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Identification of Immunostimulatory DNA-Induced Genes by Suppression Subtractive Hybridization
Biochemical and Biophysical Research Communications 286, 688 – 691 (2001) doi:10.1006/bbrc.2001.5453, available online at http://www.idealibrary.com on
Identification of Immunostimulatory DNA-Induced Genes by Suppression Subtractive Hybridization Masato Uchijima,* ,1 Eyal Raz,† Dennis A. Carson,† Toshi Nagata,* and Yukio Koide* *Department of Microbiology and Immunology, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan; and †Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093
Received July 27, 2001
Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80 –90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODNstimulated bone marrow-derived macrophages: NF-B p105, IRF-1, PA28, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms. © 2001 Academic Press Key Words: CpG; SSH; BMDM; NF-B p105; IRF-1; IRG2; MyD88; PA28.
Innate immunity enables the mammalian host to respond rapidly to invading microorganisms and provides a rapid defense and, at the same time, alters the adaptive immune responses to the pathogens. The innate immune system uses receptors which are capable of recognizing a microbial pathogen by the pathogenassociated molecular patterns (PAMPs) it displays (1). Abbreviations used: BMDM, bone marrow-derived macrophage; IRF-1, IFN regulatory factor-1; ISS-ODN, immunostimulatory oligodeoxyribo-nucleotides; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; SSH, suppression subtractive hybridization; TLR, Toll-like receptor. 1 To whom correspondence should be addressed. Fax:⫹81-53-4352335. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Two members of the Toll-like receptor (TLR) family have been demonstrated to participate in the recognition. TLR4 and TLR2 are responsible for immune responses to lipopolysaccharide (LPS) and peptidoglycan, respectively (2). Furthermore, TLR9 was recently demonstrated to be involved in the recognition of a specific pattern in bacterial DNA, consisting of an unmethylated pair of nucleotides, cytosine and guanosine (CpG), within the context of certain flanking regions (3). CpG dinucleotides are present at the expected frequency in bacterial DNA but are underrepresented (1/50 –1/60) in vertebrate DNA, where they are usually methylated (4). These differences between bacterial and vertebrate DNA are suggested to be recognized by the vertebrate immune system as a PAMP. Similar effects of those induced by bacterial DNA can be obtained with synthetic immunostimulatory oligodeoxyribonucleotides (ISS-ODN) (5). Bacterial DNA and ISS-ODN can induce macrophages, monocytes, and dendritic cells to secrete proinflammatory cytokines such as IL-6, IL-12, and TNF-␣ (6), and up-regulate MHC class II, CD40, and CD86 expressions (7). To further characterize molecular consequences of the recognition of a specific pattern in bacterial DNA, we used a sensitive PCR-based subtraction approach, termed suppression subtractive hybridization (SSH) (8), to isolate genes upregulated in ISS-ODN-stimulated murine spleen cells. MATERIALS AND METHODS Oligonucleotides. Endotoxin-free phosphorothioate ODN (Trilink, San Diego, CA) were used in all experiments. The sequences of the ODNs used in this study are as follows: ISS-ODN, 5⬘-TGACTGTGAACGTTCGAGATGA-3⬘; Control ODN, 5⬘-TGACTGTGAACCTTAGAGATGA-3⬘. The underline indicates the immunostimulatory DNA sequence. Primers: IL-6 forward, 5⬘-TATGAAGTTCCTCTCTGCAA-3⬘; IL-6 reverse, 5⬘-CTTTGTATCTCTGGAAGTTT-3⬘; NF- B p105 forward, 5⬘-CACCATGGCAGACGATGATC-3⬘; NF-B p105 reverse, 5⬘-TAGGCAAGGTCAGAATGCAC-3⬘; IRF-1 forward, 5⬘-CACCA-
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TGCCAATCACTCGAA-3⬘; IRF-1 reverse, 5⬘-AGCCCTGAGTGGTGTAACTG-3⬘; Proteasome activator (PA) 28 forward, 5⬘-ATGGCCAAGCCTTGTGGGGT-3⬘; PA 28 reverse, 5⬘-TGGAAGCCTTGGCTACATCA-3⬘; IGR2 forward, 5⬘-CACCATCATGAGTGAGGTCA-3⬘; IGR2 reverse, 5⬘-TGGAAGCCTTGGCTACATCA-3⬘; MyD88 forward, 5⬘-TAAGCAGCAGAACCAGGAGT-3⬘; MyD88 reverse, 5⬘-CATCTTCAGGGCAGGGACAA-3⬘; G3PDH forward, 5⬘-ACCACAGTCCATGCCATCAC-3⬘; G3PDH reverse, 5⬘- TCCACCACCCTGTTGCTGTA-3⬘. Cell culture and bone marrow-derived macrophages (BMDMs). Female BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Murine spleen cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS (Gemini Bioproduct, Calabasus, CA), 2 mM L-glutamine, 2 mM penicillin/streptomycin (Bio Whittaker, Walkersville, MD) and 50 M 2-mercaptoethanol. BMDMs were prepared and cultured as described previously (7). ⫹
Preparation of poly(A) RNA. Total cellular RNA was isolated from spleen cells treated with ISS-ODN, or control ODN, using a RNA isolation kit (Trizol; Gibco BRL, Rockville, MD) according to the manufacturer’s instruction. Poly(A) ⫹ RNA was prepared using Oligotex mRNA kit (Quiagen, Valencia, CA). Generation of subtracted cDNA library by SSH. SSH to generate subtracted cDNA libraries was performed between ISS-ODNstimulated cells (tester) and control ODN-stimulated cells (driver), essentially as described previously (8), using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA). Briefly, The tester poly(A) ⫹ RNA was synthesized from the mixture of murine spleen cells treated with ISS-ODN (10 g/ml) for 1, 3, and 6 h at 37°C. The driver poly(A) ⫹ RNA was prepared from murine spleen cells stimulated with control ODN (10 g/ml) in the same way. Double-stranded cDNA was synthesized from tester and driver poly(A) ⫹ RNAs, and digested with RsaI to obtain shorter blunt-ended cDNA. Two different adaptors, adaptor 1 and adaptor 2, were then ligated to 5⬘-end of each strand of tester cDNA. The adaptor 1-ligated and adaptor 2-ligated tester cDNAs were separately hybridized at 68°C for 8 h with an excess of driver cDNA after denaturation at 98°C for 90 s. The two hybridization samples were then mixed together without denaturation and hybridized at 68°C overnight with excess of driver cDNA. The resulting mixture was diluted to 1:50 and amplified by two PCRs. The primary PCR was performed with flanking primers against adaptor 1 and adaptor 2 and the amplified products were used as a template in secondary PCR with nested primers. All hybridization steps and PCR were performed on Gene Amp 9600 PCR system (Applied Biosystems, Foster City, CA). Products from the second PCR were inserted directly into pT-Adv vector (Clontech). Sequencing and database search of the subtracted cDNA. The subtracted cDNA library was transformed into an Escherichia coli strain XL-1 blue, which were then plated onto agar plates containing ampicillin, X-gal, and IPTG (Sigma). Randomly selected 87 white colonies were grown in Luria-Bertani medium with ampicillin at 37°C, from which plasmids were prepared by Ultra-Clean Miniprep kit (Mo Bio, Solana Beach, CA) and then sequenced using BigDye Terminator Cycle Sequence kit (Applied Biosystems) on an ABI 377 automated sequencer (Applied Biosystems). The program Blast was used to compare sequences of the isolated clones with all sequences present in the National Center for Biotechnology Information (NCBI) nonredundant nucleic acid database (http://www.ncbi.nlm. gov/BLAST/). Semi-quantitative reverse transcriptase (RT)-PCR. RT-PCR was performed as described previously (9). Briefly, BMDMs were stimulated with 10 g/ml of ISS-ODN or control ODN for 1, 3, and 6 h at 37°C and mixed together. Total RNA was prepared from the cells by using Trizol (Gibco BRL). Single-stranded cDNA was synthesized with SuperScript II reverse transcriptase (Gibco BRL) and then used for PCRs. The primers used were described above.
FIG. 1. Analysis of subtraction procedure efficiency. PCRs with IL-6 and G3PDH primers were performed on tester (unsubtracted) and subtracted cDNAs for 28 cycles (95°C for 60 s, 55°C for 60 s, and 72°C for 60 s). PCR products were electrophoresed on 8% polyacrylamide gels.
RESULTS AND DISCUSSION To identify ISS-ODN-immediate response genes, the tester cDNA was generated from a cell mixture of splenocytes stimulated with ISS-ODN for 1, 3, and 6 h at 37°C. First of all, the efficiency of the SSH was evaluated by PCR with two primer sets specific for IL-6 and a house-keeping gene, G3PDH, since ISS (CpG)ODN has been well known to induce IL-6 gene expression in spleen cells (10). As shown in Fig. 1, both IL-6 and G3PDH PCR products were able to be detected in the unsubtracted tester cDNA. In the subtracted cDNA, however, G3PDH PCR product was not detected and IL-6 PCR product was increased compared with the tester cDNA under the same PCR condition, suggesting that we achieved a high level of enrichment of ISS-ODN-induced cDNAs and a drastic reduction of highly abundant cDNAs. Sequence analysis of 87 clones isolated by SSH revealed that 39 clones were found to correspond exactly to known mouse mRNAs in the BLAST program (Table 1). Eleven clones were revealed to have 80 –90% homology with known mouse mRNAs. The remaining 37 clones showed no significant homology with any known sequences in the public databases. We also examined redundancy of our SSH repertoire. Eleven out of 87 clones analyzed were recovered twice (Table 1) and no clones appeared more than twice. The average fragment insert size was 459 bp, which is in close agreement with that statistically predicted by RsaI (⬇600 bp). Using reverse Northern blotting, we estimated that approximately 15% of clones recovered by SSH showed upregulated signals (data not shown). This finding is in accord with the previous findings which showed that 12.5% (11) and 11% (12) of clones recovered by SSH exhibited a differential signal. For further target validation, we used semiquantitative RT-PCR. BMDMs were employed in the
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Summary of Sequences and Clones Represented in the ISS Specific cDNA Library Match category
No. of clones represented (%)
Double hit
Exact mouse match Nonexact mouse match No data base match
39 (44.8) 11 (12.6) 37 (42.5)
7 3 1
Total
87 (100)
11
experiment to confirm the genes upregulated in ISSODN-stimulated macrophages. We confirmed overexpression of NF-B p105 (13, 14), IFN regulatory factor-1 (IRF-1) (15), PA28 (16), IRG2 (IFN-IP) (17), and MyD88 (18), which have not previously been reported as overexpressed in the ISS-ODN-stimulated cells (Fig. 2). The mRNA expressions for NF-B p105, IRF-1, IRG2 (IFN-IP), and MyD88 were detected in BMDMs as early as 1 h after stimulation with ISSODN although PA28 mRNA expression was delayed and detected 6 h after the stimulation. NF-B p105 is a precursor protein of p50 subunit which forms NF-B heterodimer with p65 subunit (13, 14). It has been reported that the promoter of the NF-B p105 gene contains a binding site for NF-B and the gene is regulated by members of the NF-B/Rel family (19). Since there is ample evidence that ISS-ODN is capable of inducing NF-B in macrophages, NK cells, and B cells (20), the NF-B p105 gene may be induced by ISS-ODN through the NF-B activation. IRF-1 is involved in the host defense mechanism through the differentiation of Th1 subsets, CD8 ⫹ T cells, and NK1.1 ⫹ T cells and the transcriptional regulation of inducible NO synthase, IL-12, and IL-5 (21). The IRF-1 gene has been shown to be induced by IFN-␣/, IFN-␥, TNF-␣, IL-1, IL-6, leukemia inhibitory factor (LIF), and prolactin (21). The IRF-1 promoter has been described as containing a composite GAS/B element and a promoter-proximal B element (22). It seems unlikely that GAS is involved in the transcription since the GAS-binding transcriptional factor, STAT1 (23), is activated by IFN-␥ which is not released from BMDMs. We infer that B element, NF-B-binding motif, may play a pivotal role in the transcription of IRF-1. The proteasome activator PA28 is a hexa- or heptamer consisting of two subunits, PA28␣ and PA28, which are inducible by IFN-␥ (24). The binding of the PA28 complex to the 20S proteasome enhances the generation of MHC class I binding peptides. It is of particular interest that PA28 is also induced by ISS-ODN without involvement of IFN-␥. IRG2 has been reported to be induced in macrophages to high levels of expression after LPS or IFN-␥ treatment and the function is not revealed yet (17). We demonstrated here that ISS-ODN
is also capable of inducing IRG2 mRNA. Recent evidence obtained by knockout mice indicates that MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), is required for cellular response to ISS-ODN through TLR9 (25). The MyD88 promoter contains overlapping binding sites for Stat and for IRF-1 and -2 (26). Given that IRF-1 is induced by ISS-ODN as described above, the transcription factor may be involved in the induction of MyD88 mRNA. Furthermore, since MyD88 is known as an IL-6 primary response gene (18) and ISS-ODN can induce IL-6 secretion from macrophages, the possibility exists that ISS-ODN may induce MyD88 gene expression through IL-6 production. Taken together, these isolated genes, which have not previously been reported as overexpressed in the ISSODN-stimulated cells, were suggested to be involved in molecular process of host defense mechanism such as transcription factors for cytokine gene expressions and antigen-processing. Further analysis of novel genes whose differential expression is proved is now being performed.
FIG. 2. Time course of mRNA expressions of selected overexpressed genes in CpG-ODN-stimulated BMDMs. BMDMs were stimulated with 10 g/ml of ISS-ODN (CpG) or control ODN (Cont) for 1, 3, and 6 h. Equal amounts of cDNAs from ODN-stimulated BMDMs were used as templates for semi-quantitative RT-PCR analysis. PCR was performed for 28 cycles. PCR products were analyzed by 8% polyacrylamide gel electrophoresis.
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ACKNOWLEDGMENTS Authors wish to thank Minh-Duc Nguyen and Tetsumichi Matsuo for their technical assistance. This work was supported in part by grants from NIH (AI40684 and AI01490), Dynavax Technologies Corporation, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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13. Kieran, M., Blank, V., Logeat, F., Vandekerckhove, J., Lottspeich, F., Le Bail, O., Urban, M. B., Kourilsky, P., Baeuerle, P. A., and Israel, A. (1990) The DNA binding subunit of NFkappa B is identical to factor KBF1 and homologous to the rel oncogene product. Cell 62, 1007–1018. 14. Ghosh, S., Gifford, A. M., Riviere, L. R., Tempst, P., Nolan, G. P., and Baltimore, D. (1990) Cloning of the p50 DNA binding subunit of NF-kappa B: Homology to rel and dorsal. Cell 62, 1019 – 1029. 15. Miyamoto, M., Fujita, T., Kimura, Y., Maruyama, M., Harada, H., Sudo, Y., Miyata, T., and Taniguchi, T. (1988) Regulated expression of a gene encoding a nuclear factor, IRF-1, that specifically binds to IFN-beta gene regulatory elements. Cell 54, 903–913. 16. Ahn, J. Y., Tanahashi, N., Akiyama, K., Hisamatsu, H., Noda, C., Tanaka, K., Chung, C. H., Shibmara, N., Willy, P. J., Mott, J. D., et al. (1995) Primary structures of two homologous subunits of PA28, a gamma- interferon-inducible protein activator of the 20S proteasome. FEBS Lett. 366, 37– 42. 17. Lee, C. G., Demarquoy, J., Jackson, M. J., and O’Brien, W. E. (1994) Molecular cloning and characterization of a murine LPSinducible cDNA. J. Immunol. 152, 5758 –5767. 18. Lord, K. A., Hoffman-Liebermann, B., and Liebermann, D. A. (1990) Nucleotide sequence and expression of a cDNA encoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 5, 1095–1097. 19. Cogswell, P. C., Scheinman, R. I., and Baldwin, A. S., Jr. (1993) Promoter of the human NF-kappa B p50/p105 gene. Regulation by NF-kappa B subunits and by c-REL. J. Immunol. 150, 2794 – 2804. 20. Yi, A. K., Tuetken, R., Redford, T., Waldschmidt, M., Kirsch, J., and Krieg, A. M. (1998) CpG motifs in bacterial DNA activate leukocytes through the pH- dependent generation of reactive oxygen species. J. Immunol. 160, 4755– 4761. 21. Taniguchi, T., Tanaka, N., Ogasawara, K., Taki, S., Sato, M., and Takaoka, A. (1999) Transcription factor IRF-1 and its family members in the regulation of host defense. Cold Spring Harbor Symp. Quant. Biol. 64, 465– 472. 22. Harada, H., Takahashi, E., Itoh, S., Harada, K., Hori, T. A., and Taniguchi, T. (1994) Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: Implications for a gene network in the interferon system. Mol. Cell Biol. 14, 1500 –1509. 23. Darnell, J. E., Jr., Kerr, I. M., and Stark, G. R. (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 264, 1415–1421. 24. Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. (1998) The proteasome: Paradigm of a self-compartmentalizing protease. Cell 92, 367–380. 25. Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S., and Wagner, H. (2000) Immune cell activation by bacterial CpGDNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J. Exp. Med. 192, 595– 600. 26. Harroch, S., Gothelf, Y., Revel, M., and Chebath, J. (1995) 5⬘ upstream sequences of MyD88, an IL-6 primary response gene in M1 cells: Detection of functional IRF-1 and Stat factors binding sites. Nucleic Acids Res. 23, 3539 –3546.
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Identification of Immunostimulatory DNA-Induced Genes by Suppression Subtractive Hybridization Masato Uchijima,* ,1 Eyal Raz,† Dennis A. Carson,† Toshi Nagata,* and Yukio Koide* *Department of Microbiology and Immunology, Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Hamamatsu 431-3192, Japan; and †Department of Medicine, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093
Received July 27, 2001
Bacterial DNA and related synthetic immunostimulatory oligodeoxyribo-nucleotides (ISS-ODN) have stimulatory effects on mammalian immune cells through a Toll-like receptor, TLR9. Genes upregulated in ISS-ODN-stimulated immune cells are obviously significant to delineate the mechanism of the induced innate immunity. Employing suppression subtractive hybridization (SSH), we have generated a profile of genes induced by ISS-ODN in spleen cells. Sequencing of 87 clones isolated by the SSH showed 39 clones corresponding to known mouse genes in the public database. Eleven clones appeared to possess 80 –90% homology with known mouse genes and the remaining 37 clones showed no significant homology with any known mouse genes. A series of known genes which have not previously been reported to be induced with ISS-ODN were confirmed to be induced in ISS-ODNstimulated bone marrow-derived macrophages: NF-B p105, IRF-1, PA28, IRG2, and MyD88. These genes were suggested to be involved in the molecular process of innate host defense mechanisms. © 2001 Academic Press Key Words: CpG; SSH; BMDM; NF-B p105; IRF-1; IRG2; MyD88; PA28.
Innate immunity enables the mammalian host to respond rapidly to invading microorganisms and provides a rapid defense and, at the same time, alters the adaptive immune responses to the pathogens. The innate immune system uses receptors which are capable of recognizing a microbial pathogen by the pathogenassociated molecular patterns (PAMPs) it displays (1). Abbreviations used: BMDM, bone marrow-derived macrophage; IRF-1, IFN regulatory factor-1; ISS-ODN, immunostimulatory oligodeoxyribo-nucleotides; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; PAMP, pathogen-associated molecular pattern; SSH, suppression subtractive hybridization; TLR, Toll-like receptor. 1 To whom correspondence should be addressed. Fax:⫹81-53-4352335. E-mail: [email protected].
0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Two members of the Toll-like receptor (TLR) family have been demonstrated to participate in the recognition. TLR4 and TLR2 are responsible for immune responses to lipopolysaccharide (LPS) and peptidoglycan, respectively (2). Furthermore, TLR9 was recently demonstrated to be involved in the recognition of a specific pattern in bacterial DNA, consisting of an unmethylated pair of nucleotides, cytosine and guanosine (CpG), within the context of certain flanking regions (3). CpG dinucleotides are present at the expected frequency in bacterial DNA but are underrepresented (1/50 –1/60) in vertebrate DNA, where they are usually methylated (4). These differences between bacterial and vertebrate DNA are suggested to be recognized by the vertebrate immune system as a PAMP. Similar effects of those induced by bacterial DNA can be obtained with synthetic immunostimulatory oligodeoxyribonucleotides (ISS-ODN) (5). Bacterial DNA and ISS-ODN can induce macrophages, monocytes, and dendritic cells to secrete proinflammatory cytokines such as IL-6, IL-12, and TNF-␣ (6), and up-regulate MHC class II, CD40, and CD86 expressions (7). To further characterize molecular consequences of the recognition of a specific pattern in bacterial DNA, we used a sensitive PCR-based subtraction approach, termed suppression subtractive hybridization (SSH) (8), to isolate genes upregulated in ISS-ODN-stimulated murine spleen cells. MATERIALS AND METHODS Oligonucleotides. Endotoxin-free phosphorothioate ODN (Trilink, San Diego, CA) were used in all experiments. The sequences of the ODNs used in this study are as follows: ISS-ODN, 5⬘-TGACTGTGAACGTTCGAGATGA-3⬘; Control ODN, 5⬘-TGACTGTGAACCTTAGAGATGA-3⬘. The underline indicates the immunostimulatory DNA sequence. Primers: IL-6 forward, 5⬘-TATGAAGTTCCTCTCTGCAA-3⬘; IL-6 reverse, 5⬘-CTTTGTATCTCTGGAAGTTT-3⬘; NF- B p105 forward, 5⬘-CACCATGGCAGACGATGATC-3⬘; NF-B p105 reverse, 5⬘-TAGGCAAGGTCAGAATGCAC-3⬘; IRF-1 forward, 5⬘-CACCA-
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TGCCAATCACTCGAA-3⬘; IRF-1 reverse, 5⬘-AGCCCTGAGTGGTGTAACTG-3⬘; Proteasome activator (PA) 28 forward, 5⬘-ATGGCCAAGCCTTGTGGGGT-3⬘; PA 28 reverse, 5⬘-TGGAAGCCTTGGCTACATCA-3⬘; IGR2 forward, 5⬘-CACCATCATGAGTGAGGTCA-3⬘; IGR2 reverse, 5⬘-TGGAAGCCTTGGCTACATCA-3⬘; MyD88 forward, 5⬘-TAAGCAGCAGAACCAGGAGT-3⬘; MyD88 reverse, 5⬘-CATCTTCAGGGCAGGGACAA-3⬘; G3PDH forward, 5⬘-ACCACAGTCCATGCCATCAC-3⬘; G3PDH reverse, 5⬘- TCCACCACCCTGTTGCTGTA-3⬘. Cell culture and bone marrow-derived macrophages (BMDMs). Female BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Murine spleen cells were cultured in RPMI 1640 supplemented with 10% (v/v) heat-inactivated FCS (Gemini Bioproduct, Calabasus, CA), 2 mM L-glutamine, 2 mM penicillin/streptomycin (Bio Whittaker, Walkersville, MD) and 50 M 2-mercaptoethanol. BMDMs were prepared and cultured as described previously (7). ⫹
Preparation of poly(A) RNA. Total cellular RNA was isolated from spleen cells treated with ISS-ODN, or control ODN, using a RNA isolation kit (Trizol; Gibco BRL, Rockville, MD) according to the manufacturer’s instruction. Poly(A) ⫹ RNA was prepared using Oligotex mRNA kit (Quiagen, Valencia, CA). Generation of subtracted cDNA library by SSH. SSH to generate subtracted cDNA libraries was performed between ISS-ODNstimulated cells (tester) and control ODN-stimulated cells (driver), essentially as described previously (8), using the PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA). Briefly, The tester poly(A) ⫹ RNA was synthesized from the mixture of murine spleen cells treated with ISS-ODN (10 g/ml) for 1, 3, and 6 h at 37°C. The driver poly(A) ⫹ RNA was prepared from murine spleen cells stimulated with control ODN (10 g/ml) in the same way. Double-stranded cDNA was synthesized from tester and driver poly(A) ⫹ RNAs, and digested with RsaI to obtain shorter blunt-ended cDNA. Two different adaptors, adaptor 1 and adaptor 2, were then ligated to 5⬘-end of each strand of tester cDNA. The adaptor 1-ligated and adaptor 2-ligated tester cDNAs were separately hybridized at 68°C for 8 h with an excess of driver cDNA after denaturation at 98°C for 90 s. The two hybridization samples were then mixed together without denaturation and hybridized at 68°C overnight with excess of driver cDNA. The resulting mixture was diluted to 1:50 and amplified by two PCRs. The primary PCR was performed with flanking primers against adaptor 1 and adaptor 2 and the amplified products were used as a template in secondary PCR with nested primers. All hybridization steps and PCR were performed on Gene Amp 9600 PCR system (Applied Biosystems, Foster City, CA). Products from the second PCR were inserted directly into pT-Adv vector (Clontech). Sequencing and database search of the subtracted cDNA. The subtracted cDNA library was transformed into an Escherichia coli strain XL-1 blue, which were then plated onto agar plates containing ampicillin, X-gal, and IPTG (Sigma). Randomly selected 87 white colonies were grown in Luria-Bertani medium with ampicillin at 37°C, from which plasmids were prepared by Ultra-Clean Miniprep kit (Mo Bio, Solana Beach, CA) and then sequenced using BigDye Terminator Cycle Sequence kit (Applied Biosystems) on an ABI 377 automated sequencer (Applied Biosystems). The program Blast was used to compare sequences of the isolated clones with all sequences present in the National Center for Biotechnology Information (NCBI) nonredundant nucleic acid database (http://www.ncbi.nlm. gov/BLAST/). Semi-quantitative reverse transcriptase (RT)-PCR. RT-PCR was performed as described previously (9). Briefly, BMDMs were stimulated with 10 g/ml of ISS-ODN or control ODN for 1, 3, and 6 h at 37°C and mixed together. Total RNA was prepared from the cells by using Trizol (Gibco BRL). Single-stranded cDNA was synthesized with SuperScript II reverse transcriptase (Gibco BRL) and then used for PCRs. The primers used were described above.
FIG. 1. Analysis of subtraction procedure efficiency. PCRs with IL-6 and G3PDH primers were performed on tester (unsubtracted) and subtracted cDNAs for 28 cycles (95°C for 60 s, 55°C for 60 s, and 72°C for 60 s). PCR products were electrophoresed on 8% polyacrylamide gels.
RESULTS AND DISCUSSION To identify ISS-ODN-immediate response genes, the tester cDNA was generated from a cell mixture of splenocytes stimulated with ISS-ODN for 1, 3, and 6 h at 37°C. First of all, the efficiency of the SSH was evaluated by PCR with two primer sets specific for IL-6 and a house-keeping gene, G3PDH, since ISS (CpG)ODN has been well known to induce IL-6 gene expression in spleen cells (10). As shown in Fig. 1, both IL-6 and G3PDH PCR products were able to be detected in the unsubtracted tester cDNA. In the subtracted cDNA, however, G3PDH PCR product was not detected and IL-6 PCR product was increased compared with the tester cDNA under the same PCR condition, suggesting that we achieved a high level of enrichment of ISS-ODN-induced cDNAs and a drastic reduction of highly abundant cDNAs. Sequence analysis of 87 clones isolated by SSH revealed that 39 clones were found to correspond exactly to known mouse mRNAs in the BLAST program (Table 1). Eleven clones were revealed to have 80 –90% homology with known mouse mRNAs. The remaining 37 clones showed no significant homology with any known sequences in the public databases. We also examined redundancy of our SSH repertoire. Eleven out of 87 clones analyzed were recovered twice (Table 1) and no clones appeared more than twice. The average fragment insert size was 459 bp, which is in close agreement with that statistically predicted by RsaI (⬇600 bp). Using reverse Northern blotting, we estimated that approximately 15% of clones recovered by SSH showed upregulated signals (data not shown). This finding is in accord with the previous findings which showed that 12.5% (11) and 11% (12) of clones recovered by SSH exhibited a differential signal. For further target validation, we used semiquantitative RT-PCR. BMDMs were employed in the
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Summary of Sequences and Clones Represented in the ISS Specific cDNA Library Match category
No. of clones represented (%)
Double hit
Exact mouse match Nonexact mouse match No data base match
39 (44.8) 11 (12.6) 37 (42.5)
7 3 1
Total
87 (100)
11
experiment to confirm the genes upregulated in ISSODN-stimulated macrophages. We confirmed overexpression of NF-B p105 (13, 14), IFN regulatory factor-1 (IRF-1) (15), PA28 (16), IRG2 (IFN-IP) (17), and MyD88 (18), which have not previously been reported as overexpressed in the ISS-ODN-stimulated cells (Fig. 2). The mRNA expressions for NF-B p105, IRF-1, IRG2 (IFN-IP), and MyD88 were detected in BMDMs as early as 1 h after stimulation with ISSODN although PA28 mRNA expression was delayed and detected 6 h after the stimulation. NF-B p105 is a precursor protein of p50 subunit which forms NF-B heterodimer with p65 subunit (13, 14). It has been reported that the promoter of the NF-B p105 gene contains a binding site for NF-B and the gene is regulated by members of the NF-B/Rel family (19). Since there is ample evidence that ISS-ODN is capable of inducing NF-B in macrophages, NK cells, and B cells (20), the NF-B p105 gene may be induced by ISS-ODN through the NF-B activation. IRF-1 is involved in the host defense mechanism through the differentiation of Th1 subsets, CD8 ⫹ T cells, and NK1.1 ⫹ T cells and the transcriptional regulation of inducible NO synthase, IL-12, and IL-5 (21). The IRF-1 gene has been shown to be induced by IFN-␣/, IFN-␥, TNF-␣, IL-1, IL-6, leukemia inhibitory factor (LIF), and prolactin (21). The IRF-1 promoter has been described as containing a composite GAS/B element and a promoter-proximal B element (22). It seems unlikely that GAS is involved in the transcription since the GAS-binding transcriptional factor, STAT1 (23), is activated by IFN-␥ which is not released from BMDMs. We infer that B element, NF-B-binding motif, may play a pivotal role in the transcription of IRF-1. The proteasome activator PA28 is a hexa- or heptamer consisting of two subunits, PA28␣ and PA28, which are inducible by IFN-␥ (24). The binding of the PA28 complex to the 20S proteasome enhances the generation of MHC class I binding peptides. It is of particular interest that PA28 is also induced by ISS-ODN without involvement of IFN-␥. IRG2 has been reported to be induced in macrophages to high levels of expression after LPS or IFN-␥ treatment and the function is not revealed yet (17). We demonstrated here that ISS-ODN
is also capable of inducing IRG2 mRNA. Recent evidence obtained by knockout mice indicates that MyD88, an adaptor protein involved in signal transduction by the Toll-like receptors (TLRs), is required for cellular response to ISS-ODN through TLR9 (25). The MyD88 promoter contains overlapping binding sites for Stat and for IRF-1 and -2 (26). Given that IRF-1 is induced by ISS-ODN as described above, the transcription factor may be involved in the induction of MyD88 mRNA. Furthermore, since MyD88 is known as an IL-6 primary response gene (18) and ISS-ODN can induce IL-6 secretion from macrophages, the possibility exists that ISS-ODN may induce MyD88 gene expression through IL-6 production. Taken together, these isolated genes, which have not previously been reported as overexpressed in the ISSODN-stimulated cells, were suggested to be involved in molecular process of host defense mechanism such as transcription factors for cytokine gene expressions and antigen-processing. Further analysis of novel genes whose differential expression is proved is now being performed.
FIG. 2. Time course of mRNA expressions of selected overexpressed genes in CpG-ODN-stimulated BMDMs. BMDMs were stimulated with 10 g/ml of ISS-ODN (CpG) or control ODN (Cont) for 1, 3, and 6 h. Equal amounts of cDNAs from ODN-stimulated BMDMs were used as templates for semi-quantitative RT-PCR analysis. PCR was performed for 28 cycles. PCR products were analyzed by 8% polyacrylamide gel electrophoresis.
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ACKNOWLEDGMENTS Authors wish to thank Minh-Duc Nguyen and Tetsumichi Matsuo for their technical assistance. This work was supported in part by grants from NIH (AI40684 and AI01490), Dynavax Technologies Corporation, and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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