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Extended Report
Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps
  1. Federico Pratesi1,
  2. Ilaria Dioni2,
  3. Cristina Tommasi1,
  4. Maria Claudia Alcaro3,
  5. Ilaria Paolini3,
  6. Francesca Barbetti3,
  7. Francesca Boscaro4,
  8. Filomena Panza1,
  9. Ilaria Puxeddu1,
  10. Paolo Rovero2,3,
  11. Paola Migliorini1,3
  1. 1Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy
  2. 2Laboratory of Peptide and Protein Chemistry and Biology, Department of Pharmaceutical Sciences, University of Florence, Florence, Italy
  3. 3Toscana Biomarkers, Siena, Italy
  4. 4Mass Spectrometry Center (CISM), University of Florence, Florence, Italy
  1. Correspondence to Professor Paola Migliorini, Clinical Immunology and Allergy Unit, Department of Internal Medicine, University of Pisa, Via Roma 67, Pisa 56126, Italy; paola.migliorini{at}


Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA).

Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs.

Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides.

Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2.

Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

  • Ant-CCP
  • Autoantibodies
  • Rheumatoid Arthritis

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