An in vitro study of ApxI from Actinobacillus pleuropneumoniae serotype 10 and induction of NLRP3 inflammasome-dependent cell death.

IF 1.3 Q2 VETERINARY SCIENCES Veterinary Record Open Pub Date : 2021-10-04 eCollection Date: 2021-12-01 DOI:10.1002/vro2.20
Eduardo Hernandez-Cuellar, Alma Lilián Guerrero-Barrera, Francisco Javier Avelar-Gonzalez, Juan Manuel Díaz, Jesús Chávez-Reyes, Alfredo Salazar de Santiago
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Abstract

Background: Actinobacillus pleuropneumoniae (AP) is the causative agent of porcine pleuropneumonia. Apx exotoxins are the most important virulence factors associated with the induction of lesions. ApxI is highly cytotoxic on a wide range of cells. Besides the induction of necrosis and apoptosis of ApxI on porcine alveolar macrophages (PAMs), its role in pyroptosis, a caspase-1-dependent form of cell death, has not been reported. The aim of this study was to analyse if NLRP3 inflammasome participates in cell death induced by ApxI.

Methods: PAMs, the porcine alveolar macrophage cell line 3D4/21 and a porcine aortic endothelial cell line were used in this study. We used Z-VAD-FMK and Ac-YVAD-cmk to inhibit caspase-1. Glyburide and MCC950 were used to inhibit the NLRP3 inflammasome. A lactate dehydrogenase release assay was used to measure the percentage of cell death. Caspase-1 expression was analysed by immunofluorescence. End-point RT-PCR was used to analyse the expression of NLRP3 mRNA.

Results: Rapid cell death in PAMs, 3D4/21 cells and the endothelial cell line were induced by ApxI. This cell death decreased by using caspase-1 and NLRP3 inflammasome inhibitors and by blocking the K+ efflux. Expression of NLRP3 mRNA was induced by ApxI in alveolar macrophages while it was constitutive in the endothelial cell line. Detection of caspase-1 in alveolar macrophages was higher after ApxI treatment and it was blocked by MCC950 or heat inactivation.

Conclusions: To the best of the authors' knowledge, we have described for the first time in vitro induction of ApxI associated pyroptosis in alveolar macrophages and endothelial cells, a rapid cell death that depends on the activation of caspase-1 via the NLRP3 inflammasome.

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来自胸膜肺炎放线杆菌血清型 10 的 ApxI 和诱导 NLRP3 炎症体依赖性细胞死亡的体外研究。
背景:胸膜肺炎放线杆菌(AP)是猪胸膜肺炎的病原体。Apx外毒素是诱发病变的最重要毒力因子。ApxI 对多种细胞具有高度细胞毒性。ApxI除了能诱导猪肺泡巨噬细胞(PAMs)坏死和凋亡外,其在热凋亡(一种依赖于caspase-1的细胞死亡形式)中的作用尚未见报道。本研究旨在分析 NLRP3 炎症小体是否参与了 ApxI 诱导的细胞死亡:本研究使用了 PAMs、猪肺泡巨噬细胞系 3D4/21 和猪主动脉内皮细胞系。我们使用 Z-VAD-FMK 和 Ac-YVAD-cmk 来抑制 caspase-1。格列本脲和 MCC950 用于抑制 NLRP3 炎症小体。乳酸脱氢酶释放试验用于测量细胞死亡的百分比。通过免疫荧光分析 Caspase-1 的表达。终点 RT-PCR 用于分析 NLRP3 mRNA 的表达:结果:ApxI诱导了PAMs、3D4/21细胞和内皮细胞系的快速细胞死亡。使用caspase-1和NLRP3炎性体抑制剂以及阻断K+外流可减少细胞死亡。在肺泡巨噬细胞中,ApxI 诱导 NLRP3 mRNA 的表达,而在内皮细胞系中则是组成型的。ApxI处理后,肺泡巨噬细胞中caspase-1的检测率较高,MCC950或热灭活可阻断caspase-1的检测:据作者所知,我们首次描述了体外诱导肺泡巨噬细胞和内皮细胞中与 ApxI 相关的热凋亡,这是一种依赖于通过 NLRP3 炎症小体激活 caspase-1 的快速细胞死亡。
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来源期刊
Veterinary Record Open
Veterinary Record Open VETERINARY SCIENCES-
CiteScore
3.00
自引率
0.00%
发文量
25
审稿时长
19 weeks
期刊介绍: Veterinary Record Open is a journal dedicated to publishing specialist veterinary research across a range of topic areas including those of a more niche and specialist nature to that considered in the weekly Vet Record. Research from all disciplines of veterinary interest will be considered. It is an Open Access journal of the British Veterinary Association.
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