Abstract
BackgroundLeflunomide is a low-molecular-weight compound that is widely used in the treatment of rheumatoid arthritis. Although leflunomide is thought to act through the inhibition of the de novo pyrimidine synthesis, the molecular mechanism of the drug remains largely unknown. We investigated the antiarthritis effects and mechanisms of action of the active metabolite of leflunomide, A77 1726, in interleukin-1 receptor antagonist-knockout (IL-1Ra-KO) mice.Methods14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30 mg/kg A77 1726 via intraperitoneal injection three times per week for 6 weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1β, tumor necrosis factor-α (TNF-α), and malondialdehyde were measured by enzyme-linked immunosorbent assay. Histologic analysis of the joints was performed using Safranin O, and immunohistochemical staining. The frequencies of interleukin-17-producing CD4+ T (Th17) cells were analyzed by flow cytometry. Heme oxygenase-1 (HO-1) expression in splenic CD4+ T cells isolated from A77 1726-treated arthritis mice were assessed by western blotting.ResultsA77 1726 treatment induced heme oxygenase-1 (HO-1) in Jurkat cells and primary mouse T cells. Interestingly, A77 1726 inhibited Th17 cell differentiation. In vivo, A77 1726 reduced the clinical arthritis severity of histological inflammation and cartilage destruction. The joints isolated from A77 1726-treated mice showed decreased expression of inducible nitric oxide synthase, nitrotyrosine, TNF-α, and IL-1β. The serum levels of TNF-α, IL-1β, and malondialdehyde were also decreased in A77 1726-treated mice. Whereas the number of Th17 cells in spleens was decreased in A77 1726-treated arthritis mice, a significant increase in the number of Treg cells in spleens was observed. Interestingly, HO-1 expression was significantly higher in splenic CD4+ T cells isolated from A77 1726-treated mice compared with those from vehicle-treated mice, whereas HO-1 expression of splenic non-CD4+ T cells did not differ between groups.ConclusionThe inhibitory effects of A77 1726 on joint inflammation and oxidative stress in autoimmune arthritis may be associated with HO-1 induction in CD4+ T cells.
Highlights
Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheuma‐ toid arthritis
A77 1726 induces nuclear-related factor 2 (Nrf2)‐HO‐1 axis and inhibited IL-17-pro‐ ducing CD4+ T (Th17) differentiation in a dose‐dependent manner in vitro First, we examined whether A77 1726 exerts a positive impact on the Nrf2-mediated heme oxygenase-1 (HO-1) induction in Jurkat T cells
Anti‐inflammatory effects of A77 1726 are associated with reduced expression of inducible nitric oxide synthase and nitrotyrosine in mice with inflammatory arthritis We investigated the expression of inflammatory cytokines in vehicle (DMSO)- or A77 1726-treated IL-1Ra-KO mic
Summary
Leflunomide is a low-molecular-weight compound that is widely used in the treatment of rheuma‐ toid arthritis. The pathogenesis of RA remains elusive, it is known to involve many cell types, including CD4+ T cells and B cells, in the inflamed hypertrophic synovium, called “pannus”. These cells play pathological roles in the development of RA by producing cytokines that perpetuate rheumatoid inflammation [1]. The major targets of RA treatment are the proinflammatory immune cells, especially CD4+ T cells, which are a pivotal player in the development and progression of RA, and their production of inflammatory cytokines, such as tumor necrosis factor-α (TNF- α), interleukin 1β (IL-1β), and IL-17. The reciprocal regulation of Th17/Treg subsets can be a novel therapeutic strategy for RA
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