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Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice
Free Radical Biology & Medicine 51 (2011) 424–431
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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice Hiroyuki Yoshida ⁎, Makiya Nishikawa, Tsuyoshi Kiyota, Hiroyasu Toyota, Yoshinobu Takakura Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho 46–29, Sakyo-ku, Kyoto 606–8501, Japan
a r t i c l e
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Article history: Received 5 February 2011 Revised 4 April 2011 Accepted 19 April 2011 Available online 22 April 2011 Keywords: 8-Hydroxydeoxyguanosine containing DNA CpG DNA TLR9 Inflammatory response
a b s t r a c t Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases. © 2011 Elsevier Inc. All rights reserved.
Introduction Progressive joint destruction with chronic inflammation is one of the chief complaints of patients with rheumatoid arthritis (RA), a disease that affects 1% of the population worldwide. Its complete pathogenic mechanism still remains to be elucidated, but the involvement of DNA in the pathogenesis has been suggested. In the inflamed joints, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) are continuously released from dead cells and the concentration of mtDNA in the joint cavity and circulation of RA patients is higher than that of healthy subjects [1]. In addition to mtDNA, various viral or bacterial DNA were also detected in the joints of arthritis patients [2–4]. mtDNA and viral/bacterial DNA contain many unmethylated CpG dinucleotides (CpG motifs), which are a known activator of dendritic
Abbreviations: CpG motif, unmethylated CpG dinucleotide; TLR, Toll-like receptor; LPS, lipopolysaccharide; ROS, reactive oxygen species; RA, rheumatoid arthritis; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; ODN, oligodeoxynucleotide; PO, phosphodiester; PS, phosphorothioate; bp, base pair; oxo-dGTP, 8-hydroxydeoxyguanosine triphosphate; oxo-dG, 8-hydroxydeoxyguanosine; oxo-G, 8-hydroxyguanosine; oxo-Gua, 8-hydroxyguanine; oxo-DNA, oxo-dG-containing DNA; ds, double-stranded; ss, singlestranded; LA2000, Lipofectamine2000; lipoplex, plasmid DNA/LA2000 complex; MFI, mean fluorescence intensity; PAGE, polyacrylamide gel electrophoresis; MyD88, myeloid differentiation factor 88. ⁎ Corresponding author. Fax: + 81 75 753 4614. E-mail address: [email protected] (H. Yoshida). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.04.035
cells, B cells, and macrophages. Upon recognition by cells through Toll-like receptor-9 (TLR9), the specific receptor for CpG motif, such DNA induces the production of TNF-α, interleukin-6 (IL-6), and interferon-γ (IFN-γ) [5]. An intraarticular injection of CpG DNA induced synovial cytokine production and aggravated RA symptoms [6,7]. Accumulation of macrophages into inflamed sites was associated with these pathological changes [6,8,9]. Loss of homeostatic balance between reactive oxygen species (ROS) and antioxidants in the cellular milieu results in oxidative stress that causes oxidative DNA damages, including chemical modification of both pyrimidine and purine bases [10]. The most general marker for oxidative DNA is 8-hydroxydeoxyguanine (oxo-dG) and the level of which in plasma is known to increase in various diseases, such as Alzheimer's disease [11], Parkinson's disease [12], cystic fibrosis [13], systemic lupus erythematosus [14], and RA [15]. mtDNA is continuously exposed to more oxidative stress than nDNA because of its location close to the respiratory chain and lack of protective histones [16], which makes mtDNA, but not nDNA, a major component for oxidation [17]. Moreover, it has been reported that a rheumatoid joint is under high oxidative stress [18,19]. These pieces of evidence will explain the finding that the synovial fluid of RA patients contains large amounts of DNA, especially oxidized DNA [1]. Although there are multiple reports of oxo-dG as a biomarker of various diseases, few studies have evaluated the immunological aspects of oxo-dG on RA. Collins et al. investigated the involvement of
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oxo-dG in the aggravation of RA and demonstrated that intraarticular injection of oligodeoxynucleotide (ODN) containing oxo-dG into mouse knee joint provoked histopathological arthritis at a high frequency despite lacking a CpG motif in the ODN [20]. Accordingly, it has been postulated that oxo-dG or oxo-dG-containing DNA (oxoDNA) is immunologically active, but its detail has not been clarified yet. Therefore, in this study, we evaluated the immunological activity of oxo-DNA by measuring the production of proinflammatory cytokines in murine macrophages. Assuming the conditions in inflamed joints, oxo-DNA was added to cells in combination with CpG DNA. Finally, the induction of experimental arthritis was examined by subcutaneous injection of a mixture of CpG DNA and oxo-DNA into the mouse footpad. Materials and methods Chemicals RPMI 1640 medium was purchased from Nissui Pharmaceutical (Tokyo, Japan). Lipofectamine2000 (LA2000) and Opti-MEM were purchased from Invitrogen (Carlsbad, CA, USA). DNase I and 20-base pair (bp) DNA ladder were purchased from Takara Bio, Inc. (Otsu, Japan). DNase II, lipopolysaccharide (LPS), poly(I:C), and polymyxin B sulfate salt were purchased from Sigma (St. Louis, MO, USA). Recombinant murine IFN-γ was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA). Triton X-114 was purchased from Nacalai Tesque (Kyoto, Japan). Imiquimod was purchased from Imgenex (San Diego, CA, USA). DNA and oxo-DNA ODNs were purchased from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). The sequences of ODNs were as follows: CpG ODN1668, 5′-TCCATGACGTTCCTGATGCT-3′; non-CpG ODN1720, 5′TCCATGAGCTTCCTGATGCT-3′; and A-type ODN2216, 5′-gggggACGATCGTCgggggG-3′. Capital letters represent phosphodiester (PO) linkages 3′ of the base and lowercases represent phosphorothioate (PS) ones. B-type ODN1668, all internucleotide linkages of which were PS, was also prepared. Double-stranded (ds-) ODN was prepared by annealing each ODN with its fully complementary ODN, and doublestrand formation was confirmed by 21% polyacrylamide gel electrophoresis (PAGE). Oxo(G9)-1668 and oxo(G15)-1668, whose deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with 8-hydroxydeoxyguanosine (oxo-dG), were purchased from Nihon Gene Research Laboratories (Sendai, Japan). Similarly, oxo(G8)-1720 and oxo(G15)-1720 were obtained from Nihon Gene Research Laboratories. ODN1668 fluorescently labeled with Alexa488 was purchased from Nihon Bioservice (Saitama, Japan). ODN1668 fluorescently labeled with ATTO488 was purchased from Nihon Gene Research Laboratories. dGTP was purchased from Sigma. 8Hydroxy-dGTP (oxo-dGTP) was purchased from Jena Bioscience (Jena, Germany). Oxo-dG was purchased from Wako Pure Chemical (Osaka, Japan). 8-Hydroxyguanosine (oxo-G) and 8-hydroxyguanine (oxo-Gua) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Plasmid vector pCMV-Luc, a CpG motif replete circular ds-DNA, was constructed as previously reported [21]. pCMV-Luc has 33 Pur-Pur-CpG-Pyr-Pyr sequences including two GACGTT, a most potent CpG motif for mice [22]. pCpG-ΔLuc, another plasmid with no CpG motifs, was constructed as previously reported [23]. Plasmid DNA/LA2000 complex (lipoplex) was prepared at a ratio of 2 μl LA2000 and 1 μg plasmid DNA according to the manufacturer's instructions. Sample preparation To minimize the activation of cells by contaminated LPS, plasmid DNA samples were extensively purified with Triton X-114, a nonionic
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detergent, before use according to a previously published method [24]. The level of contaminated LPS was checked by a Limulus amebocyte lysate assay using the Limulus F Single Test kit (Wako Pure Chemical). The level of contaminated LPS was reduced to below the detection limit of 0.01 EU/μg DNA. For poly(I:C) and imiquimod, polymyxin B, which binds to LPS, was added to cells at a final concentration of 5 μg/ml. ODNs, nucleotides, and nucleosides were used as obtained without further purification or addition of polymyxin B. Animals TLR9 −/− mice were purchased from the Oriental Yeast Company (Tokyo, Japan). C57BL/6 wild-type mice and Institute for Cancer Research (ICR) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). All mice were maintained on a standard food and water diet under conventional housing conditions. All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Institutional Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences, Kyoto University. Cell cultures Splenic macrophages were collected as previously described [25] and cultured on 96-well culture plates at a density of 3 × 10 5 cells/well in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 μg/ml), L-glutamine (292 μg/ml), and 2-mercaptoethanol (10 -5 M). These cells were used for the cytokine release experiment soon after isolation. The murine macrophage-like cell line, RAW264.7 cells, was cultured on 96-well culture plates at a density of 5 × 10 4 cells/well in RPMI 1640 supplemented with 10% FBS, penicillin G (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (292 μg/ml). These cells were used after 24-h incubation. Cytokine release from macrophages RAW264.7 cells or splenic macrophages were incubated with ODNs, oxo-ODNs, oxo-dGTP, oxo-dG, oxo-G, oxo-Gua, dGTP, lipoplex, poly(I:C), LPS, or imiquimod for 8 h (TNF-α assay) or for 24 h (IL-6 assay), then the supernatants were collected for ELISA and kept at − 80 °C until use. LPS was used as a positive control in most experiments to check the viability and reactivity of cell preparations. In the case of splenic macrophages, 10 units/ml of IFN-γ was added to the culture medium to prime cells. In addition, 5 μg/ml polymyxin B was also added to avoid cell activation by LPS in the IFN-γ sample. Separately, RAW264.7 cells were incubated with lipoplex for 2 h, the cells were washed with RPMI 1640 and incubated with fresh growth medium for an additional 6 h, and then the supernatants were collected for ELISA and kept at − 80 °C until use. The level of TNF-α and IL-6 in the media was determined by ELISA using the OptEIA set (BD Biosciences, San Diego, CA, USA). Cellular uptake of ODN in RAW264.7 cells RAW264.7 cells were incubated with ATTO488-labeled ODN1668 or ATTO488-labeled oxo(G15)-1668 for 30, 90, or 240 min and washed three times with phosphate-buffered saline (PBS). Separately, cells were incubated with Alexa488-labeled ODN1668 with or without ODN1720 or oxo(G8)-1720 for 30, 90, or 240 min and washed three times with PBS. Then, the intensity of cell fluorescence was analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (version 3.1; BD Biosciences). Cellular uptake was estimated by subtracting the mean fluorescence intensity (MFI) at 4 °C from that at 37 °C (ΔMFI) and plotted against the incubation time.
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Degradation of ODN against DNase I and DNase II ODN1668 (0.5 μg/10 μl) or oxo(G15)-1668 (0.5 μg/10 μl) were incubated with DNase I (2 units/μg DNA) or DNase II (3 units/μg DNA) at 37 °C. After 0, 5, 10, 30, or 60 min of incubation, the mixture was placed on ice and the reaction was terminated by the addition of 3 μl 0.2 M EDTA solution per 10 μl of samples. The ODN samples were run on a 21% PAGE and stained with ethidium bromide. The image of the gel was recorded using LAS 3000 (Fujifilm Life Science, Tokyo, Japan) and analyzed using Multi Gauge software (Fujifilm Life Science). Subcutaneous injection of ODN into mouse footpad Three nanomoles of ODN1668 was subcutaneously injected into the right footpad of male ICR mice with or without 10 nmol of ODN1720 or oxo(G8)-1720 in 20 μl of PBS. To different mice, 10 nmol of oxo(G8)-1720 was subcutaneously injected in a similar manner. Before and 24 h after injection of ODN, the thickness of mouse footpad was measured using a micrometer caliper with a minimum scale of 10 μm (Mitutoyo, Kawasaki, Japan). Statistical analysis Differences in the cytokine release were statistically evaluated by Student's t test. Differences in the thickness of mouse footpad were statistically evaluated by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for multiple comparisons. A P value of less than 0.05 was considered to be statistically significant. Results
TNF-α production. No significant TNF-α was produced by the addition of ODN1720, oxo(G8)-1720, or oxo(G15)-1720. Effect of DNA or oxo-DNA on CpG DNA-induced TNF-α production in macrophages Next, we evaluated the effect of DNA or oxo-DNA containing no CpG motifs on CpG DNA-induced TNF-α production. The concentration of ODN1668 was set at a relatively low level of 1 μM in RAW264.7 cells or 3 μM in splenic macrophages to avoid the saturation of TNF-α production. The addition of ODN1720 hardly affected the ODN1668induced TNF-α production in RAW264.7 cells (Fig. 2A, left panel). In strong contrast, the addition of oxo(G8)-1720 or oxo(G15)-1720 significantly increased the ODN1668-induced TNF-α production (Fig. 2A, left panel). The addition of oxo(G8)-1720 to ODN1720 did not induce TNF-α production at all, suggesting that oxo-ODN1720 upregulates the CpG DNA-induced production of TNF-α. Similar results were obtained by replacing oxo-ODN1720 with oxo(G9)-1668, another oxo-DNA with no activity to induce TNF-α (Fig. 2A, right panel). Fig. 2B shows the concentration of TNF-α in supernatants of RAW264.7 cells after the addition of ds-DNA consisting of ODN1668 and its fully complementary ODN. ds-CpG-ODN1668-induced TNF-α production was increased by the addition of ds-oxo(G8)-1720. Then, similar experiments were carried out using primary cultured macrophages. Again, oxo(G8)-1720, but not ODN1720, increased CpG motif-dependent TNF-α (Fig. 2C, hatched bar) and IL-6 (Fig. 2D, hatched bar) production in splenic macrophages from wild-type mice. Addition of oxo(G8)-1720 and ODN1668 hardly induced cytokine production in macrophages from TLR9 knockout (KO) mice (Fig. 2C, white bar, and Fig. 2D, white bar). These results using primary macrophages strongly suggest that the oxo-DNA-induced increase in the cytokine production by ODN1668 is solely dependent on the TLR9.
TNF-α production in RAW264.7 cells by DNA or oxo-DNA Fig. 1 shows the concentration of TNF-α in supernatants of RAW264.7 cells after the addition of DNA or oxo-DNA. The addition of ODN1668 induced TNF-α production in a manner dependent on the concentration of the CpG DNA. Oxo(G15)-1668, which contains oxo-dG outside the CpG motif, induced a higher amount of TNF-α than ODN1668. On the other hand, oxo(G9)-1668, whose deoxyguanine in the CpG motif was replaced with oxo-dG, hardly induced
Effect of oxo-DNA on other TLR ligand-induced TNF-α production in RAW264.7 cells To examine whether the oxo-DNA-induced increase in TNF-α production is specific to ODN1668, various types of DNAs and TLR ligands were used instead of ODN1668, a ss-PO-CpG DNA that is not a commonly used CpG ODN. The following ligands were selected and used: A-type ODN2216, a CpG ODN with PS bonds at the both ends; Btype ODN1668, a PS-type CpG ODN having the same sequence as ODN1668; pCMV-Luc, a ds-circular DNA containing many CpG motifs; non-CpG lipoplex, a complex consisting of pCpG-ΔLuc and cationic liposomes, which was proved to be a ligand for cytosolic DNA receptors; poly(I:C), a ds-RNA and a ligand for TLR3; LPS, a ligand for TLR4; and imiquimod, a ligand for TLR7. When applied alone, each ligand induced significant TNF-α production in RAW264.7 cells (Fig. 3, white bar). The addition of ODN1720 hardly increased the ligandinduced TNF-α production in all cases examined. The addition of oxo (G8)-1720 significantly increased the TNF-α production induced by A-type ODN2216, B-type ODN1668, or pCMV-Luc, all of which are ligands for TLR9. In strong contrast, TNF-α production by non-CpG lipoplex, poly(I:C), LPS, or imiquimod was not significantly affected by oxo(G8)-1720. Effect of oxo-nucleotide on the CpG DNA-induced TNF-α production in RAW264.7 cells
Fig. 1. Inflammatory cytokine production induced by oxidized ODNs in RAW264.7 cells. The cells were incubated with ODN and oxo-ODN. After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. ND, not determined. ⁎⁎P b 0.01, significantly different from the ODN1668 only-treated cells. Each result represents the mean ± SD of triplicate values.
It has been reported that oxo-dG, a component of oxo-DNA, suppresses LPS-induced inflammatory cytokine production in murine brain microglia cells [26–28], suggesting that oxo-nucleotide has potency to alter TLR4 ligand-induced cytokine production. Therefore, in order to clarify whether the oxo-nucleotide affects CpG DNAinduced TNF-α production, oxo-dGTP, oxo-dG, oxo-G, or oxo-Gua was added with ODN1668 to RAW264.7 cells. The addition of oxo-dGTP,
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Fig. 2. Increase of CpG motif- and TLR9-dependent inflammatory cytokine production by oxidized ODNs in murine macrophages. (A) RAW264.7 cells were incubated with ODN1668 (1 μM) or ODN1720 (1 μM) in the presence of oxo-ODN1720 or oxo-ODN1668. (B) RAW264.7 cells were incubated with ds-ODN1668 (1 μM) in the presence of ds-oxo(G8)-1720. (C, D) Splenic macrophages from wild-type (hatched bar) or TLR9 KO (white bar) mice were incubated with ODN1668 (3 μM) in the presence or absence of intact ODN1720 (10 μM) or oxo-ODN1720 (10 μM). After an 8-h incubation for TNF-α assay or a 24-h incubation for IL-6 assay, the supernatants were collected, and the concentration of TNF-α or IL-6 was determined by ELISA. N.D., not detected. ⁎P b 0.05, ⁎⁎P b 0.01, significantly different from the ODN1668 combination with intact ODN1720-treated cells. Each result represents the mean ± SD of triplicate values.
but not oxo-dG, oxo-G, or oxo-Gua, increased the ODN1668-induced TNF-α production in a manner dependent on the concentration (Fig. 4A). To confirm the importance of the oxidation on the amplifying activity of oxo-dGTP, oxo-dGTP was replaced with dGTP. The addition of dGTP also increased the ODN1668-induced TNF-α production even though it has no oxidized components. This finding is in a good agreement with our previous results demonstrating that deoxynucleotides with 5′-phosphate, such as dGTP, have potency to increase CpG DNA-induced cytokine production independent of the type of nucleobase [29]. To prove whether oxo-dGTP functions as an oxo-DNA or as a deoxynucleotides with 5′-phosphate, its effect on PStype CpG ODN-induced TNF-α production was examined because the cytokine production was increased by oxo-DNA (Fig. 3A), but not by deoxynucleotides with 5′-phosphate [29]. The addition of oxo-dGTP or dGTP did not increase PS-type CpG ODN-induced TNF-α production (Fig. 4B). These results suggest that oxo-dGTP exhibits its activity as a deoxynucleotide with a 5′-phosphate, but not as oxo-DNA, and that oxo-DNA becomes inactive when degraded into nucleotides. Therefore, the length of DNA is an important factor for oxo-DNA to exert the amplifying activity. Cellular uptake and degradation of ODN1668 CpG DNA-induced cytokine production depends on the cellular uptake of CpG DNA. To examine whether the increase in TNF-α production by oxo-DNA is mediated by the increased uptake of
ODN1668 by cells, fluorescently labeled ODN1668 or oxo(G15)-ODN was added to RAW264.7 cells. The ΔMFI of cells added with the labeled ODNs was increased with time, indicating that these ODNs are taken up by cells. There was no significant differences between the ΔMFI of cells added with ODN1668 and that with oxo(G15)-1668 (Fig. 5A). Then, the effects of oxo-DNA on the uptake of coexisting ODN1668 were examined using ODN1720 and oxo(G8)-1720 (Fig. 5B). Neither ODN1720 nor oxo(G8)-1720 had significant effects on the cellular uptake of fluorescently labeled ODN1668. Then, the stability of ODN1668 and oxo(G15)-1668 against DNase I (Fig. 5C) and DNase II (Fig. 5D) was evaluated. No significant differences in the stability were observed among these ODNs. These results suggest that the increase in TNF-α production by oxo-DNA was not mediated by the increased stability of ODN1668 against DNase I or DNase II.
Footpad swelling by subcutaneous injection of ODN1668 The results obtained thus far suggest that oxo-DNA amplifies the CpG DNA-induced cytokine production in macrophages. To evaluate whether oxo-DNA functions in vivo, swelling of mouse footpad was measured after subcutaneous injection of ODNs into the footpad of the right hind leg of mice. Injection of ODN1668 alone, oxo(G8)-1720 alone, or ODN1668 combination with ODN1720 hardly increased the thickness of the footpads (Fig. 6). On the other hand, an injection of
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Fig. 3. Effect of oxo(G8)-1720 on other ligand-induced TNF-α production in RAW264.7 cells. The cells were incubated with A-type ODN2216 (0.1 μM), B-type ODN1668 (0.003 μM), pCMV-Luc (10 μg/ml), or non-CpG lipoplex (pCpG-ΔLuc:LA2000 = 5:10 μg/ml) (A), poly(I:C) (5 μg/ml), LPS (1 ng/ml), or imiquimod (0.3 μg/ml) (B) in the presence of intact ODN1720 or oxo(G8)-1720. After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. ⁎⁎P b 0.01, significantly different from the ligand combination with intact ODN1720-treated cells. Each result represents the mean ± SD of triplicate values.
ODN1668 together with oxo(G8)-1720 significantly increased the thickness compared with other groups. Discussion Defense system against ROS is less perfect in patients of autoimmune diseases [30], and various biomolecules, such as lowdensity lipoprotein, IgG, glutamic acid hydroxylase, and DNA, are oxidized to immunogenic products that play important roles in the diseases as an aggravating factor [31–33]. Despite the possible importance of oxidized DNA, its immunological activity in RA has not been fully evaluated. In this study, we have demonstrated that ODN containing oxo-dG increases the CpG motif-dependent TNF-α production in a TLR9-dependent manner in murine macrophages and mice. Some early studies suggested the involvement of oxo-DNA in inflammation. Collins et al. reported that an intraarticular injection of non-CpG oxo-DNA induces histological arthritis in mice [20], suggesting that oxo-DNA is an immunostimulatory compound even when it contains no CpG motifs. Because monocytes/macrophages, but not T cells, B cells or granulocytes, were reported to be involved in the DNA-induced aggravation of RA in mice [8,20], it could be reasonable that macrophages respond to oxo-DNA to induce arthritis or other inflammatory disorders. Despite this working hypothesis, we found that the addition of oxo(G8)-1720 or oxo(G15)-1720 induced
no significant TNF-α production in macrophages (Fig. 1). Furthermore, subcutaneous injection of oxo(G8)-1720 into mouse footpad did not induce significant inflammatory responses (Fig. 6). These results indicate that the non-CpG oxo-DNA itself is immunologically inert as non-CpG DNA. Discrepancy in the in vivo results of oxo-DNA between the present study and the Collins study [20] might be attributed to the difference in the level of CpG DNA present in the site of injection: the subcutaneous tissue and the articular space for the present and the previous studies, respectively. Because bacteria-derived DNA is often detected in the articular space of healthy subjects [34], it is feasible to detect bacteria-derived DNA in mouse synovial fluid. Therefore, it is possible that intraarticular injection of non-CpG oxo-ODN increases the immune activation induced by such CpG DNA. On the other hand, the level of CpG DNA in footpad should be very low, so that a simple injection of non-CpG oxo-ODN did not induce significant inflammation without additional CpG ODN. It was reported that the activity of CpG ODN to activate NF-κB was markedly decreased when dG in the CpG motif was substituted with oxo-dG [35]. In the present study, we confirmed this finding by showing that oxo(G9)-1668 hardly induced TNF-α production in macrophages (Fig. 1). On the other hand, oxo(G15)-1668 was highly immunostimulatory. These results clearly indicate the importance of the location of oxo-dG on the immunostimulatory activity of CpG DNA, and that TLR9 cannot recognize the oxo-dG-containing CpG
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Fig. 4. Effect of oxidized-DNA component on CpG DNA-dependent TNF-α production in RAW264.7 cells. (A) The cells were incubated with ODN1668 (1 μM) in the presence of oxodGTP, oxo-dG, oxo-G, oxo-Gua, or intact dGTP (20 or 60 μM). (B) The cells were incubated with B-type ODN1668 (0.003 μM) in the presence of dGTP or oxo-dGTP (20 or 60 μM). After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. Each result represents the mean ± SD of triplicate values.
motif as a danger signal. TLR9 protein directly binds to both CpG and GpC DNA [36], but the formation of a dimer of TLR9 accessible to the adaptor protein, myeloid differentiation factor 88 (MyD88), is conducted only by CpG DNA [37]. Therefore, one possible reason why oxo(G9)-1668 induced no TNF-α production was that oxo(G9)1668 bound to TLR9 but could not transduce the signaling cascade of TLR9 because of the failure of TLR9 to form a desired dimer. The higher immunostimulatory activity of oxo(G15)-1668 than that of ODN1668 clearly demonstrates the importance of increased oxidation of DNA in the pathogenesis of inflammatory diseases. In this study, ss-ODNs with 20 bases were used but DNA released from cells and/or pathogens should be much longer. The longer the DNA, the more likely it contains both CpG motifs and oxidized nucleotides.
Therefore, DNA released from cells under oxidative conditions is more likely to be recognized through TLR9 as damage-associated molecular pattern than that released under normal conditions. The mechanism for the increasing effect of oxo-DNA on the immunostimulatory activity of CpG DNA still remains unidentified, but the present study provides helpful information for considering the mechanism. CpG DNA induces cytokine production via a series of steps: cellular uptake, intracellular trafficking to endolysosomal compartment, recognition by TLR9, and signal transduction through the MyD88-dependent pathway. The first two steps, i.e., cellar uptake and stability, were found not to be involved in the mechanism (Fig. 5). The downstream signals of MyD88 are shared with TLR9 and other receptors, including TLR7 (Fig. 3B) [38], so that it is unlikely that oxo-
Fig. 5. Cellular uptake of ODN1668 in RAW264.7 cells (A, B) and degradation of ODN1668 by DNase I (C) or DNase II (D). (A) The cells were incubated with ATTO488-labeled ODN1668 or oxo(G15)-1668 (1 or 3 μM). (B) The cells were incubated with Alexa488-labeled ODN1668 (1 μM) in the presence of intact ODN1720 (3 μM) or oxo(G8)-1720 (3 μM). After 30, 90, and 240-min incubation at 4 or 37 °C, the amount of ODN1668 or oxo(G15)-1668 associated with cells was measured by flow cytometry. The ΔMFI is expressed as the mean ± SD of triplicate values. (C, D) ODN1668 or oxo(G15)-1668 was treated with DNase I (2 units/μg ODN) (C) or DNase II (3 units/μg ODN) (D) at 37 °C for the indicated time. The reaction was terminated by adding EDTA and ODNs (0.6 μg/lane) were run on 21% PAGE at 200 V for 90 min and stained with ethidium bromide. M, 20-bp DNA ladder.
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Fig. 6. Footpad swelling produced by subcutaneous injection of CpG DNA. ICR mice were subcutaneously injected with ODN1668 alone (3 nmol), oxo(G8)-1720 alone (10 nmol), or ODN1668 (3 nmol) in combination with intact ODN1720 (10 nmol) or oxo(G8)-1720 (10 nmol) into the footpad of the right hind leg. Before and 1, 2, and 3 days after injection of ODN, the footpad swelling of the right leg was measured. #P b 0.05, significantly different from the control mice. ⁎⁎P b 0.01, significantly different from all the other groups at Day 1. Each result represents the mean ± SD of four or five mice.
DNA affects the MyD88-dependent signaling pathway. Further studies are required to clarify the mechanism for the increase in the cytokine production by oxo-DNA. Previously, Chung and co-workers have reported that oxo-dG has an anti-inflammatory effect on LPS-induced inflammation via suppression of Rac1/STATs [26–28]. On the other hand, the addition of oxo-DNA did not alter the LPS-induced TNF-α production in RAW264.7 cells (Fig. 3B), suggesting that oxo-DNA acts differently from oxo-dG. Moreover, we showed that oxo-mononucleotide is not effective as far as the increasing effect on CpG DNA-induced cytokine production is concerned. With respect to the immunostimulatory activity of mononucleotides, we have recently reported that DNase I-degraded DNA, e.g., mono- or oligonucleotides, with 5′-phosphate increases PO-CpG DNA-induced immune response in murine macrophages and mice irrespective of the sequence of nucleotides [29]. Similar increasing effects were also observed with oxo-dGTP (Fig. 4A). Therefore, even after the oxo-DNA was degraded by DNase I in the circulation or articular space, it is possible that oxo-DNA would eventually increase the CpG DNA-induced immune responses. Therefore, oxo-DNA should be taken into consideration as an exacerbating factor for CpG DNA-related immune responses before and after degradation by DNase I. There was a significant difference between oxo(G8)-1720 and oxo (G15)-1720 in terms of activity to increase CpG ODN-induced TNF-α production (Fig. 2A). These results suggest that both the position of oxo-dG and the sequence of oxo-DNA affect the activity of oxo-DNA, which would be in good agreement with the finding that oxo-DNA exhibits its activity as oligonucleotides, not as mononucleotides (Fig. 4). The reason for the difference between oxo(G8)-1720 and oxo (G15)-1720, however, needs further investigation. The combination of oxo-ODN and PS-CpG ODN induced an extremely higher amount of TNF-α production than that of oxoODN and PO-CpG ODN (Fig. 3A). This would be simply explained by the fact that the PS-ODN is much more stable than PO-ODN. In the previous studies on experimental arthritis, PS-ODNs were used to induce the arthritis [7,8,39]. However, PS-ODN is a synthetic and nonnatural ODN, so that it should not be involved in DNA-associated autoimmune diseases, including RA. Although PS-CpG ODN should induce more production of inflammatory cytokines than PO-CpG ODN, it would not be involved in the pathophysiological conditions of patients. Therefore, PO-CpG ODN, not PS-CpG ODN, was used in animal studies to examine whether oxo-ODN aggravates the CpG ODN-induced inflammatory response. In conclusion, we have shown that the addition of oxo-DNA containing oxo-dG increases CpG DNA-induced inflammatory response in macrophages and mice. The precise mechanism of this increase still remains to be elucidated, but these findings give a new and important attention that oxo-DNA has an ability to exacerbate CpG DNA-related autoimmune diseases.
Acknowledgments This work was supported by the 21st Century COE Program “Knowledge Information Infrastructure for Genome Science” and by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences, and Technology, Japan. We thank Dr. Hiroyuki Yoshitomi (Graduate School of Medicine, Kyoto University) for experimental support on subcutaneous injection of ODN into the footpad of mice.
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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d
Original Contribution
Increase in CpG DNA-induced inflammatory responses by DNA oxidation in macrophages and mice Hiroyuki Yoshida ⁎, Makiya Nishikawa, Tsuyoshi Kiyota, Hiroyasu Toyota, Yoshinobu Takakura Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshidashimoadachi-cho 46–29, Sakyo-ku, Kyoto 606–8501, Japan
a r t i c l e
i n f o
Article history: Received 5 February 2011 Revised 4 April 2011 Accepted 19 April 2011 Available online 22 April 2011 Keywords: 8-Hydroxydeoxyguanosine containing DNA CpG DNA TLR9 Inflammatory response
a b s t r a c t Unmethylated CpG dinucleotide (CpG motif) is involved in the exacerbation of DNA-associated autoimmune diseases. We investigated the effect of DNA containing 8-hydroxydeoxyguanosine (oxo-dG), a representative DNA biomarker for oxidative stress in the diseases, on CpG motif-dependent inflammatory responses. ODN1668 and ODN1720 were selected as CpG-DNA and non-CpG DNA, respectively. Deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with oxo-dG to obtain oxo(G9)-1668 and oxo(G15)-1668, respectively. Oxo(G15)-1668 induced a significantly higher amount of tumor necrosis factor (TNF)-α from RAW264.7 macrophage-like cells than ODN1668, whereas oxo(G9)-1668, oxo(G8)-1720, or oxo(G15)-1720 hardly did. CpG DNA-induced TNF-α production was significantly increased by addition of oxo(G8)-1720 or oxo(G15)-1720, but not of ODN1720. This oxo-dG-containing DNA-induced increase in TNF-α production was also observed in primary cultured macrophages isolated from wild-type mice, but not observed in those from Toll-like receptor (TLR)-9 knockout mice. In addition, TNF-α production by ligands for TLR3, TLR4, or TLR7 was not affected by oxo-dG-containing DNA. Then, the footpad swelling induced by subcutaneous injection of ODN1668 into mice was increased by coinjection with oxo(G8)-1720, but not with ODN1720. These results indicate that oxo-dG-containing DNA increases the CpG motif-dependent inflammatory responses, which would exacerbate DNA-related autoimmune diseases. © 2011 Elsevier Inc. All rights reserved.
Introduction Progressive joint destruction with chronic inflammation is one of the chief complaints of patients with rheumatoid arthritis (RA), a disease that affects 1% of the population worldwide. Its complete pathogenic mechanism still remains to be elucidated, but the involvement of DNA in the pathogenesis has been suggested. In the inflamed joints, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) are continuously released from dead cells and the concentration of mtDNA in the joint cavity and circulation of RA patients is higher than that of healthy subjects [1]. In addition to mtDNA, various viral or bacterial DNA were also detected in the joints of arthritis patients [2–4]. mtDNA and viral/bacterial DNA contain many unmethylated CpG dinucleotides (CpG motifs), which are a known activator of dendritic
Abbreviations: CpG motif, unmethylated CpG dinucleotide; TLR, Toll-like receptor; LPS, lipopolysaccharide; ROS, reactive oxygen species; RA, rheumatoid arthritis; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; ODN, oligodeoxynucleotide; PO, phosphodiester; PS, phosphorothioate; bp, base pair; oxo-dGTP, 8-hydroxydeoxyguanosine triphosphate; oxo-dG, 8-hydroxydeoxyguanosine; oxo-G, 8-hydroxyguanosine; oxo-Gua, 8-hydroxyguanine; oxo-DNA, oxo-dG-containing DNA; ds, double-stranded; ss, singlestranded; LA2000, Lipofectamine2000; lipoplex, plasmid DNA/LA2000 complex; MFI, mean fluorescence intensity; PAGE, polyacrylamide gel electrophoresis; MyD88, myeloid differentiation factor 88. ⁎ Corresponding author. Fax: + 81 75 753 4614. E-mail address: [email protected] (H. Yoshida). 0891-5849/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2011.04.035
cells, B cells, and macrophages. Upon recognition by cells through Toll-like receptor-9 (TLR9), the specific receptor for CpG motif, such DNA induces the production of TNF-α, interleukin-6 (IL-6), and interferon-γ (IFN-γ) [5]. An intraarticular injection of CpG DNA induced synovial cytokine production and aggravated RA symptoms [6,7]. Accumulation of macrophages into inflamed sites was associated with these pathological changes [6,8,9]. Loss of homeostatic balance between reactive oxygen species (ROS) and antioxidants in the cellular milieu results in oxidative stress that causes oxidative DNA damages, including chemical modification of both pyrimidine and purine bases [10]. The most general marker for oxidative DNA is 8-hydroxydeoxyguanine (oxo-dG) and the level of which in plasma is known to increase in various diseases, such as Alzheimer's disease [11], Parkinson's disease [12], cystic fibrosis [13], systemic lupus erythematosus [14], and RA [15]. mtDNA is continuously exposed to more oxidative stress than nDNA because of its location close to the respiratory chain and lack of protective histones [16], which makes mtDNA, but not nDNA, a major component for oxidation [17]. Moreover, it has been reported that a rheumatoid joint is under high oxidative stress [18,19]. These pieces of evidence will explain the finding that the synovial fluid of RA patients contains large amounts of DNA, especially oxidized DNA [1]. Although there are multiple reports of oxo-dG as a biomarker of various diseases, few studies have evaluated the immunological aspects of oxo-dG on RA. Collins et al. investigated the involvement of
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oxo-dG in the aggravation of RA and demonstrated that intraarticular injection of oligodeoxynucleotide (ODN) containing oxo-dG into mouse knee joint provoked histopathological arthritis at a high frequency despite lacking a CpG motif in the ODN [20]. Accordingly, it has been postulated that oxo-dG or oxo-dG-containing DNA (oxoDNA) is immunologically active, but its detail has not been clarified yet. Therefore, in this study, we evaluated the immunological activity of oxo-DNA by measuring the production of proinflammatory cytokines in murine macrophages. Assuming the conditions in inflamed joints, oxo-DNA was added to cells in combination with CpG DNA. Finally, the induction of experimental arthritis was examined by subcutaneous injection of a mixture of CpG DNA and oxo-DNA into the mouse footpad. Materials and methods Chemicals RPMI 1640 medium was purchased from Nissui Pharmaceutical (Tokyo, Japan). Lipofectamine2000 (LA2000) and Opti-MEM were purchased from Invitrogen (Carlsbad, CA, USA). DNase I and 20-base pair (bp) DNA ladder were purchased from Takara Bio, Inc. (Otsu, Japan). DNase II, lipopolysaccharide (LPS), poly(I:C), and polymyxin B sulfate salt were purchased from Sigma (St. Louis, MO, USA). Recombinant murine IFN-γ was purchased from Pepro Tech Inc. (Rocky Hill, NJ, USA). Triton X-114 was purchased from Nacalai Tesque (Kyoto, Japan). Imiquimod was purchased from Imgenex (San Diego, CA, USA). DNA and oxo-DNA ODNs were purchased from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). The sequences of ODNs were as follows: CpG ODN1668, 5′-TCCATGACGTTCCTGATGCT-3′; non-CpG ODN1720, 5′TCCATGAGCTTCCTGATGCT-3′; and A-type ODN2216, 5′-gggggACGATCGTCgggggG-3′. Capital letters represent phosphodiester (PO) linkages 3′ of the base and lowercases represent phosphorothioate (PS) ones. B-type ODN1668, all internucleotide linkages of which were PS, was also prepared. Double-stranded (ds-) ODN was prepared by annealing each ODN with its fully complementary ODN, and doublestrand formation was confirmed by 21% polyacrylamide gel electrophoresis (PAGE). Oxo(G9)-1668 and oxo(G15)-1668, whose deoxyguanosine in the CpG motif (G9) or outside the motif (G15) of ODN1668 was substituted with 8-hydroxydeoxyguanosine (oxo-dG), were purchased from Nihon Gene Research Laboratories (Sendai, Japan). Similarly, oxo(G8)-1720 and oxo(G15)-1720 were obtained from Nihon Gene Research Laboratories. ODN1668 fluorescently labeled with Alexa488 was purchased from Nihon Bioservice (Saitama, Japan). ODN1668 fluorescently labeled with ATTO488 was purchased from Nihon Gene Research Laboratories. dGTP was purchased from Sigma. 8Hydroxy-dGTP (oxo-dGTP) was purchased from Jena Bioscience (Jena, Germany). Oxo-dG was purchased from Wako Pure Chemical (Osaka, Japan). 8-Hydroxyguanosine (oxo-G) and 8-hydroxyguanine (oxo-Gua) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Plasmid vector pCMV-Luc, a CpG motif replete circular ds-DNA, was constructed as previously reported [21]. pCMV-Luc has 33 Pur-Pur-CpG-Pyr-Pyr sequences including two GACGTT, a most potent CpG motif for mice [22]. pCpG-ΔLuc, another plasmid with no CpG motifs, was constructed as previously reported [23]. Plasmid DNA/LA2000 complex (lipoplex) was prepared at a ratio of 2 μl LA2000 and 1 μg plasmid DNA according to the manufacturer's instructions. Sample preparation To minimize the activation of cells by contaminated LPS, plasmid DNA samples were extensively purified with Triton X-114, a nonionic
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detergent, before use according to a previously published method [24]. The level of contaminated LPS was checked by a Limulus amebocyte lysate assay using the Limulus F Single Test kit (Wako Pure Chemical). The level of contaminated LPS was reduced to below the detection limit of 0.01 EU/μg DNA. For poly(I:C) and imiquimod, polymyxin B, which binds to LPS, was added to cells at a final concentration of 5 μg/ml. ODNs, nucleotides, and nucleosides were used as obtained without further purification or addition of polymyxin B. Animals TLR9 −/− mice were purchased from the Oriental Yeast Company (Tokyo, Japan). C57BL/6 wild-type mice and Institute for Cancer Research (ICR) mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). All mice were maintained on a standard food and water diet under conventional housing conditions. All animal experiments were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocols for animal experiments were approved by the Institutional Animal Experimentation Committee of the Graduate School of Pharmaceutical Sciences, Kyoto University. Cell cultures Splenic macrophages were collected as previously described [25] and cultured on 96-well culture plates at a density of 3 × 10 5 cells/well in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin (100 μg/ml), L-glutamine (292 μg/ml), and 2-mercaptoethanol (10 -5 M). These cells were used for the cytokine release experiment soon after isolation. The murine macrophage-like cell line, RAW264.7 cells, was cultured on 96-well culture plates at a density of 5 × 10 4 cells/well in RPMI 1640 supplemented with 10% FBS, penicillin G (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (292 μg/ml). These cells were used after 24-h incubation. Cytokine release from macrophages RAW264.7 cells or splenic macrophages were incubated with ODNs, oxo-ODNs, oxo-dGTP, oxo-dG, oxo-G, oxo-Gua, dGTP, lipoplex, poly(I:C), LPS, or imiquimod for 8 h (TNF-α assay) or for 24 h (IL-6 assay), then the supernatants were collected for ELISA and kept at − 80 °C until use. LPS was used as a positive control in most experiments to check the viability and reactivity of cell preparations. In the case of splenic macrophages, 10 units/ml of IFN-γ was added to the culture medium to prime cells. In addition, 5 μg/ml polymyxin B was also added to avoid cell activation by LPS in the IFN-γ sample. Separately, RAW264.7 cells were incubated with lipoplex for 2 h, the cells were washed with RPMI 1640 and incubated with fresh growth medium for an additional 6 h, and then the supernatants were collected for ELISA and kept at − 80 °C until use. The level of TNF-α and IL-6 in the media was determined by ELISA using the OptEIA set (BD Biosciences, San Diego, CA, USA). Cellular uptake of ODN in RAW264.7 cells RAW264.7 cells were incubated with ATTO488-labeled ODN1668 or ATTO488-labeled oxo(G15)-1668 for 30, 90, or 240 min and washed three times with phosphate-buffered saline (PBS). Separately, cells were incubated with Alexa488-labeled ODN1668 with or without ODN1720 or oxo(G8)-1720 for 30, 90, or 240 min and washed three times with PBS. Then, the intensity of cell fluorescence was analyzed by flow cytometry (FACScan; BD Biosciences) using CellQuest software (version 3.1; BD Biosciences). Cellular uptake was estimated by subtracting the mean fluorescence intensity (MFI) at 4 °C from that at 37 °C (ΔMFI) and plotted against the incubation time.
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Degradation of ODN against DNase I and DNase II ODN1668 (0.5 μg/10 μl) or oxo(G15)-1668 (0.5 μg/10 μl) were incubated with DNase I (2 units/μg DNA) or DNase II (3 units/μg DNA) at 37 °C. After 0, 5, 10, 30, or 60 min of incubation, the mixture was placed on ice and the reaction was terminated by the addition of 3 μl 0.2 M EDTA solution per 10 μl of samples. The ODN samples were run on a 21% PAGE and stained with ethidium bromide. The image of the gel was recorded using LAS 3000 (Fujifilm Life Science, Tokyo, Japan) and analyzed using Multi Gauge software (Fujifilm Life Science). Subcutaneous injection of ODN into mouse footpad Three nanomoles of ODN1668 was subcutaneously injected into the right footpad of male ICR mice with or without 10 nmol of ODN1720 or oxo(G8)-1720 in 20 μl of PBS. To different mice, 10 nmol of oxo(G8)-1720 was subcutaneously injected in a similar manner. Before and 24 h after injection of ODN, the thickness of mouse footpad was measured using a micrometer caliper with a minimum scale of 10 μm (Mitutoyo, Kawasaki, Japan). Statistical analysis Differences in the cytokine release were statistically evaluated by Student's t test. Differences in the thickness of mouse footpad were statistically evaluated by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for multiple comparisons. A P value of less than 0.05 was considered to be statistically significant. Results
TNF-α production. No significant TNF-α was produced by the addition of ODN1720, oxo(G8)-1720, or oxo(G15)-1720. Effect of DNA or oxo-DNA on CpG DNA-induced TNF-α production in macrophages Next, we evaluated the effect of DNA or oxo-DNA containing no CpG motifs on CpG DNA-induced TNF-α production. The concentration of ODN1668 was set at a relatively low level of 1 μM in RAW264.7 cells or 3 μM in splenic macrophages to avoid the saturation of TNF-α production. The addition of ODN1720 hardly affected the ODN1668induced TNF-α production in RAW264.7 cells (Fig. 2A, left panel). In strong contrast, the addition of oxo(G8)-1720 or oxo(G15)-1720 significantly increased the ODN1668-induced TNF-α production (Fig. 2A, left panel). The addition of oxo(G8)-1720 to ODN1720 did not induce TNF-α production at all, suggesting that oxo-ODN1720 upregulates the CpG DNA-induced production of TNF-α. Similar results were obtained by replacing oxo-ODN1720 with oxo(G9)-1668, another oxo-DNA with no activity to induce TNF-α (Fig. 2A, right panel). Fig. 2B shows the concentration of TNF-α in supernatants of RAW264.7 cells after the addition of ds-DNA consisting of ODN1668 and its fully complementary ODN. ds-CpG-ODN1668-induced TNF-α production was increased by the addition of ds-oxo(G8)-1720. Then, similar experiments were carried out using primary cultured macrophages. Again, oxo(G8)-1720, but not ODN1720, increased CpG motif-dependent TNF-α (Fig. 2C, hatched bar) and IL-6 (Fig. 2D, hatched bar) production in splenic macrophages from wild-type mice. Addition of oxo(G8)-1720 and ODN1668 hardly induced cytokine production in macrophages from TLR9 knockout (KO) mice (Fig. 2C, white bar, and Fig. 2D, white bar). These results using primary macrophages strongly suggest that the oxo-DNA-induced increase in the cytokine production by ODN1668 is solely dependent on the TLR9.
TNF-α production in RAW264.7 cells by DNA or oxo-DNA Fig. 1 shows the concentration of TNF-α in supernatants of RAW264.7 cells after the addition of DNA or oxo-DNA. The addition of ODN1668 induced TNF-α production in a manner dependent on the concentration of the CpG DNA. Oxo(G15)-1668, which contains oxo-dG outside the CpG motif, induced a higher amount of TNF-α than ODN1668. On the other hand, oxo(G9)-1668, whose deoxyguanine in the CpG motif was replaced with oxo-dG, hardly induced
Effect of oxo-DNA on other TLR ligand-induced TNF-α production in RAW264.7 cells To examine whether the oxo-DNA-induced increase in TNF-α production is specific to ODN1668, various types of DNAs and TLR ligands were used instead of ODN1668, a ss-PO-CpG DNA that is not a commonly used CpG ODN. The following ligands were selected and used: A-type ODN2216, a CpG ODN with PS bonds at the both ends; Btype ODN1668, a PS-type CpG ODN having the same sequence as ODN1668; pCMV-Luc, a ds-circular DNA containing many CpG motifs; non-CpG lipoplex, a complex consisting of pCpG-ΔLuc and cationic liposomes, which was proved to be a ligand for cytosolic DNA receptors; poly(I:C), a ds-RNA and a ligand for TLR3; LPS, a ligand for TLR4; and imiquimod, a ligand for TLR7. When applied alone, each ligand induced significant TNF-α production in RAW264.7 cells (Fig. 3, white bar). The addition of ODN1720 hardly increased the ligandinduced TNF-α production in all cases examined. The addition of oxo (G8)-1720 significantly increased the TNF-α production induced by A-type ODN2216, B-type ODN1668, or pCMV-Luc, all of which are ligands for TLR9. In strong contrast, TNF-α production by non-CpG lipoplex, poly(I:C), LPS, or imiquimod was not significantly affected by oxo(G8)-1720. Effect of oxo-nucleotide on the CpG DNA-induced TNF-α production in RAW264.7 cells
Fig. 1. Inflammatory cytokine production induced by oxidized ODNs in RAW264.7 cells. The cells were incubated with ODN and oxo-ODN. After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. ND, not determined. ⁎⁎P b 0.01, significantly different from the ODN1668 only-treated cells. Each result represents the mean ± SD of triplicate values.
It has been reported that oxo-dG, a component of oxo-DNA, suppresses LPS-induced inflammatory cytokine production in murine brain microglia cells [26–28], suggesting that oxo-nucleotide has potency to alter TLR4 ligand-induced cytokine production. Therefore, in order to clarify whether the oxo-nucleotide affects CpG DNAinduced TNF-α production, oxo-dGTP, oxo-dG, oxo-G, or oxo-Gua was added with ODN1668 to RAW264.7 cells. The addition of oxo-dGTP,
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Fig. 2. Increase of CpG motif- and TLR9-dependent inflammatory cytokine production by oxidized ODNs in murine macrophages. (A) RAW264.7 cells were incubated with ODN1668 (1 μM) or ODN1720 (1 μM) in the presence of oxo-ODN1720 or oxo-ODN1668. (B) RAW264.7 cells were incubated with ds-ODN1668 (1 μM) in the presence of ds-oxo(G8)-1720. (C, D) Splenic macrophages from wild-type (hatched bar) or TLR9 KO (white bar) mice were incubated with ODN1668 (3 μM) in the presence or absence of intact ODN1720 (10 μM) or oxo-ODN1720 (10 μM). After an 8-h incubation for TNF-α assay or a 24-h incubation for IL-6 assay, the supernatants were collected, and the concentration of TNF-α or IL-6 was determined by ELISA. N.D., not detected. ⁎P b 0.05, ⁎⁎P b 0.01, significantly different from the ODN1668 combination with intact ODN1720-treated cells. Each result represents the mean ± SD of triplicate values.
but not oxo-dG, oxo-G, or oxo-Gua, increased the ODN1668-induced TNF-α production in a manner dependent on the concentration (Fig. 4A). To confirm the importance of the oxidation on the amplifying activity of oxo-dGTP, oxo-dGTP was replaced with dGTP. The addition of dGTP also increased the ODN1668-induced TNF-α production even though it has no oxidized components. This finding is in a good agreement with our previous results demonstrating that deoxynucleotides with 5′-phosphate, such as dGTP, have potency to increase CpG DNA-induced cytokine production independent of the type of nucleobase [29]. To prove whether oxo-dGTP functions as an oxo-DNA or as a deoxynucleotides with 5′-phosphate, its effect on PStype CpG ODN-induced TNF-α production was examined because the cytokine production was increased by oxo-DNA (Fig. 3A), but not by deoxynucleotides with 5′-phosphate [29]. The addition of oxo-dGTP or dGTP did not increase PS-type CpG ODN-induced TNF-α production (Fig. 4B). These results suggest that oxo-dGTP exhibits its activity as a deoxynucleotide with a 5′-phosphate, but not as oxo-DNA, and that oxo-DNA becomes inactive when degraded into nucleotides. Therefore, the length of DNA is an important factor for oxo-DNA to exert the amplifying activity. Cellular uptake and degradation of ODN1668 CpG DNA-induced cytokine production depends on the cellular uptake of CpG DNA. To examine whether the increase in TNF-α production by oxo-DNA is mediated by the increased uptake of
ODN1668 by cells, fluorescently labeled ODN1668 or oxo(G15)-ODN was added to RAW264.7 cells. The ΔMFI of cells added with the labeled ODNs was increased with time, indicating that these ODNs are taken up by cells. There was no significant differences between the ΔMFI of cells added with ODN1668 and that with oxo(G15)-1668 (Fig. 5A). Then, the effects of oxo-DNA on the uptake of coexisting ODN1668 were examined using ODN1720 and oxo(G8)-1720 (Fig. 5B). Neither ODN1720 nor oxo(G8)-1720 had significant effects on the cellular uptake of fluorescently labeled ODN1668. Then, the stability of ODN1668 and oxo(G15)-1668 against DNase I (Fig. 5C) and DNase II (Fig. 5D) was evaluated. No significant differences in the stability were observed among these ODNs. These results suggest that the increase in TNF-α production by oxo-DNA was not mediated by the increased stability of ODN1668 against DNase I or DNase II.
Footpad swelling by subcutaneous injection of ODN1668 The results obtained thus far suggest that oxo-DNA amplifies the CpG DNA-induced cytokine production in macrophages. To evaluate whether oxo-DNA functions in vivo, swelling of mouse footpad was measured after subcutaneous injection of ODNs into the footpad of the right hind leg of mice. Injection of ODN1668 alone, oxo(G8)-1720 alone, or ODN1668 combination with ODN1720 hardly increased the thickness of the footpads (Fig. 6). On the other hand, an injection of
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Fig. 3. Effect of oxo(G8)-1720 on other ligand-induced TNF-α production in RAW264.7 cells. The cells were incubated with A-type ODN2216 (0.1 μM), B-type ODN1668 (0.003 μM), pCMV-Luc (10 μg/ml), or non-CpG lipoplex (pCpG-ΔLuc:LA2000 = 5:10 μg/ml) (A), poly(I:C) (5 μg/ml), LPS (1 ng/ml), or imiquimod (0.3 μg/ml) (B) in the presence of intact ODN1720 or oxo(G8)-1720. After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. ⁎⁎P b 0.01, significantly different from the ligand combination with intact ODN1720-treated cells. Each result represents the mean ± SD of triplicate values.
ODN1668 together with oxo(G8)-1720 significantly increased the thickness compared with other groups. Discussion Defense system against ROS is less perfect in patients of autoimmune diseases [30], and various biomolecules, such as lowdensity lipoprotein, IgG, glutamic acid hydroxylase, and DNA, are oxidized to immunogenic products that play important roles in the diseases as an aggravating factor [31–33]. Despite the possible importance of oxidized DNA, its immunological activity in RA has not been fully evaluated. In this study, we have demonstrated that ODN containing oxo-dG increases the CpG motif-dependent TNF-α production in a TLR9-dependent manner in murine macrophages and mice. Some early studies suggested the involvement of oxo-DNA in inflammation. Collins et al. reported that an intraarticular injection of non-CpG oxo-DNA induces histological arthritis in mice [20], suggesting that oxo-DNA is an immunostimulatory compound even when it contains no CpG motifs. Because monocytes/macrophages, but not T cells, B cells or granulocytes, were reported to be involved in the DNA-induced aggravation of RA in mice [8,20], it could be reasonable that macrophages respond to oxo-DNA to induce arthritis or other inflammatory disorders. Despite this working hypothesis, we found that the addition of oxo(G8)-1720 or oxo(G15)-1720 induced
no significant TNF-α production in macrophages (Fig. 1). Furthermore, subcutaneous injection of oxo(G8)-1720 into mouse footpad did not induce significant inflammatory responses (Fig. 6). These results indicate that the non-CpG oxo-DNA itself is immunologically inert as non-CpG DNA. Discrepancy in the in vivo results of oxo-DNA between the present study and the Collins study [20] might be attributed to the difference in the level of CpG DNA present in the site of injection: the subcutaneous tissue and the articular space for the present and the previous studies, respectively. Because bacteria-derived DNA is often detected in the articular space of healthy subjects [34], it is feasible to detect bacteria-derived DNA in mouse synovial fluid. Therefore, it is possible that intraarticular injection of non-CpG oxo-ODN increases the immune activation induced by such CpG DNA. On the other hand, the level of CpG DNA in footpad should be very low, so that a simple injection of non-CpG oxo-ODN did not induce significant inflammation without additional CpG ODN. It was reported that the activity of CpG ODN to activate NF-κB was markedly decreased when dG in the CpG motif was substituted with oxo-dG [35]. In the present study, we confirmed this finding by showing that oxo(G9)-1668 hardly induced TNF-α production in macrophages (Fig. 1). On the other hand, oxo(G15)-1668 was highly immunostimulatory. These results clearly indicate the importance of the location of oxo-dG on the immunostimulatory activity of CpG DNA, and that TLR9 cannot recognize the oxo-dG-containing CpG
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Fig. 4. Effect of oxidized-DNA component on CpG DNA-dependent TNF-α production in RAW264.7 cells. (A) The cells were incubated with ODN1668 (1 μM) in the presence of oxodGTP, oxo-dG, oxo-G, oxo-Gua, or intact dGTP (20 or 60 μM). (B) The cells were incubated with B-type ODN1668 (0.003 μM) in the presence of dGTP or oxo-dGTP (20 or 60 μM). After an 8-h incubation, the supernatants were collected, and the concentration of TNF-α was determined by ELISA. Each result represents the mean ± SD of triplicate values.
motif as a danger signal. TLR9 protein directly binds to both CpG and GpC DNA [36], but the formation of a dimer of TLR9 accessible to the adaptor protein, myeloid differentiation factor 88 (MyD88), is conducted only by CpG DNA [37]. Therefore, one possible reason why oxo(G9)-1668 induced no TNF-α production was that oxo(G9)1668 bound to TLR9 but could not transduce the signaling cascade of TLR9 because of the failure of TLR9 to form a desired dimer. The higher immunostimulatory activity of oxo(G15)-1668 than that of ODN1668 clearly demonstrates the importance of increased oxidation of DNA in the pathogenesis of inflammatory diseases. In this study, ss-ODNs with 20 bases were used but DNA released from cells and/or pathogens should be much longer. The longer the DNA, the more likely it contains both CpG motifs and oxidized nucleotides.
Therefore, DNA released from cells under oxidative conditions is more likely to be recognized through TLR9 as damage-associated molecular pattern than that released under normal conditions. The mechanism for the increasing effect of oxo-DNA on the immunostimulatory activity of CpG DNA still remains unidentified, but the present study provides helpful information for considering the mechanism. CpG DNA induces cytokine production via a series of steps: cellular uptake, intracellular trafficking to endolysosomal compartment, recognition by TLR9, and signal transduction through the MyD88-dependent pathway. The first two steps, i.e., cellar uptake and stability, were found not to be involved in the mechanism (Fig. 5). The downstream signals of MyD88 are shared with TLR9 and other receptors, including TLR7 (Fig. 3B) [38], so that it is unlikely that oxo-
Fig. 5. Cellular uptake of ODN1668 in RAW264.7 cells (A, B) and degradation of ODN1668 by DNase I (C) or DNase II (D). (A) The cells were incubated with ATTO488-labeled ODN1668 or oxo(G15)-1668 (1 or 3 μM). (B) The cells were incubated with Alexa488-labeled ODN1668 (1 μM) in the presence of intact ODN1720 (3 μM) or oxo(G8)-1720 (3 μM). After 30, 90, and 240-min incubation at 4 or 37 °C, the amount of ODN1668 or oxo(G15)-1668 associated with cells was measured by flow cytometry. The ΔMFI is expressed as the mean ± SD of triplicate values. (C, D) ODN1668 or oxo(G15)-1668 was treated with DNase I (2 units/μg ODN) (C) or DNase II (3 units/μg ODN) (D) at 37 °C for the indicated time. The reaction was terminated by adding EDTA and ODNs (0.6 μg/lane) were run on 21% PAGE at 200 V for 90 min and stained with ethidium bromide. M, 20-bp DNA ladder.
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Fig. 6. Footpad swelling produced by subcutaneous injection of CpG DNA. ICR mice were subcutaneously injected with ODN1668 alone (3 nmol), oxo(G8)-1720 alone (10 nmol), or ODN1668 (3 nmol) in combination with intact ODN1720 (10 nmol) or oxo(G8)-1720 (10 nmol) into the footpad of the right hind leg. Before and 1, 2, and 3 days after injection of ODN, the footpad swelling of the right leg was measured. #P b 0.05, significantly different from the control mice. ⁎⁎P b 0.01, significantly different from all the other groups at Day 1. Each result represents the mean ± SD of four or five mice.
DNA affects the MyD88-dependent signaling pathway. Further studies are required to clarify the mechanism for the increase in the cytokine production by oxo-DNA. Previously, Chung and co-workers have reported that oxo-dG has an anti-inflammatory effect on LPS-induced inflammation via suppression of Rac1/STATs [26–28]. On the other hand, the addition of oxo-DNA did not alter the LPS-induced TNF-α production in RAW264.7 cells (Fig. 3B), suggesting that oxo-DNA acts differently from oxo-dG. Moreover, we showed that oxo-mononucleotide is not effective as far as the increasing effect on CpG DNA-induced cytokine production is concerned. With respect to the immunostimulatory activity of mononucleotides, we have recently reported that DNase I-degraded DNA, e.g., mono- or oligonucleotides, with 5′-phosphate increases PO-CpG DNA-induced immune response in murine macrophages and mice irrespective of the sequence of nucleotides [29]. Similar increasing effects were also observed with oxo-dGTP (Fig. 4A). Therefore, even after the oxo-DNA was degraded by DNase I in the circulation or articular space, it is possible that oxo-DNA would eventually increase the CpG DNA-induced immune responses. Therefore, oxo-DNA should be taken into consideration as an exacerbating factor for CpG DNA-related immune responses before and after degradation by DNase I. There was a significant difference between oxo(G8)-1720 and oxo (G15)-1720 in terms of activity to increase CpG ODN-induced TNF-α production (Fig. 2A). These results suggest that both the position of oxo-dG and the sequence of oxo-DNA affect the activity of oxo-DNA, which would be in good agreement with the finding that oxo-DNA exhibits its activity as oligonucleotides, not as mononucleotides (Fig. 4). The reason for the difference between oxo(G8)-1720 and oxo (G15)-1720, however, needs further investigation. The combination of oxo-ODN and PS-CpG ODN induced an extremely higher amount of TNF-α production than that of oxoODN and PO-CpG ODN (Fig. 3A). This would be simply explained by the fact that the PS-ODN is much more stable than PO-ODN. In the previous studies on experimental arthritis, PS-ODNs were used to induce the arthritis [7,8,39]. However, PS-ODN is a synthetic and nonnatural ODN, so that it should not be involved in DNA-associated autoimmune diseases, including RA. Although PS-CpG ODN should induce more production of inflammatory cytokines than PO-CpG ODN, it would not be involved in the pathophysiological conditions of patients. Therefore, PO-CpG ODN, not PS-CpG ODN, was used in animal studies to examine whether oxo-ODN aggravates the CpG ODN-induced inflammatory response. In conclusion, we have shown that the addition of oxo-DNA containing oxo-dG increases CpG DNA-induced inflammatory response in macrophages and mice. The precise mechanism of this increase still remains to be elucidated, but these findings give a new and important attention that oxo-DNA has an ability to exacerbate CpG DNA-related autoimmune diseases.
Acknowledgments This work was supported by the 21st Century COE Program “Knowledge Information Infrastructure for Genome Science” and by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Sciences, and Technology, Japan. We thank Dr. Hiroyuki Yoshitomi (Graduate School of Medicine, Kyoto University) for experimental support on subcutaneous injection of ODN into the footpad of mice.
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