DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Faslpr/lprmice in vivo
© Lenert et al.; licensee BioMed Central Ltd. 2009
Received: 5 February 2009
Accepted: 28 May 2009
Published: 28 May 2009
B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.
We have developed a new generation of DNA-like compounds named class R inhibitory oligonucleotides (INH-ODNs). We tested their effectiveness in autoimmune B cells and interferon-alpha-producing dendritic cells in vitro and in lupus-prone MRL-Faslpr/lprmice in vivo.
Class R INH-ODNs have 10- to 30-fold higher inhibitory potency when autoreactive B cells are synergistically activated through the BCR and associated TLR7 or 9 than when stimulation occurs via non-BCR-engaged TLR7/9. Inhibition of TLR9 requires the presence of both CCT and GGG triplets in an INH-ODN, whereas the inhibition of the TLR7 pathway appears to be sequence-independent but dependent on the phosphorothioate backbone. This difference was also observed in the MRL-Faslpr/lprmice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival.
The increased potency of class R INH-ODNs for autoreactive B cells and dendritic cells may be beneficial for lupus patients by providing pathway-specific inhibition yet allowing them to generate protective immune response when needed.
Nucleic acids, including self DNA and RNA, are recognized by a subset of Toll-like receptors (TLRs) [1–4]. To discriminate between self and non-self nucleic acids, the nucleic acid-sensing TLRs 3, 7, 8, and 9 are expressed only within the cell interior, contrasting with other TLRs (for example, TLR2 or TLR4) that are expressed on cell surfaces. Upon ligand entry into the cell, TLR9 migrates from the endoplasmic reticulum to CpG-DNA-containing endosomes [5, 6]. Interestingly, the type of endosomal compartment to which TLR9 relocates depends on cell type and the nature of the TLR ligand used for activation. For example, in the response of human dendritic cells (DCs) to linear CpG-DNA, TLR9 activation goes through late LAMP-1-positive endosomes [7, 8]. In contrast, stimulation with complex TLR9 ligands is more restricted in terms of responding cell types and, in DCs, proceeds through early endosomes instead. The uptake of these complex ligands may be facilitated by CXCL16, which may influence this differential compartmentalization . Interestingly, the outcome of the DC response to TLR9 stimulation varies greatly depending on where TLR9 meets CpG-DNA. For example, type I interferon-alpha (IFN-α) secretion is induced by complex class A(D) CpG-oligonucleotides (CpG-ODNs) via early endosomal signaling, whereas interleukin-6/tumor necrosis factor-alpha (IL-6/TNF-α) secretion requires late endosomal signaling and is induced primarily by linear TLR9 ligands .
Although bacterial DNA and double-stranded CpG-ODNs stimulate macrophages vigorously, they are very poor activators of resting B cells in both humans and mice [10–13]. In resting follicular B cells and in human naïve peripheral blood B cells, engagement of the B-cell receptor (BCR) for antigen, together with co-stimulation with either type I/II IFN or BAFF (B-cell activating factor of TNF family), may prime B cells to overcome this unresponsiveness to complex TLR ligands [13–18]. This enhancement may be due to multiple mechanisms (for example, TLR7 and 9 upregulation, increased ligand uptake, BCR-mediated delivery of TLR ligands to 'autophagosomes' where concomitant BCR and TLR signals take place, or lowered BCR signaling threshold) . It remains to be formally proven whether the same type of the crosstalk between BCR and TLR also occurs between antigen and co-delivered TLR7 ligand.
These findings have direct implications for the pathogenesis of systemic lupus erythematosus (SLE), a multisystemic disease in which autoantibodies to DNA- and RNA-containing autoantigens (for example, nucleosomes, Ku-autoantigen, Sm/RNP, or splicesosomes) are the immunologic hallmark of the disease [20–22]. These antibodies frequently antedate the clinical disease, and high levels of several lupus autoantibodies nicely correlate with either specific disease subsets (for example, lupus nephritis, congenital heart block, or subacute cutaneous lupus) or disease activity in general [20, 23]. Immune complexes between complement-fixing anti-double-stranded DNA (anti-dsDNA) antibodies and corresponding autoantigens are held responsible for the kidney damage in lupus nephritis . Complement levels frequently fall during major lupus flares, further suggesting that complement-activating immune complexes may play an important role in the tissue pathology .
It was recently found that lupus autoantigens (for example, nucleosomes or Sm/RNP) have intrinsic 'autoadjuvant' activities (endogenous mitogens) when complexed with corresponding autoantibodies, causing proliferation of autoreactive B cells and type I IFN secretion from plasmacytoid DCs [24–32]. Depending on the nature of the initiating autoantigen, the proliferation requires either the TLR7 or TLR9 pathway, including the presence of the key adaptor protein MyD88 (myeloid differentiation primary response gene 88) [25–28, 33, 34]. Thus, therapies aimed at blocking the TLRs may be beneficial for treating lupus. Indeed, promising results have been reported in animal models of lupus using TLR7- and/or TLR9-specific antagonists [35–37].
We have recently developed a new class of inhibitory ODNs that we named class R ('restricted') INH-ODNs . We show that these dsDNA-like analogues carrying the canonical TLR9-inhibitory sequence [39, 40] are selective for certain autoreactive B cells and for type I IFN-producing DCs. They are 10- to 30-fold less potent in non-autoreactive B cells stimulated with linear CpG-DNA ligands. In addition to autoreactive B cells, class R INH-ODNs are capable of blocking both DNA/anti-DNA-induced and RNA/anti-RNA-induced IFN-α secretion from DCs. Interestingly, the latter outcome is ODN sequence-independent but is dependent on a nuclease-resistant phosphorothioate (PS) backbone. Class R INH-ODNs are also active in vivo, where they preferentially block anti-dsDNA and anti-Sm/RNP antibody secretion and prolong survival of lupus-prone mice. Refinement of the class R INH-ODN structure to combine optimal TLR7/TLR9 sequences in double-stranded carrier may result in a novel class of pathway-specific therapeutics for human lupus.
Materials and methods
Creating class R inhibitory ODNs for the Toll-like receptor-9 signaling pathway
Synthetic Toll-like receptor-9 agonists and antagonists used in the study
CCT GGA TGG GAA
5' linear, shortest active
CCT GGA TGG GAA TTC CCA TCC AGG
CCT GGA TGG GAA CTT ACC GCT GCA
CCT GGA TAA AAA TTT TTA TCC AGG
CCT GAA TAA AAA CTT ACC GCT GCA
TAT GGA TGG GAA TTC CCA TCC ATA
TAT GGA TGG GAA CTT ACC GCT GCA
TAT GGA TTT TAA TTA AAA TCC ATA
TAT GGA TTT TAA CTT ACC GCG GCA
CCT GGA TGG GAA TTC CCA TCC
5' short overhang
(3 nucleotide OVHG)
5' OVHG-scr. short
CCT GGA TGG GAA CTT ACC GCT
CCT GGA TGG GAA TTC CCA
5' medium overhang
(6 nucleotide OVHG)
5' OVHG-scr. medium
CCT GGA TGG GAA CTT ACC
CCT GGA TGG GAA TTC
5' long overhang
(9 nucleotide OVHG)
5' OVHG-scr. long
CCT GGA TGG GAA CTT
TTC CCA TCC AGG CCT GGA TGG GAA
3' scr. pal.
CTT ACC GCT GCA CCT GGA TGG GAA
CCA TCC AGG CCT GGA TGG GAA
3' short overhang
(3 nucleotide OVHG)
3' OVHG-scr. short
ACC GCT GCA CCT GGA TGG GAA
TCC AGG CCT GGA TGG GAA
3' medium overhang
(6 nucleotide OVHG)
3' OVHG-scr. medium
GCT GCA CCT GGA TGG GAA
AGG CCT GGA TGG GAA
3' long overhang
(9 nucleotide OVHG)
3' OVHG-scr. long
GCA CCT GGA TGG GAA
RRR RRR RRR RRR RRR
Linear control, R = random
Lowercase PS linkages
TCC ATG ACG TTC CTG ACG TT
TCG TCG TTT TGT CGT TTT GTC GTT
Toll-like receptor agonists
CpG-ODNs 2336, 1826, and 2006 (Table 1) were obtained from Coley Pharmaceuticals (Ottawa, ON, Canada). All other ODNs were synthesized and HPLC (high-performance liquid chromatography)-purified by Integrated DNA Technologies (IDT) (Coralville, IA, USA) and used at concentrations of up to 1 μM (for PS-ODNs) or 30 μM (for phosphodiester [PO]-ODNs). PS-ODNs have a PS backbone, and PO-ODNs have a native PO backbone. TLR7/8 ligands R-837, CL-075, CL-097, and loxoribine were purchased from InvivoGen (San Diego, CA, USA). All reagents were endotoxin-free as determined by the Limulus amebocyte lysate assay (Pyrotell LAL Assay; Associates of Cape Cod, Inc., East Falmouth, MA, USA). Highly purified lipopolysaccharide (LPS) was obtained from List Biological Laboratories, Inc. (Campbell, CA, USA).
MRL-MpJ-Faslpr/lprmice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained under standard conditions in the Animal Facility at The University of Iowa. AM14 mice, expressing a BCR reactive with mouse IgG2a, were described previously [41–43] and were bred and maintained in microisolator cages at the Laboratory Animal Science Center of the Boston University School of Medicine. Several cohorts of young pre-diseased MRL-Fas lpr/lprmice were treated beginning from 4 weeks (rederived strain, MRL-MpJ-Faslpr/J) or 15 weeks of age (non-rederived strain, MRL-MpJ-Faslpr/2J). Phosphate-buffered saline (PBS), INH-1, INH-18, INH-47, or INH-48 (1 mg/kg body weight) in a final volume of 1 mL was injected intraperitoneally or subcutaneously three times weekly for 12 to 25 weeks. Each experimental group consisted of five to eight female mice. At the beginning and at the end of the treatment protocol, blood was obtained through retro-orbital bleeds and urine was collected. Serum was analyzed for cytokines, total immunoglobulin (Ig) levels, and autoantibodies. Proteinuria was semi-quantified using Multistix urinalysis strips (Bayer, Leverkusen, Germany). The study was approved by the University of Iowa animal ethics committee, and animals were euthanized according to Institutional Animal Care and Use Committee procedures. Left kidney, liver, and lungs/heart blocks were harvested and fixed in 10% buffered formalin. Paraffin-embedded organ sections were stained with periodic acid-Schiff and hematoxylin/eosin. The extent of kidney damage was graded according to published guidelines and scored in a blinded fashion . The right kidney was embedded in Sakura Finetek Tissue-Tek O.C.T. compound (Sakura Finetek U.S.A., Inc., Torrance, CA, USA) and kept frozen at -80°C before use in immunohistology for detection of C3 and IgG deposits.
INH-ODN potency studies in primary macrophages, macrophage cell lines, and human and mouse B cells
Splenic macrophages were obtained from C57BL6 mice by recovering the CD43+ fraction from the magnetic-activated cell sorting (MACS) LD columns. Cells were left to adhere to plastic for 4 hours. The adherent fraction typically contained greater than 85% CD11b+F4/80+ cells. Experiments were repeated with similar results using splenic macrophages obtained by positive selection using CD11b microbeads and two rounds of positive selection (>97% purity). For B-cell enrichment, the pass-through CD43- MACS fraction was used as a source of total B cells. The purity of B-cell fraction was typically greater than 97% as judged by CD19/B220 fluorescence-activated cell sorting staining. The ratio between the CD21intCD23+ follicular B cells and CD21highCD23low/- marginal zone B cells was approximately 8:1 to 15:1 in control strains and in young (4-week-old) MRL-lpr/lprmice. However, with age, this ratio became substantially lower in the lupus strain .
For INH-ODN potency studies, enriched primary macrophages, total splenic B cells, RAW264 macrophages, and the human B-cell line (Namalwa) were incubated for 18 to 42 hours with optimal concentrations of class A(D) (100 nM), class B(K) stimulatory CpG-ODNs (10 to 33 nM), TLR7/8 ligands R-837 (1 μg/mL), or CL-075 (0.1 μg/mL) plus increasing concentrations of INH-ODNs (from 1 to 1,000 nM). B-cell cycle entry and protection from spontaneous apoptosis was detected using acridine orange flow cytometry as described previously . Cell culture supernatants were collected and tested for cytokines in enzyme-linked immunosorbent assay (ELISA). For determination of polyclonal IgM, B cells were cultured for 6 days.
DNA – or RNA- immune complex-stimulated AM14 B cells and dendritic cells
B cells were isolated from AM14 BCR transgenic mice by positive selection using anti-B220-coupled magnetic beads . AM14 B cells were stimulated with the IgG2a monoclonal antibodies (mAbs) PL2-3, as a form of chromatin-containing immune complexes , or BWR4 (10 μg/mL) (kindly provided by Dan Eilat, Hadassah University Hospital, Jerusalem, Israel) as a form of RNA immune complexes . Results were confirmed by stimulating AM14 B with anti-Sm antibody Y2 (20 μg/mL final concentration) (kindly provided by Philip Cohen and Robert Eisenberg, University of Pennsylvania, Philadelphia, PA, USA) pre-mixed with purified endotoxin-free Sm/RNP (0.628 mg/mL). For the BWR4 and Y2 studies, B cells were pre-treated with IFN-α (PBL Laboratories) (1,000 U/mL IFN-α for 2 to 3 hours at 37°C) to upregulate TLR7 expression, resulting in markedly enhanced proliferation upon stimulation with BWR4 antibodies. In some experiments, AM14 B cells were also stimulated with linear CpG B(K) ODN-1826 (0.3 μg/mL) or ultrapure LPS (List Biological Laboratories, Inc.).
Control ODN 4173, INH-1, INH-18, and their variants were added to cultures simultaneously with the DNA or RNA immune complexes. Cell proliferation was measured after 24 hours by pulsing B cells for an additional 6 hours with [3H] thymidine. On their own, Sm/RNP particles had no activity in B cells. Non-transgenic B cells, in contrast to AM14 B cells, failed to proliferate to BWR4 antibodies, whereas TLR9-deficient AM14 B cells still proliferated, thus ruling out cross-reactivity with DNA in culture fluids.
Bone marrow-derived DCs from Balb/c mice were cultured with the Flt-3L for 8 days. Highly enriched DCs (3 × 104 cells per 200 μL volumes) were additionally cultured over the course of a 24-hour period with DNA immune complexes containing CG50 (dsDNA fragment derived from the plasmid pMCG-50 containing 50 repeats of CpG and used at 100 ng/mL ) combined with PA4 IgG2a anti-dsDNA antibodies (kindly provided by Mark Monestier, Temple University, Philadelphia, PA, USA) (10 μg/mL). INH-ODNs were pre-incubated with DCs for 30 minutes before adding immune complexes. In parallel experiments, class A(D) CpG-ODN 2336 was used for stimulation instead (at a concentration of 300 nM). Supernatants were collected and IFN-α measured in ELISA.
For TLR7-dependent stimulation of DCs, RNA immune complexes were made by using the 05-02A antibody preparation (SLE1)  from a human lupus patient at 50 μg/mL. Control ODN, INH-1 and INH-18, and their variants lacking the CCT and/or GGG were added to RNA/anti-RNA-stimulated DC cultures at a concentration of 1 μg/mL. IFN-α was measured after 24 hours.
Enzyme-linked immunosorbent assay and immunofluorescence studies for autoantibody detection
Serum samples from PBS- or INH-ODN-treated mice were diluted appropriately and tested for antinuclear and anti-dsDNA antibodies using HEp-2 or Crithidia luciliae-coated slides, respectively (Inova Diagnostics, Inc., San Diego, CA, USA). Bound IgG was revealed using fluorescein isothiocyanate-labeled anti-mouse IgG (Bethyl Laboratories, Inc., Montgomery, TX, USA). Specific staining of kinetoplasts on Crithidia slides, detected by immunofluorescence on an Olympus BX-51 microscope (Olympus, Tokyo, Japan), was used as a criterion for the presence of anti-dsDNA antibodies in lupus sera.
Autoantibody levels against dsDNA and Sm/RNP were further quantified by ELISA. Calf thymus Sm/nRNP antigen was purchased from ImmunoVision (Springdale, AR, USA). Ultrapure calf-thymus dsDNA was from Sigma-Aldrich (St. Louis, MO, USA). For anti-dsDNA detection, polystyrene plates were pre-coated with poly-L-lysine followed by S1 nuclease-treated dsDNA (coated at 5 μg/mL). Lupus sera were diluted 1:500 and incubated on dsDNA-coated plates for 30 minutes at room temperature. After extensive washings, bound IgG was detected using horseradish peroxidase (HRP)-labeled anti-mouse IgG1, IgG2a, or IgM antibodies (Bethyl Laboratories, Inc.), respectively. A similar procedure was used for the detection of anti-Sm/RNP antibodies on plates coated with 2.5 μg/mL Sm/nRNP.
Cytokine enzyme-linked immunosorbent assay
IL-6, TNF-α, and IL-12p40 were detected using pairs of antibodies obtained from eBioscience, Inc. (San Diego, CA, USA). For detection of type I IFN secretion , we used rat anti-mouse IFN-α mAb (22100-1; PBL Biomedical Laboratories, Piscataway, NJ, USA), rabbit anti-mouse IFN-α polyclonal antibody (32100-1; PBL Biomedical Laboratories), HRP-conjugated donkey anti-rabbit IgG (711-036-152; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), and mouse r-IFN-α as a standard (12100-1; PBL Biomedical Laboratories). The detection limit of the IFN-α ELISA was 80 pg/mL.
Prototypic class R and class B INH-ODNs have similar potencies and efficacies in macrophages and bone marrow-derived dendritic cells
Bone marrow-derived Flt-3L-propagated DCs secreted IFN-α in response to the class A(D) stimulatory CpG-ODN 2336 and gave a smaller response to CG50/PA4 immune complexes. Neither class of INH-ODNs nor control ODNs induced measurable IFN-α secretion (Figure 1b). When added to DC cultures simultaneously with TLR9 ligands, class R (INH-1) and class B (INH-18) INH-ODNs (but not the control ODN) showed similar inhibitory potency for TLR9 ligand-induced IFN-α production (Figure 1c–e). Figure 1e shows the dose response for inhibition of CG50+PA4-induced IFN-α secretion.
Palindromic INH-ODNs with phosphorothioate backbones show 10- to 30-fold lower potency than linear INH-ODNs for inhibiting Toll-like receptor-9 stimulation of primary mouse and human B cells
To check whether the observed difference extends to INH-ODNs synthesized with the natural PO backbone, INH-1 and INH-18 were made with the PO backbone. In this case, we used a stimulatory CpG-ODN also made with the PO backbone at a concentration of 10 μM. Various outcomes (for example, IL-6, G1-M entry, and IgM secretion) were measured, and dose-dependent inhibition of IgM secretion is shown. Again, at least a 30-fold potency difference for IgM secretion between PO backbone versions of INH-1 and INH-18 was observed (Figure 2e).
Similar to resting mouse B cells, the human B-cell line Namalwa, expressing a high level of TLR9 , was sensitive to inhibition with both class R and class B INH-ODNs (Figure 2f) when CD86 upregulation was measured as an outcome. However, again class B INH-ODNs (INH-13 is shown) were 10 times more potent than class R (INH-4) (Figure 2f). Similar results were observed in human peripheral blood B cells and with INH-1 compared with INH-18 (data not shown).
The size of the single-stranded overhang in INH-ODNs with partial palindromes determines the potency difference between class B and class R INH-ODNs
Class R INH-ODNs require canonical CCT and GGG triplets for inhibition of rheumatoid factor-specific AM14 B cells stimulated with DNA-containing immune complexes
INH-ODNs made with phosphorothioate backbone inhibit activation of dendritic cells, macrophages, and RNA/anti-RNA-stimulated AM14 B cells in a sequence-independent and backbone-dependent fashion
Class R INH-ODNs block anti-double-stranded DNA and anti-Sm/RNP secretion in vivoin lupus mice
It was first observed by Pisetsky and his group [51, 52] that several G-rich ODNs made with the PS, but not PO, backbone had inhibitory activity. We and others [39, 40, 53–61] have extended these early observations to define structural requirements for TLR9 stimulation and inhibition in the nanomolar range. Strikingly, changes at the 5' end of an ODN (either inhibitory or stimulatory), particularly those affecting the pyrimidine-rich CCT triplet, diminished both the stimulatory and inhibitory activity for the TLR9 pathway [39, 62]. We have also identified a stretch of three (four) consecutive Gs necessary for optimal inhibition in the nanomolar range [39, 40, 63].
Could DNA-like therapeutics offer a pathway-specific tool for treating systemic lupus? Variants of our prototypic INH-ODN 2114 (TCCTGGAGGGGAAGT) [44, 64] have already been tested in lupus-prone animals. For example, Patole and colleagues  found that INH-ODN 2114 was active in the MRL-lpr/lprstrain, whereas Klinman's group  discovered that an ODN containing multiple TTAGGG repeats, like those in telomeric DNA, was inhibitory in lupus-prone NZB/NZW F1 mice. Barrat and colleagues  recently obtained similar results in the NZB/W-F1 strain, with an INH-ODN that combined the canonical TLR9-inhibitory motif with a TLR7-specific TGC triplet at the 5' end. One dsDNA-based biocompound LJP 394 (Abetimus) developed by the La Jolla Pharmaceutical Company (San Diego, CA, USA) has already entered clinical trials in human lupus and showed some promising results (for example, ability to decrease anti-dsDNA antibody production in vivo) .
The current report addresses the relevance of secondary structure to INH-ODN activity. We created class R INH-ODNs by starting with the shortest strongly active 12-mer linear INH-ODN 4084-F (CCTGGATGGGAA) (Table 1) and then extending it with 12 more bases, resulting in complete palindromes (class R) or in non-palindromic linear sequences (class B). Class R INH-ODNs were between 10- to 30-fold less potent as inhibitors in resting mouse B cells than class B INH-ODNs when synthetic linear TLR9 agonists were used for stimulation (Figure 2). Similar findings were obtained in human primary B cells/B-cell lines. In contrast to resting B cells, when 'professional antigen-presenting cells' (for example, primary macrophages and bone marrow-derived DCs) were stimulated either with complex CpG-class A(D) ODNs or with DNA-containing immune complexes, classes R and B INH-ODNs showed very similar inhibitory potencies (Figure 1). Stimulation with these complex TLR9 ligands is believed to occur in DCs in an early endosomal compartment [7, 8].
This difference in inhibitory activity between class B and class R INH-ODNs in resting (follicular) B cells was clearly dependent on the ability of INH-ODNs to make double-stranded secondary structures. The biggest decrease in potency was observed with class R INH-ODNs containing either complete palindromes or short (up to three nucleotides) single-stranded 5' or 3' linear overhangs (Figure 3). Further increase in the length of the single-stranded overhang progressively reduced the difference between linear and class R INH-ODNs.
These results suggest a hypothesis that, in B cells (though not in macrophages), dsDNA (including class R INH-ODNs) has limited access to endosomal TLR9 relative to single-stranded DNA. Single-stranded CpG-ODNs have been shown to encounter TLR9 in LAMP-1-positive endosomes [8, 9]. We postulated that, if dsDNA gains access to these endosomes by BCR-mediated entry, the difference in potencies between double-stranded and single-stranded versions of the same inhibitory sequence would disappear. This hypothesis could be tested in transgenic B cells expressing a BCR either for dsDNA or for Ig by stimulating the cells with one or both of these components.
Thus, important proofs of concept emerged from studies in AM14 B cells (Figures 7 and 8). These rheumatoid factor-specific B cells express a transgenic BCR derived from hybridoma-secreting anti-Ig-specific autoantibodies . Their BCR recognizes the Fc-fragment of IgG2a antibodies expressing 'a' but not 'b' allotype . In vitro, AM14 B cells proliferate when anti-nucleosome antibodies (PL2-3) are added to spent cultures (Figure 4). This proliferation is sensitive to DNAse treatment and requires an intact MyD88/TLR9 pathway [25, 34], clearly arguing for a role of endogenous DNA in PL2-3-induced AM14 B-cell activation. Similar to other TLR9-expressing cells, AM14 B cells proliferate vigorously to stimulation with linear CpG ligands (Figure 4a).
These results suggest that PL2-3 binds nucleosome-associated chromatin released into the cultures from dying cells and that these immune complexes engage the anti-Ig BCR, deliver 'signal 1', and enter the cell . The dsDNA they contain then reaches TLR9 in the endosome ('autophagosomes' ), otherwise accessible only to single-stranded DNA. Therefore, AM14 B cells represent an excellent model that can be used to contrast data obtained through BCR-dependent and BCR-independent TLR9-mediated signaling and to test some predictions of this hypothesis. As in non-autoreactive B cells, our results show that 'BCR-independent' stimulation with linear CpG-ODN 1826 was differentially inhibited by class R and class B INH-ODNs, with class B 10-fold more potent than class R (Figure 4a). However, in 'BCR-dependent' immune complex-induced B-cell proliferation, classes B and R INH-ODNs showed similar potencies for inhibition, primarily because of the increased potency of class R INH-ODNs for BCR-mediated activation (Figure 4b).
While the exact mechanism of class R INH-ODNs action in autoreactive B cells remains to be determined, several possibilities may be considered: (a) competition between TLR9-inhibiting ODNs and endogenous DNA for binding to PL2-3 antibodies/nucleosome/HMGB1 complex, (b) competition for binding to membrane RAGE (receptor for advanced glycation endproducts)/BCR complex, (c) preferential recruitment of TLR9 into early versus late endosomes depending on the nature of the TLR9 ligand used, (d) BCR-dependent increase in passive endocytosis of class R INH-ODNs, and (e) BCR-dependent recruitment of specific helicases (DNA-unzipping enzymes) into an early endosomal compartment. These possibilities are not mutually exclusive. For example, inhibition of BCR signaling might result from both better uptake/trafficking of class R INH-ODNs into endosomes and better recruitment of helicases into TLR9-containing compartments. Helicases must have an important role in the class R INH-ODN-mediated inhibition because recent affinity studies revealed superior binding of single-stranded TLR9 ligands to chip-immobilized TLR9 , an event that may depend heavily on the sugar backbone of the TLR9 ligands used for stimulation .
Another possibility is a direct competition between endogenous DNA and INH-ODNs for binding to anti-dsDNA antibodies. While this possibility may account for some competition observed in the PL2-3 model, there are no data in the literature to suggest that natural anti-dsDNA antibodies (or anti-chromatin antibodies) preferentially bind DNA segments carrying the canonical inhibitory motif over other DNA sequences .
The possibility that the double-stranded structure of class R INH-ODNs may direct these ODNs preferentially to an early endosomal compartment in macrophages and DCs and into BCR-related 'autophagosomes' in autoreactive B cells has a precedent in a work by Guiducci and colleagues . These authors have found that IFN-α secretion from human plasmacytoid DCs induced by multimeric class A(D) CpG-ODNs occurs in transferring receptor-positive endosomes whereas linear monomeric CpG-ODNs (like CpG-1826 used in our experiments) preferentially localize to LAMP-1-positive endosomes and are poor stimulators of IFN-α secretion. However, when linear CpG ligands are complexed into microparticles, they now gain the ability to move to transferrin receptor-positive endosomes, inducing robust IFN-α production. Therefore, the most important determinant of TLR9 signaling may be its endosomal localization [7, 8] or alternatively CXCL16 engagement as suggested by the Klinman's group . For one or more of these reasons, class R INH-ODNs have higher potency for BCR-dependent TLR9 activation in autoreactive B cells than for BCR-independent activation of normal B cells.
In this article, we further question the ability of 'TLR9-specific' INH-ODNs to target additional TLR signaling pathways. While our previous data  and data from several other groups [60, 61] have clearly identified that INH-ODNs at concentrations of up to 1 μM fail to inhibit signaling through the TLR2, 3, and 4 pathways, recent literature has suggested a possibility that PS-ODNs, including INH-ODNs, may also block the TLR7 signaling pathway . It is believed that, in mice (in contrast to humans), the TLR8 pathway is non-functional since cells from TLR7-/- mice fail to respond to stimulation with synthetic TLR7/8 ligands . Here, we show that our INH-ODNs can block TLR7-dependent activation of primary macrophages, macrophage cell lines, RNA immune complex-activated DCs, and AM14 B cells in a dose-dependent but sequence-independent manner (Figure 6a–d). However, in spite of these sequence-independent inhibitory effects, palindromic INH-ODNs still have lower potency for resting B cells (Figure 6e), but classes B and R are equally potent and effective in RNA/anti-RNA-stimulated AM14 B cells (Figure 6d). Here again, BCR engagement is associated with greater potency of class R INH-ODNs.
Finally, in vivo, in the MRL-Faslpr/lprstrain, class R INH-ODNs were better than class B INH-ODNs as inhibitors of both IgG anti-dsDNA and anti-Sm/RNP antibody secretion (Figure 8). These results may have significant impact for developing novel DNA-like therapeutics for treating lupus. While TLR7-/- MRL-Faslpr/lprlupus mice have better survival, they still succumb to autoimmune disease , thus suggesting a possible role for additional DNA/RNA-triggered intracellular signaling pathways (for example, DAI and RIG-I). Interestingly, in this strain, complete lack of TLR9 results in two opposite outcomes: diminished anti-dsDNA secretion but increased anti-Sm/RNP antibody production . Moreover, the survival of these mice is reduced, likely due to insufficient number/function of regulatory T cells . One can wonder whether the ability of class R INH-ODNs to block both TLR9 and TLR7 activation in autoreactive B cells may explain the beneficial effect of these INH-ODNs on autoantibody secretion in vivo. The inferior efficacy of more in vitro potent class B INH-ODNs in lupus mice came as a surprise. While we lack a logical explanation for this result, recent literature suggests that PS-ODNs (including TLR9-specific INH-ODNs) may redirect TLR7/8 ligand-induced activation away from the TLR7 toward the TLR8 pathway, as observed in HEK cells expressing TLR7 and TLR8 and in primary human peripheral blood mononuclear cells  and mouse cells . This redirection of TLR activation may result in decreased IFN-α secretion from plasmacytoid DCs but a corresponding increase in IL-12, TNF-α, and IFN-γ secretion resulting from TLR8-expressing cells (for example, human monocytes) .
Future studies should explore the efficacy of class R INH-ODNs in different strains of lupus mice, including the BXSB male mice. This lupus strain harbors a duplication of the TLR7 gene which appears to be responsible for the phenotype [47, 48]. Moreover, refinement of the palindromic structure to generate combined selective TLR7/TLR9 inhibitors  together with anti-B-cell-depleting protocols  to re-establish critical B-cell differentiation checkpoints  may result in better treatments for human lupus.
Class R INH-ODNs have a high potency for autoreactive B cells and DCs in vitro and are effective in the MRL-Faslpr/lprmodel of lupus in vivo.
enzyme-linked immunosorbent assay
magnetic-activated cell sorting
myeloid differentiation primary response gene 88
systemic lupus erythematosus
tumor necrosis factor.
This work was supported by National Institutes of Health grants AI047374 and AI064736 (both to PL) and a grant from the Alliance for Lupus Research (to AM-R).
- Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S: A toll-like receptor recognizes bacterial DNA. Nature. 2000, 408: 740-745. 10.1038/35047123.View ArticlePubMedGoogle Scholar
- Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol. 2003, 21: 335-376. 10.1146/annurev.immunol.21.120601.141126.View ArticlePubMedGoogle Scholar
- Rutz M, Metzger J, Gellert T, Luppa P, Lipford GB, Wagner H, Bauer S: Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol. 2004, 34: 2541-2550. 10.1002/eji.200425218.View ArticlePubMedGoogle Scholar
- Haas T, Metzger J, Schmitz F, Heit A, Muller T, Latz E, Wagner H: The DNA sugar backbone 2' deoxyribose determines Toll-like receptor 9 activation. Immunity. 2008, 28: 315-323. 10.1016/j.immuni.2008.01.013.View ArticlePubMedGoogle Scholar
- Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT: TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol. 2004, 5: 190-198. 10.1038/ni1028.View ArticlePubMedGoogle Scholar
- Ewald SE, Lee BL, Lau L, Wickliffe KE, Shi G-P, Chapman HA, Barton GM: The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature. 2008, 456: 658-662. 10.1038/nature07405.PubMed CentralView ArticlePubMedGoogle Scholar
- Honda K, Ohba Y, Yannai H, Negishi H, Mizutani T, Takaoka A, Taya C, Taniguchi T: Spatiotemporal regulation of MyD88-IRF-7 signaling for robust type-I interferon induction. Nature. 2005, 434: 1035-1040. 10.1038/nature03547.View ArticlePubMedGoogle Scholar
- Guiducci C, Ott G, Chan JH, Damon E, Calacsan C, Matray T, Lee KD, Coffman RL, Barrat FJ: Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J Exp Med. 2006, 203: 1999-2008. 10.1084/jem.20060401.PubMed CentralView ArticlePubMedGoogle Scholar
- Gursel M, Gursel I, Mostowski HS, Klinman DM: CXCL16 influences the nature and specificity of CpG-induced immune activation. J Immunol. 2006, 177: 1575-1580.View ArticlePubMedGoogle Scholar
- Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman DM: Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J Immunol. 2001, 166: 2372-2377.View ArticlePubMedGoogle Scholar
- Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, Ballas ZK, Endres S, Krieg AM, Hartmann G: Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmocytoid dendritic cells. Eur J Immunol. 2001, 31: 2154-2163. 10.1002/1521-4141(200107)31:7<2154::AID-IMMU2154>3.0.CO;2-U.View ArticlePubMedGoogle Scholar
- Brummel R, Lenert P: Activation of marginal zone B cells from lupus mice with type A(D) CpG-oligodeoxynucleotides. J Immunol. 2005, 174: 2429-2434.View ArticlePubMedGoogle Scholar
- Brummel R, Roberts TL, Stacey KJ, Lenert P: Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice. Eur J Immunol. 2006, 36: 1951-1962. 10.1002/eji.200535734.View ArticlePubMedGoogle Scholar
- Goeckeritz BE, Flora M, Witherspoon K, Vos Q, Lees A, Dennis GJ, Pisetsky DS, Snapper CM, Mond J: Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynuclotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion. Int Immunol. 1999, 11: 1693-1700. 10.1093/intimm/11.10.1693.View ArticlePubMedGoogle Scholar
- Yi AK, Yoon JG, Krieg AM: Convergence of CpG-DNA- and BCR-mediated signals at the c-Jun N-terminal kinase and NF-kappa B activation pathways: regulation by mitogen-activated protein kinases. Int Immunol. 2003, 15: 577-591. 10.1093/intimm/dxg058.View ArticlePubMedGoogle Scholar
- Poeck H, Wagner M, Battiany J, Rothenfusser S, Wellisch D, Hornung V, Jahrsdorfer B, Giese T, Endres S, Hartmann G: Plasmacytoid dendritic cells, antigen, and CpG-C license human B cells for plasma cell differentiation and immunoglobulin production in the absence of T-cell help. Blood. 2004, 103: 3058-3064. 10.1182/blood-2003-08-2972.View ArticlePubMedGoogle Scholar
- Busconi L, Bauer JW, Tumang JR, Laws A, Perkins-Mesires K, Tabor AS, Lau C, Corley RB, Rothstein TL, Lund FE, Behrens TW, Marshak-Rothstein A: Functional outcome of B cell activation by chromatin immune complex engagement of the B cell receptor and TLR9. J Immunol. 2007, 179: 7397-7405.View ArticlePubMedGoogle Scholar
- Chaturvedi A, Dorward D, Pierce SK: The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNA-containing antigens. Immunity. 2008, 28: 799-809. 10.1016/j.immuni.2008.03.019.PubMed CentralView ArticlePubMedGoogle Scholar
- Uccellini MB, Busconi L, Green NM, Busto P, Christensen SR, Shlomchik MJ, Marshak-Rothstein A, Viglianti GA: Autoreactive B cells discriminate CpG-Rich and CpG-Poor DNA and this response is modulated by IFN-α. J Immunol. 2008, 181: 5875-5884.PubMed CentralView ArticlePubMedGoogle Scholar
- Tan EM: Antinuclear antibodies: diagnostic markers for autoimmune diseases and probes for cell biology. Adv Immunol. 1989, 44: 93-151. 10.1016/S0065-2776(08)60641-0.View ArticlePubMedGoogle Scholar
- Mohan C, Adams S, Stanik V, Datta SK: Nucleosome: a major immunogen for the pathogenic autoantibody-inducing T cells of lupus. J Exp Med. 1993, 177: 1367-1381. 10.1084/jem.177.5.1367.View ArticlePubMedGoogle Scholar
- Radic MZ, Weigert M: Genetic and structural evidence for antigen selection of anti-DNA antibodies. Annu Rev Immunol. 1994, 12: 487-520. 10.1146/annurev.iy.12.040194.002415.View ArticlePubMedGoogle Scholar
- Lipsky PE: Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity. Nat Immunol. 2001, 2: 764-766. 10.1038/ni0901-764.View ArticlePubMedGoogle Scholar
- Vallin H, Perers A, Alm GV, Ronnblom L: Anti-double-stranded DNA antibodies and immunostimulatory plasmid DNA in combination mimic the endogenous IFN-α inducer in systemic lupus erythematosus. J Immunol. 1999, 163: 6306-6313.PubMedGoogle Scholar
- Leadbetter EA, Rifkin IR, Hohlbaum AM, Beaudette BC, Schlomchik MJ, Marshak-Rothstein A: Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature. 2002, 416: 603-607. 10.1038/416603a.View ArticlePubMedGoogle Scholar
- Viglianti GA, Lau CM, Hanley TM, Miko BA, Shlomchik MJ, Marshak-Rothstein A: Activation of autoreactive B cells by CpG dsDNA. Immunity. 2003, 19: 837-847. 10.1016/S1074-7613(03)00323-6.View ArticlePubMedGoogle Scholar
- Boule MW, Broughton C, Mackay F, Akira S, Marshak-Rothstein A, Rifkin IR: Toll-like receptor 9 – dependent and – independent dendritic cell activation by chromatin-immunoglobulin G complexes. J Exp Med. 2004, 199: 1631-1640. 10.1084/jem.20031942.PubMed CentralView ArticlePubMedGoogle Scholar
- Lau CM, Broughton C, Tabor AS, Akira S, Flavell RA, Mamula MJ, Christensen SR, Shlomchik MJ, Viglianti GA, Rifkin IR, Marshak-Rothstein A: RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement. J Exp Med. 2005, 202: 1171-1177. 10.1084/jem.20050630.PubMed CentralView ArticlePubMedGoogle Scholar
- Rifkin IR, Leadbetter EA, Busconi L, Viglianti G, Marshak-Rothstein A: Toll-like receptors, endogenous ligands, and systemic autoimmune disease. Immunol Rev. 2005, 204: 27-42. 10.1111/j.0105-2896.2005.00239.x.View ArticlePubMedGoogle Scholar
- Barrat FJ, Meeker T, Gregorio J, Chan JH, Uematsu S, Akira S, Chang B, Duramad O, Coffman RL: Nucleic acids of mammalian origin act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. J Exp Med. 2005, 202: 1131-1139. 10.1084/jem.20050914.PubMed CentralView ArticlePubMedGoogle Scholar
- Kelly KM, Zhuang H, Nacionales DC, Scumpia PO, Lyons R, Akaogi J, Lee P, Williams B, Yamamoto M, Akira S, Satoh M, Reeves WH: 'Endogenous adjuvant' activity of the RNA components of lupus autoantigens Sm/RNP and Ro 60. Arthritis Rheum. 2006, 54: 1557-1567. 10.1002/art.21819.View ArticlePubMedGoogle Scholar
- Marshak-Rothstein A, Rifkin IR: Immunologically active autoantigens: the role of Toll-like receptors in the development of chronic inflammatory disease. Annu Rev Immunol. 2007, 25: 419-441. 10.1146/annurev.immunol.22.012703.104514.View ArticlePubMedGoogle Scholar
- Tian J, Avalos AM, Mao SY, Chen B, Senthil K, Wu H, Parroche P, Drabic S, Golenbock D, Sirois C, Hua J, An LL, Audoly L, La Rosa G, Bierhaus A, Naworth P, Marshak-Rothstein A, Crow MK, Fitzgerald KA, Latz E, Kiener PA, Coyle AJ: Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol. 2007, 8: 487-496. 10.1038/ni1457.View ArticlePubMedGoogle Scholar
- Herlands RA, Christensen SR, Sweet RA, Hershberg U, Shlomchik MJ: T cell-independent and Toll-like receptor-dependent antigen-driven activation of autoreactive B cells. Immunity. 2008, 29: 249-260. 10.1016/j.immuni.2008.06.009.PubMed CentralView ArticlePubMedGoogle Scholar
- Dong L, Ito S, Ishii KJ, Klinman DM: Suppressive oligodeoxynucleotides delay the onset of glomerulonephritis and prolong survival in lupus-prone NZB × NZW mice. Arthritis Rheum. 2005, 52: 651-658. 10.1002/art.20810.View ArticlePubMedGoogle Scholar
- Patole PS, Zecher D, Pawar RD, Grone HJ, Schlondorff D, Anders HJ: G-rich DNA suppresses systemic lupus. J Am Soc Nephrol. 2005, 16: 3273-3280. 10.1681/ASN.2005060658.View ArticlePubMedGoogle Scholar
- Barrat FJ, Meeker T, Chan JH, Guiducci C, Coffman RL: Treatment of lupus-prone mice with a dual inhibitor of TLR7 and TLR9 leads to reduction of autoantibody production and amelioration of disease symptoms. Eur J Immunol. 2007, 37: 3582-3586. 10.1002/eji.200737815.View ArticlePubMedGoogle Scholar
- Lenert P: Targeting Toll-like receptor signaling in plasmacytoid dendritic cells and autoreactive B cells as a therapy for lupus. Arthritis Res Ther. 2006, 8: 203-10.1186/ar1888.PubMed CentralView ArticlePubMedGoogle Scholar
- Lenert P, Rasmussen W, Ashman RF, Ballas ZK: Structural characterization of the inhibitory DNA motif for the type A[D]-CpG-induced cytokine secretion and NK-cell lytic activity in mouse spleen cells. DNA Cell Biol. 2003, 22: 621-631. 10.1089/104454903770238094.View ArticlePubMedGoogle Scholar
- Ashman RF, Goeken JA, Drahos J, Lenert P: Sequence requirements for oligodeoxyribonucleotides inhibitory activity. Int Immunol. 2005, 17: 411-420. 10.1093/intimm/dxh222.View ArticlePubMedGoogle Scholar
- Shlomchik MJ, Zharhary D, Camper S, Saunders T, Weigert MP: A rheumatoid factor transgenic mouse model of autoantibody regulation. Int Immunol. 1993, 5: 1329-1341. 10.1093/intimm/5.10.1329.View ArticlePubMedGoogle Scholar
- Jacobson BA, Sharon J, Shan H, Shlomchik M, Weigert MG, Marshak-Rothstein A: An isotype switched and somatically mutated rheumatoid factor clone isolated from a MRL-lpr/lpr mouse exhibits limited intraclonal affinity maturation. J Immunol. 1994, 152: 4489-4499.PubMedGoogle Scholar
- Hannum LG, Ni D, Haberman AM, Weigert MG, Shlomchik MJ: A disease-related rheumatoid factor autoantibody is not tolerized in a normal mouse: implications for the origins of autoantibodies in autoimmune disease. J Exp Med. 1996, 184: 1269-1278. 10.1084/jem.184.4.1269.View ArticlePubMedGoogle Scholar
- Stunz LL, Lenert P, Peckham D, Yi AK, Haxhinasto S, Chang M, Krieg AM, Ashman RF: Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur J Immunol. 2002, 32: 1212-1222. 10.1002/1521-4141(200205)32:5<1212::AID-IMMU1212>3.0.CO;2-D.View ArticlePubMedGoogle Scholar
- Yasuda K, Richez C, Maciaszek JW, Agrawal N, Akira S, Marshak-Rothstein A, Rifkin IR: Murine dendritic cell type I IFN production induced by human IgG-RNA immune complexes is IFN regulatory factor (IRF)5 and IRF7 dependent and is required for IL-6 production. J Immunol. 2007, 178: 6876-6885.View ArticlePubMedGoogle Scholar
- Henault M, Lee LN, Evans GF, Zuckerman SH: The human Burkitt lymphoma cell line Namalwa represents a homogenous cell system characterized by high levels of Toll-like receptor 9 and activation by CpG oligonucleotides. J Immunol Methods. 2005, 300: 93-99. 10.1016/j.jim.2005.02.012.View ArticlePubMedGoogle Scholar
- Subramanian S, Tus K, Li QZ, Wang A, Tian XH, Zhou J, Liang C, Bartov G, McDaniel LD, Zhou XJ, Schultz RA, Wakeland EK: A TLR7 translocation accelerates systemic autoimmunity in murine lupus. Proc Natl Acad Sci USA. 2006, 103: 9970-9975. 10.1073/pnas.0603912103.PubMed CentralView ArticlePubMedGoogle Scholar
- Pisitkun P, Deane JD, Diffilipantonio MJ, Tarasenko T, Satterthwaite AB, Bolland S: Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication. Science. 2006, 312: 1669-1672. 10.1126/science.1124978.View ArticlePubMedGoogle Scholar
- Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RA, Shlomchik MJ: Toll-like receptor 7 and TLR9 dictate autoantibody specificity and opposing inflammatory and regulatory roles in a murine model of lupus. Immunity. 2006, 25: 417-428. 10.1016/j.immuni.2006.07.013.View ArticlePubMedGoogle Scholar
- Andrews BS, Eisenberg RA, Theofilopoulos AN, Izui S, Wilson CB, McConahey PJ, Murphy ED, Roths JB, Dixon FJ: Spontaneous murine lupus-like syndromes: clinical and immunopathological manifestations in several strains. J Exp Med. 1978, 148: 1198-1215. 10.1084/jem.148.5.1198.View ArticlePubMedGoogle Scholar
- Halpern MD, Pisetsky DS: In vitro inhibition of murine IFNγ production by phosphorothioate deoxyguanosine oligomers. Immunopharmacology. 1995, 29: 47-52. 10.1016/0162-3109(95)00043-S.View ArticlePubMedGoogle Scholar
- Pisetsky DS, Reich CF: Inhibition of murine macrophage IL-12 production by natural and synthetic DNA. Clin Immunol. 2000, 96: 198-204. 10.1006/clim.2000.4897.View ArticlePubMedGoogle Scholar
- Shirota H, Gursel M, Klinman DM: Suppressive oligodeoxynucleotides inhibit Th1 differentiation by blocking IFN-gamma- and IL-12-mediated signaling. J Immunol. 2004, 173: 5002-5007.View ArticlePubMedGoogle Scholar
- Krieg AM, Wu T, Weeratna R, Efler SM, Love-Homan L, Yang L, Yi AK, Short D, Davis HL: Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci USA. 1998, 95: 12631-12636. 10.1073/pnas.95.21.12631.PubMed CentralView ArticlePubMedGoogle Scholar
- Lenert P, Stunz L, Yi AK, Krieg AM, Ashman RF: CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-κB activation. Antisense Nucleic Acid Drug Dev. 2001, 11: 247-256. 10.1089/108729001317022241.View ArticlePubMedGoogle Scholar
- Lenert P, Yi AK, Krieg AM, Stunz LL, Ashman RF: Inhibitory oligonucleotides block the induction of AP-1 transcription factor by stimulatory CpG oligonucleotides in B cells. Antisense Nucleic Acid Drug Dev. 2003, 13: 143-150. 10.1089/108729003768247600.View ArticlePubMedGoogle Scholar
- Klinman DM, Zeuner R, Yamada H, Gursel M, Currie D, Gursel I: Regulation of CpG-induced immune activation by suppressive oligodeoxynucleotides. Ann NY Acad Sci. 2003, 1002: 112-123. 10.1196/annals.1281.023.View ArticlePubMedGoogle Scholar
- Klinman DM, Gursel I, Klaschik S, Dong L, Currie D, Shirota H: Therapeutic potential of oligonucleotides expressing immunosuppressive TTAGGG motifs. Ann N Y Acad Sci. 2005, 1058: 87-95. 10.1196/annals.1359.015.View ArticlePubMedGoogle Scholar
- Gursel I, Gursel M, Yamada H, Ishii KJ, Takeshita F, Klinman DM: Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol. 2003, 171: 1393-1400.View ArticlePubMedGoogle Scholar
- Stacey K, Young GR, Clark F, Sester DP, Roberts TL, Naik S, Sweet MJ, Hume DA: The molecular basis for the lack of immunostimulatory activity of vertebrate DNA. J Immunol. 2003, 170: 3614-3620.View ArticlePubMedGoogle Scholar
- Duramad O, Fearon KL, Chang B, Chan JH, Gregorio J, Coffman RL, Barrat FJ: Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation. J Immunol. 2005, 174: 5193-5200.View ArticlePubMedGoogle Scholar
- Lenert P, Goeken JA, Ashman RF: Extended sequence preferences for oligodeoxyribonucleotide activity. Immunology. 2006, 117: 474-481. 10.1111/j.1365-2567.2006.02320.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Ashman RF, Lenert P: Structural requirements and applications of inhibitory oligodeoxyribonucleotides. Immunol Res. 2007, 39: 4-14. 10.1007/s12026-007-0065-4.View ArticlePubMedGoogle Scholar
- Lenert P: Inhibitory oligodeoxynucleotides – therapeutic promise for systemic autoimmune diseases?. Clin Exp Immunol. 2005, 140: 1-10. 10.1111/j.1365-2249.2004.02728.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Cardiel MH, Tumlin JA, Furie RA, Wallace DJ, Joh T, Linnik MD, LJP 394-90-09 Investigator Consortium: Abetimus sodium for renal flare in systemic lupus erythematosus: results of a randomized, controlled phase III trial. Arthritis Rheum. 2008, 58: 2470-2480. 10.1002/art.23673.View ArticlePubMedGoogle Scholar
- Wu X, Peng SL: Toll-like receptor 9 signaling protects against murine lupus. Arthritis Rheum. 2006, 54: 336-342. 10.1002/art.21553.View ArticlePubMedGoogle Scholar
- Jurk M, Kritzler A, Schulte B, Tluk S, Schetter C, Krieg AM, Volmer J: Modulating responsiveness of human TLR7 and 8 to small molecule ligands with T-rich phosphorothioate oligodeoxynucleotides. Eur J Immunol. 2006, 36: 1815-1826. 10.1002/eji.200535806.View ArticlePubMedGoogle Scholar
- Gorden KKB, Qiu XX, Binsfeld CCA, Vasilakos JP, Alkan SS: Cutting edge: activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and PolyT oligodeoxynucleotides. J Immunol. 2006, 177: 6584-6587.View ArticlePubMedGoogle Scholar
- Yurasov S, Wardemann H, Hammersen J, Tsuiji M, Meffre E, Pascual V, Nussenzweig MC: Defective B cell tolerance checkpoints in systemic lupus erythematosus. J Exp Med. 2005, 201: 703-711. 10.1084/jem.20042251.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.