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  • br Materials and methods br Acknowledgement


    Materials and methods
    Acknowledgement This work was supported by the Beijing Natural Science Foundation (Grant no. 6151001), and the National Natural Science Foundation of China (Grant no. 31572516).
    Introduction Staphylococcus aureus is an important microorganism responsible for both clinical and subclinical mastitis in lactating cows [1]. Intramammary infection of dairy cows with S. aureus is often subclinical due to the pathogen's ability to evade the innate defense mechanisms, which can lead to persistent infection, fibrosis of the mammary gland, mastoscirrhus, and even culling of the animal. Bovine mammary epithelial zj to j (BMECs) are part of the functional unit of the udder. And BMECs are responsible for synthesis of milk components offering a perfect nutritional and immunological supply to the offspring [2,3]. BMECs in culture are able to sense bacteria or bacterial products, and to react by up-regulating several sets of genes involved in interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) and other proinflammatory cytokines [4,5]. And mammary epithelial cells play an important role in the initial recognition of the microorganisms and later fibrosis by expression of cytokines and acute phase proteins that regulate some response [6,7]. Detecting the presence of a pathogen is the first step in the host response to bacterial infection. Mammary gland cells are equipped with a battery of receptors sensing the presence of pathogens. For example, Gram-positive bacteria are recognized by toll-like receptor 2 (TLR2), while toll-like receptor 4 (TLR4) binds to the lipopolysaccharides of Gram-negative bacteria [[8], [9], [10]]. Upon ligand binding, the activation of several signaling molecules leads to the release of transcription factors, such as nuclear factor-kappa B (NF-κB) and activator protein-1 (AP-1), which play crucial roles in immune defense, cell differentiation, and apoptosis [11,12]. NF-κB mediates the expression of cytokines, chemokines, and growth factors. The AP-1 family plays a key role in the synthesis of effector molecules and cytokines during innate immune responses, and its activity is controlled by various signal transduction pathways, including the TLR pathway [13]. During the early phase of S. aureus infection of the udder, the bacterium activates cells and induces secretion of inflammatory cytokines and chemokines. Subsequent phases require cell repair; and it is during this repair phase that abnormal or uncontrolled repair mechanisms can result in fibrosis, a pathological process in which excessive deposition of extracellular matrix (ECM) occurs. Transforming growth factor-beta 1 (TGF-β1) and basic fibroblast growth factor (bFGF) are important growth factors that promote the deposition of ECM. TGF-β1 is a pleiotropic cytokine that regulates cell growth and differentiation [14]. Casey et al. observed that TGF-β1 inhibited bovine epithelial cell proliferation and stimulated the ECM synthesis surrounding ducts [15]. In the mammary gland, TGF-β1 regulates ductal growth and alveolar development via its inhibitory effect on epithelial cells growth and its stimulatory effect on fibroblasts and other stromal cells [16]. The bFGF is an effective stimulator of breast epithelial cells differentiation and EMC synthesis [17]. A better knowledge of the reactions involved in BMECs infection could eventually lead to novel therapeutic approaches to combat fibrosis during S. aureus mastitis and the genetic selection of animals with improved udder health. With this in mind, an in vitro model was used in this study to analyze the mechanism of TGF-β1 and bFGF production by BMECs upon S. aureus stimulation. The molecular mechanisms, including stimulation of TLR2, TLR4, AP-1, and NF-κB transcriptional activity by S. aureus to increase TGF-β1 and bFGF production in BMECs, were investigated.
    Materials and methods
    Discussion S. aureus is known to induce mammary gland mastitis. Bovine mammary fibrosis is the pathologic regression of bovine mastitis, which occurs as a result of excessive deposition of ECM. Many of the pro-inflammatory cytokines also induce the expression of growth factors, which directly stimulate the epithelial-mesenchymal transitions, their differentiation into myofibroblasts, and the production of ECM then lead to fibrosis [22]. In our studies, BMECs were stimulated with different concentrations of heat-inactivated S. aureus to determine the expression of TGF-β1 and bFGF through the TLR-NF-kB andTLR-AP-1 signaling pathways. S. aureus infection results in the up-regulation of various pro-inflammatory cytokines and growth factors in bovine mammary epithelial cells (BMECs) and bovine udders [4,23]. Akers and Nickerson have demonstrated that the percentages of intralobular stroma were significantly higher in udders with S. aureus mastitis than in uninfected ones [24]. In this study, the expression of TLR2 and TLR4, nuclear transcription factors NF-κB and AP-1, TGF-β1 and bFGF by S. aureus-induced BMECs was measured in order to improve our knowledge of bovine mammary fibrosis during S. aureus stimulation. In order to provide standardized and reproducible conditions, we challenged the BMECs with a heat-inactivated S. aureus strain previously used in experimental infections of cattle that had resulted in mastitis.