Necessity of Oligonucleotide Aggregation for Toll-like Receptor 9 Activation
2004; Elsevier BV; Volume: 279; Issue: 32 Linguagem: Inglês
10.1074/jbc.m311662200
ISSN1083-351X
AutoresChristina Wu, Jong‐Dae Lee, Eyal Raz, Maripat Corr, Dennis A. Carson,
Tópico(s)Pediatric health and respiratory diseases
ResumoToll-like receptor 9 (TLR9), a member of the interleukin-1 (IL-1) family of pathogen-associated molecular pattern receptors, is activated by unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) in the endosomal compartment. The stimulation of an IL-1 response is thought to require the aggregation of its receptor. By analogy, we postulated that the potency of a TLR9 ligand should depend first on its ability to enter cells and gain access to TLR9 and second on its capacity to form a multimeric complex capable of cross-linking these receptors. Previously, we selected from a random library a series of phosphodiester ODNs with enhanced ability to permeate cells. Here, we studied the structural requirements for these penetrating ODNs to elicit a functional TLR9 response, as assessed by cytokine production from bone marrow-derived mouse mononuclear cells. The presence of a prototypic murine immunostimulatory DNA hexameric sequence (purine-purine-CG-pyrimidine-pyrimidine) in the ODNs was not sufficient for stimulation. In addition, the TLR9-activating ODNs had to have the ability to form aggregates and often to form secondary structures near the core CpG motifs. Multimerization was promoted by the presence of a guanine-rich 3′-terminus. The phosphodiester ODNs with CpG motifs that did not aggregate antagonized the effects of the multimeric TLR9 activators. These findings suggest that an optimal TLR9 agonist needs to contain a spatially distinct multimerization domain and a receptor binding CpG domain. This concept may prove useful for the design of new TLR9-modulating agents. Toll-like receptor 9 (TLR9), a member of the interleukin-1 (IL-1) family of pathogen-associated molecular pattern receptors, is activated by unmethylated CpG-containing sequences in bacterial DNA or synthetic oligonucleotides (ODNs) in the endosomal compartment. The stimulation of an IL-1 response is thought to require the aggregation of its receptor. By analogy, we postulated that the potency of a TLR9 ligand should depend first on its ability to enter cells and gain access to TLR9 and second on its capacity to form a multimeric complex capable of cross-linking these receptors. Previously, we selected from a random library a series of phosphodiester ODNs with enhanced ability to permeate cells. Here, we studied the structural requirements for these penetrating ODNs to elicit a functional TLR9 response, as assessed by cytokine production from bone marrow-derived mouse mononuclear cells. The presence of a prototypic murine immunostimulatory DNA hexameric sequence (purine-purine-CG-pyrimidine-pyrimidine) in the ODNs was not sufficient for stimulation. In addition, the TLR9-activating ODNs had to have the ability to form aggregates and often to form secondary structures near the core CpG motifs. Multimerization was promoted by the presence of a guanine-rich 3′-terminus. The phosphodiester ODNs with CpG motifs that did not aggregate antagonized the effects of the multimeric TLR9 activators. These findings suggest that an optimal TLR9 agonist needs to contain a spatially distinct multimerization domain and a receptor binding CpG domain. This concept may prove useful for the design of new TLR9-modulating agents. The 10 members of the Toll-like receptor (TLR) 1The abbreviations used are: TLR, Toll-like receptor; ODN, oligodeoxynucleotide; ISS, immunostimulatory sequences; MyD88, myeloid differentiation marker 88; TIR, Toll-interleukin 1 receptor; IL, interleukin; HPLC, high pressure liquid chromatography. family have attracted intense interest, because they play key roles in the initiation of innate and adaptive immune responses (1Dabbagh K. Lewis D.B. Curr. Opin. Infect. Dis. 2003; 16: 199-204Crossref PubMed Scopus (186) Google Scholar, 2Underhill D.M. Eur. J. Immunol. 2003; 33: 1767Crossref PubMed Scopus (201) Google Scholar, 3Akira S. Hemmi H. Immunol. Lett. 2003; 85: 85-95Crossref PubMed Scopus (953) Google Scholar, 4Werling D. Jungi T.W. Vet. Immunol. Immunopathol. 2003; 91: 1-12Crossref PubMed Scopus (340) Google Scholar, 5Janssens S. Beyaert R. Clin. Microbiol. Rev. 2003; 16: 637-646Crossref PubMed Scopus (467) Google Scholar, 6Beutler B. Hoebe K. Du X. Ulevitch R.J. J. Leukocyte Biol. 2003; 74: 479-485Crossref PubMed Scopus (508) Google Scholar). Many of the TLRs share common signaling pathways, mediated by the adapter proteins myeloid differentiation marker 88 (MyD88), Toll-interleukin 1 receptor (TIR) domain-containing adapter protein/MyD88 adaptor-like (7Fitzgerald K.A. Palsson-McDermott E.M. Bowie A.G. Jefferies C.A. Mansell A.S. Brady G. Brint E. Dunne A. Gray P. Harte M.T. McMurray D. Smith D.E. Sims J.E. Bird T.A. O'Neill L.A. Nature. 2001; 413: 78-83Crossref PubMed Scopus (1016) Google Scholar, 8Horng T. Barton G.M. Medzhitov R. Nat. Immunol. 2001; 2: 835-841Crossref PubMed Scopus (837) Google Scholar), or TIR domain-containing adaptor-inducing interferon-β (9Akira S. Yamamoto M. Takeda K. Biochem. Soc. Trans. 2003; 31: 637-642Crossref PubMed Scopus (171) Google Scholar, 10Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4823) Google Scholar, 11O'Neill L.A. Fitzgerald K.A. Bowie A.G. Trends Immunol. 2003; 24: 286-290Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar). However, their cellular patterns of expression and their subcellular localization characteristics differ substantially. Thus, whereas most TLRs are expressed predominantly on the external plasma membrane, TLR7 and TLR9 are found mainly on the inner surface of endosomes (12Lund J. Sato A. Akira S. Medzhitov R. Iwasaki A. J. Exp. Med. 2003; 198: 513-520Crossref PubMed Scopus (1026) Google Scholar, 13Ahmad-Nejad P. Hacker H. Rutz M. Bauer S. Vabulas R.M. Wagner H. Eur. J. Immunol. 2002; 32: 1958-1968Crossref PubMed Scopus (636) Google Scholar, 14Ishii K.J. Takeshita F. Gursel I. Gursel M. Conover J. Nussenzweig A. Klinman D.M. J. Exp. Med. 2002; 196: 269-274Crossref PubMed Scopus (126) Google Scholar, 15Takeshita F. Leifer C.A. Gursel I. Ishii K.J. Takeshita S. Gursel M. Klinman D.M. J. Immunol. 2001; 167: 3555-3558Crossref PubMed Scopus (520) Google Scholar). Hence, effective ligation of TLR7 and TLR9 might be intracellular events. The natural activating ligand for TLR9 is bacterial DNA containing unmethylated CpG dinucleotides, commonly referred to as immunostimulatory DNA or sequence (ISS) (15Takeshita F. Leifer C.A. Gursel I. Ishii K.J. Takeshita S. Gursel M. Klinman D.M. J. Immunol. 2001; 167: 3555-3558Crossref PubMed Scopus (520) Google Scholar, 16Hemmi H. Takeuchi O. Kawai T. Kaisho T. Sato S. Sanjo H. Matsumoto M. Hoshino K. Wagner H. Takeda K. Akira S. Nature. 2000; 408: 740-745Crossref PubMed Scopus (5469) Google Scholar, 17Bauer S. Kirschning C.J. Hacker H. Redecke V. Hausmann S. Akira S. Wagner H. Lipford G.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9237-9242Crossref PubMed Scopus (1302) Google Scholar). Comparative studies of CpG oligonucleotides (ODNs) with different sequences have shown that the neighboring residues of the core CpG dinucleotides strongly influence immunostimulatory potency (18Yamamoto T. Yamamoto S. Kataoka T. Tokunaga T. Antisense Res. Dev. 1994; 4: 119-122Crossref PubMed Scopus (60) Google Scholar, 19Kandimalla E.R. Yu D. Zhao Q. Agrawal S. Bioorg. Med. Chem. 2001; 9: 807-813Crossref PubMed Scopus (72) Google Scholar, 20Yu D. Kandimalla E.R. Zhao Q. Cong Y. Agrawal S. Bioorg. Med. Chem. Lett. 2001; 11: 2263-2267Crossref PubMed Scopus (45) Google Scholar, 21Zimmermann S. Heeg K. Dalpke A. Vaccine. 2003; 21: 990-995Crossref PubMed Scopus (19) Google Scholar). However, there are some differences between the optimal murine and human ISS. The central murine ISS motif for TLR9 activation has been shown to consist of the hexameric sequence purinepurine-CG-pyrimidine-pyrimidine in phosphodiester or phosphorothioate ODNs of at least 12 nucleotides (22Krieg A.M. Yi A.K. Matson S. Waldschmidt T.J. Bishop G.A. Teasdale R. Koretzky G.A. Klinman D.M. Nature. 1995; 374: 546-549Crossref PubMed Scopus (3127) Google Scholar). The prototypic ODN sequence for activation of human TLR9 contains a TCG sequence at the 5′-end of the molecule (23Hartmann G. Weeratna R.D. Ballas Z.K. Payette P. Blackwell S. Suparto I. Rasmussen W.L. Waldschmidt M. Sajuthi D. Purcell R.H. Davis H.L. Krieg A.M. J. Immunol. 2000; 164: 1617-1624Crossref PubMed Scopus (546) Google Scholar, 24Yu D. Zhao Q. Kandimalla E.R. Agrawal S. Bioorg. Med. Chem. Lett. 2000; 10: 2585-2588Crossref PubMed Scopus (68) Google Scholar). Other sequence modifications influence the abilities of the ISS to activate selectively TLR9-expressing macrophages, dendritic cells, or B lymphocytes (25Marshall J.D. Fearon K. Abbate C. Subramanian S. Yee P. Gregorio J. Coffman R.L. Van Nest G. J. Leukoc. Biol. 2003; 73: 781-792Crossref PubMed Scopus (234) Google Scholar, 26Verthelyi D. Ishii K.J. Gursel M. Takeshita F. Klinman D.M. J. Immunol. 2001; 166: 2372-2377Crossref PubMed Scopus (449) Google Scholar). The initiating events in TLR9 activation have not yet been analyzed, in part because the recombinant protein is difficult to purify in quantity and to solubilize. However, the TLRs are structurally homologous to the interleukin-1 (IL-1) receptors, which have been studied in more detail. Activation of IL-1 type I receptors correlated with IL-1-dependent receptor aggregation (27Guo C. Dower S.K. Holowka D. Baird B. J. Biol. Chem. 1995; 270: 27562-27568Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, the IL-1 receptor antagonist, which blocks IL-1 function by competing for receptor binding, did not induce IL-1 receptor aggregation. If a similar signaling mechanism applied to TLR9, one could predict that self-aggregating ISS would strongly activate the receptor by promoting its multimerization, whereas nonaggregating ISS would antagonize these effects. According to this hypothesis, the higher structure of an ISS-ODN, rather than just its primary sequence, could dictate its ability to stimulate or to inhibit innate immune system activation. To gain insight into this problem, we compared the physical properties and the TLR9-activating capacities of a series of phosphodiester ODNs that were selected from a random library for their capacity to be efficiently internalized by cells. Since all of the ODNs were selected for their ability to penetrate cells, their different functional activities were considered to be attributable mainly to TLR9 activation. The experimental results showed that a prototype ISS is not sufficient for TLR9 activation. Aggregating phosphodiester ODNs with ISS in rigid secondary structures activated the receptor effectively, whereas nonaggregating ODNs with CpG motifs functioned as receptor antagonists. Oligodeoxynucleotides—Phosphodiester ODNs from a random library with scrambled insets of 40 nucleotides were selected for their ability to penetrate cells by a repetitive selection procedure that involved (a) incubation with viable cells, (b) extensive and stringent washing to remove all external ODN binding, and (c) asymmetric PCR amplification, as described previously (28Wu C.C. Castro J.E. Motta M. Cottam H.B. Kyburz D. Kipps T.J. Corr M. Carson D.A. Hum. Gene. Ther. 2003; 14: 849-860Crossref PubMed Scopus (30) Google Scholar). After 10 rounds of selection, the retained intracellular ODNs were amplified, cloned, and sequenced. Several 40-mer ODNs corresponding to the recovered sequences as well as a random ODN of the same length (random 40) were synthesized by Integrated DNA Technologies (IDT, Corvallis, OR) (Table I). The prototype ISS-ODNs, 1018 and 1826, have been described (29Roman M. Martin-Orozco E. Goodman J.S. Nguyen M.D. Sato Y. Ronaghy A. Kornbluth R.S. Richman D.D. Carson D.A. Raz E. Nat. Med. 1997; 3: 849-854Crossref PubMed Scopus (814) Google Scholar, 30Magone M.T. Chan C.C. Beck L. Whitcup S.M. Raz E. Eur. J. Immunol. 2000; 30: 1841-1850Crossref PubMed Scopus (80) Google Scholar, 31Broide D.H. Stachnick G. Castaneda D. Nayar J. Miller M. Cho J.Y. Roman M. Zubeldia J. Hayashi T. Raz E. Hyashi T. J. Clin. Immunol. 2001; 21: 175-182Crossref PubMed Scopus (85) Google Scholar, 32Ballas Z.K. Krieg A.M. Warren T. Rasmussen W. Davis H.L. Waldschmidt M. Weiner G.J. J. Immunol. 2001; 167: 4878-4886Crossref PubMed Scopus (234) Google Scholar) and were synthesized with both phosphodiester and phosphorothioate backbones. Endotoxin contamination in ODNs was negligible as measured by a Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).Table IOligonucleotide sequences and immunostimulatory activityODNSequenceCpGISSIL-12p40/p70BALB/c(B6 × 129)F2TLR9−/−ng/mlR10-5CCAGCCACCTACTCCACCAGTGCCAGGACTGCTTGAGGGG00000R10-9CTAACGTTTAACCAGGATCCCCCAAGTCCCTGCTAGTGGG11000R10-32TGGGCGTTACCACTACAGGTCCAGATTTGTCTGTCCGGGG210.08 ± 0.0400R10-71GGGATCTACGGCTAAACATCTAACGCTCTTTGGCCCTGGG210.35 ± 0.080.22 ± 0.090R10-13CGCTCCCTTATATATCCGACGTGACTAATACTGTGGGGC310.28 ± 0.110.07 ± 0.020R10-34GCACATAAAACTTTACCCCGACGTGGAGGACGTTCTTGGC310.35 ± 0.040.06 ± 0.020R10-60CCAGTCGTACAGGAAACATGCGTTCTAGATGTTCGGGGC3010.45 ± 3.105.88 ± 1.230D-R15-8CGCAGCGTATGGATTCAGGGTTGGATCGTGTAGGGGGGG301.76 ± 0.312.45 ± 0.600R10-11CGCGTGAAGAAAAGGAGCAGTCATAAACGCTAATCGTGCC410.10 ± 0.0200R10-53TCTGCGGGGAAGAGCTACGTTACTAGTCGTGTGTCCGTG401.30 ± 0.260.38 ± 0.040R10-86GCGGCCATTCAGGAAACGTTAATGTCGATCTACGTTGGC410.44 ± 0.111.80 ± 0.940Controlsrandom40CCTGGCTGTTCCGAAACATATCCACAGTTGTTGGCCCAGG100001018*TGACTGTGAACGTTCGAGATG2116.57 ± 4.011.04 ± 0.130Lipopolysaccharide0.50 ± 0.190.39 ± 0.030.26 ± 0.04 Open table in a new tab Uptake studies were performed using fluorochrome-labeled ODNs to confirm their abilities to penetrate cells, as described (28Wu C.C. Castro J.E. Motta M. Cottam H.B. Kyburz D. Kipps T.J. Corr M. Carson D.A. Hum. Gene. Ther. 2003; 14: 849-860Crossref PubMed Scopus (30) Google Scholar). Briefly, viable cells were incubated in protein-free medium with 5′-Cy3-labeled ODNs at 0.5 μm for 2 h at 37 °C. Following washes with 3% fetal bovine serum in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA), these cells were analyzed by flow cytometry using a Becton Dickinson FAC-Scaliber. Data analysis was carried out using FlowJo 3.4 software (Tree Star, Inc., Stanford, CA). Isolation of Bone Marrow-derived Mononuclear Cells—BALB/c and C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). MyD88-/- and TLR9-/- mice were a generous gift of Dr. S. Akira (Osaka University, Japan) (33Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1743) Google Scholar). The mice were bred and maintained under standard conditions in the University of California, San Diego Animal Facility that is accredited by the American Association for Accreditation of Laboratory Animal Care. All animal protocols received prior approval by the institutional review board. Bone marrow harvested from the femurs and tibias of various strains of mice were plated in non-tissue culture-treated Petri dishes with Dulbecco's modified Eagle's medium high glucose medium supplemented with 10% fetal bovine serum, l-glutamine, penicillin/streptomycin, all from Invitrogen, and 30% L929 cell-conditioned medium. Cells were grown at 37 °C, 5% CO2 for 7 days without replacing the medium. The bone marrow-derived mononuclear cells were harvested afterward by gentle scraping, counted, and replated in medium with different conditions described below. ODN-stimulated Cytokine Release—For studies on cytokine production, 7-day-old bone marrow-derived mononuclear cells were seeded in 96-well plates at a density of 5 × 104 cells/well and grown for another 3 days. These cells were then incubated with ODNs at a final concentration of 0.2, 0.5, or 1 μm for 48 h without further supplement. In the competitive receptor binding study, the highly active ODN R10-60 (0.5 μm) was premixed with various concentrations of the inactive ODN R10-9, R10-32, or R10-13 in serum-free medium, prior to addition to the cells. Culture supernatants were collected at the end of incubation and stored at -20 °C for later determination of IL-12p40/70 by sandwich enzyme-linked immunosorbent assay (BD Biosciences). In Vitro Kinase Assays—For kinase assays, the enriched mononuclear cells were dispersed in 6-well plates at a density of 1-2 × 106 cells/ml/well and allowed to settle overnight. ODNs were then added to the mononuclear cells at 1 μm and incubated for 0.5-2 h. The cells were quickly lysed in buffer A (20 mm Hepes, pH 7.9, 1 mm EDTA, 1 mm EGTA, 1% Nonidet P-40, 1 mm glycerophosphate, 2.5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 2 mg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride) with proteinase inhibitors and 1 mm dithiothreitol on ice and centrifuged at 12,000 × g for 1 min. The aqueous phase containing cytoplasmic proteins was removed and saved. The nuclear pellet was lysed in buffer B (20 mm Hepes, pH 7.9, 1 mm EDTA, 1 mm EGTA, 0.4 m NaCl) and vortexed, and the nuclear supernatant was collected after centrifugation. Specific kinases were immunoprecipitated from cytosolic proteins with either anti-IκB kinase-β or anti-Jun NH2-terminal kinase 1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 °C overnight. Afterward, the immune complexes were washed successively in buffer A containing 0.5 m NaCl followed by kinase buffer (25 mm Tris, pH 7.5, 10 mm MgCl2, 2 mm EGTA, 1 mm dithiothreitol, 1 mm sodium orthovanadate). IκB kinase-β or Jun NH2-terminal kinase 1 kinase assays were performed using the respective recombinant glutathione S-transferase fusion protein with IκBα or c-Jun as the respective substrates in the presence of 0.1 μCi of [γ-32P]ATP at 37 °C for 30 min, as described (34Lee J. Mira-Arbibe L. Ulevitch R.J. J. Leukocyte Biol. 2000; 68: 909-915Crossref PubMed Google Scholar). The 32P-labeled products were separated by SDS-PAGE and visualized by autoradiography. DNA Secondary Structure Prediction—To predict the presence of secondary structures, the DNA mfold program (35Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10623) Google Scholar) (available on the World Wide Web at www.bioinfo.rpi.edu/applications/mfold/old/dna/) was employed. The various sequences were submitted as linear DNA and analyzed based on free energy using default program settings, assuming a temperature of 37 °C, with ionic conditions of 150 mm Na+ and 0.5 mm Mg2+. Analysis of ODN Multimerization by Size Exclusion HPLC and PAGE—A TSK-Gel G2000SWXL HPLC column with a 5-μm particle size (MAC-MOD Analytical, Montgomeryville, PA) was used to perform the size exclusion assay as previously described (28Wu C.C. Castro J.E. Motta M. Cottam H.B. Kyburz D. Kipps T.J. Corr M. Carson D.A. Hum. Gene. Ther. 2003; 14: 849-860Crossref PubMed Scopus (30) Google Scholar, 36Suzuki K. Doi T. Imanishi T. Kodama T. Tanaka T. Eur. J. Biochem. 1999; 260: 855-860Crossref PubMed Scopus (31) Google Scholar). Briefly, 50 μl of a 50 μm ODN solution in 30 mm NaCl was injected, and elution was carried out in buffer containing 10 mm sodium phosphate, pH 6.9, 0.3 m NaCl at a flow rate of 0.6 ml/min. The HPLC elution fractions were divided into a high molecular weight aggregate portion (retention time 9-12.5 min) and a low molecular weight monomer portion (retention time 12.5-15 min). To address the association of ODN multimerization with immunostimulatory activity, the two fractions were collected, equal amounts were added to bone marrow-derived mononuclear cells, and the culture supernatants were collected 48 h later for enzyme-linked immunosorbent assay, as described above. For gel analysis, phosphorothioate and phosphodiester ODNs were mixed in RPMI 1640 with and without 2% tissue culture grade bovine serum albumin (Sigma) and incubated for 10 min at 37 °C. 12 μl of the mixture were then separated on a 4-20% nondenaturing TBE polyacrylamide gel (Invitrogen). The oligonucleotides were visualized by staining with SYBR Green II (Molecular Probes, Eugene, OR) under UV light. The protein bands were then detected with Coomassie Blue staining. Circular Dichroism Spectroscopy—Oligonucleotides were resuspended in 10 mm sodium phosphate buffer, pH 7.2, containing 0.1 m KCl at a final concentration of 10 μm (final volume of 300 μl), boiled for 5 min, and annealed at 60 °C for 2 days (37Dapic V. Bates P.J. Trent J.O. Rodger A. Thomas S.D. Miller D.M. Biochemistry. 2002; 41: 3676-3685Crossref PubMed Scopus (129) Google Scholar). After slow cooling to room temperature, the samples were analyzed on an AVIV CD spectrometer (model 202, AVIV instruments, Inc., Lakewood, NJ) using a wavelength scan from 320 to 200 nm at 25 °C. Spectra were collected over three scans at 1-nm bandwidth, 1-nm wavelength step, and an average 0.5-s response time for each sample. Data are presented as the average of three scans with integrated curve fitting performed by Prism software (version 3.0; GraphPad Software, Inc., San Diego, CA). ODN Uptake Is Independent of TLR9—We previously described the selection of phosphodiester ODNs with an average length of about 40 nucleotides that displayed improved cellular uptake compared with random sequence ODNs (28Wu C.C. Castro J.E. Motta M. Cottam H.B. Kyburz D. Kipps T.J. Corr M. Carson D.A. Hum. Gene. Ther. 2003; 14: 849-860Crossref PubMed Scopus (30) Google Scholar). Because the ODNs shown in Table I were selected for uptake by human B cells, it was necessary to confirm that they also effectively penetrated murine bone marrow-derived mononuclear cells. Experiments with fluorochrome-labeled ODNs showed that they were taken up 2-14-fold better than random sequence ODNs of the same length (examples are shown in Fig. 1). In addition, bone marrow-derived cells from TLR9-/- mice displayed an uptake efficiency similar to cells from wild type mice. Sequence Requirements for Activation of Bone Marrow-derived Mononuclear Cells—To characterize the mechanisms involved in activation by these penetrating ODNs and to study the structural and functional requirements for stimulation, we carried out studies on murine bone marrow-derived mononuclear cells from different strains. A panel of ODNs containing different numbers of CpG dinucleotides and murine ISS motifs were first compared for their abilities to induce IL-12p40/p70 secretion. As expected, ODNs without any CpG dinucleotides had no ISS activity (Table I, R10-5). Unexpectedly, however, no detectable IL-12 was released by cells treated with ODN R10-9, which contained the prototype ISS sequence motif AACGTT at the 5′ terminus. Cells stimulated with ODNs that contained at least two sets of CpG dinucleotides produced detectable levels of IL-12. Furthermore, phosphodiester ODN R10-60 showed comparable or even better IL-12 stimulation than the positive control ODN 1018, with a more nuclease-resistant phosphorothioate backbone (Table I). As little as 0.2 μm ODNs R10-53, R10-60, R10-86, and D-R15-8 were sufficient to induce detectable IL-12, and the levels increased in proportion to the ODN concentration (Fig. 2). In contrast, R10-9 was not able to elicit any IL-12 secretion at concentrations up to 1 μm. Together, these data demonstrated that phosphodiester ODNs can display equivalent immunostimulatory activity toward murine bone marrow-derived mononuclear cells as phosphorothioate ODN, and that a CpG motif is necessary but not sufficient for cell activation. Role of the TLR-9 and MyD88 Pathways—Since there was no absolute correlation between an ISS motif and immunostimulatory activity among the selected ODNs, it was important to confirm that the ODNs signaled through the TLR9 and MyD88 pathway. No IL-12 production was observed from ODN-stimulated bone marrow-derived mononuclear cells from either TLR9-/- (Table I) or MyD88-/- mice (data not shown). Furthermore, the ODNs did not induce IκB kinase-β or Jun NH2-terminal kinase activities in TLR9-/- and MyD88-/- cells, whereas bacterial lipopolysaccharide clearly activated these cells through the recently described MyD88-independent alternative pathway (Fig. 3). The kinetics of cell activation revealed a maximum at 2 h after phosphodiester ODN application. Association of Multimerization with ISS Activity—As the primary ISS motif (e.g. in R10-9) was insufficient for immunostimulatory activity, we evaluated whether a higher structure of an ODN also could influence its biologic properties. Results of the DNA mfold program showed that the CpG dinucleotide sequences in the active ISS-ODN, at their predicted lowest free energy states, were often in or near rigid stem loop structures, whereas the CpG in R10-9 was not (Fig. 4A). Furthermore, point mutations of the CpG located within the predicted rigid loop structures of ISS-ODN also reduced their ability to activate murine bone marrow derived mononuclear cells (Table II, R10-53(T18G) and R10-60(T21G)). More importantly, size exclusion HPLC analysis showed that the active ISS-ODN formed multimers (Fig. 4B, R10-53, R10-60, and D-R15-8), whereas the inactive ISS-ODN R10-9 did not. Nondenaturing polyacrylamide gel fractionation of the ODNs also confirmed the presence of multimers in the biologically active ODN samples (data not shown). Removal of the guanine-rich sequences in the 3′-terminus of R10-60 and D-R15-8 (R10-60a and D-R15-8a) or near the 5′-end of R10-53 (R10-53(-7-14Pu)) abolished aggregate formation. Finally, circular dichroism spectroscopic analyses revealed absorption maxima that have been previously associated with the presence of guanine quartets, which are known to form aggregated structures (Fig. 4C).Table IIEffect of sequence modification on immunostimulatory activityNameSequenceLengthCpGAggregatesStimulationnt%%R10-53TCTGCGGGGAAGAGCTACGTTACTAGTCGTGTGTCCGTG39440,46100.0 ± 1.9Single point mutation of CpG to TGR10-53(T5G)TCTGTGGGGAAGAGCTACGTTACTAGTCGTGTGTCCGTG39323,2692.6 ± 7.7R10-53(T18G)TCTGCGGGGAAGAGCTATGTTACTAGTCGTGTGTCCGTG39329,325.3 ± 2.3R10-53(T28G)TCTGCGGGGAAGAGCTACGTTACTAGTTGTGTGTCCGTG39331,3140.0 ± 4.6R10-53(T36G)TCTGCGGGGAAGAGCTACGTTACTAGTCGTGTGTCTGTG39326,2770.8 ± 8.0R10-60CCAGTCGTACAGGAAACATGCGTTCTAGATGTTCGGGGC39364,65100.0 ± 1.6Single point mutation of CpG to TGR10-60(T6G)CCAGTTGTACAGGAAACATGCGTTCTAGATGTTCGGGGC39234,3652.2 ± 3.6R10-60(T21G)CCAGTCGTACAGGAAACATGTGTTCTAGATGTTCGGGGC39228,3524.2 ± 2.0R10-60(T34G)CCAGTCGTACAGGAAACATGCGTTCTAGATGTTTGGGGC3926,1971.4 ± 8.2Removal of G-rich tailR10-60aCCAGTCGTACAGGAAACATGCGTTCTAGATGTTCG––3533, 629.2 ± 3.8R10-60bCCAGTTGTACAGGAAACATGCGTTCTAGATGTTCG––3522, 32.9 ± 0.7Methylation of all CpG sitesmR10-6039026,296.8 ± 1.2D-R15-8CGCAGCGTATGGATTCAGGGTTGGATCGTGTAGGGGGGG39371,77100.0 ± 12.7Removal of G-rich tailD-R15-8aCGCAGCGTATGGATTCAGGGTTGGATCGTGTA–––-3231, 29.8 ± 4.0Methylation of all CpG sitesmD-R15-839045,489.3 ± 2.7 Open table in a new tab Although ODN multimerization correlated with enhanced immunostimulatory activity (Tables I and II), this observation did not prove that aggregation was responsible for the stimulation potency. To address the question directly, ODN fractions of different sizes were collected from the HPLC elutes, and equal amounts were added to bone marrow-derived mononuclear cells. The maximal IL-12 was produced by cells that were stimulated with ODN aggregates, whereas at least 5-10-fold less IL-12 was observed from cells stimulated with ODN monomers (Fig. 5). Modified ISS-ODN, which had their 3′-guanine tails removed to diminish multimerization, also lost stimulation activity (Table II). Under physiological conditions, phosphorothioate ODNs also aggregated and formed multimers by binding to plasma proteins (Fig. 6). Phosphodiester and phosphorothioate ODNs of the same sequence were incubated in medium with or without bovine serum albumin and analyzed by nondenaturing TBE PAGE. In the presence of bovine serum albumin, the prototype phosphorothioate ISS-ODNs, 1018 and 1826, were retained in the gel at higher molecular weights than the oligonucleotides in unsupplemented medium (Fig. 6A). The Coomassie Blue-stained bands suggested that these phosphorothioate ODNs co-migrated with the protein (Fig. 6B). In contrast, the phosphodiester counterparts of 1018 and 1826 were only visualized at the monomeric molecular weight and did not appreciably bind to protein. To determine whether ISS activity was retained in the absence of exogenous proteins, bone marrow-derived mononuclear cells were extensively washed to remove plasma proteins and then cultivated in completely serum/protein-free medium with the different ODNs. In the absence of serum, R10-60- and 1826-stimulated cells released ∼34 and 17% as much IL-12, respectively, as the cells stimulated in serum-containing medium (data not shown). However, ∼40% of the cells died during a 24-h cultivation, making the exact interpretation of these results uncertain. Nonetheless, the data support that aggregating phosphodiester ODNs are better immunostimulants than phosphorothioate ODNs under serumfree conditions. Biological Activities of ODN Monomers—The previous experiments demonstrated that monomeric ODNs with an ISS motif failed to effectively stimulate mouse bone marrow-derived mononuclear cells. However, the question remained whether the nonaggregating ODNs displayed other biological activities, such as the ability to antagonize cell activation by multimeric ISS-ODNs. To test this possibility, cells were stimulated with a mixture of R10-60 that forms aggregates and the nonaggregating ODN, R10-9, R10-32, or R10-13, at different ratios. A 10:1 molar excess of the monomeric ODNs reduced IL-12 production by ∼64, 55, and 46%, respectively, and even a 2:1 ratio had significant inhibitory activity (Fig. 7). The latter concentrations of nonaggregating R10-9 were i
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