Background/Purpose: Citrullination is a post translational modification of arginine catalysed by peptidyl arginine deiminases (PADs) and may be important in generating pathogenic autoantibodies to citrullinated proteins in rheumatoid arthritis. However citrullination may control inflammation by other mechanisms, including transcriptional modulation of cytokines and generation of proinflammatory extracellular proteins. In previous studies, the PAD inhibitor Cl-amidine was shown to have a modest anti-inflammatory effect, when given prophylactically at high doses. While much of this benefit was thought to be due to inhibition of PAD4, recent studies have highlighted the proinflammatory effects of PAD2. Therefore the objective of this study is to examine the PAD inhibitor (BB-Cl-amidine), which is 10 fold more active than Cl-amidine against PAD2. We chose collagen-induced arthritis (CIA) as a reproducible and predictive model of immune-mediated arthritis and used a therapeutic, rather than prophylactic, treatment protocol, which could be translated into human disease.

Methods: CIA was induced by immunization with bovine type II collagen (CII) in DBA/1 mice. After the onset of arthritis, mice were scored daily for severity (0-3 for each paw) and hind paw swelling using calipers. Mice were treated daily with vehicle (n=12) or BB-Cl-amidine (1 or 10 mg/kg, n=12) intraperitoneally for 9 days post onset. On day 10 post onset, mice were culled, and paws, blood and inguinal lymph nodes collected for further analysis. Cytokines were measured in serum and lymph node culture supernatants using a multiplex platform, and serum IgG1 and IgG2a anti-CII antibodies by ELISA. The phenotypes of lymph node T cells and their response to anti-CD3 antibody stimulation were determined via flow cytometry. 

Results: Therapeutic administration of 10 mg/kg BB-Cl-amidine after the onset of arthritis, reversed its development. Compared with vehicle treated mice, there was reduced clinical scoring (p<0.0001 from 5 days treatment), paw swelling (p<0.0001 from 6 days treatment), with a partial response in all measures to 1 mg/kg. Histological changes were almost completely normalised by BB-Cl-amidine at 10mg/kg with significant reduction in histology scores (p<0.01). These effects were accompanied by a significant increase in anti-collagen IgG1 (p<0.0001) and no change in IgG2a antibody levels. Unexpectedly, while pro-inflammatory cytokine levels in serum remained unaffected by BB-Cl-amidine, IL-4 and IL-5 were significantly elevated. In line with this, IL-4-expressing Th2 cells were increased in the lymph nodes of BB-Cl-amidine treated mice. Further analysis revealed a significant decline in Th1 and Th17 numbers with BB-Cl-amidine treatment.

Conclusion: Our study demonstrates that BB-Cl-amidine is therapeutic in CIA. The effect appears more due to immunomodulation than simple immunosuppression, by supporting anti-inflammatory Th2 responses, while inhibiting pro-inflammatory Th1 and Th17 responses. We propose that targeting PADs is now a realistic strategy for the treatment of rheumatoid arthritis and other chronic inflammatory diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/immunomodulation-by-a-second-generation-peptidyl-arginine-deiminase-inhibitor-abrogates-collagen-induced-arthritis-in-a-therapeutic-protocol/