抑制 mTORC1/STAT3 轴的致病作用促进金黄色葡萄球菌诱导的人巨噬细胞细胞焦亡。

Pathogenic effects of inhibition of mTORC1/STAT3 axis facilitates Staphylococcus aureus-induced pyroptosis in human macrophages.

机构信息

State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, 010070, China.

School of Life Sciences, Jining Normal University, Jining, 012000, China.

出版信息

Cell Commun Signal. 2020 Nov 30;18(1):187. doi: 10.1186/s12964-020-00677-9.

DOI:10.1186/s12964-020-00677-9

PMID:33256738

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7706204/

Abstract

BACKGROUND

Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established.

METHODS

Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM.

RESULTS

S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1β and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages.

CONCLUSIONS

S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages. Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.

摘要

背景

细胞焦亡是一种新发现的半胱氨酸蛋白酶-1（Caspase-1）介导的细胞程序性死亡方式，在受到微生物、脂多糖或某些化疗药物刺激时，会发生细胞焦亡。然而，细胞焦亡的调控机制尚不完全清楚。

方法

通过染色和激光共聚焦显微镜及透射电镜检测细胞内细菌；通过活/死细胞成像检测巨噬细胞死亡；通过 Western blot 和免疫组化染色检测蛋白变化；IFA 鉴定典型的细胞焦亡出芽囊泡和 STAT3 核定位；SEM 观察细胞焦亡的形态特征；ELISA 检测炎症细胞因子水平；LSCM 拍摄巨噬细胞中细胞焦亡的过程。

结果

金黄色葡萄球菌（S. aureus）被人巨噬细胞内化。细胞内 S. aureus 诱导巨噬细胞死亡。S. aureus 入侵增加了 NLRP3、Caspase1（Casp-1 p20）和 GSDMD-NT 的表达，GSDMD-NT 孔结构的积累，以及巨噬细胞中白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的释放。S. aureus 吞噬阻断可减轻 S. aureus 诱导的巨噬细胞焦亡。雷帕霉素或特异性抑制 mTORC1 或 STAT3 的 Stattic 均可促进 S. aureus 感染引起的细胞焦亡。抑制 mTORC1 或 STAT3 诱导细胞焦亡。mTORC1 通过调节 STAT3 的核定位来调节细胞焦亡基因的表达。mTORC1/STAT3 轴可能在巨噬细胞中对细胞焦亡起调节作用。