Staphylococcus pseudintermedius induces pyroptosis of canine corneal epithelial cells by activating the ROS–NLRP3 signalling pathway

<i>Staphylococcus pseudintermedius</i> (<i>S. pseudintermedius</i>) is a common pathogen that causes canine corneal ulcers. However, the pathogenesis remained unclear. In this study, it has been demonstrated that <i>S. pseudintermedius</i> invaded canine corneal epithelial cells (CCECs) intracellularly, mediating oxidative damage and pyroptosis by promoting the accumulation of intracellular reactive oxygen species (ROS) and activating the NLRP3 inflammasome. The canine corneal stroma was infected with <i>S. pseudintermedius</i> to establish the canine corneal ulcer model in vivo. The intracellular infectious model in CCECs was established in vitro to explore the mechanism of the ROS – NLRP3 signalling pathway during the <i>S. pseudintermedius</i> infection by adding NAC or MCC950. Results showed that the expression of NLRP3 and gasdermin D (GSDMD) proteins increased significantly in the infected corneas (<i>p &lt;</i> 0.01). The intracellular infection of <i>S. pseudintermedius</i> was confirmed by transmission electron microscopy and immunofluorescent 3D imaging. Flow cytometry analysis revealed that ROS and pyroptosis rates increased in the experimental group in contrast to the control group (<i>p</i> &lt; 0.01). Furthermore, NAC or MCC950 inhibited activation of the ROS – NLRP3 signalling pathway and pyroptosis rate significantly, by suppressing pro-IL-1β, cleaved-IL-1β, pro-caspase-1, cleaved-caspase-1, NLRP3, GSDMD, GSDMD-N, and HMGB1 proteins. Thus, the research confirmed that oxidative damage and pyroptosis were involved in the process of CCECs infected with <i>S. pseudintermedius</i> intracellularly by the ROS – NLRP3 signalling pathway. The results enrich the understanding of the mechanisms of canine corneal ulcers and facilitate the development of new medicines and prevention measures.