Evaluation of the TLR3 involvement during Schistosoma japonicum-induced pathology.

BACKGROUND: Despite the functions of TLRs in the parasitic infections have been extensively reported, few studies have addressed the role of TLR3 in the immune response to Schistosoma japonicum infections. The aim of this study was to investigate the properties of TLR3 in the liver of C57BL/6 mice infected by S. japonicum. METHODS: The production of TLR3+ cells in CD4+T cells (CD4+CD3+), CD8+T cells (CD8+CD3+), γδT cells (γδTCR+CD3+), NKT cells (NK1.1+CD3+), B cells (CD19+CD3-), NK (NK1.1-CD3+) cells, MDSC (CD11b+Gr1+), macrophages (CD11b+F4/80+), DCs (CD11c+CD11b+) and neutrophils (CD11b+ Ly6g+) were assessed by flow cytometry. Sections of the liver were examined by haematoxylin and eosin staining in order to measure the area of granulomas. Hematological parameters including white blood cell (WBC), red blood cell (RBC), platelet (PLT) and hemoglobin (HGB) were analyzed. The levels of ALT and AST in the serum were measured using biochemical kits. The relative titers of anti-SEA IgG and anti-SEA IgM in the serum were measured by enzyme-linked immunosorbent assay (ELISA). CD25, CD69, CD314 and CD94 molecules were detected by flow cytometry. RESULTS: Flow cytometry results showed that the expression of TLR3 increased significantly after S. japonicum infection (P < 0.05). Hepatic myeloid and lymphoid cells could express TLR3, and the percentages of TLR3-expressing MDSC, macrophages and neutrophils were increased after infection. Knocking out TLR3 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse livers.,The number of WBC was significantly reduced in TLR3 KO-infected mice compared to WT-infected mice (P < 0.01), but the levels of RBC, platelet and HGB were significantly increased in KO infected mice. Moreover, the relative titers of anti-SEA IgG and anti-SEA IgM in the serum of infected KO mice were statistically decreased compared with the infected WT mice. We also compared the activation-associated molecules expression between S.japonicum-infected WT and TLR3 KO mice. CONCLUSIONS: Taken together, our data indicated that TLR3 played potential roles in the context of S. japonicum infection and it may accelerate the progression of S. japonicum-associated liver pathology.

Characteristics of splenic PD-1+ γδT cells in Plasmodium yoelii nigeriensis infection.

Although the functions of programmed death-1 (PD-1) on αß T cells have been extensively reported, a role for PD-1 in regulating γδT cell function is only beginning to emerge. Here, we investigated the phenotypic and functional characteristics of PD-1-expressing γδT cells, and the molecular mechanism was also explored in the Plasmodium yoelii nigeriensis (P. yoelii NSM)-infected mice. Flow cytometry and single-cell RNA sequencing (scRNA-seq) were performed. An inverse agonist of RORα, SR3335, was used to investigate the role of RORα in regulating PD-1+ γδT cells. The results indicated that γδT cells continuously upregulated PD-1 expression during the infection period. Higher levels of CD94, IL-10, CX3CR1, and CD107a; and lower levels of CD25, CD69, and CD127 were found in PD-1+ γδT cells from infected mice than in PD-1- γδT cells. Furthermore, GO enrichment analysis revealed that the marker genes in PD-1+ γδT cells were involved in autophagy and processes utilizing autophagic mechanisms. ScRNA-seq results showed that RORα was increased significantly in PD-1+ γδT cells. GSEA identified that RORα was mainly involved in the regulation of I-kappaB kinase/NF-κB signaling and the positive regulation of cytokine production. Consistent with this, PD-1-expressing γδT cells upregulated RORα following Plasmodium yoelii infection. Additionally, in vitro studies revealed that higher levels of p-p65 were found in PD-1+ γδT cells after treatment with a RORα selective synthetic inhibitor. Collectively, these data suggest that RORα-mediated attenuation of NF-κB signaling may be fundamental for PD-1-expressing γδT cells to modulate host immune responses in the spleen of Plasmodium yoelii nigeriensis-infected C57BL/6 mice, and it requires further investigation.