Background/Purpose: Neutrophil extracellular traps (NETs) are webs of chromatin and proteins extruded from neutrophils during NETosis. NETs are increased and display citrullinated proteins that are targeted by autoantibodies in rheumatoid arthritis. Citrullination, the posttranslational deimination of arginines to citrullines, is catalyzed by the peptidylarginine deiminases (PADs). Thus, NETs and PADs are thought to be important in the pathophysiology of rheumatoid arthritis. However, not all stimuli induce NETs with citrullinated proteins and, further, it has been suggested that different stimuli may induce different types of NET-like structures defined in part by the presence or absence of citrullination. Also, the requirement for PAD2 and PAD4, the main PADs expressed in neutrophils, in the formation of different NET-like structures has not been clearly defined, in part because many studies assess only citrullinated NETs. Here, we determine if specific stimulants induce citrullinated and/or uncitrullinated NET-like structures in humans and mice and if those structures require PAD2 or PAD4.

Methods: Neutrophils were isolated from human peripheral blood or the bone marrow of wild type, PAD4^-/-, and PAD2^-/- mice and were treated for 4 hours with no stimulant, ionomycin, monosodium urate (MSU) crystals, phorbol myristate acetate (PMA), or Candida albicans. Cells were fixed, stained with 4’6-diamidino-2-phenylindole (DAPI) and an anti-citrulline antibody (F95) and imaged at preset locations at 400x. NET-like structures, defined as highly decondensed DNA, were quantified in a blinded manner. A t-test was used to analyze results with p<0.05 considered significant.

Results: PMA, MSU, and C. albicans were strong activators of NET-like structures in human neutrophils with MSU and C. albicans inducing primarily citrullinated NET-like structures and PMA primarily uncitrullinated. Ionomycin was a weak inducer of NET-like structures, about a third of which were citrullinated. In contrast, ionomycin and C. albicans were strong inducers of NET-like structures in murine neutrophils with ionomycin-induced NETs mostly citrullinated. MSU variably induced NET-like structures and PMA induced very few NET-like structures in mice, which were almost all uncitrullinated. Murine C. albicans- and MSU-induced NET-like structures were a mix of citrullinated and uncitrullinated. PAD4^-/- neutrophils generated almost no citrullinated NET-like structures, with similar numbers of uncitrullinated NET-like structures to PAD4^+/+ neutrophils. PAD2^-/- neutrophils demonstrated no defects in any NET-like structures.

Conclusion: Different stimuli induce varying proportions of citrullinated and uncitrullinated NET-like structures in mice and humans making it difficult to define NET subsets by citrullination status and stimulant. PAD4 is required for the generation of citrullinated NET-like structures only, whereas PAD2 is dispensable for any murine NET-like structure. These findings critically inform the design and interpretation of studies evaluating the role of citrullination, PADs, and NET-like structures in rheumatoid arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reevaluating-citrullination-and-peptidylarginine-deiminases-in-neutrophil-extracellular-trap-like-structures/