Peptidylarginine Deiminase 2 Is Required for Tumor Necrosis Factor Alpha Induced Citrullination and Arthritis, but Not Neutrophil Extracellular Trap Formation

Mandar Bawadekar¹, Daeun Shim¹, Ryan Rebernick¹, Chloe Peyton¹, Chad J. Johnson², Thomas F. Warner³, Dres Damgaard⁴, Claus Henrik Nielsen⁵, Anthony P. Nicholas⁶, Ger JM Pruijn⁷, Jeniel E. Nett⁸ and Miriam A. Shelef^1,9, ¹Department of Medicine, Division of Rheumatology, University of Wisconsin-Madison, Madison, WI, ²Department of Medicine, University of Wisconsin-Madison, Madison, WI, ³Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, ⁴Institute for Inflammation Research, Center for Rheumatology and Spine Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark, ⁵Danish Rheumatologic Biobank and DANBIO registry, Rigshospitalet, Glostrup, Gentofte and Herlev University Hospital, Copenhagen, Denmark, ⁶Neurology, University of Alabama at Birmingham and Birmingham VA Medical Center, Birmingham, AL, ⁷Biomolecular Chemistry, Institute for Molecules and Materials and Radboud Institute for Molecular Life Sciences, Radboud University, Nijmegen, Netherlands, ⁸Medicine and Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, ⁹William S. Middleton Memorial Veterans Hospital, Madison, WI

Meeting: 2016 ACR/ARHP Annual Meeting

Date of first publication: September 28, 2016

Keywords: animal models and tumor necrosis factor (TNF), citrullination, NETosis, PAD, rheumatoid arthritis

Session Information

Date: Wednesday, November 16, 2016

Title: Rheumatoid Arthritis – Animal Models II

Session Type: ACR Concurrent Abstract Session

Session Time: 9:00AM-10:30AM

Background/Purpose: The presence of anti-citrullinated protein antibodies in rheumatoid arthritis points to a potential role for citrullination in disease pathogenesis. Peptidylarginine deiminases (PADs) catalyze the conversion of peptidylarginine to peptidylcitrulline. PAD2 and PAD4 are expressed in immune cells, but it is not known if either of these PADs is required for the increased citrullination seen in inflammatory arthritis. Neutrophil extracellular traps (NETs) may be a source of citrullinated proteins in inflammation. PAD4 is critical for NETosis and inflammatory arthritis, but the role of PAD2 is unknown. Here we use mice with TNFα-induced arthritis, an inflammatory, destructive arthritis similar to rheumatoid arthritis, to determine if PAD2 and/or PAD4 are required for joint citrullination and if PAD2 is required for NETosis and inflammatory arthritis.

Methods: Joint lysates from wild type, TNFα overexpressing (TNF⁺), TNF⁺PAD4^+/+, TNF⁺PAD4^-/-, TNF⁺PAD2^+/+, and TNF⁺PAD2^-/- mice were assessed for citrullination by western blot using an anti-peptidylcitrulline antibody. PAD2 levels were determined by qPCR in PAD4^+/+ and PAD4^-/- spleens and by western blot in TNF⁺PAD4^+/+ and TNF⁺PAD4^-/- joints. NETs formed by LPS-stimulated PAD2^+/+ and PAD2^-/- neutrophils were detected by immunofluorescent staining with DAPI and anti-citrullinated histone H4 and quantified. Killing of Candida by PAD2^+/+ and PAD2^-/- neutrophils was determined by a modified XTT assay. In TNF⁺PAD2^+/+ and TNF⁺PAD2^-/- mice, bone marrow plasma cells were identified by flow cytometry, serum IgG detected by ELISA, and arthritis assessed by blinded clinical and pathological scoring.

Results: TNF⁺ ankles had increased citrullination compared to wild type. TNF⁺PAD2^-/- ankles had less citrullination than TNF⁺PAD2^+/+ ankles, but citrullination was not reduced in TNF⁺PAD4^-/- compared to TNF⁺PAD4^+/+ ankles. There was no compensatory increase in PAD2 in PAD4 deficient spleens or arthritic ankles. NETosis and killing of Candida were not reduced in PAD2^-/-compared to PAD2^+/+neutrophils. Plasma cells, IgG, and arthritis were reduced in TNF⁺PAD2^-/- compared to TNF⁺PAD2^+/+ mice.

Conclusion: PAD2 contributes to TNFα-induced arthritis, plasma cell numbers, and IgG levels. Also, PAD2 is required for TNFα-induced joint citrullination, but not NETosis. In contrast, PAD4, which has been shown to be critical for NETosis, does not play a major role in TNFα-induced joint citrullination. Thus, NETs may not be the main source of citrullinated protein in arthritic mice.

Disclosure: M. Bawadekar, None; D. Shim, None; R. Rebernick, None; C. Peyton, None; C. J. Johnson, None; T. F. Warner, None; D. Damgaard, mAbSolution, 4; C. H. Nielsen, mAbSolution, 4; A. P. Nicholas, None; G. J. Pruijn, ModiQuest, 4; J. E. Nett, None; M. A. Shelef, None.

To cite this abstract in AMA style:

Bawadekar M, Shim D, Rebernick R, Peyton C, Johnson CJ, Warner TF, Damgaard D, Nielsen CH, Nicholas AP, Pruijn GJ, Nett JE, Shelef MA. Peptidylarginine Deiminase 2 Is Required for Tumor Necrosis Factor Alpha Induced Citrullination and Arthritis, but Not Neutrophil Extracellular Trap Formation [abstract]. Arthritis Rheumatol. 2016; 68 (suppl 10). https://acrabstracts.org/abstract/peptidylarginine-deiminase-2-is-required-for-tumor-necrosis-factor-alpha-induced-citrullination-and-arthritis-but-not-neutrophil-extracellular-trap-formation/. Accessed .

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