Apoptosis-induced histone H3 methylation is targeted by autoantibodies in systemic lupus erythematosus

Abstract

ObjectivesIn systemic lupus erythematosus (SLE) apoptotic chromatin is present extracellularly, which is most likely the result of disturbed apoptosis and/or insuffi cient removal. Released chromatin, modifi ed during apoptosis, activates the immune system resulting in the formation of autoantibodies. A study was undertaken to identify apoptosis-induced histone modifi cations that play a role in SLE. Methods The lupus-derived monoclonal antibody BT164, recently established by selection using apoptotic nucleosomes, was analysed by ELISA, western blot analysis and immunofl uorescence staining using chromatin, cells, plasma and renal sections. Random peptide phage display and peptide inhibition ELISA were used to identify precisely the epitope of BT164. The reactivity of plasma samples from lupus mice and patients with SLE with the epitope of BT164 was investigated by peptide ELISA. Results The epitope of BT164 was mapped in the N-terminal tail of histone H3 (27-KSAPAT-32) and included the apoptosis-induced trimethylation of K27. siRNA-mediated silencing of histone demethylases in cultured cells resulted in hypermethylation of H3K27 and increased nuclear reactivity of BT164. This apoptosisinduced H3K27me3 is a target for autoantibodies in patients and mice with SLE and is present in plasma and in glomerular deposits. Conclusion In addition to previously identifi ed acetylation of histone H4, H2A and H2B, this study shows that trimethylation of histone H3 on lysine 27 is induced by apoptosis and associated with autoimmunity in SLE. This fi nding is important for understanding the autoimmune response in SLE and for the development of translational strategies.

Acetylated histones contribute to the immunostimulatory potential of neutrophil extracellular traps in systemic lupus erythematosus

“…NETs from primary neutrophils may therefore display a different immunostimulatory potential when compared to HL-60 or EPRO-derived NETs, as they harbour different histone modifications. Furthermore, Liu et al did not confirm reactivity of anti-histone autoantibodies with acetylated H4-K8,12,16 and tri-methylated H3-K27, as we have described [18,20]. Garcia-Romo et al reported that NETs from SLE patients, in contrast to those of healthy donors, are highly capable of activating plasmacytoid dendritic cells (pDCs), a process accompanied by an increased secretion of the proinflammatory cytokines interferon (IFN)-α, interferon gamma-induced protein 10 (IP-10), TNF-α and IL-6 [22].…”

“…We have described previously that specific histone modifications are associated with both apoptosis and the autoimmune response in patients with SLE [17][18][19][20]. In this report we investigated whether the same SLE-associated histone modifications are to be detected in NETs, as 70% of their proteins are histones.…”

“…Furthermore, the fine specificity was determined originally in peptide competition enzyme-linked immunosorbent assays (ELISAs) using specifically modified histone peptides and their unmodified counterparts. Purification of these monoclonal antibodies was performed as described previously [17][18][19][20]. The specificity for each batch of purified monoclonal antibody is routinely validated by comparing the reactivity in ELISA with the respective histone peptides.…”

“…After stimulation, neutrophils and NETs were suspended in paraformaldehyde (final concentration 1%) to a cell density of 1 × 10 6 per ml and then centrifuged at 800 g for 10 min with a cytospin cuvette (Hettich Cyto System, Tuttlingen, Germany) on glass slides. DNA was stained for 30 min with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, Eugene, OR, USA) and histones were stained employing our panel of lupus-derived monoclonal antibodies consisting of #34 (anti-H3; [17]), KM-2 (anti-H4-K8,12,16ac [18]), LG11-2 (anti-H2B-K12ac [19]) and BT164 (anti-H3-K27me3 [20]). The specificity of these four monoclonal antibodies was determined originally by epitope mapping using random peptide phage display.…”

Ocular Manifestations in Systemic Lupus Erythematosus

“…When neutrophils undergo mitosis, histones are highly susceptible to acetylation and methylation of certain residues that were reportedly associated with apoptosis [16][17][18]. The increased content of acetylated and methylated residues on histones from NETs of SLE patients may, therefore, indirectly increase the immune-stimulatory potential of NETs via an enhanced binding of antimicrobial peptides within the NETs.…”