Effect Of Six Proteins Such As Macrophage Migration Inhibitory Factor From Haemonchus Contortus On The Function Of Goat Monocyte

Haemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus(H.contortus),and it is most important and alarming challenges to the small ruminant’s production.The infection of the H.contortus could cause high economic losses worldwide.H.contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein.Modulation and suppression of the host immune response by nematode parasites have been reported extensively and the excretory and secretory proteins released by parasites were considered to play key role in this process.In the present study,we evaluated the modulating effects of six excretory and secretory proteins of H.contortus on goat monocyte/macrophage.1 Characterization of a secreted macrophage migration inhibitory factor homologue of the parasitic nematode Haemonchus Contortus acting at the parasite-host cell interfaceMacrophage migration inhibitory factor(MIF)homologs of parasitic nematodes are an important class of immunoregulatory molecules.In the present study,we cloned and produced recombinant MIF protein from the small ruminant’s nematode parasite Haemonchus contortus(rHCMIF-1),and investigated its immunomodulatory effects on goat monocyte.Enzymatic assays indicated that rHCMIF-1 possessed tautomerase activity.Immunohistochemical test demonstrated that the native HCMIF-1 protein was predominantly localized at the body surface and internal surface of the worm’s gut.We demonstrated that rHCMIF-1 could be distinguished by antisera from goats experimentally infected with H.contortus and could bind by goat monocytes.The immunomodulatory effects of HCMIF-1 on cytokine secretion,MHC molecule expression,NO production and phagocytosis were observed by co-incubation of rHCMIF-1 with goat monocytes.The results showed that the interaction of rHCMIF-1 decreased the production of TNF-α,IL-1βand IL-12p40,where as,it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes.After rHCMIF-1 exposure,the expression of MHC-II on goat monocytes was inhibited.Moreover,rHCMIF-1 could down-regulate the LPS induced NO production of goat monocytes.Phagocytotic assay by FITC-dextran internalization showed that rHCMIF-1 could inhibit the phagocytosis of goat monocytes.2 Modulation of goat monocyte function by HCcyst-2,a secreted cystatin from Haemonchus contortusCystatins secreted by parasitic nematodes can exert a wide range of regulatory effects on its host immune system.In the present study,we cloned and produced recombinant cystatin protein from nematode parasite Haemonchus contortus(rHCcyst-2)and investigated its immunomodulatory effects on goat monocyte.rHCcyst-2 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L,cathepsin B and papain.Immunohistochemical test demonstrated that the native HCcyst-2 protein was predominantly localized at the body surface and internal surface of the worm’s gut.We demonstrated that rHCcyst-2 could be distinguished by antisera from goats experimentally infected with H.contortus and could uptake by goat monocytes.The immunomodulatory effects of HCcyst-2 on cytokine secretion,MHC molecule expression,NO production and phagocytosis were observed by co-incubation of rHCcyst-2 with goat monocytes.The results showed that the interaction of rHCcyst-2 decreased the production of TNF-α,IL-1β and IL-12p40.However,it significantly increased the secretion of IL-10 in goat monocytes.After rHCcyst-2 exposure,the expression of MHC-II on goat monocytes was inhibited.Moreover,rHCcyst-2 could up-regulate the LPS induced NO production of goat monocytes.Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-2 inhibited the phagocytosis of goat monocytes.3 Characterization of a secreted cystatin of the parasitic nematode Haemonchus contortus and its immune-modulatory effect on goat monocytesIn the present study,we cloned and produced recombinant cystatin protein from Haemonchus contortus(rHCcyst-3)and investigated its immunomodulatory effects on goat monocyte.The recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli,and its inhibitory activity against cathepsin L,B,as well as papain,were identified by fluorogenic substrate analysis.Native HCcyst-3 protein was localized by an Immunohistochemical test.The immunomodulatory effects of HCcyst-3 on cytokine secretion,MHC molecule expression,NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes.The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L,cathepsin B,and papain.The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm’s gut.We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H.contortus and could uptake by goat monocytes.The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α,IL-1β and IL-12p40.However,it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes.After rHCcyst-3 exposure,the expression of MHC-Ⅱ on goat monocytes was restricted.Moreover,rHCcyst-3 could upregulate LPS induced NO production of goat monocytes.Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes.4 Cloning,expression and biological characteristics analysis of HCRD,HCETFa and HCPTPA from Haemonchus contortusIn this study,the open reading frames(ORF)of encoding Haemonchus contortus Rhodanese(HCRD),Electron transfer flavoprotein a subunit(HCETFa)and Phosphotyrosyl phosphatase activator domain containing protein(HCPTPA)were amplified by reverse transcription-polymerase chain reaction(RT-PCR),respectively.HCRD(1332 bp)contains 443 amino acid residues with a predicted molecular mass of 50.6 kDa.HCETFa(996 bp)contains 331 amino acid residues with an expected protein size of 34.5 kDa.HCPTPA(987 bp)contains 328 amino acid residues with a predicted molecular mass of 38.3 kDa.HCRD,HCETFa and HCPTPA were subcloned into pET-32a and expressed in Escherichia coli(E.coli)BL21(DE3),respectively.The expressed recombinant protein were checked by SDS-PAGE,revealing bands of about 70.6 kDa(rHCRD),about 54.5 kDa(rHCETFa)and about 58.3 kDa(rHCPTPA),respectively.Immunohistochemical test demonstrated that the native HCRD,HCETFa and HCPTPA proteins were predominantly distributed in the body surface and internal surface of the worm’s gut.We demonstrated that rHCRD,rHCETFa and rHCPTPA could be distinguished by antisera from goats experimentally infected with H.contortus and could bind by goat PBMC in vitro.5 Modulation of goat PBMC and monocyte function by three recombinant proteins of Haemonchus contortusIn this research,we investigated the immunomodulatory effects of three Haemonchus contortus recombinant proteins on goat PBMC and monocyte.The cell proliferation,migration and NO production were observed by co-incubation of goat PBMC with recombinant proteins at different concentrations,the secretion of IL-2,IL-4,IL-10,IL-17A,IFN-y,TNF-α and TGF-β1 in culture supernatant were examined by ELISA.The cell apoptosis was determined by flow cytometric analysis.Goat monocyte phagocytosis and MHC molecule expression were measured preincubated with each recombinant protein by flow cytometric analysis.Our results showed that rHCRD,rHCETFa and rHCPTPA significantly suppressed the cell proliferation of goat PBMC triggered by ConA,and rHCETFa and rHCPTPA could induce apoptosis of goat PBMC at different concentrations.After incubated with rHCETFa,the NO production of goat PBMC significantly increased,however,rHCPTPA dramatically decreased NO production.Compared to the control,20μg/ml of rHCETFa significantly promoted migration of goat PBMC,while rHCETFa could inhibit migration of goat PBMC at 20 μg/ml and 40 μg/ml concentration.After incubated with goat PBMC,rHCRD decreased the ConA induced production of IFN-y and TNF-αwhereas,rHCRD significantly increased the secretion of IL-10 at different concentrations and TGF-β1 at 40 μg/ml concentration,respectively.The engagement of rHCETFa decreased the production of,IL-2,IL-4,IL-17A and TNF-α,However,it significantly increased the secretion of TGF-β1 in goat PBMC.After rHCPTPA exposure,the secretion of IL-2 and IFN-y in goat PBMC significantly decreased,while the production of IL-4 and IL-10 significantly upregulated.rHCRD and rHCETFa significantly inhibited the phagocytosis of goat monocytes,and rHCRD simultaneously restricted the expression of MHC-II on goat monocytes.