问号钩端螺旋体对THP-1和J774A．1细胞NLRP3炎症小体活化的影响

Effects of Leptospira interrogans infection on the activation of NLRP3 in THP-1 and J774A. 1 cells

目的了解问号钩端螺旋体（钩体）对THP-1和J774A.1细胞NLRP3炎症小体活化的影响，为钩体感染不同宿主后引起不同炎症性反应的科学解释提供依据。方法分别采用real-time RT-PCR和流式细胞术检测问号钩体56601株感染的THP-1及J774A.1细胞NLRP3 mRNA及蛋白表达水平，采用ELISA结合NLRP3阻断试验检测细胞IL-1β、IL-18和IL-33表达水平。结果问号钩体感染1 h、2 h、4 h、12 h和24 h后，THP-1细胞NLRP3 mRNA水平分别为未感染细胞的4.05、0.34、0.33、0.06和1.66倍，J774A.1细胞分别为未感染细胞的12.98、16.19、10.68、5.8和0.57倍（P〈0.05）；THP-1细胞NLRP3蛋白表达率分别从感染前的9.26%上升至94.01%、89.24%、31.80%、19.74%和11.28%（P〈0.05），J774A.1细胞分别从感染前的18.71%上升至58.78%、43.64%、36.42%、76.46%和85.21% （P〈0.05）；THP-1细胞IL-1β分别上调至73.07 pg/ml、939.24 pg/ml、939.24 pg/ml、843.22 pg/ml和851.06 pg/ml（P〈0.05），J774A.1细胞IL-1β在感染后12 h出现上升，感染12 h和24 h的浓度分别为191.17 pg/ml和254.4 pg/ml（P〈0.05），THP-1细胞IL-18浓度分别为913.89 pg/ml、808.19 pg/ml、483.54 pg/ml、204.19 pg/ml和189.09 pg/ml（P〈0.05），J774A.1细胞IL-18在感染后24 h出现上升，浓度为113.37 pg/ml，THP-1细胞IL-33水平无明显变化（P〉0.05），而J774A.1细胞感染后IL-33水平在12 h和24 h后略有上升（P〈0.05）。钩体感染引起THP-1和J774A.1细胞IL-1β、IL-18和IL-33升高均能被NLRP3特异性阻断剂有效阻断（P〈0.05）。结论人和小鼠巨噬细胞感染钩体后NLRP3炎症小体均发生活化并参与介导分泌炎性细胞因子，细胞因子分泌规律存在明显差异，人巨噬细胞感染钩体后IL-1β和IL-18的分泌早于小鼠巨噬细胞，且分泌水平较小鼠巨噬细胞更高。

Objective To analyze the effects of Leptospira interrogans （L.interrogans） infection on the activation of NLRP3 in THP-1 and J774A.1 cells and to further understand the mechanism of inflammation caused by L. interrogans in different hosts.Methods Human mononuclear macrophage cell line （THP-1） and murine mononuclear macrophage cell line （J774A.1） were infected with L. interrogans strain 56601. The expression of NLRP3 at mRNA and protein levels were measured by using real-time RT-PCR and flow cytometry analysis, respectively. The NLRP3-mediated secretion of IL-1β, IL-18 and IL-33 was detected by ELISA combined with the NLRP3 inhibitory test.Results Compared with the normal cells, the expression of NLRP3 at mRNA level in L. interrogans-infected THP-1 cells was respectively increased by 4.05, 0.34, 0.33, 0.06 and 1.66 times at the time points of 1 h, 2 h, 4 h, 12 h and 24 h after infection （P〈0.05）, while that in L. interrogans-infected J774A.1 cells was respectively increased by 12.98, 16.19, 10.68, 5.8 and 0.57 times （P〈0.05）. The expression rates of NLRP3 protein in THP-1 and J774A.1 cells respectively increased from 9.26% to 94.01%, 89.24%, 31.80%, 19.74%, 11.28% and from 18.71% to 58.78%, 43.64%, 36.42%, 76.46%, 85.21% at the time points of 1 h, 2 h, 4 h, 12 h and 24 h after L. interrogans infection （P〈0.05）. The level of IL-1β in L. interrogans-infected THP-1 cells was 73.07 pg/ml, 939.24 pg/ml, 939.24 pg/ml, 843.22 pg/ml and 851.06 pg/ml at the time points of 1 h, 2 h, 4 h, 12 h and 24 h, respectively （P〈0.05）, while the level of IL-1β in L. interrogans-infected J774A.1 cells began to rise at the time point of 12 h from 191.17 pg/ml to 254.4 pg/mL at the time point of 24 h （P〈0.05）. The level of IL-18 in L. interrogans-infected THP-1 cells was 913.89 pg/ml, 808.19 pg/ml, 483.54 pg/ml, 204.19 pg/ml and 189.09 pg/ml at the time points of 1 h, 2 h, 4 h, 12 h and 24 h, respectively （P〈0.05）, while the level of IL-18 in L. interrogans-infected J774A.1 cells increased at the time point of 24 h, which was 113.37 pg/ml （P〈0.05）. A slight increase in the level of IL-33 was detected in L. interrogans-infected J774A.1 cells at the time points of 12 h and 24 h to 201.14 pg/ml and 155.68 pg/ml, respectively （P〈0.05）, but no significant change was detected in L. interrogans-infected THP-1 cells （P〉0.05）. Results of the inhibitory test showed that the up-regulation of IL-1β , IL-18 and IL-33 in THP-1 and J774A.1 cells were effectively inhibited by the specific inhibitor of NLRP3.Conclusion NLRP3 inflammasome was activated and involved in the production of specific inflammatory cytokines IL-1β and IL-18 in both THP-1 and J774A.1 cells after L. interrogans infection, but the inflammatory cytokines induced by L. interrogans infection varied in different cells. L. interrogans induced earlier and higher level of IL-1β and IL-18 production in human macrophages than in murine macrophages.