Highly virulent Aerococcus viridans activates the RIPK3 signaling pathway, inducing necroptosis and inflammation in bovine mammary epithelial cells and murine mammary tissue.

Abstract

Aerococcus viridans (A. viridans), a conditionally pathogenic bacterium widely present in the environment, has been increasingly detected in bovine mastitis; however, its pathogenic mechanisms remain unclear. Necroptosis is a form of programmed cell death associated with bovine mastitis. The objective was to elucidate molecular mechanisms underlying induction of acute infection by virulent A. viridans, in two infection models, in vitro (bovine mammary epithelial cells) and in vivo (murine mammary gland). Compared to an avirulent strain, virulent A. viridans induced significantly higher levels of late apoptotic/necrotic bMECs, accompanied by increased LDH release and markedly elevated secretions of IL-1β, TNF-α and IL-6. Furthermore, the highly virulent strain activated RIPK3 and phosphorylation of MLKL, whereas the avirulent strain failed to activate this pathway. In addition, the highly virulent strain also induced severe mammary tissue damage and inflammatory cell infiltration in vivo, with extensive acinar destruction and high expression of p-RIPK3 and p-MLKL in mammary tissues, consistent with in vitro findings. In both infection models, a specific RIPK3 inhibitor (GSK872) markedly alleviated inflammation and tissue injury, reduced cytokine release and downregulated RIPK3-MLKL signaling induced by the highly virulent strain. We concluded that the molecular mechanism by which a highly virulent strain of A. viridans induces mastitis was activation of an RIPK3-dependent necroptosis pathway, providing a theoretical basis for development of novel therapeutic strategies.