4-Octyl itaconate inhibits synovial fibroblast activation by targeting Nrf2 to exert anti-inflammatory effects in rheumatoid arthritis.

In vitro and in vivo experimental study examining the effects of 4-octyl itaconate on rheumatoid arthritis models.

“The aim of this project was to examine the anti-inflammatory effects of the nuclear factor erythroid 2–related factor 2 (Nrf2) activator 4-octyl itaconate (4-OI) on inflammatory rheumatoid arthritis (RA)-FLS–dominated pathology.”

Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), peripheral blood mononuclear cells, immortalised FLS, and mice in antigen-induced and collagen-induced arthritis models.

“Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and autologous cocultures of RA-FLS and peripheral blood mononuclear cells were cultured with 4-OI, corticosteroids or antitumor necrosis factor alpha antibodies and analysed for inflammatory activity with Western blot and enzyme-linked immunosorbent assay.”

13 patients provided samples for in vitro cultures; mouse cohorts included n=5 for the mBSA model and n=6 to 7 per group for the CIA model.

“A cross-sectional, paired set of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs), along with synovial fluid (SF) and plasma (P), was obtained from individual patients with active RA at the Department of Rheumatology, Aarhus University Hospital, from the INflammation in ARThritis biobank (n = 13).”

In vitro cultures were incubated for 48 hours; in vivo mouse studies involved treatment periods ranging from 2 days to 19 days post-induction.

“The cells were incubated at 37°C in a humidified incubator containing 5% CO2 in air for a duration of 48 hours.”

4-OI treatment increased expression of Nrf2-regulated proteins heme oxygenase-1 and NAD(P)H quinone dehydrogenase 1 while decreasing monocyte chemoattractant protein-1 and interleukin 6 secretion in RA-FLS.

“In RA-FLS in both monocultures and autolog cocultures, 4-OI treatment mediated an increase in gene expression of the Nrf2-regulated protein heme oxygenase-1 and NAD(P)H quinone dehydrogenase 1 with a concomitant decrease in monocyte chemoattractant protein-1 and specifically in monocultures also of interleukin 6 secretion.”

Local administration of 4-OI significantly reduced joint swelling in the methylated bovine serum albumin mouse model.

“In the methylated bovine serum albumin mouse model, local administration of 4-OI significantly reduced joint swelling.”

Systemic administration of 4-OI in the collagen-induced arthritis model resulted in a significant reduction of interleukin 6 in serum.

“In the collagen-induced arthritis model, a similar trend was seen accompanied by a significant reduction of interleukin 6 in serum.”

Gene silencing was performed using only one method, and in vivo results may be influenced by effects on macrophages in addition to fibroblast-like synoviocytes.

“A strength of this study is that gene silencing was performed in patient-derived immortalised RA-FLS; however, only 1 method was used and could be strengthened by other gene silencing methods such as siRNA.”