Isolation and propagation of bovine blood-derived macrophages using a mixed culture with bovine endothelial B46 cells.

Macrophages are innate immune cells with multiple functions such as phagocytosis, cytokine production, and antigen presentation. Since macrophages play critical roles in some bacterial infectious diseases in cattle, including tuberculosis, paratuberculosis, and brucellosis, the in vitro culturing of bovine macrophages is useful for evaluating host-pathogen interactions at the cellular and molecular levels. We have previously reported the establishment of two immortalized bovine liver sinusoidal cell lines, endothelial B46 cells and myofibroblast-like A26 cells (Cell Biology International, 40, 1372-1379, 2016). In this study, we investigated the use of these cell lines as feeder cells that support the proliferation of bovine blood-derived macrophages (BBMs). Notably, the B46 cell line efficiently acts as feeder cells for the propagation of BBMs. Compared with primary cultured vascular endothelial cells, the infinite proliferation ability of B46 cells is more beneficial for preparing confluent feeder layers. In conclusion, this study provides a simple and efficient protocol for the isolation and propagation of BBMs using a primary mixed culture of bovine whole blood with B46 feeder cells. Isolated BBMs are expected to be useful for developing in vitro models for studying the interactions between bovine pathogens and host immune cells.

Role of dense granule antigen 7 in vertical transmission of Neospora caninum in C57BL/6 mice infected during early pregnancy.

Neosporosis is a parasitic disease affecting the health of dogs and cattle worldwide. It is caused by Neospora caninum, an obligate intracellular apicomplexan parasite. Dogs are its definitive host, it mostly infects livestock animals, especially cattle that acts as intermediate host. It is necessary to have well-established models of abortion and vertical transmission in experimental animals, in order to determine basic control measures for the N. caninum infection. We evaluated the role of N. caninum dense granule antigen 7 (NcGRA7) in the vertical transmission of N. caninum using the C57BL/6 pregnant mouse model. We inoculated mice on day 3.5 of pregnancy with parental Nc-1 or NcGRA7-deficient parasites (NcGRA7KO). Post-mortem analyses were performed on day 30 after birth and the surviving pups were kept until day 30 postpartum. The number of parasites in the brain tissues of offspring from NcGRA7KO-infected dams was significantly lower than that of the Nc-1-infected dams under two infection doses (1 × 106 and 1 × 105 tachyzoites/mouse). The vertical transmission rates in the NcGRA7KO-infected group were significantly lower than those of the Nc1-infected group. To understand the mechanism by which the lack of NcGRA7 decreases the vertical transmission, pregnant mice were sacrificed on day 13.5 of pregnancy (10 days after infection), although parasite DNA was detected in the placentas, no significant difference was found between the two parasite lines. Histopathological analysis revealed a greater inflammatory response in the placentas from NcGRA7KO-infected dams than in those from the parental strain. This finding correlates with upregulated chemokine mRNA expression for CCL2, CCL8, and CXCL9 in the placentas from the NcGRA7KO-infected mice. In conclusion, these results suggest that loss of NcGRA7 triggers an inflammatory response in the placenta, resulting in decreased vertical transmission of N. caninum.

Toll-Like Receptor 2 is Involved in Abnormal Pregnancy in Mice Infected with Toxoplasma gondii During Late Pregnancy.

Infection with Toxoplasma gondii during pregnancy causes failure of pregnancy maintenance, resulting in fetal death, abortion, stillbirth, or premature birth, but the mechanism of disease onset remains unclear. Although Toll-like receptor 2 (TLR2) is expressed on antigen-presenting cells and trophoblasts, the role of TLR2 in T. gondii infection during pregnancy is unknown. In this study, we investigated the role of TLR2 in congenital toxoplasmosis using TLR2-deficient (TLR2-/-) mice. T. gondii infection on gestational day 12.5 (Gd12.5) induced more abnormal pregnancy, including premature birth and stillbirth, in wild-type mice than in TLR2-/- mice. Multiple calcifications were observed in the placentas of the infected wild-type mice. At Gd18.5 (6days postinfection), the parasite numbers in the placenta and uterus and the histological changes did not differ significantly between the wild-type and TLR2-/- mice. However, T. gondii infection reduced the mRNA expression of interleukin-12p40 (IL-12p40) and increased IL-4 and IL-10 mRNAs in the placentas of the wild-type mice. In contrast, the placentas of the TLR2-/- mice showed no changes in the expression of these cytokines, including IL-6 and tumor necrosis factor α, in response to T. gondii infection. Serum interferon-γ levels were significantly lower in the infected TLR2-/- mice than in the infected wild-type mice on Gd18.5. Thus, the TLR2-/- mice were less susceptible to the induction of immune responses by T. gondii infection during late pregnancy. Therefore, TLR2 signaling may play a role in the development of disease states during pregnancy, specifically placental hypofunction.

CXCR3-Dependent Immune Pathology in Mice following Infection with Toxoplasma gondii during Early Pregnancy.

Toxoplasmosis is a worldwide zoonosis caused by the obligate intracellular parasite Toxoplasma gondii The symptoms of congenital toxoplasmosis range from embryonic death and resorption to subclinical infection, but the mechanism of disease onset remains unclear. C-X-C motif chemokine receptor 3 (CXCR3) is highly expressed in Th1-associated immune cells and plays an important role in the trafficking and activation of immune cells. However, the roles of CXCR3 in T. gondii-induced fetal loss and the molecular mechanism of embryo resorption remain poorly understood. In this study, we investigated the role of CXCR3 in fetal wastage caused by T. gondii infection using CXCR3-deficient (CXCR3-/-) mice. CXCR3-/- and wild-type pregnant mice were inoculated intraperitoneally with T. gondii tachyzoites on day 3.5 of gestation (Gd3.5). Pregnancy rates decreased as the pregnancy progressed in both infected groups; however, infected CXCR3-/- mice showed a significant fetal loss at Gd13.5 compared with that at Gd7.5. All embryos of the infected groups showed necrosis, and embryo resorption was significantly increased in infected CXCR3-/- compared with wild-type mice at Gd13.5. The parasite load of fetoplacental tissues was significantly increased in CXCR3-/- mice at Gd10.5. Moreover, mRNA expression levels of inducible nitric oxide synthase were significantly increased in fetoplacental tissues from infected wild-type mice compared to infected CXCR3-/- mice following the infection. These results suggested that CXCR3-dependent immune responses provide anti-Toxoplasma activity and play an essential role in reducing embryo resorption and fetal loss caused by T. gondii infection during early pregnancy.

Toxoplasma gondii Dense Granule Proteins 7, 14, and 15 Are Involved in Modification and Control of the Immune Response Mediated via NF-κB Pathway.

Toxoplasma gondii infects almost all warm-blooded animals, including humans, leading to both cellular and humoral immune responses in the host. The virulence of T. gondii is strain specific and is defined by secreted effector proteins that disturb host immunity. Here, we focus on nuclear factor-kappa B (NFκB) signaling, which regulates the induction of T-helper type 1 immunity. A luciferase assay for screening effector proteins, including ROPs and GRAs that have biological activity against an NFκB-dependent reporter plasmid, found that overexpression of GRA7, 14, and 15 of a type II strain resulted in a strong activity. Thus, our study was aimed at understanding the involvement of NFκB in the pathogenesis of toxoplasmosis through a comparative analysis of these three molecules. We found that GRA7 and GRA14 were partially involved in the activation of NFκB, whereas GRA15 was essential for NFκB activation. The deletion of GRA7, GRA14, and GRA15 in the type II Prugniaud (Pru) strain resulted in a defect in the nuclear translocation of RelA. Cells infected with the PruΔgra15 parasite showed reduced phosphorylation of inhibitor-κBα. GRA7, GRA14, and GRA15 deficiency decreased the levels of interleukin-6 in RAW246.7 cells, and RNA-seq analysis revealed that GRA7, GRA14, and GRA15 deficiency predominantly resulted in downregulation of gene expression mediated by NFκB. The virulence of all mutant strains increased, but PruΔgra14 only showed a slight increase in virulence. However, the intra-footpad injection of the highly-virulent type I RHΔgra14 parasites in mice resulted in increased virulence. This study shows that GRA7, 14, and 15-induced host immunity via NFκB limits parasite expansion.