of induced IL-12 between the mixture and the complex. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Janeway, C. A., Jr., and Medzhitov, R. (2002) Innate immune recognition. Annu. ReV. Immunol. 20, 197–216. (2) Takeda, K., Kaisho, T., and Akira, S. (2003) Toll-like receptors. Annu. ReV. Immunol. 21, 335–76. (3) Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–5. (4) Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K. A., Monks, B. G., Knetter, C. F., Lien, E., Nilsen, N. J., Espevik, T., and Golenbock, D. T. (2004) TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. Immunol. 5, 190–8. (5) Latz, E., Verma, A., Visintin, A., Gong, M., Sirois, C. M., Klein, D. C., Monks, B. G., McKnight, C. J., Lamphier, M. S., Duprex, W. P., Espevik, T., and Golenbock, D. T. (2007) Ligandinduced conformational changes allosterically activate Toll-like receptor 9. Nat. Immunol. 8, 772–9. (6) Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A., and Klinman, D. M. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546–9. (7) Krieg, A. M. (2002) CpG motifs in bacterial DNA and their immune effects. Annu. ReV. Immunol. 20, 709–60. (8) Krieg, A. M. (2006) Therapeutic potential of Toll-like receptor 9 activation. Nat. ReV. Drug DiscoVery 5, 471–84.

Minari et al. (9) Mizu, M., Koumoto, K., Anada, T., Matsumoto, T., Numata, M., Shinkai, S., Nagasaki, T., and Sakurai, K. (2004) A polysaccharide carrier for immunostimulatory CpG DNAs to enhance cytokine secretion. J. Am. Chem. Soc. 126, 8372–3. (10) Sakurai, K., Uezu, K., Numata, M., Hasegawa, T., Li, C., Kaneko, K., and Shinkai, S. (2005) Beta-1,3-glucan polysaccharides as novel one-dimensional hosts for DNA/RNA, conjugated polymers and nanoparticles. Chem. Commun. (Camb.) 4383–98. (11) Bluhm, T. L., Deslandes, Y., Marchessault, R. H., Perez, S., and Rinaudo, M. (1982) Solid-state and solution conformation of scleroglucan. Carbohydr. Res. 100, 117–30. (12) Sakurai, K., and Shinkai, S. (2000) Molecular recognition of adenine, cytosine, and uracil in a single-stranded RNA by a natural polysaccharide: schizophyllan. J. Am. Chem. Soc. 122, 4520–4521. (13) Sakurai, K., Mizu, M., and Shinkai, S. (2001) Polysaccharide-polynucleotide complexes. 2. Complementary polynucleotide mimic behavior of the natural polysaccharide schizophyllan in the macromolecular complex with single-stranded RNA and DNA. Biomacromolecules 2, 641–50. (14) Mizu, M., Kimura, T., Koumoto, K., Sakurai, K., and Shinkai, S. (2001) Thermally induced conformational transition of polydeoxyadenosine in the complex with schizophyllan and the base-length dependence of its stability. Chem. Commun. 429–430. (15) Brown, G. D., and Gordon, S. (2001) Immune recognition. A new receptor for beta-glucans. Nature 413, 36–7. (16) Brown, G. D. (2006) Dectin-1: a signalling non-TLR patternrecognition receptor. Nat. ReV. Immunol. 6, 33–43. (17) Shimada, N., Coban, C., Takeda, Y., Mizu, M., Minari, J., Anada, T., Torii, Y., Shinkai, S., Akira, S., Ishii, K. J., and Sakurai, K. (2007) A polysaccharide carrier to effectively deliver native phosphodiester CpG DNA to antigen-presenting cells. Bioconjugate Chem. 18, 1280–6. (18) Matsumoto, T., Numata, M., Anada, T., Mizu, M., Koumoto, K., Sakurai, K., Nagasaki, T., and Shinkai, S. (2004) Chemically modified polysaccharide schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake efficiency. Biochim. Biophys. Acta 1670, 91–104. (19) Minari, J., Kubo, T., Ohba, H., Shimada, N., Takeda, Y., Karinaga, R., Anada, T., Koumoto, K., Kawazu, T., Nagasaki, T., Shinkai, S., and Sakurai, K. (2007) Delivery of antisense oligonucleotides to nuclear telomere RNA by use of a complex between polysaccharide and polynucleotide. Bull. Chem. Soc. Jpn. 80, 1091–98. (20) Koumoto, K., Kimura, T., Mizu, M., Sakurai, K., and Shinkai, S. (2001) Chemical modification of schizophyllan by introduction of a cationic charge into the side chain which enhances the thermal stability of schizophyllan-poly(C) complexes. Chem. Commun. (Camb.) 1962–3. (21) Adachi, Y., Ishii, T., Ikeda, Y., Hoshino, A., Tamura, H., Aketagawa, J., Tanaka, S., and Ohno, N. (2004) Characterization of beta-glucan recognition site on C-type lectin, dectin 1. Infect. Immun. 72, 4159–71. (22) Willment, J. A., Lin, H. H., Reid, D. M., Taylor, P. R., Williams, D. L., Wong, S. Y., Gordon, S., and Brown, G. D. (2003) Dectin-1 expression and function are enhanced on alternatively activated and GM-CSF-treated macrophages and are negatively regulated by IL-10, dexamethasone, and lipopolysaccharide. J. Immunol. 171, 4569–73. (23) Sester, D. P., Naik, S., Beasley, S. J., Hume, D. A., and Stacey, K. J. (2000) Phosphorothioate backbone modification modulates macrophage activation by CpG DNA. J. Immunol. 165, 4165–73. (24) Suzuki, K., Suda, T., Naito, T., Ide, K., Chida, K., and Nakamura, H. (2005) Impaired toll-like receptor 9 expression in alveolar macrophages with no sensitivity to CpG DNA. Am. J. Respir. Crit. Care Med. 171, 707–13. (25) Marshall, J. D., Hessel, E. M., Gregorio, J., Abbate, C., Yee, P., Chu, M., Van Nest, G., Coffman, R. L., and Fearon, K. L. (2003) Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells. Nucleic Acids Res. 31, 5122–33.

Enhanced Cytokine Due to a CpG/β-1,3-Glucan Complex (26) Wu, C. C., Lee, J., Raz, E., Corr, M., and Carson, D. A. (2004) Necessity of oligonucleotide aggregation for toll-like receptor 9 activation. J. Biol. Chem. 279, 33071–8. (27) Kerkmann, M., Costa, L. T., Richter, C., Rothenfusser, S., Battiany, J., Hornung, V., Johnson, J., Englert, S., Ketterer, T., Heckl, W., Thalhammer, S., Endres, S., and Hartmann, G. (2005) Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells. J. Biol. Chem. 280, 8086–93.

Bioconjugate Chem., Vol. 22, No. 1, 2011 15 (28) Rattanakiat, S., Nishikawa, M., Funabashi, H., Luo, D., and Takakura, Y. (2009) The assembly of a short linear natural cytosine-phosphate-guanine DNA into dendritic structures and its effect on immunostimulatory activity. Biomaterials 30, 5701– 6. (29) Minari, J., Mochizuki, S., and Sakurai, K. (2008) Enhanced cytokine secretion owing to multiple CpG side chains of DNA duplex. Oligonucleotides 18, 337–44. BC1001196