Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

www.nature.com/scientificreports OPEN Received: 11 April 2018 Accepted: 26 September 2018 Published: xx xx xxxx Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils Yebin Zhou1, Ling-Ling An1, Raghothama Chaerkady2, Nanette Mittereder1, Lori Clarke2, Taylor S. Cohen3, Bo Chen1, Sonja Hess2, Gary P. Sims1 & Tomas Mustelin1,4 Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination. Neutrophils are critically important for our defense against bacterial pathogens1,2. They are among the very first responders to sites of tissue injury and bacterial infection, where they use a large repertoire of receptors for bacterial and pro-inflammatory stimuli to trigger a range of responses that include the extrusion of neutrophil extracellular traps (NETs)3–7, phagocytosis1,8, reactive oxygen species (ROS) production9–11, and the secretion of numerous proteases and pro-inflammatory mediators12,13. Neutrophils are also attracting increasing attention as likely culprits in human diseases such as rheumatoid arthritis (RA)14–16 and systemic lupus erythematosus (SLE)17–20. In RA, neutrophils accumulate in the synovial fluid in the early stages of the disease21,22 and appear to be responsible for the increase in citrullination of numerous proteins observed in the synovial fluid of inflamed joints22–25. Approximately 70% of RA patients test positive for antibodies against citrullinated cyclic peptides (anti-CCPs)26,27, and have antibodies reactive with specific citrullinated epitopes in proteins such as filaggrin, fibrinogen, vimentin28, α-enolase, and histones14,15. Further support for an important role of protein citrullination in RA pathogenesis comes from genetic association studies, which implicated first PADI429–33 and later also PADI234 in the susceptibility to RA. These genes encode for the Ca2+-dependent protein arginine deiminases (PAD) 4 and 2, respectively, which catalyze the conversion of arginine to citrulline in protein substrates35–38. 1 Department of Respiratory, Inflammation, and Autoimmunity, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, United States. 2Antibody Discovery and Protein Engineering, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, United States. 3Department of Microbial Sciences, MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland, 20878, United States. 4Present address: Division of Rheumatology, School of Medicine, University of Washington, 750 Republican Street, Seattle, WA98109, United States. Gary P. Sims and Tomas Mustelin contributed equally. Correspondence and requests for materials should be addressed to G.P.S. (email: ) or T.M. (email: ) SCIEntIfIC REPOrTS | (2018) 8:15228 | DOI:10.1038/s41598-018-33385-z 1 www.nature.com/scientificreports/ Annotated Peptide Sequence Modifications Donors (Total =4) Protein WFDGQr(cit)AAENR R85(Citrulline) 4 NCF1 WFDGQr(cit)AAENr(cit)QGTLTEYcSTLMSLPTK R85(Citrulline) R90(Citrulline); 1 NCF1 EMFPIEAGAINPENr(cit)IIPHLPAPK R70(Citrulline 1 NCF1 FLQqr(cit)r(cit)RqARPGPQSPGSPLEEER Q334(Deamidated) R335(Citrulline) R336(Citrulline) Q338(Deamidated) 1 NCF1 GDTFIr(cit)HIALLGFEK R7(Citrulline); 1 NCF1 APGr(cit)PQLSPGQK R328(Citrulline) 2 NCF2 LFr(cit)PNEr(cit)QVAQLAK R184(Citrulline); R188(Citrulline) 1 NCF2 TPEIFr(cit)ALEGEAHR R238(Citrulline) 2 NCF2 Table 1. NCF citrullination in ionomycin/calcium treated neutrophils. Neutrophils express both PAD2 and PAD439,40. The former is found in many cell types, while the latter is more restricted to hematopoietic cells with highest levels in neutrophils. PAD4 contains a putative nuclear localization sequence41, but resides mostly in cytosolic structures in resting neutrophils40. PAD4 can also enter the nucleus to citrullinate histones42 and transcription factors43, and thereby participate in the epigenetic regulation of gene expression44 and stem cell differentiation43. Histone citrullination has also been implicated in the formation of NETs45–48, a complex process driven by multiple signaling pathways and that depends on ROS production (at least for some stimuli such as phorbol 12-myristate 13-acetate (PMA)) and involves a large transcriptional program49. Extruded NETs serve to trap bacteria3 and aid in the subsequent phagocytosis and killing process. NET formation can be induced by many stimuli48, including PMA, immune complexes, and live bacteria. Dysregulation of NET extrusion has been proposed to play a role in the pathogenesis of autoimmune conditions7,14,16–19,50, although it remains unclear if extracellular DNA and histones found in kidneys of patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis17, or in the circulation of patients with SLE18,19, originate only from NETs or also from other forms of tissue damage and cell death. In addition, a recent paper has shown that PAD4 can citrullinate NF-κB p65 to promote its nuclear translocation51. We have found that two cytosolic subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (neutrophil cytosol factor 2, NCF2) are citrullinated in primary human neutrophils upon activation of PADs by membranolytic stimuli. Furthermore, NCF1 has been found to be citrullinated in synovial fluid from RA patients24,25. NADPH oxidase is the main ROS machinery in neutrophils, and its activation is key in bacterial killing8–11. We provide evidence that PAD4 in neutrophils associates with NCF1 and NCF2, translocates with them during NADPH o (...truncated)