Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity

Abstract

Objectives: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p.

Methods: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts.

Results: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients.

Conclusions: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.

Fig. 1.

ORF1p redistributes to surface blebs in apoptotic cells and is cleaved by granzyme B but not caspases. A: Untreated (-DOX) and doxycycline-induced (+DOX) L1-RPE stained with DAPI (blue, left) and anti-ORF1p antibody (green, middle). B: Doxycycline-induced LINE-1-RPE treated with NK cell granule contents (+Gran) for 4 hours, then stained as in (A). Apoptotic cells with ORF1p-containing surface blebs are highlighted (right panels). C: Untreated MCF7 cells stained with DAPI (blue), anti-ORF1p (green) and PI (red). D: MCF7 cells treated with NK cell granule contents, then stained as in (C). Apoptotic blebs (arrowheads) demonstrating positivity for PI and ORF1p. Scale bars in A, B: 10 μm; scale bars in C, D: 5 μm. Results are representative of 3 experiments. E: MCF7 cells exposed to control buffer (–) or Granule contents (+) then harvested and probed for cleaved caspase 7 by Western blot. F: MCF7 cell lysates incubated with recombinant GrB (rGrB) and analysed by Western blotting for ORF1p. G: MCF7 cells exposed to UV light to induce apoptosis, then analysed at 16 hours for ORF1p and cleaved caspase 7 expression by Western blotting. The results are representative of 3 separate experiments.

Fig. 2.

ORF1p autoantibodies are not associated with SLE disease activity. A: Anti-ORF1p ELISA performed in healthy control (HC, n=68) and SLE (n=158) JH SPARE cohorts. Median values (solid line) and positivity threshold (dashed line) indicated. B: Anti-ORF1p results in longitudinal serum samples in 23 JH SLE patients at >1 time point (total n=29 samples). C: Corresponding SLEDAI values for the 29 longitudinal samples over the same time intervals depicted in (B). D: Individual time intervals corresponding to drop in SLEDAI of ≥4 points in the JH longitudinal cohort (n=9 intervals). E: Corresponding changes in anti-ORF1p levels observed during the same 9 intervals in (D). F: Individual time intervals corresponding to increase in SLEDAI by ≥4 points in the longitudinal JHU cohort (n=4 intervals). G: Corresponding changes in anti-ORF1p levels observed during the same 4 intervals in (F). H: Correlation between anti-ORF1p and SLEDAI in 189 total samples in the SPARE cohort. Spearman r and p-value indicated. I: EU SLE patients (n=86) with and without anti-ORF1p antibodies compared for disease activity by SLEDAI. J: Correlation between SLEDAI and anti-ORF1p level in the EU SLE cohort. AU: arbitrary units.

Fig. 3.

Anti-ORF2p antibodies are not associated with SLE disease status. Anti-ORF2p ELISA was performed using healthy control (n=69), Johns Hopkins lupus sera (SLE JH, n=158) and Emory lupus sera (SLE EU, n=90). Assay positivity was established as the mean + 2 standard deviations of healthy control values. Mann-Whitney test was used to compare median values with p-value indicated. AU: arbitrary units.