Clonorchis sinensis aggravates biliary fibrosis through promoting IL-6 production via toll-like receptor 2-mediated AKT and p38 signal pathways

Abstract

Clonorchis sinensis is an important food-borne zoonotic parasite which has been linked to biliary fibrosis and cholangiocarcinoma. However, the details of the pathogenesis of C. sinensis were unclear. To explore the role and regulatory mechanism of toll-like receptor 2 (TLR2) in C. sinensis-induced biliary fibrosis, we established the C. sinensis-infected C57BL/6 mouse model with TLR2-/- and wild type (WT) mice. The mortality rate, liver lesions, TLR2 and TGF-β1 expression, phosphorylation of Smad2/3, AKT, p38, ERK and p65, and cytokine productions were analyzed. Furthermore, similar parameters were examined in mouse biliary epithelial cells (BECs) co-cultured with C. sinensis excretory/secretory proteins (ESPs). The results showed that TLR2 expression was enhanced significantly in C. sinensis-infected WT mice and mouse BECs. C. sinensis-infected TLR2-/- mice exhibited an increased weight and a decreased mortality rate; significantly alleviated liver lesions and biliary fibrosis, reduced numbers of myofibroblasts; decreased expression of TGF-β1 and phosphorylation level of AKT, p38 and Smad2/3; significantly decreased production of IL-6, TNF-α and IL-4, while increased production of IFN-γ compared with C. sinensis-infected WT mice. Furthermore, C. sinensis ESPs could activate TLR2-mediated AKT and p38 pathways to increase the production of IL-6 in mouse BECs. In conclusion, these data indicate that C. sinensis infection activated TGF-β1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production, which resulted in myofibroblast activation and aggravating biliary fibrosis in mice.

Fig 1. C. sinensis induced prominently expressed TLR2 in mouse BECs.

(A, B) The survival rate and intrahepatic parasites for mice infected with difference C. sinensis metacercariae. WT mice (n = 10 mice per group) were orally infected with 0, 50, 100, 200, 400, 800 C. sinensis metacercariae, respectively. (A) The dead mice were recorded daily, and the survival curve was depicted. (B) Mice infected with C. sinensis were euthanized and the parasites from the livers were collected and calculated at 7 dpi, 15 dpi and 35 dpi. (C-L) TLR2 expression in mouse BECs. The WT and TLR2^-/- mice were orally administrated by 200 C. sinensis metacercariae or PBS, respectively. These mice were euthanized and livers were harvested at 7 dpi, 15 dpi and 35 dpi. The liver tissues were sliced into 5 μm and stained with antibodies against TLR2. The black arrow indicated TLR2 expression in BECs. Eggs were indicated by ‘EG’; Parasites were indicated by the letter ‘PA’. Scale bars = 20 μm. (J) The mean optical density of TLR2 expression indicated by immunohistochemical staining was digitized and quantitated by Image-Pro Plus software. (M) 3 × 10⁵ mouse BECs were stimulated with C. sinensis ESPs (50 μg/mL) at 6 h, 12 h and 24 h. TLR2 mRNA expression levels were detected by qPCR analysis. The mRNA level was normalized to β-actin.

Fig 2. TLR2^-/- mice decreased mortality rate, weight loss caused by C. sinensis.

The WT and TLR2^-/- mice were orally administrated by 200 C. sinensis metacercariae or PBS, respectively. (A) Mice were weighed every day and weight change curve was drawn. (B) The dead mice were recorded daily, and the survival curve was depicted. (C) The intrahepatic parasites were collected from C. sinensis-infected WT and TLR2^-/- mice at 35 dpi. C. sinensis adults and juvenile flukes were distinguished and counted. The data were expressed as the mean ± SD of three independent experiments.

Fig 3. C. sinensis enhanced bile duct and liver injury through TLR2 pathway.

(A) Liver lesions of C. sinensis-infected WT and TLR2^-/- mice at 7dpi, 15dpi and 35 dpi were observed. C. sinensis caused hepatomegaly, jaundice, cholestasis, connective tissue hyperplasia, and bile ducts dilated protrusions, white arrow pointed to gallbladder, and blue arrow pointed to bile ducts dilated protrusions. (B) Liver tissue sections were prepared and stained with H&E. Histopathological changes of C. sinensis-infected WT and TLR2^-/- mice at different time points were observed. The oozed inflammatory cells were indicated by the black arrows. Hepatic cells necrotic was indicated by ‘HN’; Obstruction of the biliary tract by parasites was indicated by the letter ‘PA’. Scale bars = 50 μm. (C) Immunohistochemistry for CK-19 in the liver from WT and TLR2^-/- mice at 7 dpi, 15 dpi and 35 dpi of C. sinensis infection. The BECs hyperplasia were indicated by the black arrows. Obstruction of the biliary tract by parasites was indicated by the letter ‘PA’. Scale bars = 20 μm. (D) The degree of liver inflammation and injury was calculated by hepatic HAI.

Fig 4. TLR2 deficiency alleviated liver fibrosis in C. sinensis-infected mice.

The WT and TLR2^-/- mice (n = 10) were orally administrated by 200 C. sinensis metacercariae or PBS, respectively. (A-I) liver fibrosis in C. sinensis-infected WT and TLR2^-/- mice at 7 dpi, 15 dpi and 35 dpi were visualized by Masson staining. Collagen deposition was visualized by blue stripes and indicated by the black arrows. Eggs were indicated by ‘EG’; Parasites were indicated by the letter ‘PA’. Scale bars = 50 μm. (J) Collagen depositions from each specimen were semi-quantified using Image-Pro Plus software.

Fig 5. TLR2 deficiency decreased TGF-β1 expression, phosphorylated Smad2/3 and myofibroblasts activation induced by C. sinensis.

The WT and TLR2^-/- mice (n = 10) were orally administrated by 200 C. sinensis metacercariae or PBS, respectively. These mice were euthanized and the livers were harvested at 7 dpi, 15 dpi and 35 dpi. (A) The expression level of TGF-β1 was detected by ELISA. (B, C) The phosphorylation of Smad2/3 in liver were analyzed by Western blot. (D) Immunohistochemistry for α-SMA in the liver from WT and TLR2^-/- mice following 7dpi, 15dpi and 35dpi of C. sinensis infection. α-SMA positive was shown as yellow and was pointed with black arrows. Scale bar = 50μm. (E) Quantification ofα-SMA positive cells in (D).

Fig 6. C. sinensis promoted the expressions of cytokines in liver and BECs through TLR2 pathway.

(A-D) The expression of IL-6, TNF-α, IL-4 and IFN-γ in the livers of WT and TLR2^-/- mice were detected separately by ELISA. (E) 3×10⁵ WT mouse BECs were co-cultured with ESPs (50 μg/mL) for 18 h, the cell supernatant was detected by ELISA for IL-6 expression.

Fig 7. C. sinensis ESPs increased the production of IL-6 via TLR2-mediated AKT and p38 pathways.

(A, B) The phosphorylation of AKT, p65, ERK and p38 in liver were analyzed by Western blot. (C, D) 3 × 10⁵ WT and TLR2^-/- mouse BECs were cultivated with C. sinensis ESPs (50 μg/mL) for 120 min, the cells were lysed in cells lysates and the phosphorylation of AKT, p65, ERK and p38 were analyzed by Western blot. (E) The production levels of IL-6 in the supernatant of BECs pretreated with or without AKT inhibitor, p38 inhibitor and ERK inhibitor for 1 h, then co-cultured with ESPs for 18 h were measured by ELISA.

Fig 8. C. sinensis activated TLR2 signal pathway to aggravate biliary fibrosis through promoting IL-6 production.

C. sinensis triggered AKT and p38 signal pathways dependent on TLR2 of BECs to promote the production of IL-6, which subsequently activated TGF-β1-Smad2/Smad3 pathways, myofibroblasts and up-regulated the expression of ECMs, finally resulting in biliary fibrosis in mice. In the figure, ‘↑’indicates positive regulation.