Aggregatibacter actinomycetemcomitans generates citrullinated autoantigens involved in rheumatoid arthritis through its pore-forming toxin leukotoxin A.

NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, that have been erroneously classified as “NETosis”. While these NETosis mimics share morphological similarities with NETosis (i.e. extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE. Distinguishing NETosis from LTH and defective mitophagy is paramount to understanding the role of neutrophil damage in immunity and the pathogenesis of human diseases. This provides a framework to design specific inhibitors of these distinct biological processes in human disease.

RA-associated hypercitrullination is specifically induced by immune-mediated membrane pore-forming pathways.

Purpose of review: Dysregulated citrullination is a key element that drives the production and maintenance of antibodies to citrullinated proteins, a hallmark in rheumatoid arthritis (RA). This article reviews recent literature on the origin of citrullinated antigens in RA.Recent findings: The study of synovial fluid from patients with RA has provided important insights into the identity of citrullinated proteins that accumulate in the RA joint (the RA citrullinome) and mechanisms that control their generation.Summary: Citrullinating enzymes (peptidylarginine deiminases, PADs) are tightly controlled to limit their hyperactivation. Calcium and redox conditions are important regulators of PAD activity. Studies suggest that citrullination is dysregulated both intra- and extracellularly in RA. In neutrophils, host (i.e., perforin and the membrane attack complex) and bacterial (i.e., toxins) pore-forming proteins induce prominent calcium influx, cytolysis, and hyperactivation of PADs. These factors likely drive hypercitrullination in the RA joint and at extraarticular sites of disease initiation, respectively. As oxidizing conditions present in the extracellular environment are known to inactivate PADs, extracellular citrullination in RA probably requires the constant release of active enzymes from dying cells and may be accelerated by autoantibodies that activate PADs.

Objective To define the relationship between autoantigen citrullination and different peptidylarginine deiminase (PAD) enzymes in rheumatoid arthritis (RA). Methods Citrullinated autoantigens were identified by immunoblotting control and ionomycin-activated human primary neutrophil lysate with RA sera. Autoantigen identity and citrullination sites were defined by mass spectrometry. PAD isoenzyme expression in human neutrophils was determined by immunoblotting. PAD substrate specificity was addressed in HL-60 cell lysates co-incubated with human recombinant PAD2, PAD3 and PAD4. Results Although prominent protein citrullination is observed in ionomycin-activated neutrophils, RA sera only recognised a limited number of these citrullinated molecules. Among these, the authors identified that β and γ-actins are citrullinated on at least 10 arginine residues, generating a novel 47 kDa species that is frequently recognised by RA autoantibodies. Interestingly, the authors showed that the PAD enzymes expressed in human neutrophils (ie, PAD2, PAD3 and PAD4) have unique substrate specificities, independent of their subcellular distribution. Thus, only PAD2 was able to citrullinate native β/γ-actin, while histone H3 was only citrullinated by PAD4. Conclusion These studies identified β and γ-actins as novel citrullinated autoantigens in RA, allowing enzyme specificity against intracellular substrates to be addressed. The studies provide evidence that PAD enzymes have the intrinsic capacity to select unique protein targets. The authors propose that unique PAD specificity may play a role in autoantigen selection in RA.

Objective: To address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD‐4).Methods: PAD‐4 autocitrullination was determined by anti–modified citrulline immunoblotting, using purified recombinant and endogenous PAD‐4 from activated human primary neutrophils and cell lines expressing PAD‐4. The citrullination sites in PAD‐4 were determined by mass spectrometry. Mechanisms of autocitrullination‐induced inactivation and the functional consequences of autocitrullination in PAD‐4 polymorphic variants were addressed using purified components and cell lines expressing PAD‐4 wild‐type, PAD‐4 mutant, and PAD‐4 polymorphic variants relevant to rheumatoid arthritis (RA).Results: PAD‐4 is autocitrullinated in vitro and during activation of primary cells and cell lines expressing PAD‐4. Interestingly, this modification inactivated the function of the enzyme. The efficiency of inactivation differed among genetically defined PAD‐4 variants relevant to RA. PAD‐4 was citrullinated at 10 sites, which are clustered into 3 distinct regions, including a cluster of arginines around the active site cleft where Arg‐372 and ‐374 were identified as the potential autocitrullination targets that inactivate the enzyme. Autocitrullination also modified the structure of PAD‐4, abrogating its recognition by multiple rabbit antibodies, but augmenting its recognition by human anti–PAD‐4 autoantibodies.Conclusion: Our findings suggest that autocitrullination regulates the production of citrullinated proteins during cell activation, and that this is affected by structural polymorphisms in PAD‐4. Autocitrullination also influences PAD‐4 structure and immune response.