title: Autophagy generates citrullinated peptides in human synoviocytes: a possible trigger for anti-citrullinated peptide antibodies.
abstract: Autophagy may represent a functional processing event that creates a substrate for autoreactivity. In particular, autophagy may play a role in the pathogenesis of RA, since autophagy is a key cellular event involved in the generation of citrullinated peptides, with consequent breakage of tolerance. Thus, in RA, autophagy may be the common feature in several situations (including smoking, joint injury and infection) that may drive the adaptive responses to citrullinated self-proteins. The aim of this study was the analysis, in vitro, of the role of autophagy in the generation of citrullinated peptides and, in vivo, of the relationship between autophagy and the production of anti-CCP antibodies (Abs).
  
  
  
  For autophagy induction, fibroblast-like synoviocytes, primary fibroblasts and monocytes were stimulated with tunicamycin or rapamycin. Peptidyl arginine deiminase activity was tested by enzyme-linked immunosorbent assay, and protein citrullination was evaluated by western blotting. The main citrullinated RA candidate antigens, vimentin, α-enolase and filaggrin, were demonstrated by immunoprecipitation. The relationship between autophagy and anti-CCP Abs was analysed in 30 early-active RA patients.
  
  
  
  Our results demonstrated in vitro a role for autophagy in the citrullination process. Cells treated with tunicamycin or rapamycin showed peptidyl arginine deiminase 4 activation, with consequent protein citrullination. Immunoblotting and immunoprecipitation experiments, using specific Abs, identified the main citrullinated proteins: vimentin, α-enolase and filaggrin. In vivo, a significant association between levels of autophagy and anti-CCP Abs was observed in treatment-naïve early-active RA patients.
  
  
  
  These findings support the view that the processing of proteins in autophagy generates citrullinated peptides recognized by the immune system in RA.
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Sorice
authors.affiliation: Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Roma, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Iannuccelli
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Valeria
authors.initials: V
authors.lastname: Manganelli
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Antonella
authors.initials: A
authors.lastname: Capozzi
authors.affiliation: Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Roma, Italy cristiano.alessandri@uniroma1.it.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Alessandri
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Lococo
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Tina
authors.initials: T
authors.lastname: Garofalo
authors.affiliation: Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Roma, Italy.
authors.forename: Manuela
authors.initials: M
authors.lastname: Di Franco
authors.affiliation: Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Queen Mary University of London, London, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Bombardieri
authors.affiliation: Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Queen Mary University of London, London, UK.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Nerviani
authors.affiliation: Dipartimento di Medicina Sperimentale.
authors.forename: Roberta
authors.initials: R
authors.lastname: Misasi
authors.affiliation: Reumatologia, Dipartimento di Medicina Interna e Specialità Mediche, Sapienza Università di Roma, Roma, Italy.
authors.forename: Guido
authors.initials: G
authors.lastname: Valesini
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001343:Autophagy / Q000276:immunology*; D002478:Cells, Cultured; D002956:Citrulline / Q000276:immunology; D005260:Female; D005347:Fibroblasts / Q000276:immunology; D000091344:Filaggrin Proteins; D006801:Humans; D006867:Hydrolases / Q000378:metabolism; D007381:Intermediate Filament Proteins / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000276:immunology; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000276:immunology; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D000070918:Synoviocytes / Q000276:immunology*; D014746:Vimentin / Q000378:metabolism
chemical_list: D000906:Antibodies; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D010456:Peptides, Cyclic; D014746:Vimentin; C487763:cyclic citrullinated peptide; D002956:Citrulline; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D010751:Phosphopyruvate Hydratase
keywords: PAD; anti-CCP antibodies; autophagy; citrullination; rheumatoid arthritis