Mastitis causes huge economic losses in the milk industry in worldwide. One of the popular methods for prevention and treatment of mastitis is antibiotics. However, the use of antibiotics reduces the quality of milk and sometimes causes resistant microbes. Therefore, an alternative strategy to prevent and treat mastitis is necessary for the healthy development of dairy industry. The AMP, with antimicrobial activities for a broad spectrum of bacteria, becomes valuable substitutes for antibiotics in prevention and treatment of mastitis [22].
S100A7 is one of the AMPs with a strong antimicrobial activity against E. coli. In present study, the expression sites of S100A7 in healthy and mastitis goat udder were analyzed. In both healthy and mastitis goats, S100A7 was expressed in stratified squamous epithelium of teat skin and streak canal, sebaceous gland cells, skin gland duct. This result supports the findings of previous reports in goat [21] and bovine [8]. The teat skin is consistently exposed to external environment, and streak canal may remain open for short time after milking [23]. Considering that E. coli is one of the most important environmental pathogens [24], the constitutive expression of S100A7 and other AMP, for example S100A8 [25] in teat suggested their critical roles in innate immunity in teat which as a first barrier to limit pathogens invasion.
In healthy goat mammary gland, compared with teat, S100A7 was only slightly expressed in epithelial cell of alveolus. This result is consistent with previous report that the expression level of S100A7 mRNA in alveolus is fewer than other udder regions [21]. In collapsed alveolus of mastitis goat mammary gland, the densely S100A7 immunoreactivity could be observed. As a member of AMP, predictably, the expression of S100A7 in epithelial cell of alveolus took part in local host defenses against pathogens invasion. However, in mastitis collapsed alveolus, S100A7 might not only play anti-pathogens function, but also played an important role in the fibrosis of alveolus and the destruction of blood-milk barrier. The role of S100A7 in fibrosis and epithelial barrier destruction has been reported in other pathological processes [26, 27], therefore, the relationship between S100A7 and alveolus degeneration needs to be investigated in more detail in further studies.
The expression of S100A7 is a responding to pathogens infection. The teat epithelium is considered as the main source of S100A7 in bovine [20] and goat [28]. But S100A7 concentration in milk was increased after the intramammary infusion of LPS [21], suggesting that MECs can synthesize and secrete S100A7 into the milk. In present study, goat MECs were isolated and treated with LPS. The results indicated that LPS induced the expression and secretion of S100A7 in goat MECs depended on concentration and treatment duration, providing a directed evidence to support the view that MECs is also an important source to synthesize and secrete S100A7. After LPS treatment for 3–6 h, the mRNA and secreted protein of S100A7 were significantly increased. These results suggested that the transcription, translation and secretion of S100A7 were a rapid response to infection in goat mammary gland. This rapid response played an important role in the clearance of early intramammary infection. On the other hand, these results also help to explain the previous reports in which after LPS intramammary infusion, the increased concentration of S100A7 was 48 h earlier than S100A8 [21, 25]. When bacteria invasion or LPS infusion in mammary alveolus, they firstly stimulated mammary epithelial cells to secrete S100A7, then leukocytes infiltrated mammary alveolus and secreted S100A8 into milk. Therefore, the concentration of S100A7 in milk rises earlier than S100A8. However, LPS treatment alone could not induce the continuous secretion of S100A7 in goat MECs. It is reported that sustained secretion of S100A7 is dependent on the downregulation of Caspase-8 [29].
Although LPS was reported to induce the expression and secretion of S100A7 in human breast cancer [30], its mechanism is still unclear. LPS is a cell wall component of Gram-negative bacteria, its main receptor is Toll-like receptor 4(TLR4) [31, 32]. In bovine, TLR4 was expressed in MECs [33] and its expression was up-regulated after stimulation with heat-inactivated E. coli [34]. In goat MECs, LPS treatment could increase the mRNAs level of TLR4 and MYDD88, this result is consistent with previous report [35]. Moreover, LPS treatment promoted the phosphorylation of p-65 and transfer location from cytoplasm into nuclear. If the TLR4 and NF-κB signaling pathways were inhibited by special inhibitors, the phosphorylation of p-65 was suppressed, and then the expression and secretion of S100A7 induced by LPS treatment were also inhibited. These results indicated that LPS induced the expression and secretion of S100A7 in cultured goat MECs depending on TLR4 and NF-κB signaling pathway.