Abstract
Neospora caninum is an obligate intracellular parasite, which causes significant economic losses in the cattle industry. However, the immune mechanism of the parasite–host interaction is not yet fully understood. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism by which almost all cells, especially immune and tumor cells, participate in intercellular communications. Although studies have indicated that EVs secreted by Toxoplasma gondii or Trypanosoma brucei promote exchanges of biological molecules important for the host–parasite interplay, however, EVs and their biological activities in N. caninum is not clear. Here, we used multiple methods, including electron microscopy, nanoparticle tracking analysis, RT-PCR, immunofluorescence, western blot, proteomics, and cytokine analyses, to examine the properties of N. caninum EVs. We found that N. caninum produced EVs that are similar to mammalian exosomes, which generally range from 30 to 150 nm in diameter. It was shown that N. caninum EVs could remarkably increase the production of pro-inflammatory cytokines IL-12p40, TNF-α, IL-1β, IL-6, and IFN-γ by wild-type (WT) mouse bone marrow-derived macrophages (BMDMs) whereas the secretion of IL-12p40, TNF-α, and IFN-γ was very strongly downregulated in TLR2−/− mouse BMDMs. The levels of IL-6 were not affected, but the secretion of IL-10 was upregulated. We found that the phosphorylation levels of P38, ERK, and JNK were significantly reduced in the TLR2−/− cells compared with those in WT mouse BMDMs and that treatment with chemical inhibiters of P38, ERK, and JNK resulted in upregulation of IL-6, IL-12p40, and IL-10 production. Together, these results demonstrated that N. caninum EVs could be rapidly internalized to deliver proteins to the host cells and modulate the host cell immune responses through MAPK signaling pathway in a TLR2-dependent manner. Our study is the first to reveal potential roles for N. caninum EVs in host communication and immune response in parasite–host interactions.
Highlights
Neospora caninum is an obligate intracellular parasite which naturally takes dog and cattle as hosts [1]
To test whether N. caninum produces extracellular vesicles (EVs), vesicles were isolated from parasite growth media through a series of ultracentrifugation steps, similar to that described for isolating exosomes from the parasitic protozoan Leishmania [24]
We found that the bone marrow-derived macrophages (BMDMs) from toll-like receptor 2 (TLR2)−/− exposed to N. caninum EVs showed extremely downregulated secretion of IL-12p40, TNF-α, and IFN-γ compared with the BMDMs from the WT mice, the change in IL-6 was not significant
Summary
Neospora caninum is an obligate intracellular parasite which naturally takes dog and cattle as hosts [1]. N. caninum infectivity in humans has not been identified, serological evidence suggests that humans can be exposed to N. caninum [2, 3]. The definitive hosts of N. caninum are mostly in the canid family, and infection mainly causes a variety of neurological symptoms, especially in. N. caninum EVs and Innate Immunity dogs [4]. Neosporosis is among the major causes of abortion in bovine. The calves vertically infected with the N. caninum show neurological disorders, which is costly for cattle farms and the related industry [5, 6]. Global economic loss due to neosporosis is substantial [7, 8]
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