Apoptosis‐induced acetylation of histones is pathogenic in systemic lupus erythematosus

Abstract

Objective. In systemic lupus erythematosus (SLE), inadequate removal of apoptotic cells may lead to challenge of the immune system with immunogenic self antigens that have been modified during apoptosis. We undertook this study to evaluate whether apoptosisinduced histone modifications are targets for the immune system in SLE.Methods. The epitope of KM-2, a monoclonal antihistone autoantibody derived from a lupus mouse, was mapped by random peptide phage display. The reactivity of KM-2 and plasma with (acetylated) histone H4 (H4) peptides and with nonapoptotic, apoptotic, and hyperacetylated histones was determined by immunofluorescence staining, enzyme-linked immunosorbent assay, and Western blotting.Results. KM-2 recognized apoptosis-induced acetylation of H4 at lysines 8, 12, and 16. The majority of plasma samples from SLE patients and lupus mice showed higher reactivity with triacetylated H4 peptide (residues 1-22) and with hyperacetylated and apoptotic histones than with nonacetylated H4 peptide and normal histones. Importantly, administration of triacetylated H4 peptide to lupus-prone mice before disease onset accelerated the disease by enhancing mortality and aggravating proteinuria, skin lesions, and glomerular IgG deposition, while the nonacetylated H4 peptide had no pathogenic effect. The delayed-type hypersensitivity response in lupus mice against the triacetylated H4 peptide was higher than that against the nonacetylated H4 peptide. Bone marrow-derived dendritic cells (DCs) cultured in the presence of hyperacetylated nucleosomes showed increased expression/production of CD40, CD86, interleukin-6 (IL-6), and tumor necrosis factor ␣ compared with DCs cultured in the presence of normal nucleosomes. Finally, DCs cultured in the presence of hyperacetylated nucleosomes were able to activate syngeneic T cells, because IL-2 production increased.Conclusion. Apoptosis-induced acetylation of nucleosomes may represent an important driving force in the development of lupus.

Apoptosis-linked changes in the phosphorylation status and subcellular localization of the spliceosomal autoantigen U1-70K

“…As apoptotic blebs/bodies contain many antigens recognized by lupus autoantibodies, 27,28 we isolated apoptotic bodies from Jurkat cells after incubation with CPT for 48 h as described in Materials and Methods. Western blot analysis revealed the presence of the 40 kDa fragment of the U1-70K protein in these isolated apoptotic bodies, whereas we could hardly detect any intact phosphorylated or hypophosphorylated U1-70K protein (Figure 8a).…”

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

“…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.…”

“…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.…”

“…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.…”

Apoptosis in the pathogenesis of systemic lupus erythematosus

“…Recently, we were able to show that apoptosis-induced histone acetylation is a target for autoantibodies in patients with SLE and lupus mice. 15 Histone acetylation appeared to be pathogenic in lupus-prone mice, and hyperacetylated nucleo- Figure 1 General hypothesis for the genesis of SLE. Deregulated apoptosis and/or insufficient removal of apoptotic cells/blebs leads to the release of (modified) chromatin into the circulation.…”