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The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Leflunomide 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.

Methods: 14- 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.

Results: A77 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.

Conclusion: The 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.

No MeSH data available.


Effects of A77 1726 on inflammatory molecules and oxidative stress in the joints of IL-1Ra-KO mice. a Tissue sections from the joints of IL-1Ra-KO mice treated with A77 1726 or vehicle were stained with antibodies to TNF-α, IL-1β, iNOS, or nitrotyrosine. The cells stained with each antibody are shown in brown (left). Expression of proinflammatory molecules and iNOS and nitrotyrosine (oxidative stress markers) was significantly attenuated in the joints of A77 1726-treated IL-1Ra-KO mice compared with vehicle-treated animals (right). b Levels of circulating TNF-α, IL-1β and MDA in the serum of IL-1Ra-KO mice in each group. Values are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001
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Fig3: Effects of A77 1726 on inflammatory molecules and oxidative stress in the joints of IL-1Ra-KO mice. a Tissue sections from the joints of IL-1Ra-KO mice treated with A77 1726 or vehicle were stained with antibodies to TNF-α, IL-1β, iNOS, or nitrotyrosine. The cells stained with each antibody are shown in brown (left). Expression of proinflammatory molecules and iNOS and nitrotyrosine (oxidative stress markers) was significantly attenuated in the joints of A77 1726-treated IL-1Ra-KO mice compared with vehicle-treated animals (right). b Levels of circulating TNF-α, IL-1β and MDA in the serum of IL-1Ra-KO mice in each group. Values are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Mentions: We investigated the expression of inflammatory cytokines in vehicle (DMSO)- or A77 1726-treated IL-1Ra-KO mic. TNF-α, and IL-1β are representative proinflammatory cytokines that participate in the inflammatory process in the rheumatoid synovium and have systemic effects [22, 23]. Compared with vehicle-treated IL-1Ra-KO mice, the joints from A77 7126-treated IL-1Ra-KO mice (10 and 30 mg/kg) showed markedly fewer TNF-α- and IL-1β-expressing cells (Fig. 3a). To determine the degree of oxidative stress in the joints, immunohistochemistry was used. The results showed that the expression of nitrotyrosine and iNOS was reduced in a dose-dependent manner in the joints of A77 1726-treated IL-1Ra-KO mice (Fig. 3a). Next, we used ELISA to measure the serum levels of TNF-α, IL-1β, and MDA in the different groups of mice. Although not statistically significant, the concentrations of TNF-α, IL-1β, and MDA tended to be lower in A77 1726-treated arthritis mice than in vehicle (DMSO)-treated animals (Fig. 3b). These results suggest that systemic administration of A77 1726 can inhibit joint inflammation, perhaps via inhibiting oxidative stress.Fig. 3


The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1
Effects of A77 1726 on inflammatory molecules and oxidative stress in the joints of IL-1Ra-KO mice. a Tissue sections from the joints of IL-1Ra-KO mice treated with A77 1726 or vehicle were stained with antibodies to TNF-α, IL-1β, iNOS, or nitrotyrosine. The cells stained with each antibody are shown in brown (left). Expression of proinflammatory molecules and iNOS and nitrotyrosine (oxidative stress markers) was significantly attenuated in the joints of A77 1726-treated IL-1Ra-KO mice compared with vehicle-treated animals (right). b Levels of circulating TNF-α, IL-1β and MDA in the serum of IL-1Ra-KO mice in each group. Values are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001
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Fig3: Effects of A77 1726 on inflammatory molecules and oxidative stress in the joints of IL-1Ra-KO mice. a Tissue sections from the joints of IL-1Ra-KO mice treated with A77 1726 or vehicle were stained with antibodies to TNF-α, IL-1β, iNOS, or nitrotyrosine. The cells stained with each antibody are shown in brown (left). Expression of proinflammatory molecules and iNOS and nitrotyrosine (oxidative stress markers) was significantly attenuated in the joints of A77 1726-treated IL-1Ra-KO mice compared with vehicle-treated animals (right). b Levels of circulating TNF-α, IL-1β and MDA in the serum of IL-1Ra-KO mice in each group. Values are shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001
Mentions: We investigated the expression of inflammatory cytokines in vehicle (DMSO)- or A77 1726-treated IL-1Ra-KO mic. TNF-α, and IL-1β are representative proinflammatory cytokines that participate in the inflammatory process in the rheumatoid synovium and have systemic effects [22, 23]. Compared with vehicle-treated IL-1Ra-KO mice, the joints from A77 7126-treated IL-1Ra-KO mice (10 and 30 mg/kg) showed markedly fewer TNF-α- and IL-1β-expressing cells (Fig. 3a). To determine the degree of oxidative stress in the joints, immunohistochemistry was used. The results showed that the expression of nitrotyrosine and iNOS was reduced in a dose-dependent manner in the joints of A77 1726-treated IL-1Ra-KO mice (Fig. 3a). Next, we used ELISA to measure the serum levels of TNF-α, IL-1β, and MDA in the different groups of mice. Although not statistically significant, the concentrations of TNF-α, IL-1β, and MDA tended to be lower in A77 1726-treated arthritis mice than in vehicle (DMSO)-treated animals (Fig. 3b). These results suggest that systemic administration of A77 1726 can inhibit joint inflammation, perhaps via inhibiting oxidative stress.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Background: Leflunomide 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.

Methods: 14- to 15-week-old male IL-1Ra-KO mice were treated with 10 or 30&nbsp;mg/kg A77 1726 via intraperitoneal injection three times per week for 6&nbsp;weeks. The effects of A77 1726 on arthritis severities were assessed by clinical scoring and histological analysis. The serum concentrations of IL-1&beta;, tumor necrosis factor-&alpha; (TNF-&alpha;), 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.

Results: A77 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-&alpha;, and IL-1&beta;. The serum levels of TNF-&alpha;, IL-1&beta;, 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.

Conclusion: The 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.

No MeSH data available.