Background/Purpose: Autoantibodies targeting citrullinated proteins (ACPAs) are a characteristic feature and a diagnostic marker in rheumatoid arthritis (RA). The generation of citrullinated protein autoantigens is driven by the enzymatic activity of a family of calcium-dependent enzymes known as Peptidyl Arginine Deiminases (PADs). Increased PAD2 and PAD4, PAD activity, and citrullinated proteins are present in the joints of RA patients. The release of PAD2 and PAD4 from immune cells is likely to be responsible for the generation of citrullinated antigens and the development of autoimmune response in RA patients. Although neutrophils are identified as a major source of extracellular PADs, the mechanisms regulating PAD release are not well understood. This study is aimed at elucidating the cellular events that trigger the release of PADs from primary human neutrophils.

Methods: Primary human neutrophils were isolated from whole blood and were stimulated in vitro. Expression of PADI2 and PADI4 was assessed by RT-PCR. Total protein citrullination was assessed by Western blotting using an anti-peptidyl citrulline antibody (clone F95), and NETosis was visualized by live cell imaging. PAD2, PAD4, and IL-1b concentrations were measured by ELISA. PAD activity was assessed using a histone H3 citrullination assay. Serum samples from RA patients were obtained from Sanguine Bio.

Results: Toll-like receptor – 4, 7, and 9 and cytokine (TNFa, IL-1b, and IFNa) stimulations did not trigger transcriptional induction of PADI2 and PADI4 in human neutrophils. Release of PAD2 and PAD4 from neutrophils occurred independently of protein citrullination. Phorbol-12-myristate-13-acetate (PMA) – induced NETosis negatively regulated PAD release whereas LPS treatment triggered cell death-independent release of PAD2 and PAD4. PAD release upon LPS or LPS plus nigericin stimulation was dependent on activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome and treatment with NLRP3 inhibitor MCC950 led to significant reduction in PAD release in both neutrophil and whole blood assays. Concentrations of PAD2 and PAD4 as well as PAD activity showed significant positive correlation with IL-1b in RA patient serum samples.

Conclusion: We demonstrated cell death-independent release of PAD2 and PAD4 and identified NLRP3 inflammasome activation as a trigger promoting PAD release in human whole blood and neutrophils. Our data further suggest a role for NLRP3 inflammasome in amplifying the autoimmune response in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/nlrp3-inflammasome-promotes-release-of-peptidyl-arginine-deiminases2-and-4-from-human-neutrophils/