关键词: 刚地弓形虫, 瞬时受体电位M8型通道蛋白, 巨噬细胞, 基因敲除, 基因过表达, 免疫调控

Abstract: Objective　To investigate the regulatory role of transient receptor potential cation channel subfamily M member 8 (TRPM8) in inflammatory response and migration of macrophages post⁃infection with Toxoplasma gondii, so as to provide a theoretical evidence for unraveling the immune mechanism of T. gondii infection. Methods　The transcriptional and translational levels of TRPM8 were quantified in macrophages from mice infected with T. gondii using real⁃time quantitative reverse transcription PCR (RT⁃qPCR) and Western blotting assays. TRPM8 knockout (TRPM8-/- and TRPM8+/-) mice at ages of 8 to 10 weeks and wild⁃type (WT) mice at age of 8 to 10 weeks were intraperitoneally injected with tachyzoites of the T. gondii RH strain, and the survival rate of mice and levels of cytokines in mice sera were compared between TRPM8 knockout and WT mice. TRPM8-/- and WT mice were infected with tachyzoites of the T. gondii RH strain expressing green fluorescent protein (GFP⁃RH). The effect of TRPM8 deficiency on the migration of T. gondii in mice was examined with a small animal in vivo optical imaging system, and the abundance of T. gondii internal transcribed spacer 1 (ITS1) gene was quantified in mouse brain tissues using qPCR assay. RAW264.7 cells overexpressing TRPM8 gene (RAW264.7⁃TRPM8 cells) and control cells (RAW264.7⁃Vector cells) were generated. The T. gondii ITS1 gene abundance was quantified in cells using qPCR assay, and the secretion of cytokines was detected in cell culture supernatants using enzyme⁃linked immunosorbent assay (ELISA). In addition, cell migration was examined using a scratch assay. Results　The transcriptional level of TRPM8 gene was lower in peritoneal macrophages from T. gondii⁃infected mice than from uninfected mice [(0.133 ± 0.143) vs. (1.125 ± 0.562); t = 2.962, P < 0.05]. The survival rates of WT mice, TRPM8+/-mice, and TRPM8-/- mice were 8.3%, 50.0%, and 66.7% on day 7 post⁃infection with T. gondii, respectively. Significant differences were observed among the survival curves of the three groups ([χ2] = 8.493, P = 0.014), with TRPM8-/- mice showing longer survival. The serum level of tumor necrosis factor⁃alpha (TNF⁃α) was higher in TRPM8-/- mice infected with T. gondii than in uninfected TRPM8-/- mice [(120.456 ± 15.561) pg/mL vs. (57.582 ± 3.952) pg/mL; P = 0.003], and the mean fluorescence intensity was higher in the brain of TRPM8-/- mice infected with tachyzoites of GFP⁃RH than in WT mice [(1.810 ± 0.106) × 107 (p/s/cm2/sr)/(μW/cm2) vs. (0.831 ± 0.192) × 107 (p/s/cm2/sr)/(μW/cm2); t = 8.916, P < 0.001]. The fold change of abundance of T. gondii ITS1 gene was 1.000 ± 0.004 in RAW264.7⁃Vector cells and 1.043 ± 0.009 in RAW264.7⁃TRPM8 cells following T. gondii infection; and the fold change of abundance of total (in cells and cell supernatants) T. gondii ITS1 gene in RAW264.7⁃Vector group and RAW264.7⁃TRPM8 group was 1.000 ± 0.003 and 1.018 ± 0.005, respectively. The relative abundance of TOXO ITS1 in RAW264.7⁃TRPM8 was significantly higher than that in RAW264.7⁃Vector (t = 7.515, 6.496, both P < 0.01). ELISA measured lower interleukin (IL)⁃10 secretion in RAW264.7⁃Vector cells infected with T. gondii than in RAW264.7⁃TRPM8 cells infected with T. gondii [(94.104 ± 11.277) pg/mL vs. (145.479 ± 29.156) pg/mL; t = 2.847, P < 0.05], and scratch assay measured higher migration rates of RAW264.7⁃Vector cells than those of RAW264.7⁃TRPM8 cells at 48 h [(29.2 ± 11.6)% vs. (5.5 ± 4.7)%; P < 0.01] and 72 h post⁃infection with T. gondii [(32.1 ± 10.5)% vs. (8.2 ± 4.1)%; P < 0.01]. Conclusions　T. gondii infection may downregulate TRPM8 expression in mouse macrophages. TRPM8 knockout may reduce the host susceptibility to T. gondii and promote parasite migration to brain through upregulating TNF⁃α secretion. TRPM8 overexpression may inhibit the anti⁃parasitic and migration functions of macrophages. 

Key words: Toxoplasma gondii, Transient receptor potential cation channel subfamily M member 8, Macrophage, Gene knockout, Gene overexpression, Immunoregulation