Apoptosis-derived membrane vesicles drive the cGAS-STING pathway and enhance type I IFN production in systemic lupus erythematosus

Abstract

Objective: Despite the importance of type I interferon (IFN-I) in systemic lupus erythematosus (SLE) pathogenesis, the mechanisms of IFN-I production have not been fully elucidated. Recognition of nucleic acids by DNA sensors induces IFN-I and interferon-stimulated genes (ISGs), but the involvement of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) and stimulator of interferon genes (STING) in SLE remains unclear. We studied the role of the cGAS-STING pathway in the IFN-I-producing cascade driven by SLE serum.

Methods: We collected sera from patients with SLE (n=64), patients with other autoimmune diseases (n=31) and healthy controls (n=35), and assayed them using a cell-based reporter system that enables highly sensitive detection of IFN-I and ISG-inducing activity. We used Toll-like receptor-specific reporter cells and reporter cells harbouring knockouts of cGAS, STING and IFNAR2 to evaluate signalling pathway-dependent ISG induction.

Results: IFN-I bioactivity and ISG-inducing activities of serum were higher in patients with SLE than in patients with other autoimmune diseases or healthy controls. ISG-inducing activity of SLE sera was significantly reduced in STING-knockout reporter cells, and STING-dependent ISG-inducing activity correlated with disease activity. Double-stranded DNA levels were elevated in SLE. Apoptosis-derived membrane vesicles (AdMVs) from SLE sera had high ISG-inducing activity, which was diminished in cGAS-knockout or STING-knockout reporter cells.

Conclusions: AdMVs in SLE serum induce IFN-I production through activation of the cGAS-STING pathway. Thus, blockade of the cGAS-STING axis represents a promising therapeutic target for SLE. Moreover, our cell-based reporter system may be useful for stratifying patients with SLE with high ISG-inducing activity.

Keywords: autoimmune diseases; cytokines; inflammation; systemic lupus erythematosus.

Figure 1

High IFN-I levels and ISG-inducing activities of SLE serum. (A) IFN-I bioactivities in sera. IFN-I bioactivities in SLE (n=54), SjS (n=11), SSc (n=24) and HC (n=24) were measured using HEK-Blue IFN-α/β reporter cells. (B) ISG-inducing activities in sera. Levels of ISG-inducing activity in SLE (n=60), SjS (n=11), SSc (n=24) and HC (n=24) were measured using THP1-ISG reporter cells. (C) ISG-inducing activity of SLE sera with IFN-I signalling blockade. THP1-ISG reporter cells were stimulated with sera from SLE (n=48) and HC (n=7) in the absence (IFN blockade −) or presence (IFN blockade +) of antibody cocktail (anti-IFN-α, anti-IFN-β and anti-IFNAR2). (D) IFN-I production in PBMCs induced by SLE sera. Recombinant human IFN-α(rhIFNα), SLE sera (n=12) or HC sera (n=5) were incubated for 24 hours with or without PBMCs from healthy donors. Levels of IFN-I in the culture supernatants were measured using HEK-Blue IFN-α/β reporter cells. PBMC-derived IFN-I levels were calculated by subtracting the IFN-I levels in PBMC-absent medium from the IFN-I levels in PBMC-present medium. The box chart indicates the 75th percentile (upper), median (middle) and 25th percentile (lower) (A–C). Overall statistical differences between SLE and other groups were evaluated by Steel test (A,B). Overall differences between the groups were evaluated by Mann-Whitney U test (C,D). HC, healthy control; IFN-I, type I interferon; ISG, IFN-I-stimulated genes; PBMC, peripheral blood mononuclear cells; SjS, Sjögren syndrome; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.

Figure 2

Elevated levels of dsDNA in SLE serum and involvement of TLR9. (A) dsDNA in sera. DNA was purified from SLE (n=64) and HC (n=18) sera and quantified. (B) THP1-ISG stimulated with TLR9 agonists. THP1-ISG cells were stimulated for 24 hours with STING agonist (5 µg/mL 2’3’-cGAMP), TLR9 agonist (10 µg/mL D35, 500 nM ODN2216, 10 µg/mL ODN2336, 10 µg/mL ODN2006, 10 µg/mL K3), control medium (Ctrl) or sera from SLE (n=64) and HC (n=17). (C) IFN-I-independent ISG-inducing activities and correlation with activation of TLR9. HEK-Blue TLR9 cells were stimulated with sera from SLE (n=22) and HC (n=20) for 24 hours. Levels of TLR9 pathway activation induced by sera from SLE (red circle) or HC (blue circle) are shown in the left panel. Correlation between TLR9 activation (y-axis) and ISG-inducing activity following IFN-I blockade (x-axis) is shown in the right panel. The box chart indicates the 75th percentile (upper), median (middle) and 25th percentile (lower) (A,B). Statistical analysis was performed by Mann-Whitney U test (A, B and left panel of C) and Hoeffding’s D statistic (right panel of C). cGAMP, cyclic GMP–AMP; dsDNA, double-stranded DNA; HC, healthy control; IFN-I, type I interferon; ISG, IFN-I-stimulated genes; SLE, systemic lupus erythematosus; STING, stimulator of interferon genes; TLR9, Toll-like receptor 9.

Figure 3

Serum-induced ISG expression via STING. (A) Establishment of STING-knockout reporter cells (ISG-KOSTING). Expression of STING was evaluated by western blotting. ISG-KOSTING cells did not respond to STING agonists (bar graph). Parental THP1-ISG (Parental) and ISG-KOSTING cells were stimulated with STING agonists (5 µg/mL 2’3’-cGAMP, 10 µg/mL c-diAMP), cGAS agonists (dsDNA+FuGENE), cytokine (1000 U/mL IFN-α), TLR agonists (500 ng/mL LPS, 10 µg/mL D35, or 10 µg/mL R848) or control medium (Ctrl) for 24 hours. Statistical analysis was performed by Mann-Whitney U test. (B) ISG-inducing activity in THP1-ISG-KOSTING. Parental THP1-ISG and THP1-ISG-KOSTING cells were stimulated with SLE sera (n=59) for 24 hours (upper bars). Each bar represents serum from an individual patient. %Reduction indicates the rate of reduction in reporter activity of THP1-ISG-KOSTING relative to that of parental THP1-ISG (lower bars). Dashed line indicates the mean of %reduction (C) Correlation between IFN-I-independent ISG-inducing activity and STING-dependent ISG-inducing activity. STING-dependent ISG-inducing activity was calculated by subtracting the absorbance of SEAP in THP1-ISG-KOSTING from that in parental THP1-ISG. (B). Statistical analysis was performed by Mann-Whitney U test (B) and non-parametric Spearman’s rank test (C). c-diAMP, cyclic-diAMP; cGAMP, cyclic GMP–AMP; cGAS, cyclic GMP–AMP synthase; dsDNA, double-stranded DNA; HC, healthy control; IFN-I, type I interferon; ISG, IFN-I-stimulated genes; LPS, lipopolysaccharide; SEAP, secreted embryonic alkaline phosphatase; SLE, systemic lupus erythematosus; STING, stimulator of interferon genes; TLR, Toll-like receptor.

Figure 4

Induction of IFN-I production and ISG expression by apoptosis-derived membrane vesicles from SLE serum. (A) ISG-inducing activity of SLE sera after DNase I treatment. DNase I activity in serum was confirmed by degradation of DNA added in SLE serum (left). After sera from SLE (n=24) and HC (n=11) were pretreated with or without DNase I, THP1-ISG was stimulated with pretreated sera for 24 hours (right). (B) Concentration of dsDNA in AdMVs from sera. DNA was purified from AdMVs of SLE (n=9) and HC (n=6) and quantified. (C) Degradation of dsDNA in AdMVs from SLE (n=6) using DNase I with or without membrane-rupturing treatment. Membrane of AdMVs was permeabilised with Triton X-100 prior to DNase I treatment. (D) Induction of IFN-I production in PBMCs by AdMVs from SLE sera. AdMVs isolated from SLE (n=21) and HC (n=11) sera were cocultured with PBMCs from healthy donors for 24 hours. IFN-I bioactivity in the culture supernatants was measured using HEK-Blue IFN-α/β. (E) cGAS and STING-dependent ISG induction by AdMVs. THP1-Dual, THP1-Dual-KOcGAS and Dual-KOSTING cells were cultured with AdMVs from SLE (n=29) or HC (n=15) for 24 hours. ISG-inducing activity was evaluated by measuring luciferase activity in culture supernatants. The box chart indicates the 75th percentile (upper), median (middle) and 25th percentile (lower) (right panel of A, B, D, E). Values are mean±SEM (C). Statistical analysis was performed by Mann-Whitney U test (A, B, D, E) or Steel test (C). AdMV, apoptosis-derived membrane vesicles; cGAS, cyclic GMP–AMP synthase; dsDNA, double-stranded DNA; HC, healthy control; IFN-I, type I interferon; ISG, interferon-stimulated genes; PBMC, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; STING, stimulator of interferon genes.

Figure 5

Relationships between ISG-inducing activity of SLE serum and clinical features of SLE. (A) Correlation between ISG-inducing activity and SLE disease activity, assessed according to SLEDAI-2K. (B–D) Association of ISG-inducing activity and clinical features of SLE. Patients with high levels of ISG-inducing activity manifested lymphocytopaenia (B), low serum C4 levels (C) and high anti-dsDNA antibody titres (D). All clinical data were obtained from the medical records of Osaka University Hospital. (E) ISG-inducing activity and lupus nephritis. Patients with SLE were divided into two groups according to the presence (n=35) or absence (n=29) of lupus nephritis. ISG-inducing activity of SLE sera (n=64) was evaluated using THP1-ISG cells (A–E). The box chart indicates the 75th percentile (upper), median (middle) and 25th percentile (lower) (E). Statistical analyses were performed by non-parametric Spearman’s rank test (A–D) or Mann-Whitney U test (E). dsDNA, double-stranded DNA; IFN-I, type I interferon; ISG, IFN-I-stimulated genes; SLE, systemic lupus erythematosus.