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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.


Selectively increased expression of HO-1 in splenic CD4+ T cells. a mRNA expression of HO-1 in splenic CD4+ T cells and non-CD4+ splenocytes in each group of mice (n = 5/each group). Values are shown mean ± SD. *P < 0.05. b Mouse splenic CD4+ T cells and non-CD4+ splenocytes were isolated from each group of mice. Lysates were prepared and analyzed by western blotting to detect HO-1. HO-1 expression was higher in a dose-dependent manner in splenic CD4+ T cells from A77 1726-treated mice compared with cells from vehicle-treated mice. c Representative images of HO-1-positive cells (stained in brown; original magnification ×400) are shown (left). The results are quantified as the mean ± SD for the number of HO-1-positive cells (n = 3 mice per group (right). *P < 0.05; **P < 0.01; ***P < 0.001
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Fig5: Selectively increased expression of HO-1 in splenic CD4+ T cells. a mRNA expression of HO-1 in splenic CD4+ T cells and non-CD4+ splenocytes in each group of mice (n = 5/each group). Values are shown mean ± SD. *P < 0.05. b Mouse splenic CD4+ T cells and non-CD4+ splenocytes were isolated from each group of mice. Lysates were prepared and analyzed by western blotting to detect HO-1. HO-1 expression was higher in a dose-dependent manner in splenic CD4+ T cells from A77 1726-treated mice compared with cells from vehicle-treated mice. c Representative images of HO-1-positive cells (stained in brown; original magnification ×400) are shown (left). The results are quantified as the mean ± SD for the number of HO-1-positive cells (n = 3 mice per group (right). *P < 0.05; **P < 0.01; ***P < 0.001

Mentions: To identify the mechanisms underlying the anti-inflammatory and oxidative stress-reducing properties of A77 1726 and its potential target cells, we analyzed the mRNA expression of ex vivo splenic CD4+ T cells and splenic non-CD4+ T cells isolated from each group of mice. mRNA expression of HO-1 was significantly increased in splenic CD4+ T cells isolated from A77 1726-treated IL-1Ra-KO mice (30 mg/kg) (Fig. 5a). Interestingly, the HO-1-inducing property of A77 1726 was seen only in CD4+ T cells and not in non-CD4+ splenocytes (Fig. 5a). Next, western blot analysis was conducted to confirm the HO-1 induction effects of A77 1726. The results showed that HO-1 expression in CD4+ T cells of arthritis mice was significantly induced by A77 1726 treatment in a dose-dependent manner (Fig. 5b). Immunohistochemical analysis also showed that A77 1726 increased the number of HO-1-expressing splenocytes (Fig. 5c). Taken together, these results suggest that the in vivo anti-inflammatory and oxidative stress-reducing effects of A77 1726 were related to the HO-1-inducing property and that the beneficial effects were restricted to T cells.Fig. 5


The active metabolite of leflunomide, A77 1726, attenuates inflammatory arthritis in mice with spontaneous arthritis via induction of heme oxygenase-1
Selectively increased expression of HO-1 in splenic CD4+ T cells. a mRNA expression of HO-1 in splenic CD4+ T cells and non-CD4+ splenocytes in each group of mice (n = 5/each group). Values are shown mean ± SD. *P < 0.05. b Mouse splenic CD4+ T cells and non-CD4+ splenocytes were isolated from each group of mice. Lysates were prepared and analyzed by western blotting to detect HO-1. HO-1 expression was higher in a dose-dependent manner in splenic CD4+ T cells from A77 1726-treated mice compared with cells from vehicle-treated mice. c Representative images of HO-1-positive cells (stained in brown; original magnification ×400) are shown (left). The results are quantified as the mean ± SD for the number of HO-1-positive cells (n = 3 mice per group (right). *P < 0.05; **P < 0.01; ***P < 0.001
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Fig5: Selectively increased expression of HO-1 in splenic CD4+ T cells. a mRNA expression of HO-1 in splenic CD4+ T cells and non-CD4+ splenocytes in each group of mice (n = 5/each group). Values are shown mean ± SD. *P < 0.05. b Mouse splenic CD4+ T cells and non-CD4+ splenocytes were isolated from each group of mice. Lysates were prepared and analyzed by western blotting to detect HO-1. HO-1 expression was higher in a dose-dependent manner in splenic CD4+ T cells from A77 1726-treated mice compared with cells from vehicle-treated mice. c Representative images of HO-1-positive cells (stained in brown; original magnification ×400) are shown (left). The results are quantified as the mean ± SD for the number of HO-1-positive cells (n = 3 mice per group (right). *P < 0.05; **P < 0.01; ***P < 0.001
Mentions: To identify the mechanisms underlying the anti-inflammatory and oxidative stress-reducing properties of A77 1726 and its potential target cells, we analyzed the mRNA expression of ex vivo splenic CD4+ T cells and splenic non-CD4+ T cells isolated from each group of mice. mRNA expression of HO-1 was significantly increased in splenic CD4+ T cells isolated from A77 1726-treated IL-1Ra-KO mice (30 mg/kg) (Fig. 5a). Interestingly, the HO-1-inducing property of A77 1726 was seen only in CD4+ T cells and not in non-CD4+ splenocytes (Fig. 5a). Next, western blot analysis was conducted to confirm the HO-1 induction effects of A77 1726. The results showed that HO-1 expression in CD4+ T cells of arthritis mice was significantly induced by A77 1726 treatment in a dose-dependent manner (Fig. 5b). Immunohistochemical analysis also showed that A77 1726 increased the number of HO-1-expressing splenocytes (Fig. 5c). Taken together, these results suggest that the in vivo anti-inflammatory and oxidative stress-reducing effects of A77 1726 were related to the HO-1-inducing property and that the beneficial effects were restricted to T cells.Fig. 5

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.