Project description:Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response (3 and 6 h of exposure to agonists). By using HEK 293 cells transfected with human pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and alpha-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling by a microarray and confirmation by RT-qPCR revealed an innate immune response which was common to both LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greated magnitude than those induced by SaS. Overall, the analysis of microarray data suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. Chemokines were among the most up-regulated genes, in particular ELRCXC chemokines that target neutrophils. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. This was in accordance with the much stronger up-regulation of Cxcl10, Ccl5 and Nos2 by LPS than by SaS. The higher upregulation of chemokines that target mononuclear leucocytes (CXCL10, CCL2, CCL5 and CCL20) by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. It is noteworthy that at the protein level, secretion of TNF-alpha and IL-1-beta was not induced by either stimulus. These results suggest that the response of MEC to diffusible bacterial stimuli is able to contribute to the onset of the response with differential leucocyte recruitment but does not account directly for the differential production of major pro-inflammatory cytokines. Transcriptional profiling of bovine mammary epithelial cells (Holstein breed) comparing untreated control with mammary epithelial cells (MECs) stimulated by Staphylococcus aureus (SA) versus Escherichia coli lipopolysaccharide (LPS). 16 microarrays (2 stimuli * 2 times * quadruplicate) in a two-color dye-swap experimental design. Untreated cells served as the control. One replicate per array. Log2-intensity of each dye was analyzed separately => 32 samples.

Project description:Yamoaâ¢ is marketed and sold as a dietary supplement with anecdotal positive effects in asthma and hay fever. We determined that Yamoaâ¢ (ground bark of Funtumia elastica tree) stimulated innate immunity in part by affecting gamma delta T cells. Yamoaâ¢ had distinct priming effects, very similar to, but more robust than, that of lipopolysaccharide (LPS), on bovine, mouse and human gamma delta T cells. However, the optimal effect was dependent on the presence of accessory cells. Gene expression patterns in bovine gamma delta T cells and monocytes induced by Yamoaâ¢ were very similar to those induced by ultrapure LPS, but the agonists in Yamoaâ¢ did not signal entirely through TLR4. Yamoaâ¢ stimulated human cells to produce cytokines involved innate protection. The bioactive component of Yamoaâ¢ was delineated to a complex polysaccharide fraction (Yam-I). Intraperitoneal injection of Yamoaâ¢ and very low doses of Yam-I in mice induced rapid increases peritoneal neutrophils directed by changes chemokine expression. Yamoaâ¢ and Yam-I were effective as therapeutic treatments in mice with Salmonella enterica serotype Typhimurium (ST) induced enterocolitis that resulted in decreased bacterial counts in feces. This initial characterization of the immune stimulatory properties of polysaccharides derived from Yamoaâ¢ suggests potential mechanisms for positive effects in asthma and that they have potential for application in infectious disease settings. . Experiment Overall Design: To begin to understand the effects of Yamoa in innate immunity, we investigated the global gene expression profiles of stimulated bovine gamma delta T cells. Peripheral blood from 3 neonatal bovine calves was collected. gamma delta T cells were sorted to >97% purity using a FACS Vantage. Cells were placed in culture and stimulated with either an aqueous extract of Yamoa (32.6ug/ml), ultrapure LPS [uLPS (10ug/ml)] or PBS for 4 hours after which RNA was extracted and processed for microarray analysis.

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