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CDNA microarray analysis of gene expression in blood mononuclear cells of Yorkshire pigs with SLA-DRB1 defined genotypes

ABSTRACT: The major histocompatibility complex (SLA in pigs) encodes molecules for self-nonself discrimination, constitute a major transplantation barrier and is associated with a variety of diseases. Pigs with defined SLA genotypes are a resource for the study of immune response, disease resistance and production traits, as well as an important animal model for transplantation research. The aim of the present study was to explore the influence of defined SLA genotypes on gene expression patterns of immune-related genes in blood mononuclear cells (BMCs). Using a loop design, heterologous cDNA microarray hybridizations were performed to compare Yorkshire pigs representing three defined SLA-DRB1 genotypes (including a potentially new allele). Total RNA was treated with Dnase I treated and amplified. The amplified RNA was reverse transcribed using random primers and incorporating 5-(3-aminoallyl)-dUTP for post-synthesis labelling of the cDNA with the appropriate fluor dyes. Hybridization was performed on a bovine immune-endocrine in-house cDNA microarray printed on glass slides. Microarray fold-change analysis revealed that animals carrying confirmed SLA alleles (SLA-DRB1*070701 and SLA-DRB1*050502) showed consistent differences in gene expression when compared to the two other groups. On the other hand, the group carrying a potentially new allele showed differences that varied depending on the group it was being compared to. Genes that were identified as differentially expressed include macrophage inflammatory protein 1, interleukin 1, toll-like receptor 2 and caspase 1. A better understanding of SLA gene activity can facilitate the definition of new strategies to control animal health and optimize animal production. Keywords: SLA-defined genotype comparative genomic cDNA microarray hybridizations

ORGANISM(S): Sus scrofa Bos taurus

PROVIDER: GSE7908 | GEO | 2007/05/30

SECONDARY ACCESSION(S): PRJNA100085

REPOSITORIES: GEO

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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.

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CDNA microarray analysis of gene expression in blood mononuclear cells of Yorkshire pigs with SLA-DRB1 defined genotypes

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