The Effects On Excretion Of Inflammatory Factors And Polarization Of Buffalo Peripheral Blood Mononuclear Macrophages Induced By Exosomes Of Adult Fasciola Gigantica

Fascioliasis is a zoonotic parasitic disease that seriously endangers the health of the host,causing serious economic losses worldwide.However,there is still a limited understanding of the mechanism of interaction between the pathogen Fasciola gigantica and the hosts.Exosomes play an important regulatory role in parasite-host interactions.The current study was aimed to investigate the effects of adult Fasciola gigantica exosomes(FgEXO)on the secretion of inflammatory factors and cell polarization in buffalo peripheral blood mononuclear macrophages(PBMMφ)in vitro.To preliminarily reveal the interaction between FgEXO and host immune cells,Details of the research contents and results are as follows.Firstly,the method of isolation and culture of buffalo PBMMφ was optimized based on the cell viabilities measured by MTT assay.The results showed that cells cultured with medium containing 10% buffalo autologous serum after 24 hours’ adherence,followed by further induction with GM-CSF for 5 days,achieved the highest viability;Cell characterizations were identified by microscopy,chicken erythrocyte phagocytosis test and MHC Ⅱ cellular immunostaining.The obtained cells possessed the typical morphological characteristics of macrophages and the ability to phagocytize chicken red blood cells.The rate of MHC Ⅱ positive cells reached 90.62(± 5.6)%.The optimized 12% PEG6000 precipitation method,ultracentrifugation method and the combination of the two methods for separation and purification of FgEXO were compared by BCA,TEM and MTT assay for cell viability.The results showed that FgEXO with typical characteristics of exosomes could be separated by the three methods.The exosomes separated by PEG 6000 method were the most,but the impurities were more.The exosomes separated by ultracentrifugation method were the lowest,and their sizes were different.The exosomes separated by the combination of the two methods were in the middle,and PEG 600 could be removed by ultracentrifugation method.The molecular weight of SDS-silver staining method was 15-25 KDa,and the optimum condition for preservation of exosomes was-80 ℃.The effects of FgEXO,FgESP and FgESP-exo at different time and concentration on inflammatory cytokines of PBMMφ in vitro were studied.The expression levels of IL-10 and IFN-γ were detected by qPCR.The results showed that 50 ug/mL FgEXO for 24 h could significantly increase IL-10 expression in macrophages(P < 0.05),significantly decrease IFN-gamma expression level(P < 0.05),and 200 ug/mL FgEXO for 48 h could significantly increase IL-10 expression in macrophages(P < 0.005),significantly inhibit IFN-gamma expression level(P < 0.0005).300 ug/mL FgESP-exo could significantly increase IL-10 expression in macrophages(P < 0.005),significantly inhibit IFN-gamma expression level(P < 0.005),and there was no significant difference in cytokine expression level at different time.Buffalo PBMMφ was incubated in vitro with PBS,LPS,FgEXO,LPS+FgEXO,FgESP,FgESP+LPS,FgESP-exo and FgESP-exo+LPS,respectively.Arg-1 and NO secretion level was detected by Griess method,The expression levels of phenotypic correlation gene(iNOS、Arg-1、CD86、CD206)Mrna of Mφ in the cells was detected by RT-qPCR,and cytokine content in supernatant was detected by ELISA.The results showed that FgEXO alone could significantly up-regulate the expression of Arg-2 and CD206 mRNA of PBMMφ(P < 0.0001),promote the secretion of TGF-β1(P < 0.0001),and inhibit the secretion of IL-1β(P < 0.0001),and significantly down-regulate the iNOS and CD86 mRNA of PBMM Phi compared with LPS control group.The expression level(P < 0.001)inhibited IL-1β secretion(P < 0.0001).In this study,the exosomes from adults of adult F.gigantica were successfully isolated and identified.In vitro stimulation of buffalo peripheral blood mononuclear macrophages showed that FgEXO induced PBMMφpolarization to M2 type in buffalo in vitro,promoted the expression of anti-inflammatory factor IL-10 and inhibited the expression of pro-inflammatory factor IFN-gamma,which provided a reference for further study of the molecular mechanism of interaction between Fasciola macrophages and host immune cells.