The DNA sensors AIM2 and IFI16 are SLE autoantigens that bind neutrophil extracellular traps

Abstract

Background: Nucleic acid binding proteins are frequently targeted as autoantigens in systemic lupus erythematosus (SLE) and other interferon (IFN)-linked rheumatic diseases. The AIM-like receptors (ALRs) are IFN-inducible innate sensors that form supramolecular assemblies along double-stranded (ds)DNA of various origins. Here, we investigate the ALR absent in melanoma 2 (AIM2) as a novel autoantigen in SLE, with similar properties to the established ALR autoantigen interferon-inducible protein 16 (IFI16). We examined neutrophil extracellular traps (NETs) as DNA scaffolds on which these antigens might interact in a pro-immune context.

Methods: AIM2 autoantibodies were measured by immunoprecipitation in SLE and control subjects. Neutrophil extracellular traps were induced in control neutrophils and combined with purified ALR proteins in immunofluorescence and DNase protection assays. SLE renal tissues were examined for ALR-containing NETs by confocal microscopy.

Results: AIM2 autoantibodies were detected in 41/131 (31.3%) SLE patients and 2/49 (4.1%) controls. Our SLE cohort revealed a frequent co-occurrence of anti-AIM2, anti-IFI16, and anti-DNA antibodies, and higher clinical measures of disease activity in patients positive for antibodies against these ALRs. We found that both ALRs bind NETs in vitro and in SLE renal tissues. We demonstrate that ALR binding causes NETs to resist degradation by DNase I, suggesting a mechanism whereby extracellular ALR-NET interactions may promote sustained IFN signaling.

Conclusions: Our work suggests that extracellular ALRs bind NETs, leading to DNase resistant nucleoprotein fibers that are targeted as autoantigens in SLE.

Funding: These studies were funded by NIH R01 DE12354 (AR), P30 AR070254, R01 GM 129342 (JS), K23AR075898 (CM), K08AR077100 (BA), the Jerome L. Greene Foundation and the Rheumatology Research Foundation. Dr. Antiochos and Dr. Mecoli are Jerome L. Greene Scholars. The Hopkins Lupus Cohort is supported by NIH grant R01 AR069572. Confocal imaging performed at the Johns Hopkins Microscopy Facility was supported by NIH Grant S10 OD016374.

Keywords: autoantibodies; autoimmunity; human; immunology; inflammation; medicine; neutrophil extracellular traps.

Figure 1.. Anti-AIM2 antibodies are associated with anti-IFI16 and anti-DNA antibodies in SLE.

AIM2 antibodies were detected using immunoprecipitation (IP) of ³⁵S-methionine labeled, in vitro transcribed and translated protein. Data are presented as OD units calibrated to a known positive reference serum. Dotted line indicates positive threshold value determined as the mean + 2 standard deviations of control serum samples. AIM2 autoantibodies were identified in 2/49 controls and 41/131 SLE patients. Statistical significance was determined using the Mann-Whitney test for nonparametric values (A). Relationship between anti-AIM2, -IFI16, and –DNA antibodies in the SLE cohort (B). Anti-AIM2 +SLE and control (CT) sera and AIM2 protein were each treated with or without DNase prior to being combined in the IP reaction. Coomassie stain of IgG heavy chain (HC) is shown below each IP result (C). 1 µg of Poly(dA:dT) was treated with DNase as in (C) and analyzed by SYBR Green staining in agarose gel (D). 293T cells were transfected with AIM2-PYD-GFP expression plasmid, and lysate was used in IP reaction with anti-AIM2 +SLE and CT sera (E). IP products and 293T lysates were blotted for AIM2 using anti-N terminal antibody (Cell Signaling D5X7K).

Figure 1—figure supplement 1.. MNDA autoantibodies are not enriched in SLE.

Anti-MNDA antibodies were analyzed by ELISA using 59 healthy control (HC) and 137 SLE sera. (A). MNDA protein utilized in ELISA analyzed by Coommasie staining (left) and Western botting (right) (B). AU = arbitrary units.

Figure 2.. IFI16 and AIM2 bind NETs and prevent NET degradation by DNase I.

NETs were induced in neutrophils using PMA 100 nM for 3 hr, then left untreated (A) or incubated with fluorescently labeled AIM2 (pink) and IFI16 (red) at 200 nM at RT for 1 hr (B). Following ALR incubation, samples were stained with anti-MPO-FITC antibody (green) and DAPI (blue), then imaged by confocal microscopy. NETs were treated with DNase I at 20 U/mL at RT for 1 hr (C). NETs incubated with ALRs as in (B) were then treated with 20 U/mL DNase I for 1 hr (D). Scale bars = 20 µm. NETs in 96 well plates were incubated with ALRs at 200 nM (or buffer only) for 1 hr at RT, then treated with DNase I at 0, 20, and 100 U/mL for 30 min at RT. NETs were then stained with Sytox-Green 5 µM, and samples analyzed by fluorimetry (E). RFU = fluorescence units. Mean and standard deviation of 4 replicate wells are indicated. Mann-Whitney test was used to compare groups. P > 0.05 = not significant (ns). P < 0.05 = significant (*). IFI16 and the catalytic domain of cGAS were combined with FAM-labeled 72 bp VACV dsDNA for 30 min, then DNase I added at concentration of 20 U/mL at time = 0 and the fraction of bound dsDNA was monitored via the fluorescence anisotropy of dsDNA•protein complex (F).

Figure 3.. IFI16 and AIM2 bind NETs in diffuse proliferative lupus nephritis.

Representative images of ALR expression and ALR-NETs identified in patients with class IV lupus nephritis. AIM2 (A) expression was largely detected in MPO expressing neutrophils, while IFI16 (B) was more broadly distributed. NETs (arrows) demonstrating co-localizing staining for DNA, MPO, and AIM2 (C) or IFI16 (D) visualized by confocal microscopy. Scale bars: 50 µm (A, B) 5 µm (C), 2 µm (D).

Figure 3—figure supplement 1.. Z-stack imaging of AIM2/IFI16-NETs in lupus nephritis.

Renal biopsy paraffin section stained for DNA, MPO, and IFI16/AIM2 and imaged using z-stacking to identify extracellular DNA-containing structures containing MPO and IFI16 or AIM2. Individual squares within each panel represent adjacent focal planes, proceeding sequentially from top left to bottom right in each area imaged.