Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies.
BACKGROUND
Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones.


METHODS
We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.


RESULTS
We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.


CONCLUSIONS
Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.
Department of Medicine, Division of Immunology and Rheumatology, Stanford School of Medicine, 269 Campus Drive, Stanford, California 94305, USA.
Chih Long
CL
Liu
Stephanie
S
Tangsombatvisit
Jacob M
JM
Rosenberg
Gil
G
Mandelbaum
Emily C
EC
Gillespie
Or P
OP
Gozani
Ash A
AA
Alizadeh
Paul J
PJ
Utz
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology*; D002460:Cell Line; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D057890:Epigenomics / Q000379:methods; D018922:HL-60 Cells; D006657:Histones / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D008180:Lupus Erythematosus, Systemic / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D019463:Mice, Inbred MRL lpr; D009504:Neutrophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods
D001323:Autoantibodies; D001324:Autoantigens; D002843:Chromatin; D006657:Histones; D007074:Immunoglobulin G