Roles Of MyD88-dependent TLR Signaling And ROS-induced Cell Apoptosis In Sheep Bronchial Epithelial Cells In Response To Mycoplasma Ovipneumoniae Infections

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a species of Mycoplasma bacteria that specifically infects sheep and goats, which is the cause of ovine infectious pleuropneumonia. In an effort to understand the pathogen-host interaction between the M. ovipneumoniae and bronchial epithelial cells, the Toll-like receptor-mediated host inflammatory responses induced by M. ovipneumoniae infections were evaluated using a primary air-liquid interface (ALI) epithelial culture model generated from bronchial epithelial cells of Ningxia Tan sheep (ovis aries). The ALI bronchial epithelial model of sheep bronchial epithelial cells showed a fully differentiated epithelium comprising distinct epithelial types, including the basal, ciliated and goblet cells. An exposure of M. ovipneumoniae to ALI bronchial epithelial cells led to increased expressions of Toll-like receptor (TLR) ligands, and components of a Myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling pathway, including the MyD88, TNF receptor-associated factor 6 (TRAF6), IL-1 receptor-associated kinases (IRAKs) and nuclear factor-kappa B (NF-κB), as well as the subsequent pro-inflammatory cytokines in the epithelial cells. Of interest, the infection of M. ovipneumoniae failed to induce the expressions of TANK-binding kinase 1 (TBK1), TRAF3 and interferon regulatory factor 3 (IRF3), key components of a MyD88-independent signaling pathway. The pathogenic mechanisms of M. ovipneumoniae by elucidating their role in modulating the inflammation, apoptosis, and relevant signaling pathways of ALI bronchial epithelial cells of M. ovipneumoniae treatment inhibited the growth of ALI bronchial epithelial cells. An up-regulation of cytokines, as well as an increased production of nitric oxide (NO) and superoxide anion was determined in the supernatant of M. ovipneumoniae treated ALI bronchial epithelial cells. Furthermore, flow cytometric analysis using dual staining with annexin-V-FITC and propidium iodide (PI) showed that M. ovipneumoniae induced a time-dependent apoptosis in ALI bronchial epithelial cells, which was blocked by NOS inhibitor or antioxidant. In addition, M. ovipneumoniae induced the phosphorylation of p38, the ratio of pro-apoptotic Bax protein to anti-apoptotic Bcl-2, activation of caspase-3 and caspase-8, and poly ADP-ribose polymerase (PARP) cleavage in ALI bronchial epithelial cells. These findings demonstrated that M. ovipneumoniae induced the production of proinflammatory cytokines, NO and reactive oxygen species (ROS), and apoptosis of ALI bronchial epithelial cells in vitro through p38 MAPK and Bax/Bcl-2 signaling pathways, along with an activation of caspases.These results suggest that the MyD88-dependent TLR pathway and ROS-induced cell apoptosis may play a crucial role in the sheep bronchial epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI bronchial epithelial cells system may be a reliable model for investigating the pathogen-host interaction of M. ovipneumoniae and bronchial epithelial cells.In conclusion, our results demonstrated that the MyD88-dependent TLR signaling pathway may play a crucial role in the sheep airway epithelial cells in response to M. ovipneumoniae infection, which also indicate that the ALI culture system may be a reliable model for investigating the pathogen-host interaction of M. ovipneumoniae and airway epithelial cells. Moreover, M. ovipneumoniae induced the production of proinflammatory cytokines, and ROS, and apoptosis in ALI bronchial epithelial cells in vitro, which were associated with increased Bax/Bcl-2 ratio, as well as activations of p38 MAPK pathway and caspases. These findings herein provide insights into the mechanism of M. ovipneumoniae-mediated pathogenicity in modulating the host immune system, and the mechanism underpinning the interaction of M. ovipneumoniae and sheep bronchial epithelial cells, which also warrants further study for development of novel agents for preventing M. ovipneumoniae infection in veterinary clinic settings.