title: Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps.
abstract: 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.
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Pratesi
authors.affiliation: Laboratory of Peptide and Protein Chemistry and Biology, Department of Pharmaceutical Sciences, University of Florence, Florence, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dioni
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Tommasi
authors.affiliation: Toscana Biomarkers, Siena, Italy.
authors.forename: Maria Claudia
authors.initials: MC
authors.lastname: Alcaro
authors.affiliation: Toscana Biomarkers, Siena, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Paolini
authors.affiliation: Toscana Biomarkers, Siena, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Barbetti
authors.affiliation: Mass Spectrometry Center (CISM), University of Florence, Florence, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Boscaro
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Filomena
authors.initials: F
authors.lastname: Panza
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Puxeddu
authors.affiliation: Laboratory of Peptide and Protein Chemistry and Biology, Department of Pharmaceutical Sciences, University of Florence, Florence, Italy Toscana Biomarkers, Siena, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Rovero
authors.affiliation: Clinical Immunology and Allergy Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy Toscana Biomarkers, Siena, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Migliorini
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000276:immunology / Q000378:metabolism*; D016022:Case-Control Studies; D002956:Citrulline / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology / Q000378:metabolism; D005260:Female; D006657:Histones / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000276:immunology*; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D055815:Young Adult
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D006657:Histones; D010455:Peptides; D002956:Citrulline
keywords: Ant-CCP; Autoantibodies; Rheumatoid Arthritis