NF-kappaB is involved exclusively under inflammatory conditions in the regulation of cathelicidin 3 expression in bovine mammary glands through phosphorylation of p65.

Cathelicidin 3 (CATHL3), a member of the cathelicidin family, exhibits potent resistance against a spectrum of microorganisms. Despite its importance, the expression pattern and regulatory mechanisms of CATHL3 in inflamed dairy mammary glands remain elusive. For this purpose, healthy (n†=†3) and inflamed (n†=†3) mammary gland tissues were subjected to high-throughput transcriptome sequencing and RT-qPCR analysis. Our findings revealed significant differential expression of the CATHL3 gene and significantly higher nuclear factor kappa B (NF-κB) mRNA expression in inflamed tissues compared to healthy controls. Milk somatic cells from dairy cows with varying blood glucose concentrations were extracted and analyzed. In healthy cows, a decrease in blood glucose concentration was significantly associated with an increase in CATHL3 mRNA expression in milk somatic cells, while NF-κB mRNA expression significantly decreased. Conversely, in milk somatic cells from cows with subclinical and clinical mastitis, a decrease in blood glucose concentration still led to a significant increase in CATHL3 mRNA expression, but NF-κB mRNA expression paradoxically increased, contrasting with the healthy cow group. The in vitro experiments mirrored the findings of the in vivo experiments mentioned above by culturing dairy cow mammary epithelial cells under varying glucose concentrations, with or without lipopolysaccharides (LPS). Additionally, the effects of NF-κB inhibitor (BAY 11-7082, 10 μmol/L) or NF-κB activator (NF-κB activator-1, 1 μmol/L) on cultured dairy cow mammary epithelial cells were assessed. While the addition of NF-κB activator alone upregulated the mRNA expression level of NF-κB, it did not alter the p-p65/p65 ratio and CATHL3 mRNA expression compared to the blank control group. Notably, the co-addition of LPS and the NF-κB inhibitor significantly attenuated the stimulatory effects of LPS on NF-κB mRNA expression and the p-p65/p65 ratio and also weakened the stimulatory effect of LPS on CATHL3 mRNA expression. Our results suggest that mastitis stimulates CATHL3 gene expression in mammary gland with the NF-κB signaling pathway playing a regulatory role through the phosphorylation of p65 exclusively under inflammatory conditions. Additionally, decreased blood glucose concentration promotes CATHL3 mRNA expression. These findings provide novel insights into the molecular mechanisms of bovine mastitis and potential targets for therapeutic intervention.