Autoantibodies against Modified Histone Peptides in SLE Patients Are Associated with Disease Activity and Lupus Nephritis

Abstract

Persistent exposure of the immune system to death cell debris leads to autoantibodies against chromatin in patients with systemic lupus erythematosus (SLE). Deposition of anti-chromatin/chromatin complexes can instigate inflammation in multiple organs including the kidney. Previously we identified specific cell death-associated histone modifications as targets of autoantibodies in SLE. In this study we addressed, in a large cohort of SLE patients and controls, the question whether plasma reactivities with specific histone peptides associated with serology and clinical features. Plasma from SLE patients with and without lupus nephritis, disease controls, and healthy controls, were tested in ELISA with histone H4 peptide acetylated at lysines 8, 12 and 16 (H4pac), H2B peptide acetylated at lysine 12 (H2Bpac), H3 peptide trimethylated at lysine 27 (H3pme), and their unmodified equivalents. SLE patients displayed a higher reactivity with the modified equivalent of each peptide. Reactivity with H4pac showed both a high sensitivity (89%) and specificity (91%) for SLE, while H2Bpac exhibited a high specificity (96%) but lower sensitivity (69%). Reactivity with H3pme appeared not specific for SLE. Anti-H4pac and anti-H2Bpac reactivity demonstrated a high correlation with disease activity. Moreover, patients reacting with multiple modified histone peptides exhibited higher SLEDAI and lower C3 levels. SLE patients with renal involvement showed higher reactivity with H2B/H2Bpac and a more pronounced reactivity with the modified equivalent of H3pme and H2Bpac. In conclusion, reactivity with H4pac and H2Bpac is specific for SLE patients and correlates with disease activity, whereas reactivity with H2Bpac is in particular associated with lupus nephritis.

Fig 1. Reactivity with histone peptides containing apoptosis-associated modifications and their unmodified counterparts.

Plasma samples from SLE patients were tested in ELISA with the indicated modified histone peptides and their unmodified counterparts (i.e. H4p, H2Bp, and H3p). Patients with rheumatoid arthritis (RA) or systemic sclerosis (SSc), and healthy individuals were used as controls. * p<0.01 compared to the unmodified equivalent as determined using the Mann-Whitney U test; # p<0.01 compared to healthy subjects and patients with other autoimmune diseases and ^ p<0.01 compared to patients with other autoimmune diseases using the one-way ANOVA test. H4p, histone H4 peptide; H4p^ac, histone H4 peptide acetylated at lysine 8, 12 and 16; H2Bp, histone H2B peptide; H2Bp^ac, histone H2B peptide acetylated at lysine 12; H3p, histone H3 peptide; H3p^me, histone H3 peptide trimethylated at lysine 27.

Fig 2. Reactivity with histone peptides containing apoptosis-associated modifications and with their unmodified counterparts in SLE patients with and without lupus nephritis.

(A) Plasma samples from SLE patients with or without nephritis were tested in ELISA with the indicated modified histone peptides and their unmodified counterparts (i.e. H4p, H2Bp, and H3p). (B) Ratio of the reactivity with the respective modified histone peptide divided by the reactivity with the unmodified counterpart, for SLE patients with and without nephritis. In all cases the one-way ANOVA test was used for statistical comparison. * p<0.001. H4p, histone H4 peptide; H4p^ac, histone H4 peptide acetylated at lysine 8, 12 and 16; H2Bp, histone H2B peptide; H2Bp^ac, histone H2B peptide acetylated at lysine 12; H3p, histone H3 peptide; H3p^me, histone H3 peptide trimethylated at lysine 27.

Fig 3. H4p^ac appears superior in distinguishing SLE patients from healthy and disease controls.

ROC curves for (modified) histone peptide ELISAs were calculated for SLE vs. all controls (A), SLE vs. healthy controls (B), SLE vs. disease controls (C), lupus nephritis vs. all controls (not including SLE without nephritis) (D), and lupus nephritis vs. all controls including SLE without nephritis (E). *, p<0.05 vs. unmodified; #, p<0.01 vs. H2Bp^ac; ^, p<0.01 vs. H3p^me.

Fig 4. Reactivity with (multiple) modified peptides is associated with increased SLEDAI and follow disease activity during disease flares.

(A) SLE patients with nephritis were sorted into groups depending on the number of different modified histone peptides they show reactivity with, and the mean SLEDAI and C3 levels were compared between these groups. (B) Examples of reactivity with H4p^ac and H4p along with the SLEDAI score and anti-dsDNA reactivity in 4 patients that experienced a disease flare after initial treatment. Patient characteristics are detailed in S1 Table. H4p, histone H4 peptide; H4p^ac, histone H4 peptide acetylated at lysine 8, 12 and 16; H2Bp, histone H2B peptide; H2Bp^ac, histone H2B peptide acetylated at lysine 12; H3p, histone H3 peptide; H3p^me, histone H3 peptide trimethylated at lysine 27