Insight into Neutrophil Extracellular Traps through Systematic Evaluation of Citrullination and Peptidylarginine Deiminases

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.

Reduced Anti-Histone Antibodies and Increased Risk of Rheumatoid Arthritis Associated with a Single Nucleotide Polymorphism in PADI4 in North Americans

“…First, unlike the loss of NETs in the absence of PAD4 in PAD4 −/− mice [7], the reduction in PAD4 levels associated with rs2240335 may not be sufficient to reduce NETosis. Also, recent reports suggest that PAD4 is not required for the formation of NETs in response to some stimuli, particularly stimuli that do not induce citrullination [28,29,30,31]. Thus, perhaps the NETosis that we measured simply does not require PAD4.…”

Frontline Science: Anthrax lethal toxin-induced, NLRP1-mediated IL-1β release is a neutrophil and PAD4-dependent event

“…It is important to note that the PAD4 knockout data presented here are not sufficient to suggest that LT induces NETs in vivo, as substantial questions remain on PAD4's role in NET formation 47,48,54,70 . PAD4 impacts NLRP3 inflammasome function, 71 and could thus also possibly alter NLRP1 responses.…”

Peptidylarginine deiminase 2 has potential as both a biomarker and therapeutic target of sepsis

“…Several previous studies reported that PAD2 is not required for NET formation ( 46 , 47 ). However, while they used low concentrations of PMA, ionomycin, or LPS to induce small percentage of NET formation, we used higher concentrations of PMA and A23187 to induce an abundance of NET formation on the WT group.…”