Project description:BackgroundOur purpose was to obtain 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 transcriptome profiling was carried out using microarrays enriched with immunity-related genes, and annotated with the most recent data available for the rabbit genome.ResultsThe LPS affected 15 to 20 times fewer genes than PMA-Ionomycin after both 4 hours (T4) and 24 hours (T24), of in vitro stimulation, in 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, and CCL4 at T24. Surprisingly, we could not find an up-regulation of IL8 either at T4 or at T24, and detected a down-regulation of DEFB1 and BPI at T24. A concerted up-regulation of SAA1, S100A12 and F3 was found upon stimulation by LPS. PMA-Ionomycin induced a very early expression of Th1, Th2, Treg, and Th17 responses by PBMCs at T4. The Th1 response increased at T24 as shown by the increase of the transcription of IFNG and by contrast to other cytokines which significantly decreased from T4 to T24 (IL2, IL4, IL10, IL13, IL17A, CD69) by comparison to mock-stimulation. 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 PBMC apoptosis.ConclusionsWe report new data on the responses of PBMCs to LPS and PMA-Ionomycin in the rabbit species, thus enlarging the set of mammalian species for which such reports exist. The availability of the rabbit genome assembly together with high throughput genomic tools should pave the way for more intense genomic studies for this species, which is known to be a very relevant biomedical model in immunology and physiology.

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

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: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. RESULTS: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P?<?0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. CONCLUSION: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

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Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response.

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Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response.

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