Background/Purpose: In neutrophils, peptidylarginine deiminase 4 (PAD4) citrullinates histones allowing chromatin unraveling and neutrophil extracellular trap (NET) formation. NETs are increased and display proteins targeted by autoantibodies in rheumatoid arthritis. Interestingly, antibodies against citrullinated histones are among the first autoantibodies detected in rheumatoid arthritis. Thus, PAD4 may contribute to the development of rheumatoid arthritis by inducing NETs and ultimately autoantibodies. Murine studies have demonstrated that PAD4-deficient mice have reduced NETosis, autoantibodies, and inflammatory arthritis, but the effects of a loss of PAD4 in humans is unknown. Recently it has been shown that the G allele of single nucleotide polymorphism (SNP) rs2240335 in PADI4 is associated with decreased expression of PAD4 in human neutrophils in healthy Caucasians. The purpose of this study is to determine if the G allele of rs2240335 correlates with reduced NETs and autoantibodies against citrullinated histones in humans, similar to the PAD4 knockout studies in mice.

Methods: DNA, plasma, and serum from subjects with rheumatoid arthritis, both anti-cyclic citrullinated peptide (CCP) antibody negative (n=44) and positive (n=71), and controls (n=28) were obtained from our biorepository. Subjects were ~90% Caucasian. DNA was genotyped at rs2240335. Plasma from controls homozygous at rs2240335 was subjected to an enzyme linked immunosorbent assay (ELISA) to quantify circulating NETs. Serum from rheumatoid arthritis subjects homozygous at rs2240335 was subjected to ELISA to quantify IgG that binds native histones and histones in vitro citrullinated with human PAD4. Results were compared between genotypes using a student’s t test and verified by a permutation routine.

Results: The concentration of circulating NETs is significantly lower in controls homozygous for the G allele at rs2240335 compared to homozygotes for the T allele (p=0.027). In CCP+ rheumatoid arthritis subjects, there was no significant difference in anti-native and anti-citrullinated histone H1, H2A, H2B, H3, and H4 antibody levels between genotypes. However, for CCP- rheumatoid arthritis subjects, anti-citrullinated histone H2B (p=0.036) and H3 (p=0.0041) and anti-native histone H2A (p=0.0064), H2B (p=0.0073), and H3 (p=0.030) antibody levels were decreased in subjects with the GG genotype.

Conclusion: Similar to studies in PAD4-deficient mice, the G allele of SNP rs2240335, which has been shown to be associated with reduced PAD4 expression in human neutrophils, correlates with reduced NETosis in controls and reduced anti-histone antibodies in CCP- rheumatoid arthritis. Further studies are needed to determine if this allele is associated with rheumatoid arthritis risk in Caucasians.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-nucleotide-polymorphism-in-the-gene-encoding-peptidylarginine-deiminase-4-correlates-with-reduced-neutrophil-extracellular-traps-and-anti-histone-antibodies-in-rheumatoid-arthritis/