Abstract:

Objective This study aimed to observe the dynamic expression of triggering receptor expressed on myeloid cells 1 (TREM-1) in the liver of mice infected with Schistosoma japonicum. The correlation between TREM-1 and M1 macrophage polarization was analyzed. Methods A total of 34 female C57BL/6 mice were randomly assigned to infection group (24 mice) and control group (10 mice). The mice in the control group were left untreated and were sacrificed for collection of liver tissues. The mice in the infection group were infected with (15 ± 2) S. japonicum cercaria per mouse; at 3, 6, 9 and 12 weeks after infection, six mice were randomly selected and sacrificed, and liver tissues were collected. The relative liver mRNA transcription levels of TREM-1 and IL-1β were examined with real-time quantitative PCR. Western blotting was used to observe the TREM-1 and inducible NO synthase (iNOS) protein expression in liver tissues. Frozen mouse liver sections were prepared. Immunofluorescence staining was used to observe TREM-1 expression in hepatic macrophages. The following four groups of RAW264.7 macrophage cells were examined: NC siRNA, TREM-1 siRNA, NC siRNA + SWA; and TREM-1 siRNA + SWA. The cells were transfected with synthetic TREM-1 siRNA or NC siRNA and cultured for 6 h, schistosome worm antigen (SWA, 20 μg/ml) was then added to the indicated groups for 48 h. The TREM-1 and iNOS protein expression levels were detected via Western blotting. One-way ANOVA was used to analyze the relative mRNA transcription of TREM-1 and IL-1β, and the relative protein expression of TREM-1 and iNOS. Independent samples t test was used for comparisons between two groups. Results Real-time quantitative PCR analysis indicated that the relative mRNA levels of TREM-1 in the liver in infection mice were 14.28 ± 6.26, 183.41 ± 37.37, 68.17 ± 16.19 and 106.91 ± 45.70 at 3, 6, 9 and 12 weeks after infection with respect to the control group level (1.00). A significant difference was observed among these groups (F = 6.668, P < 0.01) and between the control and infection groups 6 weeks after infection (t = -4.881, P < 0.01). The relative mRNA levels of IL-1β in the liver in infected mice were 8.16 ± 1.91, 56.12 ± 10.68, 24.41 ± 3.54 and 24.28 ± 2.98 at 3, 6, 9 and 12 weeks after infection with respect to the control (1.00). A significant difference was observed among these groups (F = 16.943, P < 0.01) and between the control and infection groups (t = -3.740, P < 0.05; t = -5.159, -6.606, -7.799, P < 0.01). Western blotting revealed that the relative expression levels of TREM-1 protein in the liver in infected mice were 1.24 ± 0.38, 1.50 ± 0.13, 1.13 ± 0.28 and 1.17 ± 0.60 at 3, 6, 9 and 12 weeks after infection. No significant difference was observed among these groups (F = 1.547, P > 0.05), and between the control and infection groups at 3, 9, 12 weeks after infection (t = -1.247, -0.745, -0.559, P > 0.05). However, a significant difference was found between the control and infection groups 6 weeks after infection (t = -6.011, P < 0.01). The relative expression of iNOS protein in liver tissues in infected mice were 5.27 ± 3.66, 23.27 ± 14.72, 10.16 ± 4.97 and 2.69 ± 1.65 at 3, 6, 9 and 12 weeks after infection. Significant differences were found among these groups (F = 9.384, P < 0.01) and between the control and infection groups (t = -2.893, -3.716, -4.537 and -2.571, P < 0.05). Immunofluorescence staining indicated that TREM-1 (green) and F4/80 (red) expression was low in the control group but was elevated around hepatic granulomas in infected mice 6 weeks after infection. The number of cells with co-localization (yellow) of TREM-1 and F4/80 increased around the mouse hepatic granulomas. Western blotting revealed that the relative expression levels of TREM-1 protein in the NC siRNA + SWA, TREM-1 siRNA and TREM-1 siRNA + SWA groups were 1.35 ± 0.13, 0.58 ± 0.09 and 1.09 ± 0.03 (F = 46.689, P < 0.01). A significant difference was found between the NC siRNA + SWA, TREM-1 siRNA and NC siRNA (1.00) groups (t = -4.716, 7.858, P < 0.05), and between the TREM-1 siRNA + SWA and TREM-1 siRNA groups (t = 9.000, P < 0.01). The relative expression levels of of iNOS in the NC siRNA + SWA, TREM-1 siRNA and TREM-1 siRNA + SWA groups were 3.69 ± 1.04, 0.77 ± 0.12 and 2.74 ± 0.86 (F = 12.714, P < 0.01). A significant difference was observed between the NC siRNA + SWA, TREM-1 siRNA and NC siRNA (1.00) groups (t = -4.451, 3.254, P < 0.05). No significant difference was observed between the TREM-1 siRNA + SWA and TREM-1 siRNA groups (t = 3.913, P > 0.05). Conclusion The expression of TREM-1 in the liver of mice infected with S. japonicum is significantly up-regulated. Inhibition of TREM-1 expression may suppress iNOS expression in SWA-treated macrophages.

Key words: Schistosoma japonicum, Triggering receptor expressed on myeloid cells 1, Macrophage