| Trypanosoma evansi(T.evansi)is bloodborne protozoan that can cause Surra in many species.T.evansi could infect most mammals exhibiting different clinical characteristics.In horses and dogs,the parasites cause acute infection causing high fever,weight loss,anemia,genital inflammation,neurological symptoms,and eventually death.On the contrary,in cattle and buffalo,the parasites cause chronic infection and produce symptoms of emaciation,anorexia,anemia,and decreased productivity,causing huge economic losses to the livestock industry.Several cases of human infection by T.evansi have been reported.At present,the treatment of trypanosomiasis is mainly using chemical drugs such as Diminazene,and there is no effective vaccine to prevent the disease.It has been reported that T.evansi evades host immunity through antigen variation,body surface binding to host plasmin and lymphocyte death,etc.Further understanding of the mechanism of host immune evasion by T.evansi will help us to explore new strategies for trypanosomiasis control.Pathogen-associated molecular patterns were recognized by Toll-like receptors(TLRs)and activate a series of signaling pathways to produce effector molecules,which ultimately regulate the host immune response.A variety of parasites such as Neospora caninum,Toxoplasma gondii,Leishmania and Plasmodium could be recognized by TLRs and induced protective,regulatory or pathogenic responses.However,the role and mechanism of TLRs in T.evansi infection were still poorly understood.Extracellular vesicles(EVs)are vesicles with membrane structure released by cells,and play a vital role in information transmission between cells.It has been reported that T.brucei transfers variable surface glycoproteins into host red blood cells through EVs,causing host anemia,and it can also deliver serum resistance-related proteins to trypanosomes that do not contain the protein.Whether T.evansi could release EVs to regulate host immune responses in T.evansi infection has not been reported.Therefore,this study addresses the problem that the immune evasion mechanism of T.evansi is not yet clear.We investigated the role of TLRs pathway in T.evansi infection,the isolation and identification of T.evansi EVs(Te EVs)and proteomic analysis,the role and molecular mechanism of Te EVs and its components in the transmission of information between the parasites and the host cells and the pathogenic process of T.evansi from the perspective of TLRs signaling pathways and EVs.The main contents are as follows.1.T.evansi activated the TLR2 pathway and the effect of TLR2 on the pathogenicity of T.evansi.(i)Screening TLRs involved in the identification of T.evansi.Fluorescence quantitative PCR(q PCR)was used to determine which TLRs of mouse bone marrow-derived macrophages(BMDMs)could be activated by T.evansi.The results showed that T.evansi could activate TLR2,TLR3 and TLR4 in BMDMs.(ii)The role of TLRs in T.evansi infection.After wild-type(C57BL/6,WT)and TLR2-/-,TLR3-/-and TLR4-/-(C57BL/6 background)mice were infected with T.evansi,the number of peripheral blood parasites,the survival time of the mice,the secretions of IL-12p40 and IFN-?in the mice serum were detected and the pathological changes were observed.The result showed that TLR2-/-mice infected with T.evansi showed milder trypanosomiasis compared with WT mice,with a significantly lower infection rate,longer survival time and less parasite load,and increased secretion of IL-12p40 and IFN-?,while TLR3-/-and TLR4-/-mice showed no significant difference in resistance to T.evansi compared with WT mice.(iii)T.evansi activates the TLR2 signaling pathway and affects the secretion of cytokines.BMDMs of WT or TLR2-/-mice were stimulated with T.evansi,Western blot was used to detect TLR2,ERK and AKT signaling pathways,and ELISA was used to detect inflammatory cytokine secretion.The results showed that T.evansi could inhibit the secretion of IL-12p40,IL-6 and TNF-?through the TLR2-AKT signaling pathway.(iv)The role of AKT pathway in T.evansi infection.AKT inhibitor(MK-2206)was used in animal experiments,the number of peripheral blood parasites,the survival time of the mice,the secretions of IL-12p40 and IFN-?in the mice serum were detected.The results showed that the mice in the AKT inhibition group showed milder trypanosomiasis compared with WT mice,with a significantly lower infection rate,longer survival time and less parasite load,and increased secretion of IL-12p40 and IFN-?.2.The role of Te EVs in activating TLR2 pathway and T.evansi infection.(i)Isolation and identification of Te EVs.T.evansi extracellular vesicles(Te EVs)were separated by differential ultracentrifugation and identified using transmission electron microscopy,scanning electron microscopy(SEM)and particle size analysis.Te EVs were partially flat ball-shaped,with a double-layer membrane structure and the average diameter was 110±22.12 nm.SEM and ultra-thin section results showed that the T.evansi could release EVs through nanotubes dissociation and Te EVs had similar membrane structures with the cell plasma membrane.The labeled Te EVs were co-incubated with BMDMs and found that Te EVs could be internalized into the cytoplasm of BMDMs.(ii)Te EVs activates the TLR2 signaling pathway and affects the secretion of cytokines.BMDMs of WT or TLR2-/-mice were stimulated with Te EVs,Western blot was used to detect TLR2,ERK and AKT signaling pathways,and ELISA was used to detect inflammatory cytokine secretion.The results showed that Te EVs could inhibit the secretion of IL-12p40,IL-6 and TNF-?through the TLR2-AKT signaling pathway.(iii)The role of Te EVs in T.evansi infection.Te EVs were injected intravenously(tail vein)into the mice and infected with T.evansi,the number of peripheral blood parasites,the survival time of the mice,the secretions of IL-12p40 and IFN-?in the mice serum were detected.The results showed that Te EVs could inhibit the secretion of IL-12p40 and IFN-?,increase the number of T.evansi in the blood,and shorten the survival time.3.The role of Te EVs component kinetoplastid membrane protein-11 in T.evansi infection.(i)Proteomics analysis of Te EVs.746 proteins were identified in Te EVs by proteomics analysis,of which at least one unique peptide accounted for57.5%.Heat shock protein 70(HSP-70),translation elongation factor-1?(EF-1?),kinetoplastid membrane protein 11(KMP-11)and aldolase were selected to verify proteomics results.The results of KEGG analysis showed that 595 proteins were involved in 78 signal pathways,including PI3K-AKT,MAPK,NOD-like and Toll-like pathways.Immunofluorescence assay showed that Te KMP-11 protein was detected in BMDMs stimulated with Te EVs,indicating that EVs could release their cargo into BMDMs.(ii)r Te KMP-11 activates the BMDMs signaling pathway and regulates the secretion of cytokines.BMDMs of WT mice were stimulated with r Te KMP-11,Western blot was used to detect MAPK and AKT signaling pathways,and ELISA was used to detect inflammatory cytokine secretion.The results showed that r Te KMP-11 could inhibit the secretion of IL-12p40,IL-6 and TNF-?through the AKT signaling pathway.(iii)The role of r Te KMP-11 in T.evansi infection.r Te KMP-11 proteins were coated with DOTAP liposome and injected intravenously(tail vein)into the mice and infected with T.evansi,the number of peripheral blood parasites,the survival time of the mice,the secretions of IL-12p40 and IFN-?in the mice serum were detected.The results showed that r Te KMP-11 could inhibit the secretion of IL-12p40 and IFN-?,increase the number of T.evansi in the blood,shorten the survival time,and accelerate the disease progression in infected mice.In conclusion,this study revealed the immune evasion mechanism of T.evansi by activating the TLR2-AKT signaling pathway to inhibit the secretion of IL-12p40and IFN-?.Te EVs were isolated and identified for the first time,and the proteomics data of Te EVs were obtained.It was determined that the Te EVs and its component Te KMP-11 were involved in the immune evasion process of T.evansi.In this study,the immune evasion mechanism of T.evansi was studied from the perspective of TLR2 and EVs for the first time,providing a new theoretical basis for the prevention and treatment of trypanosomiasis. |
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