Abstract:

Objective To investigate the expression of Tsc22 domain family member 3 (Tsc22d3) expression in natural killer (NK) cell subsets in liver of mice infected with Schistosoma japonicum and its effect on the cytotoxicity of NK cells. Methods Forty-two female C57BL/6 mice were infected with S. japonicum cercariae (20 ± 1 per mouse) by abdominal patch method. Six mice were sacrificed before infection and at the 4th and 6th weeks after infection respectively. NK cells in the liver were enriched by magnetic bead sorting and single-cell sequencing was performed to analyze the expression of Tsc22d3 in NK cells and the activated signaling pathways. The remaining 24 mice were randomly divided into an infection group and a control group, with 12 mice in each group. At the 4th and 6th weeks after infection, 6 mice in each group were anesthetized and non-parenchymal cells in the liver were isolated. The Tsc22d3 expression was detected in mouse hepatic NK cells in the control and infection groups using flow cytometry. Empty plasmid and Tsc22d3 plasmid were transfected into NK92 cells to obtain the empty plasmid control group (NC group) and the Tsc22d3 overexpression NK92 cell group (Tsc22d3⁺NK92 group) by using liposome transfection method. NK92 cells in the Tsc22d3⁺NK92 group and the NC group were stimulated with soluble egg antigen (SEA) for 24 or 48 h, and the expression of interferon-γ (IFN-γ) and perforin 1 (Prf1) was detected in NK cells using flow cytometry and real-time quantitative fluorescence PCR (qPCR) assay. All statistical analyses were performed with the software GraphPad Prism 9, and differences of means between the two groups was tested for statistical significance with t test. Results Single-cell sequencing revealed that compared with uninfected mice (2.41), the expression of Tsc22d3 continued to increase at 4 weeks (3.16) and 6 weeks after infection (3.76), and Tsc22d3 was highly expressed in the C3 subset of NK cells. The pathway of natural killer cell mediated cytotoxicity in the C3 subgroup were significantly activated. Flow cytometry showed that the proportions of Tsc22d3⁺NK92 cells were (0.24 ± 0.01)% and (1.40 ± 0.14)% in mouse hepatic NK cells 4 weeks and 6 weeks post-infection in the infection group, which were both higher than in the control group (0.12% ± 0.02%) (t = 12.110 and 22.010, both P values < 0.01). The proportions of IFN-γ and Prf1 positive cells were (1.66 ± 0.15)% and (53.23 ± 0.81)% in the Tsc22d3⁺NK92 group 24 h post-stimulation with SEA, which higher than in the NC group [(0.88 ± 0.13)% and (29.93 ± 1.85)%, respectively] (t = 6.800 and 20.010, both P values < 0.01), and the relative mRNA expression of IFN-γ and Prf1 was (1.53 ± 0.24) and (1.41 ± 0.04) in the Tsc22d3⁺NK92 group 24 h post-stimulation with SEA, which was higher than in the NC group [(1.00 ± 0.07) and (1.00 ± 0.140), respectively] (t = 3.573 and 4.973, P < 0.05 and 0.01). The relative mRNA expression of IFN-γ and Prf1 was (2.06 ± 0.39) and (1.54 ± 0.26) in the Tsc22d3⁺NK92 group 48 h post-stimulation with SEA, which was higher than in the NC group [(1.06 ± 0.44) and (1.00 ± 0.04), respectively] (t = 2.953 and 3.588, both P values < 0.05). Conclusion Tsc22d3 is highly expressed in the C3 subset of hepatic NK cells during the course of S. japonicum infection, and high Tsc22d3 expression may increase the cytotoxicity of NK cells.

Key words: Schistosoma japonicum, Tsc22d3, Liver fibrosis, Natural killer cell