Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus

Abstract

Raised serum levels of interferon (IFN)-alpha have been observed in systemic lupus erythematosus (SLE) patients, and these levels are correlated with both disease activity and severity. The origin of this IFN-alpha is still unclear, but increasing evidence suggests the critical involvement of activated plasmacytoid predendritic cells (PDCs). In SLE patients, DNA and RNA viruses, as well as immune complexes (ICs), that consist of autoantibodies specific to self-DNA and RNA protein particles can stimulate production of IFN-alpha. We have developed three series of oligonucleotide (ODN)-based inhibitors of Toll-like receptor (TLR) signaling. These ODNs include inhibitors of TLR9, inhibitors of TLR7 but not TLR9, and sequences that inhibit both TLR7 and TLR9. Specificity of these inhibitors is confirmed by inhibition of IFN-alpha production by PDCs in response to DNA or RNA viruses. We show that mammalian DNA and RNA, in the form of ICs, are potent self-antigens for TLR9 and TLR7, respectively, and induce IFN-alpha production by PDCs. This work suggests that TLRs may have a critical role in the promotion of lupus through the induction of IFN-alpha by PDCs. These inhibitors of TLR signaling thus represent novel therapeutic agents with potential for the treatment of lupus.

Figure 1.

Identification of inhibitors specific for mouse TLR7 and TLR9 signaling. Splenocytes from BALB/c mice were stimulated for 48 h with (A) 0.7 μM 1018 ISS or (B) 1 μM R848 alone or in the presence of IRS 869 (open squares), IRS 661 (open circles), or IRS 954 (closed triangles) at different concentrations. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS (A) or R848 alone (B). Maximum levels of IL-6 varied between 1,663 and 4,266 pg/ml for 1018 ISS and between 924 and 2,721 pg/ml for R848. Values represent the means ± SEM of four independent experiments.

Figure 2.

IRS do not affect signaling through other TLRs. Purified CD11c-positive splenocytes from BALB/c mice were stimulated for 48 h with either 5 μg/ml LPS, 0.1 μg/ml Pam3Cys, 1 μg/ml Flagellin, or 50 μg/ml Poly I:C alone or in the presence of IRS 954 at different concentrations. IL-6 production was measured by ELISA and plotted as a percentage of the stimuli alone. Means of maximum levels were 1,620 pg/ml (LPS), 2,463 pg/ml (Pam3Cys), 880 pg/ml (Flagellin), and 678 pg/ml (Poly I:C). Values represent means ± SEM of five independent experiments.

Figure 3.

IRS are active in vivo to block TLR7 stimulation. BALB/c mice were injected i.p. with inactive control ODN or various amounts of IRS 954 as indicated. 2 h later, mice were injected i.p. route R848. 2 h after the R848 injection, the serum was collected, and IL-12 was measured by immunoassay. Values represent means ± SEM of a group of 10 mice. ***, P < 0.001.

Figure 4.

IRS retain similar specificity in human cells. Purified human B cells were stimulated with 0.7 μM 1018 ISS (left) or 1 μM R848 (right) alone or in the presence of IRS 869, IRS 661, or IRS 954 at 0.7 μM and 2.8 μM, respectively. IL-6 production was measured by ELISA and plotted as a percentage of 1018 ISS or R848 alone. Means of maximum levels of IL-6 were 2,904 pg/ml (R848) and 851 pg/ml (1018 ISS). Values represent means ± SEM of 10 (1018 ISS) and 6 (R848) independent donors. ***, P < 0.001.

Figure 5.

IRS specific for TLR7 act independently of TLR9. Splenocytes from C57BL/6 (closed bars) and TLR9-deficient (hatched bars) mice were stimulated for 48 h with R848 (A) or 1018 ISS (B) alone or in the presence of IRS 661 or IRS 954 at different concentrations. IL-6 production was measured by ELISA. Values represent means ± SEM of three independent experiments. **, P < 0.01; ***, P < 0.001.

Figure 6.

IRS inhibit IFN-α from human PDCs after stimulation with DNA or RNA viruses. Purified human PDCs were stimulated with (A and B) UV-irradiated HSV (MOI = 5) or (C and D) heat-inactivated influenza virus (MOI = 2) alone or in the presence of different concentrations of IRS 954, IRS 661, or a control sequence. After 48 h of culture, supernatants were harvested and IFN-α was measured by ELISA and plotted as a percentage of virus stimulation alone. Means of maximum levels of IFN-α were 34,308 pg/ml (HSV) and 40,599 pg/ml (Influenza). Values represent means ± SEM of 13 (A), 12 (B), 8 (C), and 13 (D) donors. *, P < 0.1; ***, P < 0.001.

Figure 7.

IRS inhibit IFN-α from human PDCs after stimulation with either anti-dsDNA or anti-RNP ICs. Purified human PDCs were cultured for 48 h with UV-irradiated U937 cells and (A) serum (10% final concentration) from six different anti-dsDNA–positive SLE patients, (B) serum (10% final concentration) from an anti-dsDNA–positive SLE patient (260 IU/ml of anti-dsDNA antibodies and negative for anti-RNP autoantibodies), (C) purified IgG (0.5 mg/ml final concentration) from three different anti-RNP–positive SLE patients, or (D) purified IgG (0.5 mg/ml final concentration) from an anti-RNP–positive SLE patient (142 U/ml of antiautoantibodies and negative for anti-dsDNA antibodies). (B and D) Cells were stimulated with ICs alone or in the presence of control ODN, IRS 661, or IRS 954. Patient D (B) and patient A (D) were used as sources of ICs. IFN-α was measured by ELISA. Values represent means ± SEM of eight (B) and six (D) PDC donors. *, P < 0.1; **, P < 0.01; ***, P < 0.001.