Study On Macrophage Polarization And Cellular Immune Response Against Fasciola Gigantica And Its Excretory-secretory Products

Fasciola gigantica and F.hepatica,pathogenic species of genus Fasciola,are both known to infect human and mammal species and cause fascioliasis.The different susceptibility to diseases among species could be attributed by escape mechanism or immunomodulation ability of fluke against the host immune responses.Fasciola hepatica and its excretory-secretory products(FhESP)had been proven to induce M2 polarization of macrophages in mice and cattle.However,whether there is similar immune function caused by F.gigantica is still unclear.Our study is to investigate the change of macrophage polarization and innate immune response induced by F.gigantica or FgESP in vivo and in vitro,and to investigate innate cell recruitment and cytokine profile in afferent lymphatic system affected by Fasciola ESP,with the aid of man-made lymphatic cannulation models on sheep,in the aims of providing a reliable theoretical basis for better understanding of interactions between Fasciola and its hosts.The researches include the following parts:1.Buffaloes,Kun Ming(KM)mice and C57BL/6 Nju(B6)mice were experimentally infected by F.gigantica metacercarial cysts,executed at 4 wpi,10 wpi and 14 wpi for buffalo,and 3 wpi,4 wpi and 7?8 wpi(depended on the period of natural death)for mice.Enrichment of inflammatory cells and subsequent hyperplasia of connective tissues were observed from liver tissue sections from both mice and buffaloes,adult flukes and granulation were found by 14 wpi of infection in buffalo liver tissue.Realtime Q-PCR was applied to detect mRNA expression of Kupffer cells(macrophage)markers and cytokines in liver tissues.ELISA kits were employed to measure IFN-y and IL-4 concentration in serum.The results inidicated the phenotype of buffalo KCs was shifted from M2 to a mixture of M1/M2 phenotype during the infection,while in KM mice from a mixed M1/M2 phenotype to MO,KCs of B6 mice mainly showed a typical M2 during the progress.The result suggested that,the infection of F.gigantica in different host species could induce dissimilar trends of KC polarization and cytokine profiles.We suspect that these differences are closely associated with the worm-loading capacity,wound repair capacity,and lesion degree of the host liver,as well as the intrahepatical migration and growth activities of parasite.2.Both KM mice and B6 mice received continuous peritoneal FgESP injection 4 day,7days or 14 weeks prior to execution for short-or long-term studies.The injection of PBS was set as control.No significant histological change had observed in any mouse liver from short-term study regardless of injection,while long-term continuous injection of FhESP leaded to the increase of cell membrane penetrability,connective tissue proliferation and a tendency of fibrosis in mouse liver.The result of Q-PCR showed a down-regulation of iNOS mRNA level in the liver of KM,while upregulated CD206,Arg-2 and YM-1 mRNA level in the liver of B6 induced by FhESP during the short-term study,which both reflecting M2 polarization.However,in the long-term study,different trend was observed.Long-term injection of FgESP induced a mixed M1/M2 phenotype of KCs in KM but a MO-like phenotype lacking any trend to M1 nor M2 in B6.3.200?g/mL of FgESP was used to stimulated buffalo primary BLM?or B6 mice-sourced immortalized passage M?(iPMO)in vitro.Q-PCR was applied to detect mRNA expression of macrophage markers and cytokines.ELISA and Griess assay were employed to measure IFN-y,IL-4 and NO concentration in cell culture supernatant respectively.A chenmical assay was applied to detect the production of Arg-1 in the cells.A downregulated expression of CD68 gene and upregulated expression of Arg-2 gene were found in buffalo BLM?,as well as the increase of Arg-1 and NO production.The supernatant concentration of IFN-? but not IL-4 was found increased.The results obtained from buffalo BLM? suggest the M2 polarization and the inhibition of M?activation or apotosis might exist.When stimulating mouse wildtype iPM?,increase of Arg-1 and IL-4 were found but not the production of IFN-? and NO,also suggesting the plorization of M2M?.These results together indicated that FgESP stimulation could induce M2M? polarization in vitro regardless of host species.4.Different gene knocked-out mouse iPM?(MyD88-/-,MyD88/TRIF-/-,and TLR-2/4-/-)were stimulated with 200 ?g/mL of FgESP in vitro.The concentrations of NO,IFN-? and IL-4 in the culture supernatant as well as the concentration of Arg-1 in the cell lysates were measured and compared with those from the wildtype iPM? after stimulation.The result showed no difference of IL-4 production in MyD88-/-compared to the control,decrease of IL-4 production in MyD88/TRIF-/-,and both of which had lower amount of IL-4 in wildtype iPM?.None of the above knocked-out iPM? showed increase Arg-1 production.It appear that M2 phenotype of macrophage could be activated by FgESP in vitro via a MyD8 8-dependent pathway,promoting Th2 cytokine and Arg-1 but inhibiting NO,whereas TRIF,TLR2 or TLR4 might somehow play a role during the progress.5.Prefemoral pseudo-afferent lymphatic cannulation and hepatic afferent cannulation were surgically established,and the appreciated success rates were 70%and 33%respectively.6.200?g of fluorescent dye labeled FhESP was given by multiple-points subcutaneous injection into prefemoral area of sheep(OVA was setted as control antigen),and lymph was collected during 0-7 days pre-or post-injection via the successfully implanted pseudo-afferent prefemoral cannulation,followed by cell isolation from the lymph collections.FCM was applied for immune cell type and antigen uptaking analysis.Q-PCR was used for detection of mRNA expression for M? markers.ELISA was employed to measure IFN-y and IL-4 in lymph.The results showed that,the percentage of neutrophils and monocytes versus total cells were both found specifically increase induced by FhESP stimulation during 4-8 hpi and 8?12 hpi,respectively.Lymphocytes decreased during 4-8 hpi was also FhESP-Specific.There was also an increase of DCs during 4?8 hpi however with no statistical difference compared to that agaist OVA injection.No significant percentage change was found on MLCs or eosinophils regardle of timepoints.Almost all phagocytes shortly increased their early antigen uptake abilities during 4-12 hpi.Among which,A647-FhESP+ eosinophils,monocytes and DCs were detected increase with antigenic specificity,while the percentage change of either DC subset was lack of antigenic specificity.The results of Q-PCR and ELISA indicated the the expression of CD206,Arg-2,and YM-1 were all specifically up-regulated within 24 hpi,and there was also a specific increase of IL-4 in lymph during 0-8 hpi.No any alter of IFN-y,IL-10 or IL-12 production was found in the lymph fluid after FhESP injection.These results suggested the operation of ovine pseudo-afferent lymphatic cannulation was relatively simple with higher successful rate during model establishment,and proved applicable for studies on immune response kinetics.This study also confirmed monocytes and neutrophils recruitment induced by FhESP,as well as the promotion of phagocytosis ability of phagocytes,especially their mature subsets.The activation of M2M? by FhESP had again been proved here,associated with Th2 cytokine excretion.Conclusions:the infection of F.gigantica could lead to different tendency of KC phenotype polarization and the feature of cellular immune response among species,which is possibly depend on worm-loading capacity,wound repair capacity,and lesion degree of host liver.FgESP produced by F.gigantica could induce a MyD8 8-dependent M2M? activation in vivo and in vitro,associated with Th2 cytokine secretion,up-regulation of Arg-1 synthesis,and suppression of NO production.This could be a pathogen clearance mechanism,and possibly one of the strategy of Fasciola in escape from macrophage immune response.Moreover,a pseudo-afferent lymphatic cannulation model was successfully established on sheep and was used to investgate host immune response against F.hepatica products.The early recruitment of neutrophils and monocytes,and rapid phagocytosis promotion capability,as well as M2M? and Th2 cellular response induced by injection of FhESP was firstly noted.