Background/Purpose: Inhibitor of DNA binding-1 (Id1) is a nuclear protein actively transcribed in endothelial progenitor cells (EPCs) and synovial fibroblasts. We previously identified Id1 as a soluble inflammatory protein that displays elevated expression in rheumatoid arthritis (RA) synovial tissues (STs) and effusions, contributing to angiogenesis, vasculogenesis and cellular migration. We and others have shown that RA fibroblasts display unchecked growth and are a major source of cytokine production. In this study, we investigated the mechanisms of Id1 regulation of synovial fibroblast cytokine production, trans-cellular activity and cellular growth in the RA joint.

Methods:  RA STs and K/BxN mouse ankles were analyzed histologically for correlation of Id1 expression and disease severity. Exosomes from fibroblast supernatants and synovial fluids (SFs) were purified via differential centrifugation. Exosome fractions were then subjected to 0.5% Triton X-100 for lysis and measured for Id1 by ELISA. RA fibroblasts were transfected with a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 plasmid, containing a GFP insert, via electroporation to induce error-prone non-homologous end joining repair (NHEJ), causing a nonsense mutation and removal of the targeted Id1 gene. To validate transfection efficiency, Tracking of Indels by Decomposition (TIDE) analysis was performed to verify CRISPR/Cas9 activity in the transfected fibroblasts. GFP-positive cells deleted of Id1 were then sorted by FACS, cultured for 24 hours, and measured for cytokine release and cell proliferation.

Results: Histologic analysis revealed positive correlations of Id1 expression and disease severity by Pearson’s Correlation Coefficient in RA STs (r=0.74, p<0.05, n=12) and K/BxN mouse ankles (r=0.87, p<0.05, n=10). RA compared to osteoarthritis (OA) fibroblast culture supernatants contained greater amounts of exosomes, and >80% of the Id1 released by RA fibroblasts was encapsulated within exosomes. Correspondingly, exosomes isolated from SFs have abundant levels of Id1 with a two-fold higher amount of Id1 in RA compared to OA SFs. We targeted Id1 to determine the function of Id1 on RA fibroblast growth and cytokine production. TIDE analysis confirmed CRISPR/Cas9-mediated NHEJ at the Id1 gene, resulting in Id1 deleted cells. Id1 deleted RA fibroblasts showed a 40% decrease in cell proliferation, and four and 100 fold increases in IL-6 and IL-8 production respectively, compared to transfected control plasmids and sham transfected (no plasmid) cells. Epithelial-derived neutrophil-activating peptide 78 (ENA-78)/CXCL5 did not show an increase in the Id1 deleted RA fibroblasts. The attenuated fibroblast growth in the RA fibroblasts can be explained, in part, by a significant increase in IL-6, a known inhibitor of fibroblast growth.

Conclusion:  We show that Id1 is upregulated and packaged within RA fibroblast exosomes for trans-cellular distribution. Id1 deletion by CRISPR/Cas9 transfection significantly slows RA fibroblast growth and induces elevated production of IL-6 and IL-8. Our data indicate that Id1 is an active regulator of both fibroblast growth and proinflammatory cytokine production in the RA joint.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/regulation-of-rheumatoid-arthritis-synovial-fibroblast-cytokine-production-by-inhibitor-of-dna-binding-1-via-crisprcas9-transfection/