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

摘要： 目的　探究弓形虫感染后瞬时受体电位M8型通道蛋白（transient receptor potential cation channel subfamily M member 8，TRPM8）对巨噬细胞炎症应答及迁移的调控作用，为阐明弓形虫感染免疫机制提供理论依据。方法　采用实时荧光定量PCR（reverse transcription quantitative PCR，RT⁃qPCR）和蛋白免疫印迹实验（Western blotting）检测弓形虫感染小鼠巨噬细胞中TRPM8的转录和蛋白水平。分别向TRPM8敲除（TRPM8-/-、TRPM8+/-）小鼠（8 ~ 10周龄）和野生型（wild⁃type，WT）小鼠（8 ~ 10周龄）腹腔注射弓形虫RH株速殖子，观察并比较两组小鼠生存率和血清中的细胞因子水平。用带有绿色荧光蛋白（green fluorescent protein，GFP）标签的弓形虫RH株（GFP⁃RH）速殖子感染TRPM8-/-小鼠和WT小鼠，结合小动物活体成像系统分析TRPM8缺失对弓形虫在小鼠体内迁移的影响，并利用qPCR检测小鼠脑部弓形虫内转录间隔区1（internal transcribed spacer 1，TOXO ITS1）的丰度。构建TRPM8过表达的RAW264.7细胞系（RAW264.7⁃TRPM8）及对照细胞系（RAW264.7⁃Vector），通过qPCR检测虫体基因丰度，采用酶联免疫吸附试验（enzyme⁃linked immunosorbent assay，ELISA）检测细胞因子分泌水平，采用划痕实验评估细胞迁移功能。结果　弓形虫感染小鼠腹腔巨噬细胞中TRPM8基因的转录水平（0.133 ± 0.143）显著低于未感染小鼠转录水平（1.125 ± 0.562）（t = 2.962，P < 0.05）。生存分析结果显示，弓形虫感染第7天，WT小鼠、TRPM8+/-小鼠和TRPM8-/-小鼠生存率分别为8.3%、50.0%和66.7%；3组生存曲线差异有统计学意义（[χ2] = 8.493，P = 0.014），TRPM8-/-小鼠生存时间更长。弓形虫感染后，TRPM8-/-小鼠血清中肿瘤坏死因子（tumor necrosis factor，TNF）⁃α含量为（120.456 ± 15.561） pg/mL，显著高于未感染TRPM8-/-小鼠［（57.582 ± 3.952） pg/mL］（P = 0.003）。GFP⁃RH株速殖子感染后，TRPM8-/-小鼠脑部GFP平均荧光强度为（1.810 ± 0.106） × 107 （p/s/cm2/sr）/（μW/cm2），WT小鼠为（0.831 ± 0.192） × 107 （p/s/cm2/sr）/（μW/cm2）；TRPM8-/-小鼠脑部荧光信号更强（t = 8.916，P < 0.001）。弓形虫感染后，RAW264.7⁃Vector和RAW264.7⁃TRPM8细胞内TOXO ITS1丰度的变化倍数分别为1.000 ± 0.004和1.043 ± 0.009，细胞内及上清中总TOXO ITS1丰度的变化倍数为1.000 ± 0.003和1.018 ± 0.005；感染后RAW264.7⁃TRPM8细胞内及总TOXO ITS1相对丰度均显著高于RAW264.7⁃Vector（t = 7.515、6.496，P均< 0.01）。弓形虫感染RAW264.7⁃Vector和RAW264.7⁃TRPM8细胞上清中白细胞介素（interleukin，IL）⁃10分泌水平分别为（94.104 ± 11.277） pg/mL和（145.479 ± 29.156） pg/mL；RAW264.7⁃TRPM8细胞IL⁃10分泌水平显著高于RAW264.7⁃Vector（t = 2.847，P < 0.05）。划痕实验结果显示，弓形虫感染48 h时，RAW264.7⁃Vector细胞和RAW264.7⁃TRPM8细胞的迁移率分别为（29.2 ± 11.6）%和（5.5 ± 4.7）%；72 h时，迁移率分别为（32.1 ± 10.5）%和（8.2 ± 4.1）%；RAW264.7⁃TRPM8迁移率均显著低于RAW264.7⁃Vector（P均< 0.01）。结论　弓形虫感染可下调小鼠巨噬细胞TRPM8的表达。TRPM8敲除可上调促炎细胞因子TNF⁃α的分泌，降低宿主对弓形虫的易感性，但同时会促进虫体向脑部迁移；TRPM8过表达则可抑制巨噬细胞抗虫和迁移功能。

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