Involvement of NF-κB in regulation of Actinobacillus pleuropneumoniae exotoxin ApxI-induced proinflammatory cytokine production in porcine alveolar macrophages

Actinobacillus pleuropneumoniae is a crucial respiratory pathogen that causes fibrinous, hemorrhagic, necrotizing pleuropneumonia in pigs. A. pleuropneumoniae exotoxins (ApxI to IV) are the major virulence factors contributing to A. pleuropneumoniae pathogenesis. Previously, we-demonstrated that ApxT induces the expression of proinflammatory cytokines in porcine alveolar macrophages (PAMs) via the mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK). Nonetheless, the role of nuclear factor (NF)-kappa B-a transcription factor widely implicated in immune and inflammatory responses in ApxI-elicited cytokine production has yet to be defined. In the present study, we examined the involvement of NF-kappa B in ApxI-elicited production of interleukin (IL)-1 beta, IL-8, and tumor necrosis factor (TNF)-alpha in PAMs and investigated the correlation between NF-kappa B and MAPK (p38 and JNK) pathways in this event. The results of Western blot analysis, confocal microscopy, and a DNA binding activity assay revealed that the classical NF-kappa B pathway was activated by ApxI, as evidenced by the decreased levels of IKB and subsequent NF-kappa B translocation and activation in ApxI-stimulated PAMs. Moreover, the blocking of ApxI-induced NF-kappa B activation significantly attenuated the levels of mRNA and protein secretion of IL-1 beta, IL-8, and TNF-alpha in PAMs. Notably, the attenuation of JNK activation by a specific inhibitor (SP600125) reduced ApxI-induced NF-kappa B activation, whereas a p38 blocker (SB203580) had no effect on the NF-kappa B pathway. Further examination revealed that the level of phosphorylation at serine 536 on the NF-kappa B p65 subunit was dependent on JNK activity. Collectively, this study, for the first time, demonstrates a pivotal role of NF-kappa B in ApxI-induced IL-beta, IL-8, and TNF-alpha production; JNK, but not p38, may positively affect the activation of the classical NF-kappa B pathway. (C) 2016 Elsevier B.V. All rights reserved.