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

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. [ABSTRACT FROM AUTHOR]

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