Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of dendritic cells derived from bone marrow (BM-DC) in response to myxoma virus infection.

ABSTRACT: Background : Poxviruses have been shown to be efficient vectors for the production of protective immune responses in host species but also in on host species. Myxoma virus belongs to poxviridae family. In order to evaluate the capacity of (MYXV) as a vaccine vector in ruminants, we invistigated its interactions with bone marrow-derived dendritic cells (BM-DC). DCs are the most potent antigen-presenting cells and play a crucial role during the priming and reactivation of antigen-specific immune responses . Following infection by a pathogen, the functional changes of DC are essential since priming and polarization of the immune response depend on these changes . Therefore a better understanding of the modifications in gene expression of proinflammatory cytokines, chemokines and stimulatory molecules expression may prove useful in predicting whether or not a vaccine vector will be effective. Moreover, it may help identifying modifications for improving its efficacy. Methodology/Principal Findings: We investigated in vitro the interactions between recombinant MYXV expressing GFP protein, SG33-GFP and ovine BM-DCs. To gain a global view of the gene remodelling induced by MYXV, we infected BM-DCs from three sheep with SG33-GFP at different multiplicity of infection (MOI) (0, 1, 3) during different time (0, 3, 8 hours). We measured the expression of 15K probes in BM-DCs after each kind of conditions with ovine Agilent microarrays. Furthermore, a selected number of genes were confirmed by RT-qPCR. MYXV infection induces a strong reprogramming of the cells leading to the expression of pro-inflammatory cytokines and mobilisation of type I IFN pathways, cellular death and features associated with the activation of the adaptive immune response. Conclusion/Significance: Microarray profiling of a poxvirus-DC interaction help to delineate some interesting features of a vector candidate in ovine, and pave the way for additional improvements of a promising platform. Keywords : Myxoma virus ; bone marrow-derived dendritic cells ; transcriptome ; sheep; vaccine 9 samples: 3 time points and 3 replicates of MOI=1

ORGANISM(S): Ovis aries

SUBMITTER: Gilles FOUCRAS

PROVIDER: E-GEOD-32656 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background : Poxviruses have been shown to be efficient vectors for the production of protective immune responses in host species but also in on host species. Myxoma virus belongs to poxviridae family. In order to evaluate the capacity of (MYXV) as a vaccine vector in ruminants, we invistigated its interactions with bone marrow-derived dendritic cells (BM-DC). DCs are the most potent antigen-presenting cells and play a crucial role during the priming and reactivation of antigen-specific immune responses . Following infection by a pathogen, the functional changes of DC are essential since priming and polarization of the immune response depend on these changes . Therefore a better understanding of the modifications in gene expression of proinflammatory cytokines, chemokines and stimulatory molecules expression may prove useful in predicting whether or not a vaccine vector will be effective. Moreover, it may help identifying modifications for improving its efficacy. Methodology/Principal Findings: We investigated in vitro the interactions between recombinant MYXV expressing GFP protein, SG33-GFP and ovine BM-DCs. To gain a global view of the gene remodelling induced by MYXV, we infected BM-DCs from three sheep with SG33-GFP at different multiplicity of infection (MOI) (0, 1, 3) during different time (0, 3, 8 hours). We measured the expression of 15K probes in BM-DCs after each kind of conditions with ovine Agilent microarrays. Furthermore, a selected number of genes were confirmed by RT-qPCR. MYXV infection induces a strong reprogramming of the cells leading to the expression of pro-inflammatory cytokines and mobilisation of type I IFN pathways, cellular death and features associated with the activation of the adaptive immune response. Conclusion/Significance: Microarray profiling of a poxvirus-DC interaction help to delineate some interesting features of a vector candidate in ovine, and pave the way for additional improvements of a promising platform. Keywords : Myxoma virus ; bone marrow-derived dendritic cells ; transcriptome ; sheep; vaccine

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: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:Estradiol (E2) is a well-known modulator of fetal neuroendocrine activity, and has been proposed as a critical endocrine signal readying the fetus for birth and postnatal life. To investigate the modulatory role of E2 on fetal stress responsiveness and the response of the fetal brain to asphyxic stress, we subjected chronically catheterized fetal sheep to a transient (10 min) brachiocephalic occlusion or sham occlusion. Half of the fetuses received subcutaneous pellets that released E2 (5 days@~250 M-NM-<g/day, increasing plasma E2 from 62M-BM-16 to 102M-BM-112 pg/mL). Hypothalamic mRNA was analyzed using the Agilent 8x15k ovine array (019921), processed and annotated as previously reported by our laboratory. Analysis of the data by ANOVA revealed that E2 differentially regulated (DR) 561 genes, and BCO DR 894 genes compared to control and estradiol+BCO DR 1153 genes compared to BCO alone (all p<0.05). E2 upregulated epigenetic pathways and downregulated local steroid biosynthesis, but did not significantly involve genes known to directly respond to the estrogen receptor (ie., contain ERE sequences). BCO upregulated kinase pathways as well as genes associated with lymphocyte infiltration into the brain, and downregulated neuropeptide synthesis. E2 upregulated immune- and apopotosis- related pathways after BCO, and reduced kinase and epigenetic pathway responses to the BCO. We conclude that array results are consistent with the results of more limited hypotheses previously published from these experiments. Chronically catheterized fetal sheep; 4 groups: control (no treatment); estradiol treatment (250 ug/day x 5 days); brachiocephalic occlusion of fetal sheep (BCO, 10 min, no other treatment); BCO plus estradiol (combination of estradiol treatment and BCO). All fetuses had blood drawn from chronically implanted catheters at 0, 5, 10, and 30 min after start of BCO or sham-BCO period. Fetal sheep 124-128 days gestation (term=147 days) at the time of study. Fetuses euthanized and fetal hypothalami collected for gene array studies 1 hour after first blood sample drawn (and start of BCO or sham-BCO period).

Project description:Many modern vaccines use defined adjuvants to stimulate the innate immune system and shape the adaptive immune response. The exact nature of these innate signals and whether immune differentiation can originate within the periphery is not known. In the present study we used an ovine lymphatic cannulation model to characterise the cellular and transcriptomic profile of the afferent lymph following injection of a liposomal vaccine formulation incorporating diphtheria toxoid and the innate stimulator poly (I:C) over a 78 h period. The response to this vaccine featured an early activation of broad proinflammatory pathways (e.g. TLR signalling and inflammasome pathways) and the transient recruitment of granulocytes into the lymph. At 24hrs a more monocytic cellular profile arose coinciding with a transition to a specific antiviral response characterised by the up-regulation of genes associated with the receptors typical for the viral mimic, poly (I:C) (e.g. TLR3, RIG-I and MDA5). At the latest time points the up-regulation of IL-17A and IL-17F suggested that Th17 cells may participate in the earliest adaptive response to this vaccine. Together these data provide the most comprehensive picture of the cellular and molecular mechanisms that link the periphery to the draining lymph node following vaccination and indicate that the immune response is capable of specialising within the periphery. This study employed an ovine model of pseudoafferent lymphatic cannulation (see de Veer et al. 2010, Vaccine) to characterise the innate immune response within the afferent lymph to vaccination with liposomes+poly (I:C)+ diptheria toxoid. The sheep used in this study had both their pre-femoral lymph nodes removed at 1 year of age. Approximately 1 year after the lymph node removal, a second surgery was performed to insert a 0.96_0.58mm heparin-coated polyvinyl chloride cannula into the pseudoafferent (previous efferent) lymphatic duct of both sides. At least seven days were allowed for healing to occur after surgery before vaccinations were administered and experimental lymph samples were collected. Handling of animals and experimental procedures were all approved by the Monash University Animal Ethics Committee in accordance with the relevant licensing agreement. Vaccinations were injected subcutaneously in the area drained by the prefemoral lymph node. Afferent lymphatic samples were collected prior to vaccination as a control (PRE) and at 4-6, 26-28, 51-53 and 76-78 hours post vaccination with liposomes+poly (I:C)+ diptheria toxoid. Lymphocytes were removed from the afferent lymph using immunomagnetic separation. Next-generation sequencing was performed on RNA derived from the remaining innate immune cells. Samples from three sheep were used at all time points except 4-6 and 26-28 hours, where only two samples yielded sufficient RNA for sequencing.

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Gene expression profiling of dendritic cells derived from bone marrow (BM-DC) in response to myxoma virus infection.

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