Isolation And Identification Of Extracellular Vesicles Of Neospora Caninum And Its Immunoregulatory Mechanism On Host Mediated By TLR2 Signaling Pathway

Neospora caninum is an obligate intracellular apicomplexan parasite,the etiologic agent of neosporosis,major causes of abortion and neurological symptoms in dogs.Neosporosis leads to significant economic losses in both the dairy and beef industries worldwide.There is still a lack of effective and safe prevention and treatment strategies because of unclear understanding the pathogenic mechanism of the N.caninum with the host immune response.Host innate immunity is a major player against protozoal infections via inhibiting parasite replication and triggering appropriate adaptive immune responses,which controls the active infections and overcomes subsequent re-exposures.The recognition of highly conserved sets of molecular patterns(pathogen-associated molecular patterns,PAMPs)by pattern-recognition receptors(PRRs)of host cells leads to the production of inflammatory cytokines,but the activation mechanism is not fully understood.One recently described mechanism for release of proteins or lipids as well as infectious agents from parasites is through EVs.EVs have been shown to be important in communication and genetic exchange among microbes.However,whether N.caninum can produces EVs and whether it participates in the host innate immune response has not been reported yet.Our study will help us to further understand the pathogenesis of neosporosis and the immune response of host,provide new diagnostic and treatment ideas for the prevention and treatment neosporosis.Isolation,Identification and Proteomic Analysis of N.caninum extracellular vesicles.We show herein that N.caninum continuously secretes EVs,and NEVs were isolated from parasite growth media through a series of ultracentrifugation steps.NEVs were rounded or cup-shaped,limited by a membrane bilayer and approximately 124±20.89 nm in diameter by TEM and SEM.The proteome results shown that 705 proteins were identified in the NEVs,including the 14-3-3 and HSP70 and HSP90,which have been extensively used for exosome definition.Among these proteins,705 were grouped into 97 pathways,the top five of which were Ribosome,Carbon metabolism,Spliceosome,RNA transport,and Proteasome.The pathways also included the PI3K-Akt signaling pathway,MAPK signaling pathway,NOD-like receptor signaling pathway and Toll signaling pathway,which suggested that NEVs are involved in the host cell immune responses.The mechanism of innate immune response mediated through TLR2 receptor of N.caninum extracellular vesicles.Firstly,we examined the relationship between NEVs and mouse bone marrow macrophages(BMDMs)by confocal microscope and flow cytometry.NEVs were labeled in vitro with the green fluorescent lipid dye PKH67 prior to exposure,the mouse BMDMs were examined at various times,the results showed that NEVs entered BMDMs were time-dependent.To test whether uptake was occurring by an active process,we co-treated BMDMs with NEVs and cytochalasin D(Cyt D),the results showed that Cytochalasin D treatment blocked EV uptake.BMDMs were incubated with NEVs,a significant upregulation of TLR2,TLR3 and TLR4 resulted compared with the negative control cells.In our study,NEVs were found to induce IL-12p40,TNF-α,IL-1β and IFN-γ responses after stimulation of BMDMs,while the BMDMs from TLR2-/-exposed to NEVs showed extremely downregulated secretion of IL-12p40,TNF-α and IFN-γ compared with the WT group.We observed that NEVs could induce the phosphorylation of major MAPK pathway proteins(P38,ERK and JNK)and significantly increased production of IL-6,IL-12 and IL-10 when BMDMs were separately pretreated with P38,ERK and JNK inhibitors.In conclusion,we demonstrated here that NEVs were recognized by Toll-like receptor 2 and modulate host cell innate immunity through the MAPK signaling pathway.The expression and function study of NEVs protein.Proteins involved in EV biogenesis and trafficking,such as 14-3-3,HSP70,and HSP90 were highly enriched in the purified NEVs,in accordance with the results of the identification of EVs using anti-14-3-3,anti-HSP70 and anti-enolase antibodies.NEVs were labeled in vitro with the green fluorescent lipid dye PKH67 prior to exposure,and BMDMs were examined by confocal microscope at various times,the results shown that green fluorescence was found in the cytoplasm of BMDMs as early as 3 h,and the fluorescence intensity increased steadily at time points later than 3 h.These results provide strong evidence that NEVs can deliver their contents to BMDMs.Furthermore,we present the first characterization and immunomodulatory investigation of the N.caninum 14-3-3 protein.Immunofluorescence and immunogold electron microscopy studies of tachyzoites or N.caninum-infected cells suggested that 14-3-3 was localized in the cytosol and the membrane.Western blot analysis indicated that r Nc14-3-3 induced cytokine expression by activating the MAPK and AKT signaling pathways,and inhibitors of p38,ERK,JNK and AKT could significantly decrease the production of IL-6,IL-12p40 and IFN-γ,but had little relationship with TLR2.In addition,our results indicated that r Nc14-3-3 could induce the translocation of NF-κB/p65 from the cytosol to the nucleus.NEVs and r Nc14-3-3 immunized mice could reduced mortality in mice,suggesting that NEVs and r Nc14-3-3 is a novel vaccine candidate against neosporosis.In summary,we first isolated extracellular vesicles from the NC-1 strain of N.caninum and described the composition and characteristics of these NEVs.Furthermore,our results indicated that NEVs could modulate the cytokines expression in BMDMs through TLR2 and MAPK signaling pathway since they delivered their contents to BMDMs.We established that N.caninum 14-3-3 protein in NEVs can induce effective immune responses and stimulate cytokine expression by activating the MAPK,AKT and NF-κB signaling pathways not by TLR2.Our study revealed a new mechanism of interaction between N.caninum and host,and suggesting that NEVs and r Nc14-3-3 is a novel vaccine candidate against neosporosis.