CRISPR/Cas9-mediated knockout of SLC15A4 gene involved in the immune response in bovine rumen epithelial cells

The objective of this study was to determine the role of SLC15A4 in the response to muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs). First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL-1 MDP treatments was examined. RT-qPCR results showed that the mRNA expressions of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) were significantly increased under MDP stimulation (P < 0.001). Moreover, SLC15A4-KO cells were obtained through lentivirus packaging, transfection, screening, and cell monoclonal culture. In order to gain further insight into the potential function of SLC15A4, we utilized transcriptome data and revealed a change of genes between WT-BRECs and SLC15A4-KO. Five down-regulated pro-inflammatory genes and thirteen down-regulated chemokine genes related to the inflammatory response were identified. Meanwhile, the down-regulated genes were mostly enriched in NF-κB and MAPK signaling pathway. The results of RT-qPCR also verified these detected changes. To further determine the mechanism of how WT and SLC15A4-KO BRECs involved inflammatory responses, we investigated the inflammatory response of cells exposed to MDP. WT-BRECs and SLC15A4-KO were treated with a culture medium increase of 10 μg mL-1 MDP, in comparison to a control without MDP. Our results show that SLC15A4-KO BRECs decreased the expression of genes (IL-6, TNF-α, CXCL2, CXCL3, CXCL9, and CCL2) and proteins (p-p65 and p-p44/42) from MDP-mediated inflammatory response compared to WT-BRECs (P < 0.05). In this experiment, CRISPR-Cas9 was used to KO the di/tripeptide transporter SLC15A4, and its role was confirmed via MDP-induced inflammatory response in BRECs. This work will provide a theoretical basis for the study of the pro-inflammatory mechanism of MDP and its application in the prevention and treatment work of subacute rumen acidosis in dairy cows.