OBJECTIVE: Posttranslational modifications (PTMs) of proteins are crucial for regulating various biological processes. However, protein alteration via PTMs, and consequently, the creation of new epitopes, can induce abnormal autoimmune responses in predisposed individuals. Immunopathogenesis of several rheumatic diseases, including the most common childhood form, juvenile idiopathic arthritis (JIA), is associated with the generation of autoantibodies against such modified proteins. Dysregulated generation of neutrophil extracellular traps (NETs) can be a source of post-translationally altered proteins. Thus, we investigated the role of PTMs and the presence of NET-associated markers in JIA patients. METHODS: We recruited 30 pediatric patients with JIA (20 with active disease and 10 in remission) and 30 healthy donors. The serum concentrations of citrullinated histone H3 (citH3), peptidyl arginine deiminases (PADs), and NET-related products were detected using ELISA, and the number of citH3+ neutrophils was assessed using flow cytometry. RESULTS: The serum levels of citH3 and PADs were higher in active as well as in remission JIA patients than in healthy donors. Similarly, the number of citH3+ neutrophils was higher in the peripheral blood of patients with JIA, implying an enhanced process of NETosis. This was effectively reflected by elevated serum levels of NET-associated products, such as neutrophil elastase, LL37, and cell-free DNA-histone complexes. Additionally, 16.7% of active JIA patients were seropositive for carbamylated autoantibodies, the levels of which declined sharply after initiation of anti-TNFα therapy. CONCLUSION: Collectively, our data suggest that the accelerated process of NETosis and PTMs in JIA may result in the generation of anti-citrullinated/carbamylated autoantibodies against various epitopes later in life, which could be prevented by effectively regulating inflammation using immune therapy.

Department of Immunology 2nd Faculty of Medicine Charles University University Hospital Motol Prague Czechia

Department of Paediatric and Adult Rheumatology University Hospital Motol Prague Czechia

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Mahmud SA, Binstadt BA. Autoantibodies in the pathogenesis, diagnosis, and prognosis of juvenile idiopathic arthritis. Front Immunol. (2019) 10:3168. 10.3389/fimmu.2018.03168 PubMed DOI PMC

Glerup M, Rypdal V, Arnstad ED, Ekelund M, Peltoniemi S, Aalto K, et al. . Long-term outcomes in juvenile idiopathic arthritis: eighteen years of follow-up in the population-based nordic juvenile idiopathic arthritis cohort. Arth Care Res. (2020) 72:507–16. 10.1002/acr.23853 PubMed DOI

Matsumoto T, Matsui T, Hirano F, Tohma S, Mori M. Disease activity, treatment and long-term prognosis of adult juvenile idiopathic arthritis patients compared with rheumatoid arthritis patients. Mod Rheumatol. (2020) 30:78–84. 10.1080/14397595.2018.1554228 PubMed DOI

Shi J, Van De Stadt LA, Levarht EWN, Huizinga TWJ, Hamann D, Van Schaardenburg D, et al. . Anti-carbamylated protein (anti-CarP) antibodies precede the onset of rheumatoid arthritis. Ann Rheum Dis. (2014) 73:780–3. 10.1136/annrheumdis-2013-204154 PubMed DOI

Carubbi F, Alunno A, Gerli R, Giacomelli R. Post-translational modifications of proteins: novel insights in the autoimmune response in rheumatoid arthritis. Cells. (2019) 8:657. 10.3390/cells8070657 PubMed DOI PMC

Hissink Muller PCE, Anink J, Shi J, Levarht EWN, Reinards THCM, Otten MH, et al. . Anticarbamylated protein (anti-CarP) antibodies are present in sera of juvenile idiopathic arthritis (JIA) patients. Ann Rheum Dis. (2013) 72:2053–5. 10.1136/annrheumdis-2013-203650 PubMed DOI

Pratesi F, Dioni I, Tommasi C, Alcaro MC, Paolini I, Barbetti F, et al. . Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps. Ann Rheum Dis. (2014) 73:1414–22. 10.1136/annrheumdis-2012-202765 PubMed DOI

Verheul MK, Böhringer S, van Delft MAM, Jones JD, Rigby WFC, Gan RW, et al. . Triple positivity for anti-citrullinated protein autoantibodies, rheumatoid factor, and anti-carbamylated protein antibodies conferring high specificity for rheumatoid arthritis: implications for very early identification of at-risk individuals. Arthritis Rheumatol. (2018) 70:1721–31. 10.1002/art.40562 PubMed DOI

Frasca L, Palazzo R, Chimenti MS, Alivernini S, Tolusso B, Bui L, et al. . Anti-LL37 antibodies are present in psoriatic arthritis (PsA) patients: new biomarkers in PsA. Front Immunol. (2018) 9:1936. 10.3389/fimmu.2018.01936 PubMed DOI PMC

Stoop JN, Liu BS, Shi J, Jansen DTSL, Hegen M, Huizinga TWJ, et al. . Antibodies specific for carbamylated proteins precede the onset of clinical symptoms in mice with collagen induced arthritis. PLoS ONE. (2014) 9:102163. 10.1371/journal.pone.0102163 PubMed DOI PMC

van Delft MAM, Verheul MK, Burgers LE, Derksen VFAM, van der Helm-van Mil AHM, van der Woude D, et al. . The isotype and IgG subclass distribution of anti-carbamylated protein antibodies in rheumatoid arthritis patients. Arthritis Res Ther. (2017) 19:1–12. 10.1186/s13075-017-1392-z PubMed DOI PMC

Shi J, Knevel R, Suwannalai P, Van Der Linden MP, Janssen GMC, Van Veelen PA, et al. . Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage. Proc Natl Acad Sci U S A. (2011) 108:17372–7. 10.1073/pnas.1114465108 PubMed DOI PMC

Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. . Neutrophil extracellular traps kill bacteria. Science. (2004) 303:1532–5. 10.1126/science.1092385 PubMed DOI

Saitoh T, Komano J, Saitoh Y, Misawa T, Takahama M, Kozaki T, et al. . Neutrophil extracellular traps mediate a host defense response to human immunodeficiency virus-1. Cell Host Microbe. (2012) 12:109–16. 10.1016/j.chom.2012.05.015 PubMed DOI

Urban CF, Reichard U, Brinkmann V, Zychlinsky A. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol. (2006) 8:668–76. 10.1111/j.1462-5822.2005.00659.x PubMed DOI

Klocperk A, Vcelakova J, Vrabcova P, Zentsova I, Petruzelkova L, Sumnik Z, et al. . Elevated biomarkers of NETosis in the serum of pediatric patients with type 1 diabetes and their first-degree relatives. Front Immunol. (2021) 12:699386. 10.3389/fimmu.2021.699386 PubMed DOI PMC

Vecchio F, Lo Buono N, Stabilini A, Nigi L, Dufort MJ, Geyer S, et al. . Abnormal neutrophil signature in the blood and pancreas of presymptomatic and symptomatic type 1 diabetes. JCI Insight. (2018) 3:122146. 10.1172/jci.insight.122146 PubMed DOI PMC

Jariwala MP, Laxer RM. NETosis in rheumatic diseases. Curr Rheumatol Rep. (2021) 23:1–12. 10.1007/s11926-020-00977-6 PubMed DOI

Warnatsch A, Ioannou M, Wang Q, Papayannopoulos V. Neutrophil extracellular traps license macrophages for cytokine production in atherosclerosis. Science. (2015) 349:316–20. 10.1126/science.aaa8064 PubMed DOI PMC

Qiu S-L, Zhang H, Tang Q, Bai J, He Z-Y, Zhang J-Q, et al. . Neutrophil extracellular traps induced by cigarette smoke activate plasmacytoid dendritic cells. Thorax. (2017) 72:1084–93. 10.1136/thoraxjnl-2016-209887 PubMed DOI

Zentsova I, Parackova Z, Kayserova J, Palova-Jelinkova L, Vrabcova P, Volfova N, et al. . Monocytes contribute to DNA sensing through the TBK1 signaling pathway in type 1 diabetes patients. J Autoimmun. (2019) 105:0–1. 10.1016/j.jaut.2019.06.005 PubMed DOI

Parackova Z, Zentsova I, Vrabcova P, Klocperk A, Sumnik Z, Pruhova S, et al. . Neutrophil extracellular trap induced dendritic cell activation leads to Th1 polarization in type 1 diabetes. Front Immunol. (2020) 11:661. 10.3389/fimmu.2020.00661 PubMed DOI PMC

Khandpur R, Carmona-Rivera C, Vivekanandan-Giri A, Gizinski A, Yalavarthi S, Knight JS, et al. . NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis. Sci Transl Med. (2013) 5:178ra40–178ra40. 10.1126/scitranslmed.3005580 PubMed DOI PMC

Sur Chowdhury C, Giaglis S, Walker UA, Buser A, Hahn S, Hasler P. Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility. Arthritis Res Ther. (2014) 16:R122. 10.1186/ar4579 PubMed DOI PMC

Corsiero E, Pratesi F, Prediletto E, Bombardieri M, Migliorini P. NETosis as source of autoantigens in rheumatoid arthritis. Front Immunol. (2016) 7:485. 10.3389/fimmu.2016.00485 PubMed DOI PMC

Wang Y, Li M, Stadler S, Correll S, Li P, Wang D, et al. . Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol. (2009) 184:205–13. 10.1083/jcb.200806072 PubMed DOI PMC

Romero V, Fert-Bober J, Nigrovic PA, Darrah E, Haque UJ, Lee DM, et al. . Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in rheumatoid arthritis. Sci Transl Med. (2013) 5: 209ra150. 10.1126/scitranslmed.3006869 PubMed DOI PMC

Darrah E, Rosen A, Giles JT, Andrade F. Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis. Ann Rheum Dis. (2012) 71:92–8. 10.1136/ard.2011.151712 PubMed DOI PMC

Zhou Y, Chen B, Mittereder N, Chaerkady R, Strain M, An LL, et al. . Spontaneous secretion of the citrullination enzyme PAD2 and cell surface exposure of PAD4 by neutrophils. Front Immunol. (2017) 8:1200. 10.3389/fimmu.2017.01200 PubMed DOI PMC

Consolaro A, Ruperto N, Bazso A, Pistorio A, Magni-Manzoni S, Filocamo G, et al. . Development and validation of a composite disease activity score for juvenile idiopathic arthritis. Arthritis Rheum. (2009) 61:658–66. 10.1002/art.24516 PubMed DOI

Perdomo J, Leung HHL, Ahmadi Z, Yan F, Chong JJH, Passam FH, et al. . Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia. Nat Commun. (2019) 10:1–14. 10.1038/s41467-019-09160-7 PubMed DOI PMC

Holzer M, Zangger K, El-Gamal D, Binder V, Curcic S, Konya V., et al. . Myeloperoxidase-derived chlorinating species induce protein carbamylation through decomposition of thiocyanate and urea: novel pathways generating dysfunctional high-density lipoprotein. Antioxid. Redox Signal. (2012) 17:1043–52. 10.1089/ars.2011.4403 PubMed DOI PMC

Tilvawala R, Nguyen SH, Maurais AJ, Nemmara V V, Nagar M, Salinger AJ, et al. . The rheumatoid arthritis-associated citrullinome. Cell Chem Biol. (2018) 25:691. 10.1016/j.chembiol.2018.03.002 PubMed DOI PMC

Van Venrooij WJ, Pruijn GJM. How citrullination invaded rheumatoid arthritis research. Arthritis Res Ther. (2014) 16:1–5. 10.1186/ar4458 PubMed DOI PMC

Ling S, Cline EN, Haug TS, Fox DA, Holoshitz J. Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling. Arthritis Rheum. (2013) 65:618–26. 10.1002/art.37814 PubMed DOI PMC

Quirke A-M, Fisher BAC, Kinloch AJ, Venables PJ, Williams R, Flügel A, et al. . Citrullination of autoantigens: Upstream of TNFα in the pathogenesis of rheumatoid arthritis. FEBS Lett. (2011) 585:3681–8. 10.1016/j.febslet.2011.06.006 PubMed DOI

Andrade F, Darrah E, Gucek M, Cole RN, Rosen A, Zhu X. Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation. Arthritis Rheum. (2010) 62:1630–40. 10.1002/art.27439 PubMed DOI PMC

Sokolove J, Zhao X, Chandra PE, Robinson WH. Immune complexes containing citrullinated fibrinogen costimulate macrophages via toll-like receptor 4 and Fcγ receptor. Arthritis Rheum. (2011) 63:53–62. 10.1002/art.30081 PubMed DOI PMC

Sørensen OE, Borregaard N. Neutrophil extracellular traps: the dark side of neutrophils. J Clin Invest. (2016) 126:1612–20. 10.1172/JCI84538 PubMed DOI PMC

Thieblemont N, Wright HL, Edwards SW, Witko-Sarsat V. Human neutrophils in auto-immunity. Semin Immunol. (2016) 28:159–73. 10.1016/j.smim.2016.03.004 PubMed DOI

Hu X, Xie Q, Mo X, Jin Y. The role of extracellular histones in systemic-onset juvenile idiopathic arthritis. Ital J Pediatr. (2019) 45:1–9. 10.1186/s13052-019-0605-2 PubMed DOI PMC

Knight JS, Carmona-Rivera C, Kaplan MJ. Proteins derived from neutrophil extracellular traps may serve as self-antigens and mediate organ damage in autoimmune diseases. Front Immunol. (2012) 3:380. 10.3389/fimmu.2012.00380 PubMed DOI PMC

Lewis HD, Liddle J, Coote JE, Atkinson SJ, Barker MD, Bax BD, et al. . Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat Chem Biol. (2015) 11:189–91. 10.1038/nchembio.1735 PubMed DOI PMC

Darrah E, Giles JT, Ols ML, Bull HG, Andrade F, Rosen A. Erosive rheumatoid arthritis is associated with antibodies that activate PAD4 by increasing calcium sensitivity. Sci Transl Med. (2013) 5: 186ra65–186ra65. 10.1126/scitranslmed.3005370 PubMed DOI PMC

Neumann A, Berends ETM, Nerlich A, Molhoek EM, Gallo RL, Meerloo T, et al. . The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps. Biochem J. (2014) 464:3–11. 10.1042/BJ20140778 PubMed DOI

Lande R, Palazzo R, Gestermann N, Jandus C, Falchi M, Spadaro F, et al. . Native/citrullinated LL37-specific T-cells help autoantibody production in systemic lupus erythematosus. Sci Rep. (2020) 10:1–14. 10.1038/s41598-020-62480-3 PubMed DOI PMC

Lande R, Gregorio J, Facchinetti V, Chatterjee B, Wang Y-H, Homey B, et al. . Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature. (2007) 449:564–9. 10.1038/nature06116 PubMed DOI

Expanded population of low-density neutrophils in juvenile idiopathic arthritis