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-Fas(lpr/lpr) mice in vivo

Abstract

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.

Figure 1

Class R and B inhibitory oligonucleotides (INH-ODNs) have similar inhibitory potencies for Toll-like receptor-9 (TLR9)-activated primary macrophages and dendritic cells (DCs). (a) Enriched primary macrophages were stimulated with class A(D) CpG-ODN (100 nM) for 18 to 24 hours. INH-ODNs were added over the concentration range shown. Interleukin (IL)-12p40 and tumor necrosis factor-alpha (TNF-α) were measured in enzyme-linked immunosorbent assay (ELISA). Flt-3-propagated bone marrow-derived DCs were stimulated for 24 hours either with class A(D) CpG-ODN 2336 or CG50+PA4 immune complexes or with various combinations of TLR9 ligands and class R or class B INH-ODNs or control ODNs. INH-ODNs and control ODNs were used either at a concentration of 1 μg/mL (b-d) or over the concentration range shown (e). Interferon-alpha (IFN-α) secretion was measured in ELISA (n = 3 to 5). *P < 0.05.

Figure 2

Class R inhibitory oligonucleotides (INH-ODNs) show lower potency for resting mouse and human B cells. Total CD43^- B cells from C57BL6 mice were stimulated with class B(K) CpG-ODN 1826 for either 18 to 42 hours (a-c) or 6 days (d) in the presence of increasing concentrations of INH-ODNs (1 to 1,000 nM). Percentage of cells with hypodiploid DNA content and cells entering the G₁-M phase of the cell cycle was determined in acridine orange flow cytometry. Interleukin-6 (IL-6) and polyclonal IgM were measured in enzyme-linked immunosorbent assay (ELISA) (n = 3 to 7). (e) Total mouse B cells were stimulated with PO-CpG-ODN for 6 days. (f) Total human Namalwa B cells were stimulated with human PS-CpG-2006 for 42 hours. CD86 expression and polyclonal IgM secretion were measured. Indicated class R and B INH-ODNs were added over the concentration range shown (n = 3 or 4). *P < 0.05. PO, phosphodiester; PS, phosphorothioate.

Figure 3

The size of the linear overhang determines the potency difference between class R and class B inhibitory oligonucleotides (INH-ODNs). Total mouse B cells were stimulated with 33 nM CpG-1826 together with indicated class R or class B INH-ODNs added simultaneously and used over the concentration range shown. (a) Palindromic Class R INH-4 and linear INH-13 ODNs with CCT/GGG blocks at the 3' were used. (b) Palindromic Class R INH-1 and linear INH-18 ODNs with CCT/GGG blocks at the 5' end were used. (c) INH-ODNs with short, medium or long 3' linear overhangs or linear INH-ODNs of the equal length were used. (d) INH-ODNs with short, medium or long 5' linear overhangs or linear INH-ODNs of the equal length were used. Inhibition of CpG-1826-induced rescue from apoptosis is shown. Sequences of INH-ODNs are shown in Table 1 (n = 3 to 5). *P < 0.05. OVHG, overhang; scr., scrambled.

Figure 4

Higher potency of class R inhibitory oligonucleotides (INH-ODNs) for B-cell receptor-dependent activation of AM14 B cells induced with DNA-containing immune complexes. AM14 B cells were stimulated with (a) linear CpG-1826, (b) anti-nucleosome antibody PL2-3, or (c) lipopolysaccharide (LPS), and class R and class B INH-ODNs were added simultaneously. Proliferation of AM14 B cells was determined by measuring the [³H] thymidine incorporation for the last 6 hours. Results are expressed as percentage of maximal stimulation induced with the particular Toll-like receptor ligand (n = 3). *P < 0.05 (INH-1 versus INH-18). **P < 0.05 (INH-1 versus control).

Figure 5

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.

Figure 6

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 [³H] thymidine incorporation for the last 6 hours. CD86 expression was determined by flow cytometry (n = 3 to 5). *P < 0.05 (ODN-treated versus medium-treated samples). FITC, fluorescein isothiocyanate.

Figure 7

Inhibitory oligonucleotides (INH-ODNs) prolong survival and decrease morbidity in MRL-Fas^lpr/lpr mice in vivo. Pre-diseased MRL-Fas^lpr/lpr (2J strain) mice were treated with INH-1, INH-18, or vehicle starting from week 15 for 25 consecutive weeks. ODNs were injected intraperitoneally three times weekly at the concentration of 1 mg/kg body weight. Surviving mice were sacrificed at week 40. Similar results were obtained in three additional cohorts of lupus mice (J and 2J strains). Effects of INH-ODNs on survival (a), total lymph node weight (b), proteinuria (c), composite renal score (d), and IgG deposits in kidneys (e, f) are shown. *P < 0.05 (ODN-treated versus vehicle-treated). PBS, phosphate-buffered saline.

Figure 8

Class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantibody secretion in MRL-Fas^lpr/lpr mice in vivo. Pre-diseased MRL-Fas^lpr/lpr mice were treated with INH-1, INH-18, or vehicle for 25 weeks as in Figure 7. Surviving mice were sacrificed and their sera tested for autoantibodies. (a) The presence of anti-double-stranded DNA (anti-dsDNA) antibodies binding native DNA was determined semi-quantitatively by staining the Crithidia lucillae kinetoplasts. (b) The concentrations of anti-DNA and anti-Sm/RNP antibodies were further measured in enzyme-linked immunosorbent assay. *P < 0.05 (ODN-treated versus vehicle-treated). PBS, phosphate-buffered saline.