Introduction: 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.
Bibliographical noteFunding Information:
This work was supported in part by grants from the NIH to PJU (5 U19-AI082719, G. Fathman as primary investigator), from the NIH to OG (R01 GM079641), by the National Heart, Lung, and Blood Institute Proteomics contract (HHSN288201000034C, G. Nolan as primary investigator), by the Canadian Institute of Health Research grant to PJU (2 OR-92141), by the European Commission FP Grant #261382 to PJU, and by a gift of the Floren Family Trust to PJU. This publication was made possible by T32 AR050942 from NIAMS/NIH to CLL. CLL is a recipient of an NIH National Research Service Award Fellowship (AI-080086-02). AAA is a Special Fellow of the Leukemia & Lymphoma Society and recipient of a Doris Duke Clinician Scientist Development Award. OG is a recipient of an Ellison Senior Scholar in Aging Award.