Project description:Background: Our purpose was to obtain non-existing genome-wide expression data for the rabbit species on the responses of peripheral blood mononuclear cells (PBMCs) after in vitro stimulation by lipopolysaccharide (LPS) or phorbol myristate acetate (PMA) and ionomycin. This transcriptomic study was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome. Results: The LPS affected 15 to 20 less genes than PMA/Ionomycin after 4 hours (T4) or 24 hours (T24), of in vitro stimulation, by comparison with mock-stimulated PBMCs. LPS induced an inflammatory response as shown by a significant up-regulation of IL12A and CXCL11 at T4, followed by an increased transcription of IL6, IL1B, IL1A, IL36, IL37, TNF, CCL4 and SAA1 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24. PMA/ionomycin induced a very early expression of Th1, Th2, iTreg, and Th17 responses by PBMCs at T4. The Th1 response was increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines that significantly decreased from T4 to 24 (IL2, IL4, IL10, IL13, IL17A, CD69). The granulocyte-macrophage colony-stimulating factor (CSF2) was by far the most over-expressed gene at both T4 and T24 by comparison to mock-stimulated cells, confirming a major impact of PMA/ionomycin on cell growth and proliferation. A significant down-regulation of IL16 was observed at T4 and T24, in agreement with a role of IL16 in in PBMC apoptosis. Conclusion: We report new data on the responses of PBMCs to LPS and PMA/ionomycin for the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with highly performing genomic tools should pave the way for more intense genomic studies for this species known to be a very relevant animal model in immunology and physiology. To characterize gene expression changes in peripheral blood mononuclear cells (PBMCs), we collected blood from 4 adult rabbits. PBMCs were isolated and stimulated with LPS or a mixture of PMA/Ionomycin, versus a control group (C). Cells were harvested either 4h or 24h after activation.

Project description:Background: During the past 25 years, selection of production traits highly increased pig production but diseases have emerged that may cause economic loss of extreme importance. Designing sustainable animal production that better balances productivity with resistance to disease is a major concern and challenge for the next decade. In order to address questions related to immunity and resistance to disease, it is necessary to increase knowledge on the pig immune system and to produce efficient tools dedicated to this species. In this context we produced a generic array enriched in immunity genes and validated its relevance by studying innate immune response of pigs by stimulating porcine mononuclear cells with lipopolysaccharide (LPS) or a mixture or phorboml myristate acetae (PMA) and ionomycin for 24 hours. Results: A long oligonucleotide-based chip was produced by combining a generic set of 13K probes targeting 8541 genes to a newly designed SLA-RI set that targets all genes and pseudogenes of the pig major histocompatibility complex region (SLA complex) in both orientations (906 probes) and immunity genes outside the SLA complex (2957 porbes). The porcine chip was referred to as SLA-RI/NRSP8-13K. Transcripotme analysis of PBMCs stimulated by LPS or PMA/ionomycin was carried out. Ten times more genes were up regulated after PMA/ionomycin stimulation by comparison to LPS stimulation. The LPS response was more related to the catalog Diseases and Disorders and the PMA/ionomycin response to the catalog Molecular and Cellular Function. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 genes. In addition, a very intense repression of THBS1 was observed, suggesting a predominant role of this matricellular glycoprotein during T/B cell stimulation by tumor inducers. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing and repression of MHC class II genes was observed after both stimulations. A significant reduction of antigen presentation to T cells was shared by the two types of responses that is likely to be due to separate mechanisms that need further elucidation. In addition, our results provided preliminary data suggesting a role of antisense transcripts mapping to the SLA complex during immune response. Conclusion: The SLA-RI/NRSP8-13K was highly relevant to. On the one hand the SLA-RI/NRSP8-13K was shown to be relevant and accurate to decipher two distinct innate immune responses by PBMCs indicating that this chip will constitute a valuable tool to further study immunity and resistance to disease in pig. On the other hand, the transcriptom analysis revealed specific and common features of the innate immune response according to stimulation that increase knowledge on pig immunity. Keywords: immune response activation in pig PBMCs Two-condition experiment, LPS stimulated PBMCs vs. mock-stimulated PBMCs, PMA_ionomycine stimulated PBMCs vs.mock-stimulated PBMCs. Biological replicates: 1 control, 1 LPS stimulated and 1 PMA_ionomycine stimulated from 7 animals independently grown and harvested. Dye-swap design. One replicate per array. 28 slides.

Project description:Background: During the past 25 years, selection of production traits highly increased pig production but diseases have emerged that may cause economic loss of extreme importance. Designing sustainable animal production that better balances productivity with resistance to disease is a major concern and challenge for the next decade. In order to address questions related to immunity and resistance to disease, it is necessary to increase knowledge on the pig immune system and to produce efficient tools dedicated to this species. In this context we produced a generic array enriched in immunity genes and validated its relevance by studying innate immune response of pigs by stimulating porcine mononuclear cells with lipopolysaccharide (LPS) or a mixture or phorboml myristate acetae (PMA) and ionomycin for 24 hours. Results: A long oligonucleotide-based chip was produced by combining a generic set of 13K probes targeting 8541 genes to a newly designed SLA-RI set that targets all genes and pseudogenes of the pig major histocompatibility complex region (SLA complex) in both orientations (906 probes) and immunity genes outside the SLA complex (2957 porbes). The porcine chip was referred to as SLA-RI/NRSP8-13K. Transcripotme analysis of PBMCs stimulated by LPS or PMA/ionomycin was carried out. Ten times more genes were up regulated after PMA/ionomycin stimulation by comparison to LPS stimulation. The LPS response was more related to the catalog Diseases and Disorders and the PMA/ionomycin response to the catalog Molecular and Cellular Function. LPS stimulation induced a general inflammation response with over-expression of SAA1, pro-inflammatory chemokines IL8, CCL2, CXCL5, CXCL3, CXCL2 and CCL8 as well as genes related to oxidative processes (SOD2) and calcium pathways (S100A9 and S100A12). PMA/ionomycin stimulation induced a stronger up-regulation of T cell activation than B cell activation with dominance toward a Th1 response, including IL2, CD69 and TNFRSF9 genes. In addition, a very intense repression of THBS1 was observed, suggesting a predominant role of this matricellular glycoprotein during T/B cell stimulation by tumor inducers. Repression of MHC class I genes was observed after PMA/ionomycin stimulation despite an up-regulation of the gene cascade involved in peptide processing and repression of MHC class II genes was observed after both stimulations. A significant reduction of antigen presentation to T cells was shared by the two types of responses that is likely to be due to separate mechanisms that need further elucidation. In addition, our results provided preliminary data suggesting a role of antisense transcripts mapping to the SLA complex during immune response. Conclusion: The SLA-RI/NRSP8-13K was highly relevant to. On the one hand the SLA-RI/NRSP8-13K was shown to be relevant and accurate to decipher two distinct innate immune responses by PBMCs indicating that this chip will constitute a valuable tool to further study immunity and resistance to disease in pig. On the other hand, the transcriptom analysis revealed specific and common features of the innate immune response according to stimulation that increase knowledge on pig immunity. Keywords: immune response activation in pig PBMCs

Project description:A greater understanding of the proteins involved in reproduction can benefit animal production. New advances in proteomics are having a major impact on our understanding of how spermatozoa acquire their capacity for fertilization [1]. Sperm proteomics aims at the identification of the proteins that compose the sperm cell and the study of their function [2]. The sperm cell is one of the most highly differentiated cells and is composed of a head with a highly compacted chromatin structure and a large flagellum with midpiece that contains the required machinery for movement and therefore to deliver the paternal genetic and epigenetic content to the oocyte [3]. By being so highly differentiated, spermatozoa are advantageous cells to study proteomics of specific compartments such as the membrane, which basically is the area of major importance for its role in interacting with the surroundings and the oocyte [4]. The fusion of a sperm and an oocyte is a sophisticated process that must be preceded by suitable changes in the sperm's membrane composition [5]. Recent studies of spermatozoa from the proteomic point of view have allowed the identification of different proteins in spermatozoa that are responsible for the regulation of normal/defective sperm functions [6]. While several techniques are available in proteomics, LC-MS based analysis of complex protein/peptide mixtures has turned out to be a mainstream analytical technique for quantitative proteomics [7]. Using this method, detailed proteomic data are now available for human [8], macaque [9,10], mouse [11], rat [12], bull [13-15], stallion [16], fruit fly [17], Caenorhabditis elegans [18], carp [19], rainbow trout [20], mussel [21], ram [22], honeybee [23] and rooster [24] sperm membrane proteins. Rabbit (Oryctolagus cuniculus) is an important mammalian species worldwide, being at the same time of commercial interest and a research model animal. European rabbit meat production is approximately 500 thousand tons, corresponding to a 30% share of world production [25]. Besides, rabbits account for the seventh highest number of animals slaughtered per year in the European Union-27, with 347,603 × 1000 head in 2014 [26]. In a previous work, we identified and quantified rabbit seminal plasma proteins between two different genotypes [27], concluding the clear effect of genotype in the abundance of certain seminal plasma proteins. However, it is unknown at present whether these differences also exist at sperm proteome level. Therefore, the aim of the present study was to characterise rabbit sperm membrane proteins through NanoLC-MS/MS analysis focusing on the influence of the genetic origin.

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Oryctolagus cuniculus

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