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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/lpr mice in vivo

Introduction 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. Methods 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/lpr mice in vivo. Results 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/lpr mice in vivo, where the prototypic class R INH-ODN was more effective in curtailing abnormal autoantibody secretion and prolonging survival. Conclusions 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.


Introduction
Nucleic acids, including self DNA and RNA, are recognized by a subset of Toll-like receptors (TLRs) [1][2][3][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 inte-rior, 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 [9]. Interestingly, the outcome of the DC response to TLR9 stimulation varies greatly depending on where TLR9 meets CpG-DNA. For example, type I interferonalpha (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 [8].
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][11][12][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][14][15][16][17][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) [19]. 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][21][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 [20]. Complement levels frequently fall during major lupus flares, further suggesting that complement-activating immune complexes may play an important role in the tissue pathology [20].
We have recently developed a new class of inhibitory ODNs that we named class R ('restricted') INH-ODNs [38]. We show that these dsDNA-like analogues carrying the canonical TLR9inhibitory 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 sequenceindependent 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 lupusprone 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 We used INH-ODN 4084-F, with a PS backbone, as a template for creating class R INH-ODNs (Table 1). INH-ODN 4084-F is the shortest active 12-mer INH-ODN that contains both previously identified suppressive elements (CCT and GGG), appropriately spaced from each other (four nucleotides apart) and properly oriented in a single-stranded ODN molecule (5'-CCT → GGG-3') [39]  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 [35]. 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 CD21 int CD23 + follicular B cells and CD21 high CD23 low/marginal zone B cells was approximately 8:1 to 15:1 in control strains and in young (4-week-old) MRL-lpr/lpr mice. However, with age, this ratio became substantially lower in the lupus strain [13].
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 [44]. 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 [17]. AM14 B cells were stimulated with the IgG2a monoclonal antibodies (mAbs) PL2-3, as a form of chromatin-containing immune complexes [25], or BWR4 (10 μg/mL) (kindly provided by Dan Eilat, Hadassah University Hospital, Jerusalem, Israel) as a form of RNA immune complexes [28].

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 precoated with poly-L-lysine followed by S 1 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 antimouse 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.

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
We next created INH-ODNs with partial palindromes and single-stranded linear segments at their 3' or 5' ends ranging from three to nine nucleotides in length. We reasoned that the selectivity favoring linear INH-ODNs in resting B cells may be lost if 5'→3' or 3'→ 5' helicases are recruited to the TLR9 signalosome. As predicted, creating INH-ODNs with progressively longer linear overhangs attached to their 3' or 5' ends increased the potency of such class R INH-ODNs in TLR9stimulated B cells, eventually abrogating the difference between the class B and class R INH-ODNs. Figure 3 shows results of apoptosis protection; however, very similar data were observed with other B-cell outcomes (for example, G 1 -M entry, IL-6, and IgM secretion). (page number not for citation purposes) 5). The two elements were equally important for inhibition, as observed both in class B and in class R INH-ODN variants tested, suggesting that the requirement for both CCT and GGG elements previously observed in non-autoreactive B cells also applies to AM14 B cells.

INH-ODNs made with phosphorothioate backbone inhibit activation of dendritic cells, macrophages, and RNA/anti-RNA-stimulated AM14 B cells in a sequenceindependent and backbone-dependent fashion
Recent literature suggests that some TLRs may bind their ligands through their sugar backbone residues and that possession of a PS backbone not only protects from nucleases but also increases affinity for the relevant TLR [3,4]. As TLR7 has restricted cell distribution in humans (plasmacytoid DCs and B cells) and has been linked to lupus pathogenesis in mice [47][48][49], we next tested the ability of class R and B INH-ODNs to block TLR7-induced activation of macrophages, DCs,

Class R INH-ODNs block anti-double-stranded DNA and anti-Sm/RNP secretion in vivo in lupus mice
To address whether class R INH-ODNs preferentially affect autoreactive B cells in vivo more than normal B cells, we used the MRL-Fas lpr/lpr model of lupus. In this model, mice develop massive lymphoproliferation (lymphadenomegaly and splenomegaly) due to the mutation in the Fas and additionally produce numerous autoantibodies, including anti-dsDNA and anti-Sm/RNP [50]. Several lines of evidence suggest that autoreactive B cells play a primary role in the pathogenesis of SLE, not only as autoantibody secreting effector cells but as key antigen-presenting cells [23]. When pre-diseased MRL-Fas lpr/lpr mice (2J strain) were treated with either class R or class B INH-ODNs intraperitoneally at a dose of 1 mg/kg three times weekly for 25 weeks, INH-ODN-treated lupus mice survived longer than vehicle-treated controls (Figure 7a). INH-1treated, but not INH-18-treated, mice also showed less lymphadenomegaly (Figure 7b), less proteinuria (Figure 7c), and decreased composite renal score (Figure 7d). As shown in Figures 7e and 7f, INH-1-treated mice displayed less prominent glomerular and peritubular IgG deposits. Furthermore, palindromic class R INH-ODNs were more effective than linear  Both classes of inhibitory oligonucleotides (INH-ODNs) require CCT and GGG triplets for the full inhibitory activity Both classes of inhibitory oligonucleotides (INH-ODNs) require CCT and GGG triplets for the full inhibitory activity. AM14 B cells were stimulated with PL2-3 immune complexes. Indicated INH-ODNs and control ODN-4173 (all at a concentration of 1 μg/mL) were added simultaneously. Proliferation was measured. One of two similar experiments is shown. class B INH-ODNs as inhibitors of both anti-dsDNA antibody secretion (detected by immunofluorescence and by ELISA) and of anti-Sm/RNP antibody secretion (as detected by ELISA) (Figure 8).

Discussion
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][54][55][56][57][58][59][60][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 [36] found that INH-ODN 2114 was active in the MRL-lpr/lpr strain, whereas Klinman's group [35] 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 [37] recently obtained similar results in the NZB/W-F1 strain, with an INH-ODN that com- Class R and B inhibitory oligonucleotides (INH-ODNs) inhibit Toll-like receptor-7 (TLR7)-dependent activation of macrophages, dendritic cells (DCs), AM14 B cells, and primary mouse B cells in a sequence-independent but backbone-dependent manner Class R and B inhibitory oligonucleotides (INH-ODNs) inhibit Toll-like receptor-7 (TLR7)-dependent activation of macrophages, dendritic cells (DCs), AM14 B cells, and primary mouse B cells in a sequence-independent but backbone-dependent manner. RAW264 .7 macrophages (a, b), Flt-3L-propagated bone marrow-derived DCs (c), AM14 B cells (d), and primary mouse resting B cells (e) were stimulated with TLR7/8 ligands (CL-075, R-837, or RNA immune complexes as indicated) with INH-ODNs or control ODNs added simultaneously. Tumor necrosis factor-alpha (TNF-α) and interferon-alpha (IFN-α) were measured in enzyme-linked immunosorbent assay. AM14 proliferation was determined by measuring the [ 3 H] thymidine incorporation for the last 6 hours. CD86 expression was determined by flow cytometry (n = 3 to 5). *P < 0.05 (ODN-treated versus mediumtreated samples). FITC, fluorescein isothiocyanate. bined 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) [65].
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  (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 [43]. Their BCR recognizes the Fc-fragment of IgG2a antibodies expressing 'a' but not 'b' allotype [25]. 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 PL2-3induced AM14 B-cell activation. Similar to other TLR9expressing 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 [25]. The dsDNA they contain then reaches TLR9 in the endosome ('autophagosomes' [18]), 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 BCRindependent 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 TLR9inhibiting 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) BCRdependent 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 [3], an event that may depend heavily on the sugar backbone of the TLR9 ligands used for stimulation [4].
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 [33].
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 [8]. 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 [9]. 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 [44] 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 [28]. 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 [2]. 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-Fas lpr/lpr strain, 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-Fas lpr/lpr lupus mice have better survival, they still succumb to autoimmune disease [49], 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 [49]. Moreover, the survival of these mice is reduced, likely due to insufficient number/function of regulatory T cells [66]. 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 [67] and mouse cells [68]. 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) [67].
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 [37] together with anti-B-cell-depleting protocols [23] to re-establish critical B-cell differentiation checkpoints [69] may result in better treatments for human lupus.