Project description:Background: Staphylococcus aureus is a major pathogen of humans and animals and rapidly emerging antibiotic-resistant strains have further increased the severity of this health issue. Host genetics influence susceptibility to S. aureus infections, and genes determining infection outcome should be identified to analyze immune-based therapies as an alternative to antibiotics. Here, we used outbred animals from a divergent selection on susceptibility towards Staphylococcus infection to explore host immunogenetics. Methodology/Principal Findings: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals with different degree of susceptibility have distinct gene expression profiles. We measured expression levels of 15K probes from in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hours) by using Agilent microarrays. Furthermore, a selected number of genes were confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and genes involved in inflammatory process and T helper cell polarization were highly up regulated upon stimulation. Moreover, a set of 204 genes were statistically different between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, a role of classical pathway of Complement and early regulation of inflammation is observed in the resistant line through over expression of C1q and Ido1 genes respectively. On the opposite, over expression of genes in the IL1R pathway in the susceptible animals was noticed. Furthermore, leucocyte extravasation pathway was also found dominant in the susceptible line. Conclusion/Significance: we successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in 8 hour kinetics experiment. Distinct transcriptional profiles of dendritic cells obtained from resistant and susceptible lines may explain susceptibility towards S. aureus infections in a broad context. Keywords: Staphylococcus aureus, dendritic cells, mammalian, transcriptome, immunity, mastitis 44 sample records; two-colour dye-swap experimental design

Project description:Background: Staphylococcus aureus is a major pathogen of humans and animals and rapidly emerging antibiotic-resistant strains have further increased the severity of this health issue. Host genetics influence susceptibility to S. aureus infections, and genes determining infection outcome should be identified to analyze immune-based therapies as an alternative to antibiotics. Here, we used outbred animals from a divergent selection on susceptibility towards Staphylococcus infection to explore host immunogenetics. Methodology/Principal Findings: We investigated how dendritic cells respond to heat-inactivated S. aureus and whether dendritic cells from animals with different degree of susceptibility have distinct gene expression profiles. We measured expression levels of 15K probes from in vitro S. aureus-stimulated bone marrow-derived dendritic cells at three different time points (0, 3 and 8 hours) by using Agilent microarrays. Furthermore, a selected number of genes were confirmed by RT-qPCR. Gene signatures of stimulated DCs were obtained and genes involved in inflammatory process and T helper cell polarization were highly up regulated upon stimulation. Moreover, a set of 204 genes were statistically different between susceptible and resistant animals, and grouped them according to their predisposition to staphylococcal infection. Interestingly, a role of classical pathway of Complement and early regulation of inflammation is observed in the resistant line through over expression of C1q and Ido1 genes respectively. On the opposite, over expression of genes in the IL1R pathway in the susceptible animals was noticed. Furthermore, leucocyte extravasation pathway was also found dominant in the susceptible line. Conclusion/Significance: we successfully obtained Staphylococcus aureus associated gene expression of ovine BM-DC in 8 hour kinetics experiment. Distinct transcriptional profiles of dendritic cells obtained from resistant and susceptible lines may explain susceptibility towards S. aureus infections in a broad context. Keywords: Staphylococcus aureus, dendritic cells, mammalian, transcriptome, immunity, mastitis

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: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:Staphylococci are major pathogens in humans and animals and emerging antibiotic-resistant strains have further increased the importance of this health issue. The existence of a genetic basis of host response to bacterial infections has been widely documented but the underlying mechanisms and genes are still largely unknown. Previously, two divergent lines of sheep selected on their milk somatic cell count called high and low SCS lines, have been showed to be respectively more and less susceptible to intra mammary infections (IMI). Transcriptional profiling of milk somatic cells (MSC) of high and low SCS sheep infected successively by S. epidermidis and S. aureus was performed to provide enhanced knowledge about the genetic basis of differential host response to IMI with Staphylococci. Gene expression in MSC of high and low SCS sheep collected 12h post-challenge was performed on a 15K gene ovine oligoarray (Agilent). MSC were mainly neutrophils. The high number of differentially expressed genes between the two bacterial strains indicated, among others, increased number of T-cells in MSC after S. aureus, compared to S. epidermidis challenge. Differential regulation of 366 genes between resistant and susceptible animals was largely associated with immune and inflammatory response (including pathogen recognition TLR2 pathway and cell migration), signal transduction, cell proliferation and apoptosis. Close biological connection between most of differentially expressed genes into Ingenuity Pathway Analysis networks further indicated consistency between the genes that were differentially-expressed between resistant and susceptible animals. Gene profiling in high and low SCS sheep provided strong candidates for biological pathway and gene underlying genetically determined resistance and susceptibility towards Staphylococci infections opening new fields for further investigation. Keywords: Staphylococcus epidermidis, Staphylococcus aureus, milk somatic cells, mammalian, transcriptome, immunity, mastitis 22 samples in a two-colour dye-swap experimental design

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. Furthermore, 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.

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Gene expression profiling of mammary epithelial cells and their role in the predisposition to Staphylococcus aureus infections

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