Effect Of The Expression Of Mice Toll Like Receptors By Fasciola Gigantica Infection And Excretory-secretory Products

Toll-like receptors(TLRs)can recognize and interact with pathogen-associated molecular patterns(PAMPs)and damage-associated molecular patterns(DAMPs),is the first defence in host innate immune defense.Parasites can evade or regulate the host immune response through affecting the expression and function of TLRs.Researches on host TLR signaling pathways against Fasciola gigantica and its Excretory-Secretory Products(FgESP),could be helpful to reveal the innate immunity mechanism of the early infection with F.gigantica.In this study,BALB/c and C57BL/6 mice were experimentally infected with F.gigantica metacercaria.Serum antibody levels at diffenrent timepoints post-infection were determined by ELISA.The mRNA levels of TLR 1-9 and inflammatory cytokines in liver tissues were detected by Real-time quantitative PCR(RT-qPCR).The results showed that F.gigantica infection could induce immune types of liver in a mixed type of Th1/Th2 immune response in BALB/c but a Th1 response tendency in C57BL/6 during the tested timepoints.Infection of two groups of mice with F.gigantica could induce the dynamic changes of TLR1-9 in liver.Results showed that TLR1,TLR6,TLR7 and TLR8 were up-regulated(P<0.05)and TLR6 earliestly which was possibly related to the specific recognition of lipopeptides molecules.TLR1 was significantly up-regulated when both groups of mice were in a Th1/Th2 mixed immune response,indicating that it may play an important role.Naive FgESP(nFgESP)was administered into both groups of mice by intraperitoneal injection.The mRNA levels of TLR1-9 and inflammatory cytokines in liver tissues were detected by RT-qPCR.The results showed that there was a difference immune types of liver by intraperitoneal injection of nFgESP in two groups of mice(BALB/c mice tend to be Th2,C57BL/6 mice tend to Th1);FgESP could induce TLR1,TLR2,TLR8 up-regulation.Naive FgESP and heat-inactiviated FgESP(hiFgESP)were used to stimulate the murine macrophages cell line RAW264.7 in vitro.The mRNA levels of TLR1-9 and inflammatory cytokines was detected by RT-qPCR.The results showed that both nFgESP and hiFgESP stimulated RAW264.7 cells can induced significant up-regulation of TLR1,TLR7,and TLR8(P<0.05)and significant down-regulation of TLR2,TLR3,and TLR9(P<0.05).The significant up-and down-regulation of TLRs were related to the non-active enzyme substances in FgESP.However,no significant regulation of TLR4 or TLR6 was observed when heat inactivation,which may relate to active enzyme substances that should be in heated FgESP.By comparing the results from both types of experiments of FgESP(in vivo/in vitro)stimulation with that of F.gigantica infection,we found that the mRNA levels of TLR1 and TLR8 were up-regulated,suggesting a selective reaction between host TLR1 and TLR8 with F.gigantica through FgESP which could be important to mice immune response.The present study was based on gene transcription dynamics of TLRs and cytokines,thus there is still further investigation at protein levels to be done as validation.Screening of TLRs and their specific helminth-derived ligands,which may be presumed to have an important role,further study of anti-Fasciola innate immuninty mechanism of host,will provide theoretical basis for vaccine candidate screening and drug development,and help for the prevention and control of fascioliasis.