Abstract
Bovine mastitis is a common infectious disease which causes huge economic losses in dairy cattle. Bovine mammary epithelial cell (BMEC) damage usually directly causes the decrease of milk production, which is one of the most important causes of economic loss. NETs, novel effector mechanisms, are reported to exacerbate the pathogenesis of several inflammatory diseases. NETs formation has also been observed in the milk and mammary glands of sheep. However, the effects and detailed mechanisms of NETs on BMEC damage remain unclear. Thus, we aim to examine the effects of NETs on BMECs in vitro, and further to investigate the detail mechanism. In this study, the cytotoxicity of NETs on BMECs was determined using lactic dehydrogenase (LDH) levels in culture supernatants. Histone-induced BMEC damage was examined by flow cytometry and immunofluorescence analysis. The activities of caspase 1, caspase 3, caspase 11, and NLRP3 was detected using western blotting and immunohistochemical analysis. The results showed that NETs and their component histone significantly increased cytotoxicity to BMECs, suggesting the critical role of NETs, and their component histone in BMEC damage. In addition, histone could also induce necrosis, pyroptosis, and apoptosis of BMECs, and the mechanisms by which histone leads to BMEC damage occurred via activating caspase 1, caspase 3, and NLRP3. Altogether, NETs formation regulates inflammation and BMEC damage in mastitis. Inhibiting excess NETs formation may be useful to ameliorate mammary gland damage associated with mastitis.
Highlights
Mastitis is one of the most prevalent and costly diseases in dairy herds
To investigate the effects of neutrophil extracellular traps (NETs) on Bovine mammary epithelial cell (BMEC), BMECs were incubated with NETs, NETs treated with DNase I or DNase I for 16 h
It was found that NET (484 ng/mL) stimulation increased Lactate dehydrogenase (LDH) release, but NETs treated with DNase I significantly decrease the cytotoxicity of BMECs (Figure 2A, n = 5)
Summary
Mastitis is one of the most prevalent and costly diseases in dairy herds. It causes enormous economic losses due to reduced milk production and quality, and treatment costs [1, 2]. A large number of studies have shown that many microorganisms can induce the occurrence of mastitis [3, 4]. It is the result of attempts of the host to eliminate invading microorganisms. Neutrophils are most abundant leukocyte cells in the innate immune system of the host [6] They have the ability to eliminate invading pathogens through phagocytosis and death [7]. The formation of NETs has been observed in milk and in the mammary alveoli of mastitic sheep and cows [9, 10], but the effects of NETs on mammary epithelial cells has not being reported
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