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:Outcome and resolution of an udder inflammation (mastitis) depends on the type of the invading pathogen. Gram-negative pathogens, such as Escherichia coli mostly trigger strong inflammation and full local activation of the immune defense eventually resulting in pathogen elimination. Staphylococcus aureus and other Gram-positive pathogens elicit only weak immune reactions possibly allowing for pathogen persistence. Little is known about the very early molecular determinants of this pathogen-species specific immune response of the udder. We therefore infected healthy mid-lactating heifers with a high dose of defined E. coli1303 or S. aureus1027 pathogens, sampled udder tissues at 1, 2, and 3 h post infection and globally profiled the transcriptome of samples from the gland cistern. We expectedly found that the E. coli infection elicited a fast and strong immune response. Bioinformatics analyses suggested that pathogen recognizing receptors triggered the activation of the IkB/NF-kB signaling cascade inducing the expression of a wealth of inflammation related genes, but also many immune dampeners. S. aureus infection failed to elicit inflammation and activate IkB/NF-kB signaling. Instead, it triggered immune suppressive mechanisms which were, however not outweighed by strong induction of inflammatory genes. Moreover, it activated the wnt/b-catenin signaling cascade resulting in active suppression of NF-kB signaling and rearrangement of the actin-cytoskeleton through modulating expression of the Rho GTPases. These alterations facilitate invasion of pathogens into host cells. We validated experimentally that pathogenic S. aureus, but not E. coli may invade mammary epithelial cells (MEC). Microscopy revealed an altered structure of the actin-cytoskeleton of primary MEC having internalized live GFP-tagged S. aureus pathogens. Our study reveals for the first time the earliest host responses in the udder differentiating E. coli from S. aureus mastitis. The latter is characterized by eliciting immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host.

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:Establishment of an in vitro system to explore molecular mechanisms of mastitis susceptibility in cattle by comparative expression profiling of Escherichia coli and Staphylococcus aureus inoculated primary cells sampled from cows with different genetic predisposition for somatic cell score Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows that were selected for high and low mastitis susceptibility by applying a marker assisted selection strategy considering QTL and molecular marker information of a repetitively confirmed QTL for SCS in the telomeric region on BTA18 The cells were cultivated and subsequently inoculated with heat inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 hours the cells were harvested and comparatively analyzed using microarray expression chip technology to identify differences in mRNA expression profiles attributed to cultivation, inoculation or to genetic predisposition. Six heifers inheriting the favorable paternal QTL allele and five heifers inheriting the unfavorable QTL allele were selected by applying a marker assisted selection strategy. All heifers were kept under the same environmental conditions, had no clinical mastitis and did not show any indication of bacterial infection at slaughter. Primary bovine mammary gland epithelial cell cultures were established from cells sampled from the udder parenchyma of each cow. The cells were challenged with heat inactivated Escherichia coli and Staphylococcus aureus or with PBS for control. After 1, 6 and 24 hours the cells were harvested and mRNA expression was comparatively analyzed between time points for each treatment and each paternally inherited SCS-BTA18-QTL allele, respectively. In addition, the differences in gene expression at time points 1, 6 and 24h between inoculated and respective uninoculated control cells were investigated using the short time series expression miner STEM for co-expression profiling and GO cattegory enrichment analyses.

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Expression data from primary bovine mammary epithelial cells (pbMEC) challenged with heat inactivated E. coli and S. aureus particles

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