Pos1032 itaconate – a glucose metabolite with anti-inflammatory properties in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is the most prevalent autoimmune mediated joint disease. A complex dysfunction of the immune response is considered central for its pathogenesis as well as a dysregulated crosstalk between fibroblast-like synoviocytes (FLS) and monocytes. Objectives This project aims to examine and validate the anti-inflammatory effects of the natural glucose metabolite itaconate-derivative 4-octyl itaconate (4-OI) on stromal and immune cells from patients with active RA and evaluate the downstream effector molecule Heme-Oxygenase 1 (HO-1) as a potential biomarker. Methods Synovial fluid mononuclear cells (SFMC) from patients with chronic RA (cRA) were used to harvest RA FLS. Monocultures of synovial fluid derived FLS (SF-FLS) and autologous co-cultures of SF-FLS and peripheral blood mononuclear cells (PBMC) were cultured with and without 4-OI, corticosteroids, and anti-TNF (n=7). Subsequently, analyses by flow cytometry, Western Blotting, MTT assay, and ELISA were performed. The 4-OI target protein Nrf2 was knocked out in immortalized FLS (FLS-KO) generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Furthermore, we measured HO-1 by ELISA in plasma samples from newly diagnosed, active and treatment-naïve early RA (eRA) patients at baseline and after 6 months of intensive treatment (the OPERA trial, n=80) [1] with a follow-up period of 24 months and plasma and in synovial fluid samples from patients with active, chronic RA (cRA, n=20) during disease activity flares. Plasma samples from age approximated and gender matched healthy controls (HC) (n=35) were also included. Results In vitro 4-OI significantly inhibited MCP-1 secretion in cultured SFMC, THP-1 (monocyte cell line), and FLS and resulted in a concomitant increased HO-1 production. 4-OI reduced the MCP-1 secretion by 84%, anti-TNF by 41% and corticosteroids by 54% (all p<0.01) in co-cultures consisting of autologous RA SF-FLS and PBMCs. In FLS we observed a linear decrease in pro-inflammatory cytokine production. 4-OI did not decrease collagen production, even in dosage of 500 µn FLS we observed a linear decrease in pro-inflammatory cytokine production. 4-OI did not decrease collagen production, even in dosage of 500 1 production. 4-OI reduced the MCP. In eRA plasma levels of HO-1 was significantly increased at baseline (2039 pg/mL, 95% CI [1619, 2460]) vs HC (1209 pg/mL, 95% CI [1047, 1370]). Treatment with MTX or MTX + anti-TNF, lowered levels of HO-1 (1704 pg/mL, 95% CI [1377, 2031]) after 6 months irrespective addition of anti-TNF treatment, reducing the difference to a no longer significantly different level from HCs. HO-1 was associated with number of swollen joints (r=0.25, p<0.02), tender joints (r=0.24, p<0.02) and Clinical Disease Activity Index (r=0.23, p<0.02). Baseline HO-1 predicted radiographic joint damage progression (change in Total Sharp-van der Heijde score (Δn eRA plasma levels of HO-1 was significantly increased at baseline (2039 pg/mL, 95% CI [1619, 2460]) vs HC (1209 pg/mL, 95% CI [1047, 1370]). Treatment with MTX or MTX + anti-TNF, lowered levels of HO-1 (1704 pg/mL, 95% CI [1377, 2031]) after 6 months irund (r=0.75, p<0.001). High levels of HO-1 in SF from inflamed joints were associated with CRP (r=0.52, p<0.05) during flares in cRA. Conclusion In vitro 4-OI decreased MCP-1 secretion and increased the HO-1 expression in cultures of monocytes and in RA FLS and PBMC without decrease in collagen production or cell growth. In total, 4-OI decreased MCP-1 in both immune and stromal dominated RA pathobiology. In eRA patients, pre-treatment-plasma HO-1 levels were increased and correlated with parameters for disease activity, treatment-effect after 6 months, and predicted radiographic progression after 24-month. Reference [1] Hørslev-Petersen K et al. Ann Rheum Dis. 2013 Acknowledgements We thank medical doctors and nurses at the Department of Rheumatology, Aarhus University Hospital for helping to collect the patient samples. The authors kindly acknowledge a generous grant from the Danish Rheumatoid Association (R177-A6156). Disclosure of Interests None Declared.