Carmelo Carmona-Rivera et al. Sci. Immunol. 2018;3:eaar3701

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NETosis is not a source of cellular hypercitrullination.

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Carmelo Carmona-Rivera et al. Sci. Immunol. 2018;3:eaar3701 (A and B) Citrullinated protein patterns present in NETs vary depending on the disease state and stimulus. (A) NETs were induced in control (Ctrl) neutrophils by RF or spontaneously generated in lupus low-density granulocytes (LDGs). NETs were purified, and citrullinated proteins were detected using rhodamine-phenylglyoxal probes. The citrullination pattern varies when comparing the two samples. (B) Spontaneously formed NETs were purified from lupus low-density granulocytes isolated from a different patient as in (A), incubated in the presence or absence of 1000 IU of recombinant IFN-α for 2 hours. Citrullinated proteins were detected using rhodamine-phenylglyoxal. Total histone H3 (Abcam) and MPO (Dako) were used as loading controls. Increased citrullination of proteins is evident after IFN treatment. (C) Serum from RA patients preferentially recognizes citrullinated over native MPO. Human recombinant MPO (Lee BioSolutions) was citrullinated in vitro with purified PAD4. Purified PAD4, native MPO, and citrullinated MPO (cit-MPO) were resolved in a gradient 4 to 12% SDS–polyacrylamide gel electrophoresis gel. Proteins were transferred onto a nitrocellulose membrane and probed with high-titer ACPA+ serum from a subject with RA (1:250 dilution). Anti-human IgG coupled to horseradish peroxidase was used to develop the membrane. Same samples were probed against MPO to demonstrate the presence of recombinant MPO. RA serum can differentially recognize the citrullinated version of MPO over the native one. Results are representative of five RA subjects. Carmelo Carmona-Rivera et al. Sci. Immunol. 2018;3:eaar3701 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works