TLR4 Deficiency Exacerbates Biliary Injuries and Peribiliary Fibrosis Caused by Clonorchis sinensis in a Resistant Mouse Strain.

Mice with different genetic backgrounds have various susceptibilities to infection with Clonorchis sinensis, although the mechanisms underlying are largely unknown. Toll-like receptor 4 (TLR4) as one of the most important pattern recognition receptors (PPRs) is essential for the invasion, survival, pathogenesis, and elimination of worms. The roles played by TLR4 in C. sinensis infection may vary due to the different genetic backgrounds of mice. In the present study, a relatively resistant mouse strain-C57BL/10 to C. sinensis was used for investigation on the possible roles of TLR4 in the biliary injuries and peribiliary fibrosis. TLR4 wild type (TLR4 wild ) and TLR4 defective (TLR4 def ) mice were orally infected with 45 metacercariae of C. sinensis, and all C. sinensis-infected mice and non-infected groups were anesthetized on day 28 post-infection. The liver and serum from each mouse were collected for assessment of the biliary injuries and biliary fibrosis. Meanwhile, hepatic leukocytes were isolated and detected for the activation of M1 or M2 macrophage using flow cytometry. The hepatic type 1 immune response and type 2 immune responses -relative molecules were also evaluated using ELISA and quantitative PCR. The data showed that TLR4 def aggravated liver inflammatory cell infiltrations, bile duct proliferation, biliary and hepatocellular injuries, and ECM deposition in C. sinensis-infected mice, compared with TLR4 wild mice when they were intragastrically administered with the same amounts of C. sinensis metacercaria. Furthermore, the M2-like macrophages and type 2 immune responses were significantly predominant induced in TLR4 def mice, compared with that of TLR4 wild mice following C. sinensis infection. But the type 1 immune response were significantly decreased in TLR4 def mice, compared with TLR4 wild mice after C. sinensis infection. These data demonstrate that TLR4 deficiency exacerbates biliary injuries and peribiliary fibrosis caused by C. sinensis in C57BL/10 strain mice, which is contributed by augments of type 2 immune responses and decrease pro-inflammatory responses.

The roles of Toll-like receptor 4 in the pathogenesis of pathogen-associated biliary fibrosis caused by Clonorchis sinensis.

Pathogen-associated biliary fibrosis (PABF) is a type of liver fibrosis characterized by injuries of cholangiocytes and extra cellular matrix (ECM) deposition around bile ducts caused by various bacteria, fungi, virus and parasites. Recent studies show that TLR4 plays an important role in several other types of liver fibrosis, but the mechanism of TLR4 in PABF is yet really unclear. In the present study, a PABF mouse model was established by a trematode infection-Clonorchis sinensis which dwells in the bile ducts and causes severe biliary fibrosis of mice. The results showed that the levels of collagen depositions, α-SMA and hydroxyproline (Hyp) contents in TLR4 mut mice infected by C. sinensis were significantly lower than in those of TLR4 wild ones. Furthermore, we found that the activation of TGF-ß signaling was impaired in the TLR4 mut mice, compared with wild mice when they were challenged to the same dose of C. sinensis metacercariae. Moreover, the mice with TLR4 mutation showed a decreased activation of hepatic stellate cells indicated by the expression of α-SMA, when compared with TLR4 wild mice. These data demonstrate that TLR4 contributes to PABF caused by C. sinensis and TLR4 signaling may be a potential medical target for treatment of PABF.

Expression and potential roles of IL-33/ST2 in the immune regulation during Clonorchis sinensis infection.

During clonorchiasis, immune responses of hosts are responsible for the removal of the worms and also are involved in the progress of the pathological damage caused by Clonorchis sinensis. Interleukin-33 (IL-33), a recently described cytokine signaling through the ST2 receptor, has emerged as a potent inducer to bile duct proliferation and fibrosis; however, little is known of this signaling in the pathogen-caused periductal inflammation and fibrosis. In the present study, using immunohistochemistry, real-time PCR, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, we studied the expression of IL-33/ST2 during C. sinensis infection, as well as their potential roles in C. sinensis-induced host immune responses. The results showed that a higher level of IL-33 was detected in the sera of patients of clonorchiasis (n = 45), compared with in those of healthy donors (n = 16). Similarly, in FVB mice experimentally infected with C. sinensis, a higher level of IL-33 was detected at latent stage both in the serum and in the liver, as well as the up-regulated expression of ST2 receptor on the inflammatory cells, especially on CD4(+) T cells in the liver of infected mice. Our results, for the first time, indicated that the increased IL-33/ST2 may be involved in the regulation of immunopathology induced by C. sinensis.

Clonorchis sinensis excretory/secretory products promote the secretion of TNF-alpha in the mouse intrahepatic biliary epithelial cells via Toll-like receptor 4.

BACKGROUND: Toll-like receptor 4 (TLR4), as one of the most important pathogen pattern recognitions (PPRs) plays a central role in elicitation of innate immunity and mediation of adaptive responses against foreign antigens. However, little is known of the roles of TLR4 in the immune responses of biliary epithelial cells (BECs) induced by Clonorchis sinensis, a parasite of significance in human health. METHODS: In the present study, the primary mouse intrahepatic biliary epithelial cells (MIBECs) were pre-treated with TLR4 inhibitor peptide or control peptide and then stimulated by excretory/secretory products (ESP) of C. sinensis, respectively. The expressions of TLR4 and relative cytokines were determined using western blot and a bead-based analytic detection system, respectively. RESULTS: The results showed that ESP of C. sinensis significantly increased the expression of TLR4 which promoted the expression of MyD88 and NF-κB in BECs; the levels of TNF-α but not IL-6 from MIBECs stimulated by ESP alone were also considerably increased, compared with the group of the medium stimulated. However, the concentration of TNF-α was significantly decreased when MIBECs were pre-treated with TLR4 inhibitor. In addition, ESP could depress the level of IL-6 in MIBECs which was elevated by LPS. CONCLUSIONS: Our data for the first time demonstrate that ESP of C. sinensis can potently induce secretion of pro-inflammatory cytokines via TLR4 in MIBECs, which suggests that TLR4 plays an important role in host defenses against C. sinensis and the pathogenesis of clonorchiasis.