Identification Of Binding Proteins To T Lymphocytes And Monocytes From Haemonchus Contortus Excretory And Secretory Products And Their Functions To Corresponding Immune Cells

The gastrointestinal nematode Haemonchus contortus,a voracious bloodsucker residing in the abomasum,is one of the most economically important parasites for small ruminants.Infection with H.contortus can lead to severe anemia,diarrhea,dehydration and even death of the hosts and,therefore,causes major economic losses in the livestock industry worldwide.Recent studies revealed that gastrointestinal nematodes could release factors into their environment,called excretory and secretory products or proteins,which interacted with host immune cells or molecules to suppress or subvert host immune response to ensure their survival.T lymphocytes and monocytes were multifunctional cells engaged in host immune response against gastrointestinal nematodes.The identification of potential molecules,derived from H.contortus excretory and secretory products,which inhibit the immunomodulatory functions of T lymphocytes and monocytes will improve our understanding of the regulation of the host immune response mediated by H.contortus and continue to illustrate the mechanism underlying parasitic immune evasion.Meanwhile,the identified inhibitory factors acting as candidate vaccine antigens may have applications for clinical research against H.contortus infections.Thus,related research were listed below.1 The N-and C-terminal carbohydrate recognition domains of Haemonchus contortus galectin bind to distinct receptors of goat PBMC and contribute differently to its immunomodulatory functions in host-parasite interactionsHco-gal-m is a tandem-repeat galectin isolated from the adult worm of Haemonchus contortus.A growing body of studies have demonstrated that Hco-gal-m could exert its immunomodulatory effects on host peripheral blood mononuclear cells(PBMC)to facilitate the immune evasion.Our previous work revealed that C-terminal and N-terminal carbohydrate recognition domains(CRD)of Hco-gal-m had different sugar binding abilities.However,whether different domains of Hco-gal-m account differently for its multiple immunomodulatory functions in the host-parasite interaction remains to be elucidated.In this study,we found that the N-terminal CRD of Hco-gal-m(MNh)and the C-terminal CRD(MCh)could bind to goat peripheral blood mononuclear cells by distinct receptors:transmembrane protein 63A(TMEM63A)was a binding receptor of MNh,while transmembrane protein 147(TMEM147)was a binding receptor of MCh.In addition,MCh was much more potent than MNh in inhibiting cell proliferation and inducing apoptosis,while MNh was much more effective in inhibiting NO production.Moreover,MNh could suppress the transcription of interferon-y(IFN-y),but MCh not.Our data suggested that these two CRDs of Hco-gal-m bind to distinct receptors and contributed differently to its ability to downregulate host immune response.These results will improve our understanding of galectins from parasitic nematodes contributing to the mechanism of parasitic immune evasion and continue to illustrate the diverse range of biological activities attributable to the galectin family.2 Proteomic analysis of Haemonchus contortus excretory and secretory proteins(ESP)binding to goat T lymphocytes in vitro revealed potential suppressors of T cell functionsWe have identified 114 proteins binding to goat T lymphocytes from Haemonchus contortus excretory and secretory products and 15 binding receptors of T lymphocytes through co-immunoprecipitation and shotgun LC-MS/MS analysis.To validate the results from gene ontology and KEGG orthology,we have investgated the relugation of immune response mediated by T lymphocytes with the stimulation of Haemonchus contortus ESP in vitro.The results showed that ESP significantly affected cell viability and inhibited cell proliferation of T lymphocytes.Flow cytometric analysis and transcriptional analysis revealed that ESP promoted intrinsic and extrinsic T cell apoptosis mediated by FasL,which engaged by Caspase-8 and Caspase-9,respectively.In addition,ESP induced cell cycle arrest at G0/G1 phase through the down-regulation of mRNA transcription of CCNE1,CCND1,CDK4/6 and CDK2 gene.Meanwhile,the treatment of ESP predominantly promote the secretion of IL-10,IL-17 and TGF-?1 in goat T lymphocytes in vitro,as well as inhibting the secretion of IL-2,IL-4 and IFN-?.All these results presented herein will further elucidate the mechanism underlying H.contortus-engaged regulation of host T lymphocytes immune response and continue to provide new clues into the findings of antagonists against T lymphocytes.3 Proteomic analysis of Haemonchus contortus excretory and secretory proteins(ESP)binding to goat monocytes in vitro revealed potential suppressors of monocyte functionsWe have identified 108 proteins,interacted with goat monocytes,from Haemonchus contortus excretory/secretory products and 33 receptors through co-immunoprecipitation and shotgun LC-MS/MS analysis.To investigate the effects of ESP on goat monocytes,we had stimulated monocytes through the incubation of various concentration of ESP in vitro.The results showed that ESP had no impacts on monocyte apoptosis,as well as the expression of MHC-I.However,ESP significantly suppressed nitric oxide production and inhibited MHC-II expression of goat monocytes,as well as restraining macrophage phagocytosis.Meanwhile,the treatment of ESP predominantly inhibited the secretion of IL-1?,IL-6,IL-12 and TNF-? in monocytes,as well as inducing the secretion of IL-10.However,ESP had no significant effects on the secretion of IL-8 in monocytes.All these results presented here will further elucidate the mechanism underlying H.contortus-engaged regulation of host monocytes immune response and continue to provide new clues into the findings of antagonists against monocytes.4 Identification and characterization of Hc-ABHD,Hc-GHD and Hc-ADRM from H.contortus and their potential effects on goat T lymphocytes functionsHc-ABHD gene,Hc-GHD gene and Hc-ADRM gene of H.contortus were cloned by RT-PCR,expressed in Escherichia coli BL21 cells and the recombinant proteins(rHc-ABHD,rHc-GHD and rHc-ADRM were obtained,respectively.All these recombinant proteins were identified as single bands by Western blotting using serum from goats experimentally infected with H.contortus and they also reacted with rat anti-rHcABHD IgG,rat anti-rHcGHD IgG and rat anti-rHcADRM IgG,respectively,as indicated by specific bindings of these recombinant proteins to the surface of goat T lymphocytes.Immunohistochemical studies were performed to figure out the distribution of native Hc-ABHD,Hc-GHD and rHc-ADRM proteins,respectively,within the worms by checking sections of the partial-length adult female H.contortus.Real-time PCR analysis revealed that transcriptional expressions of Hc-ABHD,Hc-GHD and Hc-ADRM were considerably different in different life-cycle stages(eggs,L3,xL3,female and male adults)of H.contortus.The highest expression of Hc-ABHD and Hc-ADRM were in the host stage,while the highest expression of Hc-GHD was in the free-living third-stage larvae.The stimulation of rHc-ABHD and rHc-ADRM significantly suppressed cell viability,inhibited cell proliferation and induced intrinsic and extrinsic apoptosis of T lymphocytes mediated by Caspase-8,Caspase-9 and Caspase-3.Meanwhlie,rHc-ABHD induced cell cycle arrest at G0/G1 phase through the down-regulation of mRNA transcription of CCNE1,CCND1,CDK4/6 and CDK2 gene,and rHc-ADRM caused cell cycle arrested via the down-regulation of mRNA transcription of CCNE1 and CDK2.In addtion,the treatment of rHc-GHD significantly enhanced T cell viability,promotes cell proliferation and cell cycle through the up-regulation of mRNA transcription of CDK4,CDK6 and CDK2.Our data also showed that goat T lymphocytes exposed to these recombinant proteins change their cytokine production profile:rHc-ABHD predominantly inhibited the secretion of IL-4,TGF-?1 and IFN-?y and promoted the secretion of IL-10;rHc-GHD significantly promoted the secretion of IL-2,IL-4 and IFN-y;while rHc-ADRM predominantly suppressed the secretion of IL-4,IL-10 and IFN-y.All these results presented here suggested that these three molecules were involved in the immunomodulation engaged by T lymphocytes and Hc-ABHD or Hc-ADRM,as repressive factors against T lymphocytes-mediated immune responses,could be candidate vaccine antigens for further clinical research.5 Identification and characterization of Hc-Rab33,Hc-Makl and Hc-ATPD from H.contortus and their potential effects on goat monocytes functionsHc-Rab33 gene,Hc-Makl gene and Hc-ATPD gene of H.contortus were cloned by RT-PCR,expressed in Escherichia coli BL21 cells and the recombinant proteins rHc-Rab33,rHc-Makl and rHc-ATPD were obtained,respectively.All these recombinant proteins were identified as single bands by Western blotting using serum from goats experimentally infected with H.contortus and they also reacted with rat anti-rHcRab33 IgG,rat anti-rHcMakl IgG and rat anti-rHcATPD IgG,respectively,as indicated by specific binding of these recombinant proteins to the surface of goat monocytes.Immunohistochemical assays were performed to figure out the distribution of native Hc-Rab33,Hc-Mak1 and Hc-ATPD proteins,respectively,within the worms by checking sections of the partial-length adult female H.contortus.Real-time PCR analysis revealed that transcriptional expressions of Hc-Rab33,Hc-Makl and Hc-ATPD were considerably different in different life-cycle stages(eggs,L3,xL3,female and male adults)of H.contortus.The highest expression of Hc-Rab33 and Hc-ATPD were in the host stage,while the highest expression of Hc-Makl was in the first stage larva.In addition,the treatments of rHc-Rab33 significantly reduced nitric oxide production by monocytes,suppressed cell phagocytosis and downregulated the expression of MHC-II.While rHc-Makl predominantly induced apoptosis of monocytes.In addition,the stimulation of rHc-ADRM decreased nitric oxide production of monocytes.Our data also revealed that monocytes exposed to these recombinant proteins changed their cytokine production profile:rHc-Rab33 predominantly inhibited the secretion of IL-12 and TNF-? and promoting the secretion of IL-10 and TGF-?1;rHc-Makl significantly promoted the secretion of IL-6,IL-10 and TGF-?1;while rHc-ATPD predominantly suppressed the secretion of IL-10,as well as promoting the secretion of IL-10.Results presented here suggested that all these three recombinant proteins were involved in the immunomodulation engaged by monocytes and Hc-Rab33,as a suppression molecule against monocytes-mediated immune response,could be a candidate vaccine antigen for further clinical research.6 Studies on the potential roles of Hc-ABHD,Hc-ADRM and Hc-Rab33 in H.con tortus infectionPotential roles of goat anti-rHcABHD serum,goat anti-rHcADRM serum and goat anti-rHcRab33 serum in anti-H.contortus infection were investigated in this study.25 healthy goats were allocated into five groups with five each.The goats injected with normal goat serum without infection of H.contortus set as Control group.The goats injected with normal goat serum,infecting with H.contortus set as Infection group.The goats injected with goat anti-rHcABHD serum.infecting with H.contortus,set as Immunized group A.The goats injected with goat anti-rHcADRM serum,infecting with H.contortus,set as Immunized group B.The goats injected with goat anti-rHcRab33 serum,infecting with H.contortus,set as Immunized group C.All these groups injected twice with relevant goat serum according to experimental protocol at day 6 and day 3 before challenge.All the goats in Infection group,Immunized group A,Immunized group B,Immunized group C were inoculation with 8,000 L3.Fecal egg counts(FECs)were performed post challenge at day 22,24,26,28,30,32 and 34.Quantity of neutrophils,eosinophils,monocytes,basophils,RBC,packed cell volume,and hemoglobin in goat peripheral venous blood were analysed post challenge at day 0,7,14,21,28 and 35,as well as the concentration of IgG1,IgA,IgE,IL-2,IL-4,IL-10,IL-17,TGF-?1,IFN-? and TNF-? in goat peripheral venous blood.At post-mortem on day 35,abomasum worm burden were determined.The results indicated that FECs and worm burden in Immunized group A reduced by 50.1%and 66.7%compared to Infection group,respectively,as well as in Immunized group B with 37.2%and 44.4%.No significant changes of worm burden were observed between Immunized group C and Infection group and FECs were reduced by 32.1%.Meanwhile,quantity of neutrophils,eosinophils and monocytes in Immunized group A and Immunized group B significantly decreased compared to Infection group at day 35 post challenge,with a tendency to the counts in Blank group.While no significant counting changes of basophils,RBC,packed cell volume and hemoglobin were observed between Immunized group A and Infection group,as well as Immunized group B and Infection group.In addition,secretion of IL-2 and IL-4 in goat peripheral venous blood of Immunized group A,as well as IL-2 of Immunized group B,significantly increased as compared to Infection group at day 35 post challenge.Whilst no significant changes were observed in the concentration of IgG1,IgA,IgE and secretion of IL-10,IL-17,TGF-?1,IFN-y and TNF-? in goat peripheral venous blood among challenged groups.All these results present herein indicated that injected with goat anti-rHcABHD serum or goat anti-rHcADRM serum confer protection against H.contortus infection to a certain extent.