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: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.

			Action	DRS
	KK1321_1_Control.raw	Raw
	KK1321_2_LPS.raw	Raw
	KK1321_3_LPS_SAMC.raw	Raw
	KK1321_Pool_Library_GPF1.fasta	Fasta
	KK1321_Pool_Library_GPF1.mgf	Mgf

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Inhibitory effect of S-Allylmercaptocysteine on TLR4-mediated inflammation in mammary epithelial cells

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