Heat-Killed Ligilactobacillus salivarius FFIG56 Modulates Innate Immune Response in Bovine Mammary Epithelial Cells

Immunomodulatory lactic acid bacteria (immunobiotics) have been proposed to prevent bovine mastitis through the enhancement of host immune responses. Furthermore, accumulating evidence suggests that non-viable immunobiotics (postimmunobiotics) can be as effective as viable bacteria in modulating immunity in the bovine host. However, the molecular mechanisms underlying the immunomodulatory effects of heat-inactivated lactic acid bacteria in bovine mammary epithelial cells remain incompletely understood. In the present study, we evaluated the effects of heat-killed Ligilactobacillus salivarius FFIG56 (hk-FFIG56) on the Toll-like receptor (TLR)-4-mediated innate immune response in bovine mammary epithelial cells (BMECs) in vitro. BMECs were pre-stimulated with hk-FFIG56 and subsequently challenged with Escherichia coli-derived lipopolysaccharide (LPS). Transcriptomic analyses revealed that, compared with cells stimulated with LPS alone, hk-FFIG56-pretreated BMECs exhibited 743 upregulated and 881 downregulated genes, indicating substantial changes in the LPS-induced transcriptional response. Among the inflammatory mediators evaluated, hk-FFIG56 pretreatment significantly reduced LPS-induced interleukin-6 (IL-6) and interleukin-8 (IL-8) expression at 12 h, as well as CCL2 expression at 6 and 12 h, compared with cells challenged with LPS alone at the corresponding time points. Differential regulation of the negative regulators of TLR signaling, IRAK-M, Tollip, and Mitogen-activated protein kinase phosphatase 1 (MKP-1), was also detected in BMECs pretreated with hk-FFIG56. In addition, hk-FFIG56 pretreatment prevented LPS-induced reduction in IκBα protein levels, whereas no significant changes were detected in the p-ERK/ERK ratio at the evaluated time points. Furthermore, hk-FFIG56 pretreatment attenuated the LPS-induced reduction in CSN3 (κ-casein) mRNA expression in BMECs. Overall, these results suggest that hk-FFIG56 modulates LPS-induced inflammatory responses and CSN3 mRNA expression in BMECs, supporting its potential as a postimmunobiotic candidate for further investigation in the context of bovine mastitis prevention.