Xiaocen Wang

Canine Neutrophil Extracellular Traps Release Induced by the Apicomplexan Parasite Neospora caninum In Vitro

Neosporosis is considered as one of the main causes of abortion and severe economic losses in dairy industry. The Canis genus serving as one of the confirmed definitive hosts of the apicomplexan parasite Neospora caninum (N. caninum) plays a critical role in its life cycle. However, the effects of N. caninum on its definitive hosts of neutrophils extracellular traps (NETs) formation remain unclear. In the present study, N. caninum tachyzoite-induced canine NETs formation was observed by scanning electron microscopy (SEM). Visualization of DNA decorated with H3, neutrophil elastase (NE), and myeloperoxidase (MPO) within N. caninum tachyzoite-induced NETs were examined using fluorescence confocal microscopy analyses. Furthermore, the formation of canine NETs was quantified using Sytox Green staining, and the LDH levels in supernatants were examined by an LDH Cytotoxicity Assay® kit. The results clearly showed that NETs-like structures were induced by N. caninum tachyzoites, and the major components within these structures induced by N. caninum tachyzoite were further confirmed by fluorescence confocal microscopy visualization. These results suggest that N. caninum tachyzoites strongly induced NETs formation in canine polymorphonuclear neutrophils (PMN). In functional inhibition assays, the blockings of NADPH oxidase, NE, MPO, SOCE, ERK 1/2, and p38 MAPK signaling pathways significantly inhibited N. caninum tachyzoite-induced NETs formation. To our knowledge, this study is the first to report the formation of NETs in canine PMN against N. caninum infection.

NLRP3 inflammasome activation in murine macrophages caused by Neospora caninum infection

BackgroundNeospora caninum is an intracellular parasite that causes significant economic losses in cattle industry. Understanding the host resistance mechanisms in the innate immune response to neosporosis could facilitate the exploration of approaches for controlling N. caninum infection. The NLR inflammasome is a multiprotein platform in the cell cytoplasm and plays critical roles in the host response against microbes.MethodsNeospora caninum-infected wild-type (WT) macrophages and Nlrp3−/− macrophages, and inhibitory approaches were used to investigate inflammasome activation and its role in N. caninum infection. Inflammasome RT Profiler PCR Arrays were used to identify the primary genes involved in N. caninum infection. The expression of the sensor protein NLRP3, processing of caspase-1, secretion of IL-1β and cell death were detected. Neospora caninum replication in macrophages was also assessed.ResultsMany NLR molecules participated in the recognition of N. caninum, especially the sensor protein NLRP3, and further study revealed that the NLRP3 distribution became punctate in the cell cytoplasm, which facilitated inflammasome activation. Inflammasome activation-mediated caspase-1 processing and IL-1β cleavage in response to N. caninum infection were observed and were correlated with the time of infection and number of infecting parasites. LDH-related cell death was also observed, and this death was regarded as beneficial for the clearance of N. caninum. Treatment of N. caninum-infected macrophages with caspase-1, pan-caspase and NLRP3 inhibitors led to the impaired release of active IL-1β and a failure to restrict parasite replication. And Neospora caninum infected peritoneal macrophages from Nlrp3-deficient mice displayed greatly decreased release of active IL-1β and the failure of caspase-1 cleavage.ConclusionsThe NLRP3 inflammasome can be activated in N. caninum-infected macrophages, and plays a protective role in the host response to control N. caninum.

An anti-tumor protein produced by Trichinella spiralis induces apoptosis in human hepatoma H7402 cells

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic myeloid leukemia cell line (K562) and a human hepatoma cell line (H7402) using the display library. The protein encoded by the A200711 gene was identified and analyzed using protein analysis software. To test the antitumor effects of A200711, variations in cell proliferation and apoptosis were monitored after recombinant pEGFP-N1-A200711 was transfected into H7402 cells. The results show that the expressed target gene successfully induced apoptosis in H7402 cells as measured by Hoechst-PI staining, MTT assay (p<0.05). This study warrants further investigation into the therapeutic use of A200711 for anti-hepatocellular carcinomas.

Caprine Monocytes Release Extracellular Traps against Neospora caninum In Vitro

Neospora caninum is an obligate intracellular apicomplexan parasite that causes reproductive loss and severe economic losses in dairy and goat industry. In the present study, we aim to investigate the effects of N. caninum tachyzoites on the release of extracellular traps (ETs) in caprine monocytes and furthermore elucidated parts of its molecular mechanisms. N. caninum tachyzoite-induced monocytes-derived ETs formation was detected by scanning electron microscopy. H3 and myeloperoxidase (MPO) within monocyte-ETs structures were examined using laser scanning confocal microscopy analyses. The results showed that N. caninum tachyzoites were not only able to trigger ETs formation in caprine monocytes, but also that monocyte-released ETs were capable of entrapping viable tachyzoites. Histones and MPO were found to be decorating the DNA within the monocytes derived-ETs structures thus proving the classical components of ETs. Furthermore, inhibitors of NADPH oxidase-, MPO-, ERK 1/2-, or p38 MAPK-signaling pathway significantly decreased N. caninum tachyzoite-triggered caprine monocyte-derived ETosis. This is the first report of ETs release extruded from caprine monocytes after N. caninum exposure and thus showing that this early innate immune effector mechanism might be relevant during the acute phase of caprine neosporosis.

Bovine macrophage-derived extracellular traps act as early effectors against the abortive parasite Neospora caninum

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

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

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.

NLRP3 Inflammasome Participates in Host Response to Neospora caninum Infection

Neospora caninum is an intracellular protozoan parasite closely related to Toxoplasma gondii that mainly infects canids as the definitive host and cattle as the intermediate host, resulting in abortion in cattle and leading to financial losses worldwide. Commercial vaccines or drugs are not available for the prevention and treatment of bovine neosporosis. Knowledge about the hallmarks of the immune response to this infection could form the basis of important prevention strategies. The innate immune system first responds to invading parasite and subsequently initiates the appropriate adaptive immune response against this parasite. Upon infection, activation of host pattern-recognition receptors expressed by immune cells triggers the innate immune response. Toll-like receptors, NOD-like receptors, and C-type lectin receptors play key roles in recognizing protozoan parasite. Therefore, we aimed to explore the role of the NLRP3 inflammasome during the acute period of N. caninum infection. In vitro results showed that N. caninum infection of murine bone marrow-derived macrophages activated the NLRP3 inflammasome, accompanied by the release of IL-1β and IL-18, cleavage of caspase-1, and induction of cell death. K+ efflux induced by N. caninum infection participated in the activation of the inflammasome. Infection of mice deficient in NLRP3, ASC, and caspase-1/11 resulted in decreased production of IL-18 and reduced IFN-γ in serum. Elevated numbers of monocytes/macrophages and neutrophils were found at the initial infection site, but they failed to limit N. caninum replication. These findings suggest that the NLRP3 inflammasome is involved in the host response to N. caninum infection at the acute stage and plays an important role in limiting parasite growth, and it may enhance Th1 response by inducing production of IFN-γ. These findings may help devise protocols for controlling neosporosis.

A novel dense granule protein NcGRA23 in Neospora caninum

Neospora caninum is an intracellular parasite first discovered by Bjerkas in 1984 in young canine suffering from myositis and meningitis [1]. Neospora caninum can infect canines, bovines, and other animals, leading to abortion or stillborn foetuses in bovines and motor nervous system diseases in nascent calves, severely damaging the animal agricultural industry. Tranas et al. [2] showed that N. caninum could also be detected in human serum, but no human N. caninum infections have been reported. Neospora caninum has brought great losses to the animal husbandry industry; thus, it has received much attention from veterinarians and is a subject of many studies. Dense granule proteins (GRAs) are secreted by Toxoplasma gondii and N. caninum [3–6], and play important roles in forming parasitophorous vacuoles (PV) [7,8]. GRAs are thought to modify the microenvironment within the PV, and influence intracellular survival and replication [9]. In 2013, Liu et al. [10] expressed NcGRA14 and showed that it was associated with PV membrane (PVM) formation. However, the functions of most other granule proteins remain unknown. In this study, the N. caninum GRA23 (NcGRA23) gene (NCLIV_006780) was analyzed by BLAST based on the homologous sequence of the T. gondii GT1 GRA23 gene (TGGT1_297880) in GenBank. BLAST analysis of nucleotide and amino acid sequences of N. caninum and T. gondii GRA23 gene showed that nucleotide homology was 63.96%, and amino acid homology was 57.92% (Supplementary Fig. S1). To obtain the NcGRA23 protein and its polyclonal antibodies, cDNA of NcGRA23 was amplified from RNA (Supplementary data) by PCR and then cloned into Escherichia coli. The primers used to amplify the full-length NcGRA23 gene were PF (5′-ATAGAATTCC TCGCGTCCGCCGACGAAGCTTT-3′) and PR (5′-ATACTCGAG TTAGTTCTTTCGCGCGAGCACTGA-3′). The purified protein (0.6 mg/ml) was verified by SDS-PAGE (Supplementary Fig. S2A) and western blot analysis using sera immunized with N. caninum NC-1 tachyzoites (Supplementary Fig. S2B). Rabbits were immunized subcutaneously three times with the GRA23 protein, at 10-day intervals. Freund’s complete adjuvant was used (1:1) in the first immunization (500 μg/per rabbit), and Freund’s incomplete adjuvant was used (1:1) in the second and third immunizations (300 μg/per rabbit). Blood was collected from the heart 10 days after the last immunization. After blood was centrifuged at 3000 g for 15min, the serum was separated, and the antibody titer was detected by indirect ELISA and stored at −20°C for future use. The NC-1 tachyzoites were lysed and analyzed by western blot analysis using anti-recombinant NcGRA23 antibodies. The result showed that anti-recombinant NcGRA23 antibodies are specific (Supplemented Fig. S2C). NcGRA6 is located in the PV network [11]. To determine the location of NcGRA23, we co-localized GRA6 with GRA23. MDBK cells (Cell Bank of the Shanghai Institutes for Biological Sciences, Shanghai, China) were inoculated with NC-1 tachyzoites (MOI = 3) for 48 h and washed three times (15min each) with pre-cooled PBS. Then, cells were treated in 0.5% Triton-X-100 at room temperature for 15min and washed with PBS. Subsequently, cells were blocked with 5% BSA at room temperature for 30min and then incubated with diluted primary rabbit anti-GRA23 and rat anti-GRA6 polyclonal antibodies overnight at 4°C. After wash with PBS, cells were incubated with FITC-labeled goat anti-rabbit IgG and Cy3-labeled goat anti-rat IgG (Earthox, Millbrae, USA) for 1 h. Finally, intracellular parasites were observed and mapped on a laser scanning confocal microscope (Fv1000; Olympus, Tokyo, Japan). The results showed that N. caninum GRA23 was distributed in the front of the parasite, while GRA6 was located in the back of the parasite (Fig. 1A). The GRA6 was co-localized with the newly discovered compact granule protein GRA23 by immunofluorescence (Fig. 1A). GRA6 was mainly distributed in the PV. After N. caninum invasion, GRA6 was nearly absent from the PVM (Fig. 1B). GRA23 was distributed on the edge of the PV and existed in the PVM (Fig. 1B). The co-localization with GRA6 revealed that GRA23 is secreted in the PV and partially secreted at the PV margins.