lipoteichoic-acid and Mastitis

Mastitis is one of the most common diseases of dairy cattle and can be caused by physical stress, chemicals and microbial infection. Staphylococcus aureus is the most common pathogens that induce mastitis in dairy cattle. In this study, bovine mammary epithelial cells (BMECs) were treated either with lipoteichoic acid (LTA, 30 µg/ml) or 1 × phosphate-buffer saline (PBS, control) and RNA-Seq was applied to explore the effect of LTA on the expression microRNAs (miRNAs) in BMECs. Compared to the control group, 43 miRNAs were significantly up-regulated and eight miRNAs were significantly down-regulated. Additionally, 724 genes were significantly up-regulated and 13 genes were significantly down-regulated in LTA group relative to the control group. Bta-miR-196a, bta-miR-2285aj-5p, bta-miR-143, bta-miR-2433, bta-miR-2284f and bta-miR-2368-3p were selected from 51 differentially expressed miRNAs and are discussed in this manuscript. Target gene prediction revealed that the target genes of these six miRNAs were all differentially expressed, including MT1E, SPDYA, FGL1, TLR2, PAPOLG, ZDHHC17 and SMC4. Subsequently, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the target genes with differentially expressed miRNAs were enriched in mitogen-activated protein kinase (MAPK) signalling pathway, rheumatoid arthritis and cancer. Therefore, the results of this study provided new evidences for the molecular mechanism of LTA-induced mastitis, which may provide new targets for the diagnosis and treatment of mastitis in dairy cattle.

Topics: Animals; Cattle; Cattle Diseases; Epithelial Cells; Female; Gene Expression Profiling; Mastitis; MicroRNAs

Mastitis is a common disease of the dairy cattle, which affects the development of the dairy industry and leads to huge economic losses. Forsythoside A (FTA) has anti-inflammatory, antioxidant, antiviral and anti-apoptotic effects. However, the therapeutic effect and molecular mechanism of FTA on dairy cow mastitis remain unclear. In this study, bovine mammary epithelial cells (BMECs) were stimulated with lipoteichoic acid (LTA), a key virulence factor of Staphylococcus aureus (S. aureus), to construct in vitro models, and then treated with FTA. Subsequently, the differentially expressed genes (DEGs) in different groups were determined by RNA sequencing (RNA-Seq) analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyse the possible function of the DEGs, real-time quantitative PCR (RT-qPCR) was used to verify whether the expression levels of these DEGs were consistent with RNA-Seq results. The results showed that cell division cycle 20B (CDC20B), endothelial cell surface expressed chemotaxis and apoptosis regulator (ECSCR), complement factor H-related 5 (CFHR5) and phospholipase A2 group IVA (PLA2G4A) were down-regulated after FTA treatment. In contrast, Kruppel-like factor 15 (KLF15) and Metallothionein 1E (MT1E) were up-regulated. These DEGs are involved in processes such as apoptosis, inflammation and development of cancer. This study provides valuable insights into the transcriptome changes in BMECs after FTA treatment. Further analysis may help identify the underlying molecular mechanisms.

Topics: Animals; Cattle; Cattle Diseases; Epithelial Cells; Female; Mammary Glands, Animal; Mastitis; Staphylococcus aureus

Mastitis is an inflammatory disease caused by microbial infection. Chlorogenic acid (CGA), one of the major phenolic acids in Taraxacum officinale, has natural antioxidant and anti-inflammatory properties in various cell types; however, the effects of CGA on Lipoteichoic acid (LTA)-induced bovine mammary epithelial cells (BMECs) have not been investigated. In this study, the CGA content in T. officinale was determined by High-performance liquid chromatography (HPLC). BMECs were infected with LTA to induce the mastitis model. Different concentrations of CGA were administered after establishing the LTA infection. The results showed that the T. officinale contained CGA 1.36 mg/g. CGA significantly reduced the pro-inflammatory gene and protein expression of TNF-α, IL-6, and IL-1β. In addition, CGA downregulated the NO, TLR2, and NF-κB signaling pathways in LTA-infected bovine mammary epithelial cells. Our results indicate that CGA reduced the expression of TNF-α, IL-6, IL-1β, and TLR2 by inhibiting the phosphorylation of proteins in the NF-κB signaling pathways in a dose-dependent manner. This finding suggests that CGA may be a potential agent for the treatment of mastitis in dairy cows.

Topics: Animals; Anti-Inflammatory Agents; Cattle; Chlorogenic Acid; Epithelial Cells; Female; Interleukin-6; Lipopolysaccharides; Mammary Glands, Animal; Mastitis; NF-kappa B; Taraxacum; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

Staphylococcus aureus causes subclinical mastitis; lipoteichoic acid (LTA) from S. aureus causes mastitis-like adverse effects on milk production by mammary epithelial cells (MECs). Here, we investigated the early effects of LTA from S. aureus on mouse MECs using a culture model, in which MECs produced milk components and formed less permeable tight junctions (TJs). In MECs of this model, Toll-like receptor 2 (receptor for LTA), was localized on the apical membrane, similar to MECs in lactating mammary glands. LTA weakened the TJ barrier within 1 h, concurrently with localization changes of claudin 4. LTA treatment for 24 h increased αS1-casein and decreased β-casein levels. In MECs exposed to LTA, the activation level of signal transducer and activator of transcription 5 (major transcriptional factor for milk production) was low. LTA activated signaling pathways related to cell survival (extracellular signal-regulated kinase, heat shock protein 27, and Akt) and inflammation (p38, c-Jun N-terminal kinase, and nuclear factor κB). Thus, LTA caused abnormalities in casein production and weakened the TJs by affecting multiple signaling pathways in MECs. LTA-induced changes in signaling pathways were not uniform in all MECs. Such complex and semi-negative actions of LTA may contribute to subclinical mastitis caused by S. aureus.

Topics: Animals; Caseins; Claudin-4; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; HSP27 Heat-Shock Proteins; Humans; JNK Mitogen-Activated Protein Kinases; Lactation; Lipopolysaccharides; Mammary Glands, Animal; Mastitis; Mice; Milk; NF-kappa B; Proto-Oncogene Proteins c-akt; Signal Transduction; Staphylococcus aureus; STAT5 Transcription Factor; Teichoic Acids; Toll-Like Receptor 2

Mastitis is usually caused by a variety of pathogenic bacteria that include both Gram-positive and Gram-negative bacteria. Lipopolysaccharide (LPS) is the pathogen-associated molecular pattern (PAMP) of Gram-negative bacteria, and peptidoglycan (PGN) and lipoteichoic acid (LTA) are those of Gram-positive bacteria. The effects of LPS, PGN and/or LTA on inflammatory response and lactation in bovine mammary epithelial cells (BMECs) are well studied, but the epigenetic mechanisms of their effects received less attention. Furthermore, since the three PAMPs are often simultaneously present in the udder of cows with mastitis, it has implications in practice to study their additive effects. The results show that co-stimulation of bovine mammary epithelial cells with PGN, LTA, and LPS induced a higher number of differentially expressed genes (DEGs) and greater expressions of inflammatory factors including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), chemokine (C-X-C motif) ligand (CXCL)1, and CXCL6. In addition, co-stimulation further increased DNA hypomethylation compared with sole LPS stimulation. Co-stimulation greatly decreased casein expression but did not further decrease histone acetylation levels and affect the activity of histone acetyltransferase (HAT) and histone deacetylase (HDAC), compared with sole LPS stimulation. Collectively, this study demonstrated that PGN, LTA, and LPS had an additive effect on inducing transcriptome changes and inflammatory responses in BMECs, probably through inducing a greater decrease in DNA methylation. Co-stimulation with PGN, LTA, and LPS decreased casein expression to a greater degree, but it might not be linked to histone acetylation and HAT and HDAC activity.

Topics: Animals; Cattle; Cell Line; Cytokines; DNA Methylation; Drug Synergism; Epigenesis, Genetic; Epithelial Cells; Female; Inflammation Mediators; Lactation; Lipopolysaccharides; Mammary Glands, Animal; Mastitis; Pathogen-Associated Molecular Pattern Molecules; Peptidoglycan; Teichoic Acids; Transcriptome