Mycoplasma hyopneumoniae-derived lipid-associated membrane proteins induce apoptosis in porcine alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3 activation

Abstract

Mycoplasma hyopneumoniae is the primary etiological agent of enzootic pneumonia in swine. Lipid-associated membrane proteins (LAMP) of mycoplasma are the main pathogenicity factors in mycoplasma diseases. In this study, we investigated the effects of M. hyopneumoniae LAMP on porcine alveolar macrophage (PAM) 3D4/21 cell line. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in LAMP-treated PAM 3D4/21 cells. Moreover, LAMP significantly increased the number of TUNEL positive apoptotic cells in PAM 3D4/21 cells compared with the untreated control. In addition, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that LAMP of M. hyopneumoniae induced a time-dependent apoptosis in PAM 3D4/21 cells. Moreover, increased levels of superoxide anion production and activated caspase-3 in PAM 3D4/21 cells were observed after exposure to LAMP. Increased production of nitric oxide (NO) was also confirmed in the cell supernatants. Besides, apoptotic rates increase and caspase-3 activation were suppressed by NOS inhibitor or antioxidant. It is suggested that LAMP of M. hyopneumoniae induced apoptosis in porcine alveolar macrophage via NO production, superoxide anion production, and caspase-3 activation.

Keywords: 4′-6-diamidino-2-phenylindole; Apoptosis; Caspase-3; DAPI; IL; LAMP; Lipid-associated membrane proteins; Mycoplasma hyopneumoniae; N-acetylcysteine; N-monomethyl-l-arginine; NAC; NO; Nitric oxide; PAM; Porcine alveolar macrophage; ROS; TNF; TUNEL; interleukin; l-NMMA; lipid-associated membrane proteins; nitric oxide; porcine alveolar macrophage; reactive oxygen species; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; tumor necrosis factor.