Past studies have demonstrated that triptolide can induce anti-inflammatory responses in several assay systems [14
]. However, the direct molecular targets of triptolide have remained elusive. In our previous report, we showed that triptolide significantly inhibited the secretion of inflammatory cytokines that occurred with RAW 264.7 cells when stimulated with LPS [13
]. Subsequently, we performed a gene array analysis (Tables , , ) to elucidate additional members of the immune signaling cascade that could be potential targets of triptolide activity. The results of this analysis showed that 44 genes, which were known to regulate inflammation were differentially expressed in stimulated RAW 264.7 cells following triptolide treatment. These 44 genes represented key immune signaling pathways such as TLR signaling, MAPK signaling, Jak-STAT signaling and cytokine-cytokine receptor interaction. In our present study we undertook further investigation on cellular mechanism of triptolide activity that focused on TLRs, given their upstream location in the immune signaling cascade and their increasingly recognized importance in inflammatory and autoimmune diseases [24
LPS induces TLR4 dimerization to trigger the activation of downstream signaling pathways [25
]. This receptor dimerization activates transcription factor NFκB, leading to the induction of inflammatory gene products such as COX-2 and iNOS [25
]. Many studies have demonstrated that triptolide and its synthetic derivatives inhibited NFκB activation induced by TLR4 agonist LPS [13
] but effect of triptolide on any TLR expression was never reported. The present study shows that triptolide suppressed ligand (LPS)-induced expression of TLR4 at mRNA and protein levels (Figure ). We also observed for the first time that triptolide suppressed Poly(I:C) (TLR3 agonist) and Zymosan (TLR2 agonist) induced expression of COX-2 and iNOS (Figure ). These observations suggest that triptolide may offer protection against wide range of infections that occurs by different TLR inductions.
The TLR-ligand activities are transduced through specific intracellular adaptor molecules, most notably MyD88 and TRIF. The importance of MyD88 and TRIF lies in finding that each leads to a distinct profile of immune mediators that in turn determines the phenotype of the cells that primarily are responsible for the development of adaptive immune responses [1
]. By studying the expression of well-characterized cytokine/chemokine target genes downstream of MyD88 (e.g., CCL3) and TRIF (e.g., IRG-1) [21
] we demonstrated differential regulation exerted by triptolide in ligand-induced 264.7 RAW macrophages. We observed that triptolide downregulated the expression of both MyD88-dependent cytokine such as CCL3 induced by MyD88-dependent ligands (Zymosan/TLR2 ligand, LPS/TLR4) and TRIF-dependent cytokine IRG-1 (Poly I:C/TLR3 and LPS/TLR4) in a concentration dependent manner (Figure and ). To further confirm that both MyD88 and TRIF mediated signaling are involved in triptolide activity, we showed that triptolide suppressed the NFκB translocation (p65 molecule) to the nucleus and also the downstream expression of COX-2 and iNOS mRNA in MyD88 KO (when only TRIF is present) and TRIF-KO (when only MyD88 is present) macrophages as well as in wild-type macrophages induced with TLR specific ligand, LPS (Figure and ). These results show that inhibition of NFκB activation and COX-2 and iNOS expression by triptolide could be achieved in presence of MyD88 or TRIF alone or when both are present as in wild type macrophages. These results also suggest that triptolide can block both MyD88- and TRIF-dependent pathways that lead to NFκB transactivation. Since MyD88 and TRIF are the only adapters exclusively mediating all TLR signaling, it is possible that triptolide may suppress wide-range of TLR signaling through other TLRs in addition to TLR4. Although in the current study we focused on TLR4 pathway, our gene array data (Tables and ) as well as the observations presented in Figure and may support the suggestion that triptolide has activities against other TLRs. When the changes in the expression of TRIF and MyD88 in response to triptolide were tested (Figure ), triptolide inhibited the mRNA and protein expression of TRIF, but not MyD88 in a dose dependent manner. A study by Yamamoto et al.
] using TRIF knockout mice has revealed that TRIF is physiologically essential for TLR-3 mediated signaling and that TRIF is involved in the LPS-induced MyD88-independent pathway.
Triptolide blocked early signaling pathways of TLRs suggesting that its inhibitory action was at the receptor and adapter molecule levels. Thus, a direct interaction of triptolide with TLR4 may be hypothesized. This study suggests that plant compounds, such as triptolide, can modulate TLR-mediated inflammatory responses and can reduce the risk of chronic diseases, associated with exaggerated TLR activation. Macrophages are the key antigen presenting cells in the pathogenesis of RA and involvement of TLR4 has been shown to play role in joint destruction in RA [29
]. Therefore, the present study showing interaction of triptolide with components of TLR signaling, such as TLR4, are particularly relevant and support the recent promise shown in clinics against RA by botanical extracts containing triptolide [30