Abstract
In rheumatoid arthritis, an autoimmune inflammatory arthritis, citrullinated proteins are targeted by autoantibodies and thus thought to drive disease. Neutrophil extracellular traps (NETs) are a source of citrullinated proteins and are increased in rheumatoid arthritis and therefore also implicated in disease pathogenesis. However, not all NETs are citrullinated. One theory aiming to clarify the intersection of citrullination, NETs, and rheumatoid arthritis suggests that specific stimuli induce different types of NETs defined by citrullination status. However, most studies do not evaluate uncitrullinated NETs, only citrullinated or total NETs. Further, the requirement for peptidylarginine deiminase (PAD) 2 and 4, two important citrullinating enzymes in neutrophils and rheumatoid arthritis, in the formation of different NETs has not been clearly defined. To determine if specific stimulants induce citrullinated or uncitrullinated NETs and if those structures require PAD2 or PAD4, human and murine neutrophils, including from PAD4−/− and PAD2−/− mice, were stimulated in vitro and NETs imaged and quantified. In humans, phorbol myristate acetate (PMA), ionomycin, monosodium urate (MSU), and Candida albicans induced NETs with MSU and C. albicans inducing primarily citrullinated, PMA primarily uncitrullinated, and ionomycin a mix of NETs. Only ionomycin and C. albicans were strong inducers of NETs in mice with ionomycin-induced NETs mostly citrullinated and C. albicans-induced NETs a mix of citrullinated and uncitrullinated. Interestingly, no stimulus induced exclusively citrullinated or uncitrullinated NETs. Further, PAD4 was required for citrullinated NETs only, whereas PAD2 was not required for either NET in mice. Therefore, specific stimuli induce varying proportions of both citrullinated and uncitrullinated NETs with different requirements for PAD4. These findings highlight the complexity of NET formation and the need to further define the mechanisms by which different NETs form and their implications for autoimmune disease.
Highlights
Neutrophil extracellular traps (NETs) are complex webs of chromatin and proteins extruded from neutrophils during the programmed cell death process of NETosis [1]
NETs, human neutrophils were isolated from peripheral blood and incubated with no treatment or each stimulant for 4 hours followed by fixation, staining to detect DNA and citrullinated proteins, imaging, and quantification
Some differences may be due to the location from which the neutrophils were purified and maturation level, these findings suggest that NET production varies with the source of neutrophils, which may contribute to conflicting reports about the ability of different stimuli to induce NETs [21]
Summary
Neutrophil extracellular traps (NETs) are complex webs of chromatin and proteins extruded from neutrophils during the programmed cell death process of NETosis [1]. A categorization of NETs has been hypothesized with NETosis induced by several stimuli including PMA, fungi, and monosodium urate (MSU) without citrullination and LTH induced by pore-forming molecules with citrullination [18]. Such a categorization is helpful for understanding different types of NETs, their mechanisms of formation, their functions, and their potentially different roles in autoimmune disease. We evaluate if PAD2 or PAD4 is required for the NETs induced by these stimuli
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