Project description:Infections of the udder by Escherichia coli very often elicit acute inflammation, while Staphylococcus aureus infections tend to cause mild, subclinical inflammation and persistent infections. The molecular causes undercovering the different disease patterns are poorly understood. We therefore profiled kinetics and extent of global changes in the transcriptome of primary bovine mammary epithelia cells (MEC) subsequent to challenging them with heat inactivated preparations of E. coli or S. aureus pathogens. E. coli swiftly and strongly induced expression of cytokines and bactericidal factors. S. aureus elicited a retarded response and failed to quickly induce expression of bactericidal factors. Both pathogens induced a similar pattern of chemokines for cell recruitment into the udder, but E. coli stimulated their synthesis much faster and stronger. The genes which are exclusively and most strongly up-regulated by E. coli may be clustered into a regulatory network with Tumor necrosis factor alpha (TNF-a) and Interleukin 1 (IL-1) in a central position. In contrast, the expression of these master cytokines is barely regulated by S. aureus. Both pathogens quickly trigger enhanced expression of IL-6. This is still possible after completely abrogating MyD88 dependent TLR-signalling in MEC. The E. coli specific strong induction of TNF-a and IL-1 expression may be causative for the severe inflammatory symptoms of animals suffering from E. coli mastitis while avoidance to quickly induce synthesis of bactericidal factors may support persistent survival of S. aureus within the udder. We suggest that S. aureus subverts MyD88-dependent activation of immune gene expression in MEC. We challenged pbMEC cultures with 1E+07 particles per ml of heat inactivated E. coli strain 1303 for 1, 3, 6, and 24 h and compared their transcriptomes to that of untreated control cells. The experiment included three biological replicas (rep1, rep2, rep3), each from a different cow We challenged pbMEC cultures with 1E+07 particles per ml of heat inactivated S. aureus strain 1027 for 1, 3, 6, and 24 h and compared their transcriptomes to that of untreated control cells. The experiment included three biological replicas (rep1, rep2, rep3), each from a different cow

Project description:Background: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the diary industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of an immune stimulant like lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes / macrophages are known to develop tolerance to the endotoxin (ET) LPS as adaptation strategy to prevent exuberant inflammation. We have recently observed that application of 1 µg of LPS/udder quarter effectively protects the cow for several days from an experimentally elicited mastitis. We have modelled this process in primary cultures of Mammary Epithelial Cells (MEC) from the cow. This is by far the most abundant cell type in the udder coming into contact with invading pathogens and little is known about the role of MEC in establishing ET in the udder. Results: We primed primary MEC cultures for 6 h with LPS (100 ng/ml) and stimulated some cultures 12 h or 42 h later with 107/ml of E. coli particles. Affymetrix microarrays were used to profile priming related alterations in the global transcriptome of those cells. Corner stone data were validated with quantitative real time PCR. LPS-priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (ß-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1,-3) as well as mediators of the sentinel function of MEC (CCL5, IL-8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL-1B, IL-6, TNF-alpha and immune effectors (iNOS, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL-1B and of IRF7 as key to this modulation. Conclusion: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells.

Project description:Background: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the diary industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of an immune stimulant like lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes / macrophages are known to develop tolerance to the endotoxin (ET) LPS as adaptation strategy to prevent exuberant inflammation. We have recently observed that application of 1 M-BM-5g of LPS/udder quarter effectively protects the cow for several days from an experimentally elicited mastitis. We have modelled this process in primary cultures of Mammary Epithelial Cells (MEC) from the cow. This is by far the most abundant cell type in the udder coming into contact with invading pathogens and little is known about the role of MEC in establishing ET in the udder. Results: We primed primary MEC cultures for 6 h with LPS (100 ng/ml) and stimulated some cultures 12 h or 42 h later with 107/ml of E. coli particles. Affymetrix microarrays were used to profile priming related alterations in the global transcriptome of those cells. Corner stone data were validated with quantitative real time PCR. LPS-priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (M-CM-^_-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1,-3) as well as mediators of the sentinel function of MEC (CCL5, IL-8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL-1B, IL-6, TNF-alpha and immune effectors (iNOS, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL-1B and of IRF7 as key to this modulation. Conclusion: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells. Each experiment was performed in triplicate using separate pbMEC cultures (80% confluence) prepared from the udders of udders of three healthy, pregnant (d130 of gestation) cows in the mid of their first lactation. Controls (C.) were cultured for 12 h in GM, washed 3x with PBS and cultivated for additional 18 h (short waiting experiment) or 48 h (long waiting experiment). For the priming experiment (P.) the pbMEC cultures were stimulated (M-bM-^@M-^\primedM-bM-^@M-^]) at time 0 h with 100 ng/ml LPS , washed three times with PBS and cultivated for another 18 h or 48 h. For the induction trial (I.) cells were challenged with 107/ml particles of heat inactivated E. coli1303 for 6 h. The cultures were handled exactly like the control group (C.) prior to the E. coli challenge. For the induction post priming experiment (I.p.P.) cultures were primed for 12 h with LPS, just as described for the priming group (P.). After 3x washing with PBS and waiting for 12 h or 42 h in GM the I.p.P. cultures were also challenged with 107/ml particles of E. coli1303. Cells were harvested at time 30 h (short waiting experiments) or 60 h (long waiting experiments) and total RNA was prepared.

Project description:Mastitis is an inflammation of the mammary gland (MG), usually due to bacterial infection. Although considerable attention has been paid to this economically important disease, the early stages of the host response remain poorly defined. In particular, it is unclear how mammary epithelial cells (MEC), a first barrier against pathogens, respond to infection. Indeed, it is difficult to differentiate between the contributions of MEC and infiltrating immune cells to gene expression profiles of mammary tissue during mastitis. The current investigation examines the response of MEC at the early stage of infection using a non invasive RNA sampling method taking advantage of the presence of cytoplasmic crescents contained in milk fat globules. We have recently shown that, in goats, Milk Fat Globules (MFG) provide a unique source of RNA to study the in vivo regulation of gene expression in MEC. This non invasive RNA sampling method was used during the time course of an experimental intra mammary infection (IMI) with S. aureus. Experiments were performed using ovine microarrays (Agilent) to compare gene expression patterns before infection, at 12h, 18h and 24h post-infection (PI). Furthermore, we combined this approach with laser capture microdissection of MEC isolated from frozen slides of mammary tissue to study some specific genes at the late stage of infection (30h PI). We show that at 18h PI, before the burst of somatic cells in milk, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signaling pathways. Then, at the late stage of infection (30h PI), the contribution of MEC in immune response changes to produce different acute phase proteins, including SAA3, serpin A1 and PTX3. These cells also express factors that contribute directly to fighting infection, such as S100A12. In summary, we demonstrate for the first time in vivo how MEC orchestrate innate immune response during an IMI with S. aureus in the goat species. We report here new opportunities to assess the dynamics of gene expression in the mammary gland, thus providing significant advances in the understanding of MEC immune capacity.M-bM-^@M-^BFurthermore, the production of some molecules by MEC, in the early stages of IMI, could provide sensitive biomarkers for early detection and therefore, treatment of mastitis. Experiments were performed using ovine microarrays (Agilent) to compare gene expression patterns before infection, at 12h, 18h and 24h post-infection (PI). 20 sample records; mono-colour experimental design

Project description:Purpose: The goal of this study is to explore the role of miRNAs in dairy cow response to E. coli and S. aureus, mastitis causing pathogens, is not well understood. Results: The global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing. A total of 231 known bovine miRNAs were identified with more than 10 counts per million (CPM) in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the total reads of known bovine miRNAs. One hundred and fifty novel miRNAs were identified and half of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P<0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to one for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (Bta-miR184, miR-24-3p, miR-148, miR-486 and bta-let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG (Kyoto encyclopedia of genes and genomes) pathways of the immune system, signal transduction, cellular process, nervous system, development and pathways in human diseases, especially cancer. Conclusion: Using next-generation sequencing, our study identified 150 novel bovine miRNAs and revealed a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. E. coli elicited an earlier differential regulation of miRNAs as opposed to a delayed regulation by S. aureus. Furthermore, target gene prediction showed significant enrichments for functions in different biological and cellular processes as well as KEGG pathways in immunity, development and human diseases. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis of mastitis and development of control measures. Bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing, no replicates, using illumina HiScanSQ platform.

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Differentiating Staphylococcus aureus from Escherichia coli mastitis: S. aureus triggers unbalanced immune-dampening and host cell invasion immediately after udder infection

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