Opisthorchis viverrini excretory/secretory products induce toll-like receptor 4 upregulation and production of interleukin 6 and 8 in cholangiocyte

Abstract

Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma--a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses is unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of pro-inflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma.

Fig. 1

Expression of TLR 1, TLR2, TLR3, TLR4 and TLR 6 in normal cholangiocytes (H69) and cholangiocytes cocultured with OVES. Semi-quantitative RT-PCR shows increased TLR4 mRNA (A). Significant increases in copy numbers of TLR4 transcript and protein are shown in cholangiocytes following exposure to OVES by qRT-PCR (B) and Western blot (C). Each value represents the mean ± SE of three independent experiments.

Fig. 2

Western blot analysis of IκB-α in control (neg), O. viverrini ES cocultured for 2 h and 8 h in H69 cells (H69) or H69-MyD88 dominant negative (H69-MyD88-DN). Densitometric analysis normalized to actin is shown in the lower panel. Significant (P < 0.05) degradation of IκB-α, compared to control, is shown at 8 h following incubation with the OVES. Each value represents the mean ± SE of three independent experiments.

Fig. 3

Nuclear translocation of NF-κB p65 in OVES cocultured cholangiocytes (H69+OVES) and control (H69) using imunofluorescence confocal microscopy. Average intensity of nuclear fluorescence analyzed from 200 cells is shown in the lower panel. Significant increase (P < 0.05) of fluorescence intensity is seen in the nucleus of OVES cocultured H69 cells. Each value represents the mean ± SE of three independent experiments.

Fig. 4

Cytokine levels of IL-6 and IL-8 in culture supernatants of H69 (control) and H69 cocultured with OVES (H69+OVES) by bead-based flow cytometery. Bar graph shows the levels of IL-6 or IL-8 in pg/mL. Note that OVES induces significant increased of IL-6 (**, P<0.001) and IL-8 (*, P<0.05) compared to control. Each value represents the mean ± SD of three independent experiments.

Fig. 5

Relative luciferase activity in OVES induced IL-6 and IL-8 promoter activation in H69. LPS is used as positive control for IL-6 and IL-8 activation. The IL-6 or IL-8 promoter driven luciferase activity, relative to transfection control Renilla luciferase expression, is significantly increased in OVES cocultured H69 biliary cells. However, polymyxin B inhibits LPS stimulated H69 cells but not OVES. Each value represents the mean ± SE of three independent experiments. * = P < 0.01 vs control (Ctrl), # = P<0.01 vs treatment without polymyxin B.