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.