Selenium ameliorates Staphylococcus aureus-induced inflammation in bovine mammary epithelial cells by inhibiting activation of TLR2, NF-κB and MAPK signaling pathways

Abstract

Background: Staphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMECs) is considered an important pathogenic mechanism for the establishment of mastitis. Given the interesting link between selenium (Se) status and mastitis, our objective was to prove that Se was essential to suppress pro-inflammatory mediators, in part, by modulation of Toll-like receptor2 (TLR2), nuclear factor kappaB (NF-κB) and mitogen activated protein kinase (MAPK) signal transduction pathway in bMECs.

Results: Results showed that Se (0~ 16 μM) did not affect the growth of bMECs. The mRNA expression of TLR2, Myeloid differentiation factor 88 (Myd88), Interleukin-1 receptor-associated kinase4 (Irak4), Interleukin-1 receptor-associated kinase1 (Irak1) and TNF receptor-associated factor6 (Traf6) in TLR2 signal pathway were increased or significantly increased by S. aureus. Se played an important role in regulating the genes expression of TLR2, Myd88, Traf6 but not in controlling the expression of Irak4 and Irak1. In addition, Se exerted strong inhibitory effects on the genes expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) induced by S. aureus. To further investigate the possible signaling mechanisms involved in the processes, we analyzed the role of MAPK and NF-κB signaling pathway in inflammation response in S. aureus-stimulated bMECs in vitro. Results showed that the phosphorylation of inhibitory kappaB alpha (IκBα), p65, p38 and extracellular regulated protein kinase (Erk) were significantly increased in S. aureus-stimulated bMECs. It indicated that S. aureus activated NF-κB and MAPK signaling pathway. We also examined the effects of Se on the phosphorylation of IκBα, p65, p38 and Erk in NF-κB and MAPK signaling pathway, which have well been proved to control the synthesis and release of pro-inflammatory mediators during inflammation. The findings are exciting, that pretreatment with Se (4, 8 μM) significantly suppressed the phosphorylation of IκBα, p65, p38 and Erk.

Conclusions: These results suggest that Se down-regulates inflammatory mediators TNF-α, IL-1β and IL-6 gene expressions via TLR2, NF-κB and MAPK signaling pathway in S. aureus-stimulated bMECs, which may be responsible for the anti-inflammatory effect of Se.

Keywords: MAPK; Mammary; NF-κB; Se; TLR2.

Fig. 1

The cytotoxicity of Se on bMECs. Cell viability was measured by MTT following treatment with various concentrations (0, 1, 2, 4, 8, 16, 32 and 64 μM) of Se for 12 h. Cell proliferation was not inbitited by Se in concentration lower than 16 μM. The data are mean ± sem (n = 6). ^***p < 0.001 vs. 0 μM

Fig. 2

The effect of Se on TLR2 signaling pathway associate genes induced by S. aureus in bMECs. Cells were incubated with different concentrations of Se or serum-free medium for 12 h and subsequently treated with S. aureus (MOI = 1:1) for 0, 6, 8 and 10 h. Total RNA was prepared at the indicated time points after S. aureus treated. The TLR2 (a) , Myd88 (b) , Irak4 (c) , Irak1 (d) and Traf6 (e) mRNA expression levels were assayed using qRT-PCR. con = control cells without any treatment; mod = cells treated with S. aureus (MOI = 1:1) only; low = Se (2 μM) + S. aureus (MOI = 1:1); mid = Se (4 μM) + S. aureus (MOI = 1:1); high = Se (8 μM) + S. aureus (MOI = 1:1). The data are shown as mean ± sem (n = 3). ^###: p < 0.001 vs. Con; ^*: p < 0.05 vs. Mod; ^**: p < 0.01 vs. Mod; ^***: p < 0.001 vs. Mod

Fig. 3

The effect of Se on gene expression of pro-inflammatory cytokines induced by S. aureus in bMECs. Cells were incubated with various concentrations of Se or serum-free medium for 12 h and subsequently challenged with S. aureus (MOI = 1:1) for 0, 6, 8, and 10 h. Total RNA was prepared at the indicated time points after S. aureus injection. The TNF-α, IL-1β and IL-6 mRNA expression were quantified using qRT-PCR. con = control cells without any treatment; mod = cells treated with S. aureus (MOI = 1:1) only; low = Se (2 μM) + S. aureus (MOI = 1:1); mid = Se (4 μM) + S. aureus (MOI = 1:1); high = Se (8 μM) + S. aureus (MOI = 1:1). The data are shown as mean ± sem (n = 3). ^##: p < 0.01 vs. Con; ^###: p < 0.001 vs. Con; ^*: p < 0.05 vs. Mod; ^**: p < 0.01 vs. Mod; ^***: p < 0.001 vs. Mod

Fig. 4

Effect of Se on S. aureus-induced IκBα and p65 phosphorylation in bMECs. Cells were pretreated with various concentrations (0, 2, 4 and 8 μM) of Se or serum-free medium for 12 h before stimulated with S. aureus (MOI = 1:1) for 0.5 h and then washing twice with PBS. Total proteins were prepared at the indicated time points and subjected to Western blotting. Con = control cells without any treatment; mod = cells treated with S. aureus (MOI = 1:1) only; low = Se (2 μM) + S. aureus (MOI = 1:1); mid = Se (4 μM) + S. aureus (MOI = 1:1); high = Se (8 μM) + S. aureus (MOI = 1:1). The data are shown as mean ± sem (n = 3). ^###: p < 0.001 vs. Con; ^*: p < 0.05 vs. Mod; ^**: p < 0.01 vs. Mod; ^***: p < 0.001 vs. Mod. One out of 3 independent experiments is shown

Fig. 5

Effect of Se on S. aureus-induced p38 and Erk phosphorylation in bMECs. Cells were pretreated with various concentrations (0, 2, 4 and 8 μM) of Se or serum-free medium for12 h before stimulated with S. aureus (MOI = 1:1) for 0.5 h and then washing twice with PBS. Total proteins were prepared at the indicated time points and subjected to Western blotting. Con = control cells without any treatment; mod = cells treated with S. aureus (MOI = 1:1) only; low = Se (2 μM) + S. aureus (MOI = 1:1); mid = Se (4 μM) + S. aureus (MOI = 1:1); high = Se (8 μM) + S. aureus (MOI = 1:1). The data are shown as mean ± sem (n = 3). ^###: p < 0.001 vs. Con; ^*: p < 0.05 vs. Mod; ^**: p < 0.01 vs. Mod; ^***: p < 0.001 vs. Mod. One out of 3 independent experiments is shown