Background/Purpose: Citrullination is a post-translational modification mediated by peptidyl arginine deiminase (PAD) enzyme in which arginine is converted to citrulline. Inhibitor of DNA binding 1 (Id1) is known to be actively transcribed in endothelial progenitor cells (EPCs) and cells that exhibit hyperproliferative responses. Previously, we showed that Id1 is expressed in rheumatoid arthritis synovial tissues (RA STs) and upregulated in RA synovial fluids (SFs), suggesting that Id1 may be post-translationally modified in RA. A more detailed analysis of the structure of Id1 reveals a relatively small protein of approximately 16kDa, but containing 10 modifiable arginines. We show that Id1 can be citrullinated, and that RA STs and SFs contain citrullinated Id1 (cit-Id1), suggesting that cit-Id1 may be pathogenic in the course of RA. We also show that by immunodepletion of Id1, we can reduce the angiogenic potency and overall amount of citrullinated protein in RA SF.

Methods: Id1 concentrations in RA SFs were measured by enzyme-linked immunosorbent assay (ELISA). To further explore the role of Id1 in RA SF, we immunodepleted Id1 with a neutralizing antibody or non-specific IgG (“sham depleted”), and used this in the severe combined immunodeficient (SCID) mouse chimera. Mice grafted with human RA ST were injected i.v. with fluorescently dye-tagged EPCs while receiving simultaneous intragraft injections with the treated SFs. To examine Id1 citrullination, we modified recombinant human (rhu) Id1 by incubation with rabbit PAD enzyme. Cit-Id1 was measured for deimination by a cit-ELISA using an anti-modified citrulline antibody. Citrullinated bovine serum album (BSA) was used as a relative standard. RA SFs imunodepleted with either Id1 or sham depleted were also measured by cit-ELISA to determine the total amount of citrullinated protein contained in RA SFs before and after Id1 depletion. Finally, we homogenized RA ST and performed immunoprecipitation (IP) of Id1 to determine the presence of Id1 and cit-Id1 by Western blotting using anti-human Id1 and anti-modified citrulline antibodies. 

Results:

We found a 50% reduction in EPC recruitment to intragraft injections of RA SF immunodepleted of Id1 compared to sham depleted RA SF (*p<0.05). We measured the amounts of total citrullinated protein in RA SF, and found that by removal of Id1, we could significantly reduce the total amount of citrullinated protein in RA SF (*p<0.05). Western blot analysis of Id1 and cit-Id1 confirmed that we could discriminate cit-Id1 from non-cit-Id1, and that we could successfully modify Id1 in vitro. Similarly, Id1 isolated by IP from RA ST homogenates showed that we could detect both Id1 and cit-Id1 in RA ST homogenates, and that a significant portion of the total Id1 in RA ST was in modified form.

Conclusion: We identify Id1 as an angiogenic factor, capable of recruiting EPCs to RA ST. We also show for the first time that Id1 can be modified using rabbit PAD enzyme. The significant shift downward in total citrullination in RA SFs depleted of Id1 indicates that cit-Id1 is present and elevated in the RA SF. We also show that Id1 in the RA ST is also citrullinated. Taken together, we show that Id1 functions as an angiogenic mediator and is robustly deiminated in RA tissues.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/evidence-for-citrullination-of-the-nuclear-transciption-factor-inhibitor-of-dna-binding-1-id1-in-rheumatoid-arthritis/