MyD88-dependent and independent pathways of Toll-Like Receptors are engaged in biological activity of Triptolide in ligand-stimulated macrophages

Abstract

Background: Triptolide is a diterpene triepoxide from the Chinese medicinal plant Tripterygium wilfordii Hook F., with known anti-inflammatory, immunosuppressive and anti-cancer properties.

Results: Here we report the expression profile of immune signaling genes modulated by triptolide in LPS induced mouse macrophages. In an array study triptolide treatment modulated expression of 22.5% of one hundred and ninety five immune signaling genes that included Toll-like receptors (TLRs). TLRs elicit immune responses through their coupling with intracellular adaptor molecules, MyD88 and TRIF. Although it is known that triptolide inhibits NFkappaB activation and other signaling pathways downstream of TLRs, involvement of TLR cascade in triptolide activity was not reported. In this study, we show that triptolide suppresses expression of proinflammatory downstream effectors induced specifically by different TLR agonists. Also, the suppressive effect of triptolide on TLR-induced NFkappaB activation was observed when either MyD88 or TRIF was knocked out, confirming that both MyD88 and TRIF mediated NFkappaB activation may be inhibited by triptolide. Within the TLR cascade triptolide downregulates TLR4 and TRIF proteins.

Conclusions: This study reveals involvement of TLR signaling in triptolide activity and further increases understanding of how triptolide activity may downregulate NFkappaB activation during inflammatory conditions.

Figure 1

Effect of triptolide on gene expression of COX-2, iNOS and chemokines in response to various TLR ligand activation in RAW macrophages. The effect of triptolide treatment (three replicates) on a specific gene expression was measured by the mRNA quantity relative to the response to ligand activation only (positive control) that was normalized to a value of 1.00; lower values represent greater inhibitory effects with 0.00 corresponding to a complete inhibition of the induced gene expression. The value of the negative control (no induction) was normalized to 0.00. Values are mean ± S.D. *, p < 0.05; (post-ANOVA comparison with Ligand-treated positive control). A. Effect of triptolide on mRNA levels of COX-2, iNOS and CCL3 following zymosan stimulation. B. Effect of triptolide on mRNA levels of COX-2, iNOS, and IRG-1 following Poly I:C stimulation. C. Effect of triptolide on mRNA levels of COX-2, iNOS, CCL3 and IRG-1 following LPS stimulation.

Figure 2

Effect of triptolide on NFκB p65 nuclear translocation. Representative immunoblots of NFκB (p65). β-actin used as internal control. The protein expression was measured by densitometric analysis (Total Labs software v 2.01). The untreated control T^- was normalized to a value of 100. Each value represents mean ± SD of three experiments performed in triplicate. A. Effect of triptolide on NFκB translocation in MyD88KO RAW macrophages B. Effect of triptolide on NFκB translocation in TRIFKO RAW macrophages C. Effect of triptolide on NFκB translocation in Wild-type RAW macrophages. * Significantly different from control (p < 0.05) ANOVA followed by LSD

Figure 3

Effect of triptolide on gene expression of COX-2 and iNOS (mean ± S.D). The expression of specific genes was measured by the mRNA quantity relative to the response to LPS activation (positive control) that was normalized to a value of 1.00; lower values represent greater inhibitory effects with 0.00 corresponding to a complete inhibition of the induced gene expression. The value of the negative control (no induction) was normalized to 0.00. A. Expression of COX-2 and iNOS genes in MyD88KO macrophages; B. Expression of COX-2 and iNOS genes in TRIFKO macrophages; C. Expression of COX-2 and iNOS genes in WT macrophages. * Significantly different from control (p < 0.05) ANOVA followed by LSD.

Figure 4

Effect of triptolide on mRNA and protein expression of TRIF (mean ± S.D). The mRNA expression of TRIF was measured by the mRNA quantity relative to the response to LPS activation (positive control) that was normalized to a value of 1.00; lower values represent greater inhibitory effects with 0.00 corresponding to a complete inhibition of the induced gene expression. The value of negative control (no induction) was normalized to 0.00. A. mRNA expression of TRIF; B. Protein expression of TRIF; C. Densitometric analysis for protein expression. * Significantly different from control (p < 0.05) ANOVA followed by LSD.

Figure 5

Effect of triptolide on mRNA and protein expression of TLR 4 (mean ± S.D.). The mRNA expression of TLR 4 was measured by the mRNA quantity relative to the response to LPS activation (positive control) that was normalized to a value of 1.00; lower values represent greater inhibitory effects with 0.00 corresponding to a complete inhibition of the induced gene expression. The value of negative control (no induction) was normalized to 0.00. A. mRNA expression of TLR 4; B. Protein expression of TLR 4; C. Densitometric analysis for protein expression. * Significantly different from control (p < 0.05) ANOVA followed by LSD