The Effect And Mechanism Of TLR2 And TLR3 In Liver Fibrosis Caused By Clonorchis Sinensis

Clonorchis sinensis,an important food-borne zoonotic parasite,parasitizing in the bile ducts of humans and other animals induces clonorchiosis,has been linked to liver fibrosis and cholangiocarcinoma.Over 15 million people are infected by this liver fluke disease in the east Asia,including about 13 million in China,and has been listed as a neglected tropical disease.People infected with C.sinensis are six times more likely to develop cirrhosis than uninfected people,and C.sinensis has been classified as a Class I carcinogen by the International Agency for Research on Cancer.The current prevention and control of clonorchiasis is based on the promotion of no raw fish and prawns,and using praziquantel.There is no effective vaccine for clonorchiosis and there are no drugs or therapies to reverse liver fibrosis caused by C.sinensis.An in-depth understanding of the pathogenesis of C.sinensis-induced liver fibrosis will lay the foundation for the new drugs development and strategies for the prevention and treatment of clonorchiosis and the liver fibrosis caused by C.sinensis.As important innate immune pattern recognition receptors,Toll-like receptors（TLRs）can recognize a variety of endogenous and exogenous pathogen-associated molecular patterns,trigger signaling pathways and induce the release of inflammatory cytokines.TLR2,TLR3,TLR4 and TLR9 have been reported to be associated with liver fibrosis.However,the role and specific mechanisms of TLRs in C.sinensis-induced liver fibrosis and resistance to C.sinensis infection have not been fully elucidated.To address these scientific problems,this study first conducted a survey on the prevalence of C.sinensis and other fish-borne zoonotic trematodes（FZTs）in Jilin province;the C.sinensis-infected mouse model was established,to investigate the roles of TLR2 and TLR3 in C.sinensis-induced liver fibrosis,and analyze the molecular mechanisms of the two receptors-regulated signaling pathways and cytokine expression.In addition,the roles of C.sinensis excreted/secreted proteins（ESPs）and extracellular vesicles（CSEVs）in recognizing TLR2 and TLR3were studied.Finally,the effect of Poly（I:C）,which targets TLR3,was investigated in the treatment of clonorchiasis.The main research contents and results are as follows:1.Prevalence of FZTs infection in Yitong river basin of Jilin provinceFZTs are the most serious food-borne parasites in Asia,including C.sinensis,which is predominantly endemic in China.In Jilin province,the epidemiological background of FZTs in the intermediate and definitive hosts has not been explored extensively.From July to November in 2020,a total of 132 fresh water snails,4122wild freshwater fishes and 143 fecal samples from canines,ducks and swine were collected from Yitong river basin of Jilin province.The FZT species were identified by morphological observation combined with internal transcribed spacer（ITS）sequences analysis.Results showed that the prevailing species of FZTs in Jilin province were Clonorchis sinensis,Metorchis orientalis and Echinochasmus japonicus.The total prevalence of FZTs was 29.74%（1226/4122）in fish,2.27%（3/132）in snails,75%（21/28）in canines and 37.18%（29/78）in ducks.The co-infection rates of the two trematodes were 13.39%（552/4122）in fish,35.71%（10/28）in canines and 7.69%（6/78）in ducks.The co-infection rate of the three flukes was 2.60%（107/4122）in fish.9 of the 12 fish species examined were infected with FZTs metacercariae.2.Establishment of C.sinensis-induced liver fibrosis mouse model（1）Determination of the optimal number of C.sinensis infections.The C57BL/6 WT mouse model with different numbers of C.sinensis metacercariae was established,the mortality rate and the number of intrahepatic parasites in the mice were observed and detected.The results showed that the optimal number of metacercariae for C.sinensis-induced liver fibrosis in C57BL/6 mouse model was 200metacercariae/mouse.（2）Detection of liver inflammation and injury in C.sinensis-infected mice.The liver lesions were observation,the liver inflammation and injury were analyzed by immunohistochemistry and histomorphology.The results showed that C.sinensis infection caused enlargement of the gallbladder,bile stasis,connective tissue protrusions with intrahepatic inflammatory cell infiltration,foci of hepatic necrosis,bile duct degeneration and ulceration.（3）Detection of liver fibrosis indicators in C.sinensis-infected mice.The liver fibrosis model was established,the expression of TGF-β/Smad pathway factors,liver fibrosis markerα-SMA,type I collagen（Col1a）and type III collagen（Col III）were detected by RT-PCR,the collagen deposition in the liver was detected by Masson staining.The results showed that TGF-β/Smad pathway factors,andα-SMA,Col1a,Col III were significantly increased in C.sinensis infected mice,with persistent accumulation of collagen fibers around bile ducts and fibrosis formed at 35days（d）.3.The effects and mechanism of TLR2 in liver fibrosis caused by C.sinensisTo investigate the effect and mechanism of TLR2 in liver fibrosis induced by C.sinensis,the C.sinensis-infected TLR2^-/-and WT mouse model was established.（1）TLRs expression in liver of C.sinensis infected mice.The expression of TLRs in liver was detected by RT-PCR and the expression of TLR2 in biliary epithelial cells was detected by immunohistochemistry.The results showed that expressions of TLR2,TLR3,TLR4 and TLR9 were significantly increased by C.sinensis in liver of WT mice.Immunohistochemistry results showed that TLR2 expression level in BECs of C.sinensis-infected WT mice was significantly increased compared to that in PBS control group.（2）The effect of TLR2 on body weight,survival and numbers of parasites in C.sinensis-infected mice.The weight,percent survival of C.sinensis-infected WT and TLR2^-/-mice were recorded daily,and the numbers of intrahepatic parasites in C.sinensis-infected WT and TLR2^-/-mice was counted at 35d.The results showed that the weight of C.sinensis-infected WT mice was significantly decreased,with an overall mortality rate of 40%,and the number of intrahepatic parasites was mean 13.Compared with C.sinensis-infected WT mice,the weight loss,mortality rate（25%）and intrahepatic parasites（7）were reduced in C.sinensis-infected TLR2^-/-mice.（3）The effect of TLR2 on liver inflammation and injury in C.sinensis infected mice.The liver inflammation and injury were analyzed by immunohistochemistry and histomorphology,the expressions of cytokines were analyzed by ELISA.The results showed that C.sinensis infection caused significant liver inflammation and injury in mice,with a significant increase in intrahepatic IL-6,TNF-α,IFN-γand IL-4 secretion.TLR2 deficiency reduced liver injury and bile duct degeneration,and decreased IL-6,TNF-αand IL-4 secretion in C.sinensis-infected mice.（4）The effect of TLR2 on liver fibrosis in C.sinensis-infected mice.The expression of TGF-β1 was detected by ELISA,the phosphorylation level of Smad2/3was analyzed by Western blot,the expression ofα-SMA and GFAP were analyzed by immunohistochemistry,and the collagen deposition was detected by Masson staining.The results showed that TLR2 deficiency reduced the expression of TGF-β1 and phosphorylation levels of Smad2/3 in mice,decreased the number ofα-SMA-positive myofibroblasts,and slowed liver fibrosis.（5）C.sinensis ESPs activated BECs TLR2 pathway to regulate the secretion of cytokine.The phosphorylation levels of AKT,p65,p38 and ERK in BECs were detected by Western blot and the secretion of cytokine was detected by ELISA.The results showed that C.sinensis ESPs activated the TLR2-mediated AKT and p38 pathways and increased the production of IL-6 in mouse BECs.4.The effects and mechanism of TLR3 in liver fibrosis caused by C.sinensisTo investigate the effect and mechanism of TLR3 in liver fibrosis induced by C.sinensis,the C.sinensis-infected TLR3^-/-and WT mouse model was established.（1）The effect of TLR3 on wight,percent survival and parasites numbers in C.sinensis-infected mice.The weight,percent survival of C.sinensis-infected WT and TLR3^-/-mice were recorded daily,and the numbers of intrahepatic parasites in C.sinensis-infected mice was counted at 35 d.Compared with C.sinensis-infected WT mice,results showed the weight loss,mortality rate and intrahepatic parasites were increased in C.sinensis-infected TLR3^-/-mice.The RT-PCR results showed that C.sinensis significantly increased the expression of TLR3 in WT mice liver.（2）The effect of TLR3 on liver inflammation and injury induced by C.sinensis.The liver inflammation and injury were analyzed by immunohistochemistry and histomorphology,the expressions of cytokines were detected by ELISA.The results showed that liver injury,bile duct degeneration was aggravated,secretions of IL-6,TNF-αand IL-4 were increased,but IFN-γsecretion was decreased in C.sinensis-infected TLR3 deficiency mice.（3）The effect of TLR3 on liver fibrosis in C.sinensis-infected mice.The expression of TGF-β1 was detected by ELISA,the phosphorylation level of Smad2/3 was analyzed by Western blot,the expression ofα-SMA was analyzed by immunohistochemistry,the collagen deposition was detected by Masson staining.The results showed that TLR3 deficiency further augmented the expression of TGF-β1 and phosphorylation levels of Smad2/3 in mice,increased the number ofα-SMA-positive myofibroblasts,and aggravated liver fibrosis.（4）C.sinensis ESPs and CSEVs activated BECs TLR3 pathway to regulate the secretion of IL-6.BECs was stimulated by ESPs or CSEVs,TLR3 m RNA level was detected by RT-PCR,phosphorylation levels of AKT,P65,P38 and ERK were detected by Western blot,and cytokine secretion was detected by ELISA.The results showed that TLR3 m RNA expression,p38 and ERK phosphorylation and IL-6 and TNF-αsecretion was promoted by C.sinensis ESPs and CSEVs in BECs;When TLR3 deficiency,the phosphorylation levels of P38 and ERK and the secretion of IL-6 and TNF-αwere further increased.Dot immunobinding assay（DIBA）showed that the high-concentration ds RNA was found in C.sinensis ESPs and CSEVs.Poly（I:C）treatment inhibited IL-6 and TNF-αsecretion in BECs induced by ESPs or CSEVs.5.The effects of TLR3 agonist Poly（I:C）on liver fibrosis induced by C.sinensisThe C.sinensis-infected WT mouse model was established,and was treated or untreated with Poly（I:C）.（1）The effect of Poly（I:C）on wight,percent survival and parasites numbers in mice infected with C.sinensis.The body weight and mortality were recorded daily during infection.The number of intrahepatic parasites was counted and analyzed at 35 d.The results showed that body weight were significant increased,mortality was completely blocked,and the number of parasites was decreased in C.sinensis-infected mice with the Poly（I:C）treatment.（2）The effect of Poly（I:C）on liver inflammation and injury in C.sinensis infected mice.The expressions of cytokines were analyzed by ELISA,the liver inflammation and injury were analyzed by immunohistochemistry and histomorphology.These results showed that Poly（I:C）treatment significantly decreased the secretions of IL-6,TNF-αand IL-4,significantly increased IFN-γsecretions;Poly（I:C）treatment significantly alleviated the liver injury and bile duct degeneration caused by C.sinensis;（3）The effect of Poly（I:C）on liver fibrosis induced by C.sinensis.The expression ofα-SMA were analyzed by immunohistochemistry,and the collagen deposition was detected by Masson staining.The results showed that the number ofα-SMA-positive myofibroblasts was decreased,and liver fibrosis was significantly alleviated after Poly（I:C）treatment.In summary,this study investigated the prevalence of C.sinensis and other FZTs in the Yitong river basin of Jilin province,and identified the Yitong river basin of Jilin province as the epidemic focus of C.sinensis,M.orientalis and E.japonicus.The role and mechanism of TLR2 and TLR3 in liver fibrosis of C.sinensis were investigated for the first time.C.sinensis infection activated TGF-β1-Smad2/3 through TLR2-mediated AKT and p38 pathways to promote IL-6 production,which resulting in myofibroblasts activation,aggravating liver fibrosis in mice.Besides,TLR3recognized ds RNA,which was in C.sinensis ESPs and CSEVs,reduced IL-6 and TNF-αsecretion by regulating the phosphorylation of p38 and ERK to decreased liver inflammation and injury,thereby relieving C.sinensis-induced liver fibrosis.Treatment with the TLR3 agonist Poly（I:C）alleviated C.sinensis-induced liver fibrosis.In addition,this study first affirmed that myofibroblasts were main derived from portal fibroblasts in C.sinensis-induced liver fibrosis.Based on the above study,we obtained epidemiological data on C.sinensis and other FZTs in Jilin province,revealed the different roles and mechanisms of TLR2 and TLR3 in liver fibrosis caused by C.sinensis,and provided candidate drugs for the treatment of C.sinensis-induced liver fibrosis.The results provide a scientific basis for further development of prevention and control strategies for clonorchiosis using TLRs as targets.