Triptolide ameliorates IL-10-deficient mice colitis by mechanisms involving suppression of IL-6/STAT3 signaling pathway and down-regulation of IL-17

Abstract

Triptolide is an active component of extracts derived from the medicinal vine Tripterygium Wilfordii Hook. f. (TWHF) whose extracts have been used to treat inflammatory bowel disease (IBD). We have reported that triptolide showed therapeutic activity in a murine IBD model, but the potential mechanism of action of this agent in IBD remains elusive. Accumulated data showed that both T-helper (Th) 1 and Th17 response may contribute to pathogenesis of human IBD and animal colitis. Interleukin (IL)-6/signal transducer and activator of transcription-3 (STAT3) signaling pathway play an important role in Th17 response as well as pathophysiology of IBD. We hypothesized that triptolide would attenuate the experimental colitis by repressing IL-17 and that this would involve down-regulation of IL-6/STAT3 signaling pathway. Histological examination demonstrated that triptolide significantly reduced the severity of colitis in C3H/HeJBir.IL-10-deficeint mice. Triptolide suppressed the IL-6/STAT3 signaling pathway, as well as repressed gene expression of IL-17 in vivo. In addition, triptolide (20 ng/ml) in vitro was able to down-regulate the IL-6/STAT3 pathway and reduce IL-17 expression in cultured colonic explants from patients with Crohn's disease (CD).

Introduction

Inflammatory bowel disease (IBD) is comprised of ulcerative colitis (UC) and Crohn's disease (CD) (Kucharzik et al., 2006). Based on studies in human and animal colitis model, it was considered the immune response in CD to be dominated by T-helper (Th) 1 cells. But this concept has been challenged since a novel T cell subtype, named Th17 cells, which play dominant role in autoimmune diseases, has been gradually recognized (Neurath, 2007, Tesmer et al., 2008). It was reported that Th17 cells are highly pathogenic and elicit IL-17-dependent inflammation (Langrish et al., 2005). Data showed that, T cell-mediated colitis correlates with increased appearance of IFN-γ-secreting Th1 cells and high levels of IL-17-secreting Th17 cells, implying that both Th1 and Th17 cells may contribute to pathogenesis of IBD (Hue et al., 2006).

Interleukin (IL)-6 is a cytokine that could combine with soluble IL-6 receptor (sIL-6R) which was found to be released via shedding from the surface of macrophages. The IL-6/sIL-6R complex in turn activates gp130-positive cells via trans-signaling (Scheller et al., 2006). IL-6 trans-signaling further induces signal transducer and activator of transcription-3 (STAT3), a downstream instrument of IL-6 signaling pathway (Rose-John et al., 2009). Recently, it was reported that the retinoid orphan receptor γ-T (RORγt) which is critical for Th17 differentiation, could be induced by IL-6 signaling in a STAT3-dependent way (Ivanov et al., 2006). It was indicated that the STAT3-dependent expression of RORγt provides the molecular basis for the critical role played by IL-6–gp130–STAT3 signaling in promoting the development of Th17 from CD4⁺ T cells (Nishihara et al., 2007). Thereby, the IL-6/STAT3 signaling pathway that is well characterized in IBD plays an important role in Th17 response, providing a new function of this pathway (Weaver et al., 2007). In summary, the IL-6–gp130–STAT3 signaling pathway plays predominant role in autoimmune diseases at least in part through the induction of Th17 response.

In addition to spontaneously developing Th1-mediated chronic enterocolitis (Spencer et al., 2002), IL-10-deficient mice (IL-10⁻/⁻ mice) which have many similarities to human CD, also showed high expression of IL-17 (Lytle et al., 2005). Emerging evidence suggests that Th17 cells play an important role in pathogenesis of IL-10⁻/⁻ mice as well as in other models of chronic inflammation (Yen et al., 2006). Additionally, elevated IL-6 and activated STAT3 have been observed in both human IBD and IL-10⁻/⁻ mice colitis (Atreya et al., 2000, Berg et al., 1996). Recently, it has been discovered that Th17 cells are the major effector cells mediating disease progression in animal IBD, and the author suggested the early disease in mice colitis is likely mediated by the Th1 subset but disease progression is due to Th17 cells (Elson et al., 2007). It was also reported that blockade of IL-6 trans-signaling reduces STAT3 activation, and approves IL-10⁻/⁻ mice colitis in a Th17-independent manner (Noguchi et al., 2007). These accumulated data indicated that similar to that in human IBD, Th1 and Th17 response may contribute together to pathogenesis of animal colitis.

Triptolide, a diterpene triepoxide, is an active component of extracts derived from the medicinal vine Tripterygium Wilfordii Hook. f. (TWHF) whose extracts have been used widely to treat autoimmune diseases in China for many years. It has been demonstrated to be effective for the treatment of autoimmune diseases (Lin et al., 2007, van Eden, 2009). The immunosuppressive activity of triptolide can be partly attributed to its potent inhibitory effects on monocytes activation (Liu et al., 2007). Recently, it was reported that extracts of TWHF showed therapeutic activity in mildly or moderately active CD (Ren et al., 2007). Additionally, triptolide could inhibit IL-1β-induced chemokine and MMP-3 expression in subepithelial myofibroblasts which play an important role in wound repair in CD (Tao et al., 2007). Studies carried out in our laboratory also showed that treatment with triptolide improves the spontaneous colitis of IL-10⁻/⁻ mice in a Th1-cytokines dependent way, indicating that triptolide has therapeutic activity in this model (Wei et al., 2008a, Wei et al., 2008b), but the potential mechanism of action of triptolide in IBD still needs further study.

Th17 immune response plays an important role both in human IBD and IL-10⁻/⁻ mice, additionally, IL-6/STAT3 signaling pathway participates in the Th17 response and IBD. Prompted by these important findings, we hypothesized that triptolide would ameliorate the experimental colitis by repressing IL-17 and that this would involve down-regulation of IL-6/STAT3 signaling pathway. Our results provided evidence that triptolide suppresses the IL-17 expression in IBD by mechanisms involving inhibiting of IL-6/STAT3 signaling pathway.