Hernandez-Cuellar Eduardo, Guerrero-Barrera Alma Lilián, Avelar-Gonzalez Francisco Javier, Díaz Juan Manuel, Chávez-Reyes Jesús, Salazar de Santiago Alfredo
Laboratorio de Biología Celular y Tisular Departamento de Morfología Universidad Autónoma de Aguascalientes (UAA) Aguascalientes Mexico.
Laboratorio de Ciencias Ambientales Departamento de Fisiología y Farmacología Universidad Autónoma de Aguascalientes (UAA) Aguascalientes Mexico.
Vet Rec Open. 2021 Oct 4;8(1):e20. doi: 10.1002/vro2.20. eCollection 2021 Dec.
(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.
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.
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.
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.
胸膜肺炎放线杆菌（AP）是猪胸膜肺炎的病原体。Apx外毒素是与病变诱导相关的最重要的毒力因子。ApxI对多种细胞具有高度细胞毒性。除了诱导ApxI对猪肺泡巨噬细胞（PAM）的坏死和凋亡外，其在焦亡（一种半胱天冬酶-1依赖性细胞死亡形式）中的作用尚未见报道。本研究的目的是分析NLRP3炎性小体是否参与ApxI诱导的细胞死亡。
本研究使用了PAM、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抑制半胱天冬酶-1。使用格列本脲和MCC950抑制NLRP3炎性小体。采用乳酸脱氢酶释放试验测定细胞死亡百分比。通过免疫荧光分析半胱天冬酶-1的表达。采用终点RT-PCR分析NLRP3 mRNA的表达。
ApxI诱导PAM、3D4/21细胞和内皮细胞系快速细胞死亡。使用半胱天冬酶-1和NLRP3炎性小体抑制剂以及阻断钾外流可减少这种细胞死亡。ApxI在肺泡巨噬细胞中诱导NLRP3 mRNA表达，而在内皮细胞系中其表达是组成性的。ApxI处理后肺泡巨噬细胞中半胱天冬酶-1的检测水平更高，并且被MCC950或热灭活所阻断。
据作者所知，我们首次在体外描述了ApxI在肺泡巨噬细胞和内皮细胞中诱导的焦亡，这是一种依赖于通过NLRP3炎性小体激活半胱天冬酶-1的快速细胞死亡。
