钩端螺旋体56606v对小鼠中性粒细胞凋亡的影响

doi:10.3969/j.issn.1674-8115.2022.11.001

摘要/Abstract

摘要：

目的·探究钩端螺旋体56606v对小鼠中性粒细胞凋亡的影响及相关作用机制。方法·用钩端螺旋体56606v在感染复数（multiplicity of infection，MOI）分别为100、300、500时感染小鼠中性粒细胞0.5、6、12、24 h；以未被感染的中性粒细胞为对照。将含有4 μmol/L星形孢菌素（Staurosporine，STS）的等量细胞培养基处理的细胞作为凋亡阳性对照。采用流式细胞术检测细胞的凋亡情况；采用Western blotting技术检测钩端螺旋体56606v感染小鼠中性粒细胞后caspase-3的切割情况和核因子κB（NF-κB）通路的激活情况，并验证NF-κB抑制剂BAY 11-7082对NF-κB通路的抑制作用；通过添加NF-κB抑制剂BAY 11-7082，进一步检测NF-κB通路在钩端螺旋体56606v导致的中性粒细胞凋亡抑制中是否发挥作用；将巨噬细胞与钩端螺旋体56606v感染或未感染的中性粒细胞共孵育，通过流式细胞术检测巨噬细胞对中性粒细胞的吞噬情况。结果·流式细胞术检测结果显示，钩端螺旋体感染组与未感染对照组相比，小鼠中性粒细胞凋亡率降低；随MOI的增大，钩端螺旋体对中性粒细胞的凋亡抑制作用增强；不同MOI条件下，2组凋亡率的差异均有统计学意义（均P=0.000）。MOI=100的钩端螺旋体感染中性粒细胞0.5、6和12 h时，其凋亡率均较相应的未感染对照组降低（均P<0.05）；而使用促凋亡剂STS处理后，则增强了小鼠中性粒细胞的凋亡程度。Western blotting结果显示caspase-3的切割在钩端螺旋体56606v感染后明显减少，NF-κB磷酸化水平在感染后显著上升，添加NF-κB抑制剂BAY 11-7082能够显著抑制感染后和未感染条件下NF-κB通路的激活。在钩端螺旋体56606v感染或未感染条件下，添加NF-κB抑制剂BAY11-7082均显著提高了小鼠中性粒细胞凋亡率（均P=0.000）。钩端螺旋体56606v感染的中性粒细胞与未感染对照组相比，被巨噬细胞吞噬的比例显著降低（P=0.027）。结论·钩端螺旋体56606v通过激活NF-κB通路，抑制小鼠中性粒细胞的凋亡，进一步降低巨噬细胞对中性粒细胞的胞葬作用。

关键词: 钩端螺旋体, 中性粒细胞, 细胞凋亡, 核因子κB

Abstract:

Objective ·To investigate the effect of Leptospira interrogans 56606v on apoptosis of murine neutrophils and related mechanisms. Methods ·Murine neutrophils were infected with Leptospira interrogans 56606v at indicated multiplicity of infection (MOI was 100, 300 and 500 respectively) and time points (0.5,6,12 and 24 h), with uninfected neutrophils as control. Cells treated with an equal amount of cell culture medium containing 4 μmol/L astrosporine (STS) were used as positive control for apoptosis. The apoptosis was analyzed by using flow cytometry. The cleavage of caspase-3 and activation of nuclear factor-κB (NF-κB) pathway were detected in murine neutrophils infected with Leptospira interrogans 56606v by Western blotting, and the inhibitory effect of NF-κB inhibitor BAY 11-7082 on NF-κB pathway was also verified by Western blotting. The role of NF-κB pathway in the inhibition of neutrophil apoptosis induced by Leptospira interrogans 56606v was further explored by adding NF-κB inhibitor BAY 11-7082. Macrophages were co-incubated with Leptospira-infected or-uninfected carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled neutrophils, and flow cytometry was used to detect the phagocytosis of neutrophils by macrophages to reflect the effect of the neutrophil apoptosis inhibition induced by Leptospirainterrogans 56606v infection on efferocytosis. Results ·The results of flow cytometry showed that the apoptosis rate of neutrophils in Leptospira infection group was decreased compared with the uninfected control group. The inhibition of Leptospirainterrogans 56606v on neutrophil apoptosis was enhanced with the increase of MOI. Compared with the uninfected control group, the difference was statistically significant under different MOIS (P=0.000). The apoptosis rate was lower than that of the corresponding uninfected control group when Leptospirainterrogans 56606v with MOI=100 infected neutrophils for 0.5, 6 and 12 h (P<0.05), and the apoptosis of neutrophils was enhanced after treatment with STS. The cleavage of caspase-3 was significantly reduced after infection with Leptospira interrogans 56606v and the phosphorylation of NF-κB was significantly increased after infection demonstrated by Western blotting. The activation of NF-κB pathway in both post-infection and non-infection conditions was significantly inhibited by BAY 11-7082. The apoptosis rate of murine neutrophils infected with or without Leptospira interrogans 56606v was significantly increased by BAY 11-7082 (P=0.000). The neutrophils infected with Leptospira interrogans 56606v were significantly less phagocytosed by macrophages than those uninfected (P=0.027). Conclusion ·Leptospira interrogans 56606v inhibits the apoptosis of murine neutrophils by activating the NF-κB pathway, which further reduces efferocytosis of macrophages on neutrophils.

Key words: Leptospirainterrogans, neutrophil, apoptosis, nuclear factor-κB

中图分类号:

杜琳, 何平. 钩端螺旋体56606v对小鼠中性粒细胞凋亡的影响[J]. 上海交通大学学报（医学版）, 2022, 42(11): 1517-1523.

DU Lin, HE Ping. Effect of Leptospira interrogans 56606v on apoptosis of murine neutrophil[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(11): 1517-1523.

图/表 6

图1 流式细胞术检测感染不同浓度钩体6 h后的小鼠中性粒细胞凋亡情况Note： A. The representative results of neutrophil apoptosis detected by flow cytometry. B. The statistical results of neutrophil apoptosis. ①P=0.000, compared with the control (MOI=0) group.

Fig 1 Detection of apoptosis of murine neutrophils infected with different concentrations of Leptospira at 6 h post infection by flow cytometry

图2 流式细胞术检测感染钩体（MOI=100）不同时间的小鼠中性粒细胞凋亡情况Note： A. The representative results of neutrophil apoptosis at indicated time points detected by flow cytometry. B. The statistical results of neutrophil apoptosis. ①P=0.031, ②P=0.000, ③P=0.048, compared with the control group (Annexin+PI-) at the same time; ④P=0.000, compared with the control group (Annexin+PI+) at the same time.

Fig 2 Detection of apoptosis of murine neutrophils infected with Leptospira (MOI=100) for different periods by flow cytometry

图3 感染钩体的小鼠中性粒细胞caspase-3的激活情况

Fig 3 Activation of caspase-3 in murine neutrophils infected with Leptospira

图4 钩体感染对小鼠中性粒细胞NF-κB通路的激活情况

Fig 4 Activation of NF-κB in murine neutrophils by Leptospira infection

图 5 抑制NF-κB通路对小鼠中性粒细胞凋亡的影响Note： ①P=0.000, compared with untreated cells; ②P=0.000, compared with 56606v-treated cells without BAY 11-7082.

Fig 5 Effect of inhibition of NF-κB pathway on apoptosis of murine neutrophils

图6 巨噬细胞对中性粒细胞的胞葬作用检测Note： A. Gating strategy for assessing phagocytosis of neutrophils by macrophages by flow cytometry. B. Leptospira (MOI=100)-infected or-uninfected CFSE-labeled neutrophils were incubated with macrophages and then processed for flow cytometry after staining with anti-F4/80.

Fig 6 Efferocytosis of neutrophils by macrophages

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