The Regulation of Staphylococcus aureus-Induced Inflammatory Responses in Bovine Mammary Epithelial Cells

Abstract

Mastitis, an inflammatory disease, causes severe economic loss in the dairy industry, which is mainly infected by bacteria. Staphylococcus aureus (S. aureus), the major pathogenic microorganism, derived from lipoteichoic acid (LTA) has been identified to activate inflammatory responses, but the cellular or intercellular regulatory mechanism is unclear. This study mainly focused on the effects of LTA in bovine mammary epithelial cells (Mac-T) and elaborated the regulation of microRNAs (miRNAs). The results showed that LTA enhanced the messenger RNA (mRNA) expression and production of tumor necrosis factor α (TNF-α) and interleukin (IL)-6. Furthermore, LTA could activate Toll-like receptor (TLR)2/MyD88-mediated phosphoinositide 3-kinase (PI3K)/AKT pathway, and TLR2 plays a pivotal role in LTA-induced inflammatory responses. The results of qRT-PCR showed that miRNA levels increased and reached the highest at 3 h and then gradually decreased over time in Mac-T cells. In exosomes, the levels of 11 and three miRNAs were upregulated and downregulated at 24 h, respectively. In addition, miR-23a showed the highest increase in Mac-T cells treated with LTA and targeted PI3K to regulate inflammatory responses. Furthermore, Mac-T cell-derived exosomes were identified to play a cell-cell communication by promoting M1 polarization of bovine macrophages. In summary, our study demonstrated that LTA could activate inflammatory responses via TLR2/MyD88/PI3K/AKT signaling pathway, and miR-23a inhibited it by targeting PI3K. Furthermore, we found that Mac-T cell-derived exosomes might be associated with inflammatory responses by promoting M1 polarization of bovine macrophages.

Keywords: LTA; exosome; mammary epithelial cells; mastitis; miR-23a.

Figure 1

Effects of lipoteichoic acid (LTA) on the expression and production of pro-inflammatory cytokines in Mac-T cells at different time points (compared with 0 h, n = 3). (A) The mRNA expression of tumor necrosis factor (TNF)-α. (B) The secretion of TNF-α (pg/ml) in cell culture medium. (C) The mRNA expression of interleukin (IL)-6. (D) The secretion of IL-6 (pg/ml) in cell culture medium. **p < 0.01.

Figure 2

The activation of Toll-like receptor (TLR)2/MyD88-mediated phosphoinositide 3-kinase (PI3K)/AKT signaling pathway in lipoteichoic acid (LTA)-treated Mac-T cells at different time points (compared with 0 h, n = 3). (A) The mRNA expression of TLR2. (B) The mRNA expression of MyD88. (C) Western blot analysis of TLR2, MyD88, PI3K, and AKT. (D) The expression of TLR2 protein. (E) The expression of MyD88 protein. (F) The expression of PI3K protein. (G) The expression of p-AKT protein. *p < 0.05, **p < 0.01.

Figure 3

Effects of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (OxPAPC) on lipoteichoic acid (LTA)-induced inflammatory response (compared with 0 h, n = 3). (A) The expression of Toll-like receptor (TLR)2 protein. (B) Western blot analysis of phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. (C) The expression of MyD88 protein. (D) The expression of PI3K protein. (E) The expression of p-AKT protein. (F) The mRNA expression of tumor necrosis factor (TNF)-α. (G) The mRNA expression of interleukin (IL)-6. **p < 0.01.

Figure 4

qRT-PCR of 14 miRNAs in Mac-T cells and exosomes. (A) TEM analysis of exosomes. (B) Nanosight depicting the size distribution of exosomes. (C) Western blot analysis of exosomal markers (n = 3). (D) The expression of miRNAs in Mac-T cells (n = 3). (E) The expression of miRNAs in Mac-T-derived exosomes (n = 3).

Figure 5

The regulation of miR-23a in lipoteichoic acid (LTA)-induced inflammatory responses. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of target genes of miR-23a. (B) The sequence information of miR-23a and target genes. (C) Dual-luciferase reporter analysis of miR-23a and phosphoinositide 3-kinase (PI3K) 3′-UTR (n = 3). (D) The expression of miR-23a in mimic-transfected cell (n = 3). (E,F) The expression of PI3K and AKT proteins after the transfection of miR-23a mimics (n = 3). (G) The expression of miR-23a in inhibitor-transfected cell (n = 3). (H,I) The expression of PI3K and AKT proteins after the transfection of miR-23a inhibitor (n = 3). *p < 0.05, **p < 0.01.

Figure 6

The polarization of bovine macrophages induced by exosomes (n = 3). (A,B) The expression of CD11b in macrophages after treatment with control-exo and inflammation-exo. (C–E) The expression of M1 and M2 macrophage markers, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), and ARG-1, were detected by qRT-PCR. *p < 0.05, **p < 0.01.