Extracellular Vesicles Secreted by Neospora caninum Are Recognized by Toll-Like Receptor 2 and Modulate Host Cell Innate Immunity Through the MAPK Signaling Pathway

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.

Neospora caninum infection induces an isolate virulence-dependent pro-inflammatory gene expression profile in bovine monocyte-derived macrophages

“…Studies in murine and human MØs have identified signalling pathways implicated in host resistance against N. caninum and mechanisms used by the parasite to evade the immune responses mounted by these cells [5][6][7][8][9][10][11][12]. Here, the transcriptional analysis of bovine monocytederived MØs infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) N. caninum isolates has been used as an approach to study if these mechanisms may be also implicated in N. caninum interaction with his natural host.…”

“…Studies in murine MØs have identified the TLR2-MAPK, TLR3-TRIF and TLR11-MEK/ERK pathways and NLRP3-inflammasome activation as signalling pathways implicated in host resistance against N. caninum , that trigger the production of pro-inflammatory cytokines [ 5 – 8 ]. Several studies have demonstrated that N. caninum has evolved mechanisms to evade the immune response mounted by murine MØs.…”

N6-methyladenosine modification modulate in the heat stress of sheep (Ovis aries)

“…Following external stimulation, Rap1 transfers information to MAPK and PI3K-Akt (phosphatidylinositol 3-kinase and protein kinase B) signaling pathways [38,39], and the latter activates downstream signaling pathways such as NF-κB (nuclear factor κB), FoxO, HIF-1 (hypoxia-inducible factor 1), and mTOR (mammalian target of rapamycin) to respond to stress [40][41][42][43]. Sheep generate a series of immunoreactions under heat stress; for instance, the Toll-like receptor signaling pathway induces the transcription of genes participating in immunoreactions by activating NF-κB or MAPK signaling pathways [44,45]. The Toll-like receptor signaling pathway also participates in immunoreactions by recognizing HSPs [46].…”

Perils and Promises of Pathogenic Protozoan Extracellular Vesicles

“…In respect to PPEV internalization by immune cells, stained EVs of Neospora caninum have been found to randomly accumulate within the cytoplasm of macrophages (Li et al, 2018c ), while fluorescent P. falciparum -iRBC-derived EVs were observed in the perinuclear region of the human bone marrow-derived endothelial cell (Mantel et al, 2016 ). Importantly, temperature could have facilitated this endocytotic internalization because P. falciparum -iRBC-derived EVs were significantly detected in monocytes at 37°C (Sisquella et al, 2017 ), as well as G. intestinalis MV internalization by immature dendritic cells (iDCs) which were later inhibited almost completely at 4°C and by the addition of cytochalasin D (Evans-Osses et al, 2017 ).…”

“…Prior exposure of T. cruzi Y strain trypomastigote EVs to human macrophages transfected with TLR2 expressed CD25 and activated NF-κB via TLR2 (Cronemberger-Andrade et al, 2020 ). Meanwhile, TLR2 might be activated by N. caninum EVs in BMDMs because it contains some pathogen-associated molecular patterns (PAMPs) (Li et al, 2018c ). A similar work showed that T. gondii exosomes induced elevated expression of JNK mRNA, activated the nuclear translocation of phosphorylated JKN-protein, and eventually activated the MAPK pathway (Li et al, 2018b ).…”