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    • TLRs play a key role in pathogen recognition

    2024-02-21

    TLRs play a key role in pathogen recognition and early response. In our study, we showed an increase of TLR2 mRNA and protein expression after BMECs were stimulated by S. aureus, as seen for BMECs in a previous study [25]. Although most studies describe TLR4 as a primary receptor for Gram-negative bacteria [26], our measurements indicated that TLR4 is also upregulated in BMECs by Gram-positive S. aureus. Similar findings have been reported by Yang et al., although their study was carried out in mammary epithelial RGDfK isolated from naturally infected udders. Goldammer et al. experimentally induced Staphylococcus aureus mastitis in bovine mammary gland and observed a coordinated up-regulation of the mRNA expression of both TLR2 and TLR4 [27]. Yang et al. also found comparable signal intensities of TLR2 and TLR4 in bovine mammary parenchymal cells in the presence of S. aureus [25]. Likewise, in human conjunctival cells, S. aureus was recognized by TLR2 and induced a variety of transcription factors, such as NF-κB and AP-1, which then resulted in cytokine secretion [28,29]. An upregulation of NF-κB mRNA expression was shown in BMECs by Griesbeck-Zilch et al. following stimulation with heat-inactivated S. aureus, indicating the involvement of the NF-κB pathway in signaling transduction in mastitis [4]. Meanwhile, Kim et al. demonstrated that heat-inactivated S. aureus induced NF-κB and AP-1 mRNA expression in BMECs [30]. In the present study, we also demonstrated that heat-inactivated S. aureus could upregulate the production of p-NF-κB-p65 and p-c-jun in BMECs. The most striking finding of this study was the upregulation of TGF-β1 and bFGF expression in BMECs following heat-inactivated S. aureus stimulation via activation of AP-1 and NF-κB. During the tissue repair process, several growth factors are involved, such as TGF-β1 and bFGF, which play an important role in cell proliferation and differentiation as they are potent mitogens for epithelial cells [31,32]. TGF-β regulates ductal growth and alveolar development via its inhibitory effect on epithelial cell growth and its stimulatory effect on fibroblasts and other stromal cells [16]. Musters et al. have demonstrated that TGF-β1 selectively acts on the stromal compartment of the bovine mammary gland by increasing cell proliferation and gene expression of the ECM protein fibronectin [33]. For TGF-β1, our measurements showed a very strong increase in mRNA expression and protein secretion in BMECs. Similarly, a previous study has reported S. aureus is able to evoke increased production of TGF-β1 during the course of intramammary infection [23]. The highest production of TGF-β1 was reached at 12 h following S. aureus stimulation. The induction of TGF-β1 expression activated by S. aureus was significantly decreased in treated cells to which the NF-κB inhibitor NAC and the AP-1 inhibitor Sp-600125 were added. The observation that NF-κB and AP-1 mediate TGF-β1 gene expression is in accordance with previous reports. Lee et al. have demonstrated that IL-β induces TGF-β1 protein and mRNA production via the activation of NF-κB and AP-1 in a human alveolar epithelial cell line [34]. In addition, a fluid shear stress response element activated the TGF-β1 promoter, which consists of functional NF-κB and AP-1 transcription factor binding sites [35]. These results further supported the concept that regulation of gene expression by NF-κB-p65 activity is complex and dependent on the cell type. Nevertheless, the data in our study suggest that NF-κB and AP-1 are involved in the transcriptional activation of TGF-β1 in BMECs. For bFGF, mRNA expression and protein secretion were significantly higher in S. aureus-stimulated cells than in untreated controls. Faris et al. found that inflammatory cytokines induced bFGF production by increasing AP-1 in Kaposi's sarcoma cells [36]. The mechanism by which S. aureus-induced activation of AP-1 regulates the transcription factors involved in the induction of cytokine expression in BMECs remains to be elucidated. Furthermore, we demonstrated that the expression of bFGF is dependent on the NF-κB and AP-1 signaling pathways. In BMECs, S. aureus-induced bFGF expression was blocked by treatment with NF-κB and AP-1 inhibitors, as seen for TGF-β1.