Regulation of Inflammatory Responses of Cow Mammary Epithelial Cells through MAPK Signaling Pathways of IL-17A Cytokines

Cow mastitis is the most common and important economic problem among ruminant cows in the world, with bacterial infection constituting one of the main causes of the disease. Lipopolysaccharides (LPS) are one prototype Pathogen-Associated Molecular Pattern (PAMP) of Gram-negative bacteria and an important risk factor of cow mastitis caused by Gram-negative bacteria. Interleukin-17A (IL-17A), which is a signature cytokine of auxiliary T-cell 17, can be secreted by immune and non-immune cells. It has been proved that IL-17A plays a key role in the resistance of host animals against various bacterial infections. However, its role in the resistance of host animals against mastitis is not clear. The aim of this study is to explore the mechanism of IL-17A infection in the development of bacterial mastitis in dairy cows. In this study, RT-qPCR and ELISA were used to measure the promoting effect of IL-17A on the generation of pro-inflammatory cytokines (TNF-alpha, IL-1 beta, and IL-6) and chemokine (IL-8). In addition, Western blot (WB) was applied to measure the influences of IL-17A on the inflammation-related ERK and p38 proteins in the MAPK pathways. The results show that under the stimulation of LPS on cow mammary epithelial cells (CMECs), cytokines IL-1 beta, IL-6, IL-8, TNF-alpha, and IL-17A will exhibit significantly increased expression levels (p < 0.05). With inhibited endogenous expression of IL-17A, cytokines IL-1 beta, IL-6, IL-8, and TNF-alpha will present reduced genetic expression (p < 0.01), with reduced phosphorylation levels of ERK and p38 proteins in the MAPK signaling pathways (p < 0.001). Upon the exogenous addition of the IL-17A cytokine, the genetic expression of cytokines IL-1 beta, IL-6, IL-8, and TNF-alpha will increase (p < 0.05), with increased phosphorylation levels of the ERK and p38 proteins in the MAPK signaling pathways (p < 0.001). These results indicate that under the stimulation of CMECs with LPS, IL-17A can be expressed together with relevant inflammatory cytokines. Meanwhile, the inflammatory responses of mammary epithelial cells are directly proportional to the expression levels of IL-17A inhibited alone or exogenously added. In summary, this study shows that IL-17A could be used as an important indicator for assessing the bacterial infections of mammary glands, indicating that IL-17A could be regarded as one potential therapeutic target for mastitis.