Analysis of gene expression patterns by microarray hybridization in blood mononuclear cells of SLA-DRB1 defined Canadian Yorkshire pigs
© Nino-Soto1 et al; licensee BioMed Central Ltd. 2008
Received: 10 April 2008
Accepted: 23 June 2008
Published: 23 June 2008
The Swine Leukocyte Antigen (SLA) system encodes molecules for self-nonself discrimination and is associated with immune responses and disease resistance. Three lines of pigs defined by their SLA-DRB1 alleles were developed at the University of Guelph for xenotransplantation and immune response studies. The aim of this project was to explore the potential association between defined SLA-DRB1 alleles and gene transcriptional patterns of other immune-related genes in blood mononuclear cells.
Three SLA-DRB1 alleles were characterized using a RT-PCR-based sequencing method. The loci represented included a new allele, DRB1*04ns01. Next, microarray heterologous (bovine-porcine) hybridization together with qPCR were used to explore differential gene expression between SLA-DRB1-defined groups. Microarray analysis showed significant (p < 0.01) differential expression for 5 genes, mostly related to inflammation. Genes varied according to the comparison analyzed. Further testing with qPCR revealed the same trend of differential expression for 4 of the genes, although statistical significance was reached for only one.
A new SLA-DRB1 allele was characterized. A potential association was found between SLA-DRB1 alleles and inflammation-related genes. However, the influence of other genes cannot be ruled out. These preliminary findings agree with other studies linking MHC haplotypes and inflammation processes, including autoimmune disease. The study provides an initial view of the biological interactions between the SLA complex and other immune-related genes. Future studies will focus on characterization of SLA-haplotypes associated with these particular alleles and the dynamics of the immune response to antigenic challenges.
The highly polymorphic MHC-encoded molecules are crucial for self-nonself discrimination in vertebrates. They constitute the major barrier for transplantation, contain numerous genes involved in immunological and non-immunological functions and are associated with resistance or susceptibility to various diseases. The two main classes, I and II, are involved in antigen presentation to T-cells. However, a large number of the genes in the MHC, like class III genes, are not directly related to this function [1, 2]. A total of 152 loci have been annotated within this region. In pigs, known as the SLA, the DRB genes show extensive polymorphism in exon 2 and the 135 available sequences identified to date are distributed into at least 10 confirmed allele groups .
Different SLA haplotypes have been associated with variation in immune response and disease, as well as reproduction and production traits . Therefore, SLA-defined pigs constitute an invaluable resource to study immune response, disease resistance and production traits, as well as an important large animal model for biomedical research [5, 6]. Three lines of commercial Yorkshire pigs with defined SLA-DRB1 genotypes were produced at the University of Guelph for xenotransplantation and immune response research [7, 8]. The aims of this study were to characterize the SLA-DRB1 alleles in these three pig lines and explore differential transcriptional activity between the three groups using heterologous (bovine probes – porcine targets) cDNA microarray and qPCR.
Animals and samples
Animal use was approved by the Animal Care Committee of the University of Guelph. Thirty-five pigs were included in the study (n = 6 for microarray analysis and n = 29 for qPCR). Pigs came from crossings of an outbred population selected for only by specific SLA-DRB1 alleles. Age of pigs ranged between 3–6 months and all pigs were in good general health at the time of sampling. Venous blood was collected in EDTA coated BD Vacutainer® collection tubes (BD – Canada, Oakville, ON, Canada) and processed immediately after collection. MNCs were isolated using Histopaque-1077 (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) and total RNA was extracted using TRIzol™ reagent (Invitrogen Canada Inc., Burlington, ON, Canada). Total RNA was treated with DNA-free (Ambion Inc., TX, USA) to eliminate genomic DNA contamination. Concentration and quality were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA).
SLA-DRB1 alleles characterization
cDNA Microarray experiments
Summary of results from cDNA microarray and qPCR data analysis
Comparison A: SLA-DRB1 alleles 0502/04ns01
Comparison B: SLA-DRB1 alleles 0502/0701
Comparison C: SLA-DRB1 alleles 0701/04ns01
Numerous associations have been established in swine between SLA haplotypes and features such as immune response and disease [16, 17], reproduction  and production traits . Many of these traits are not directly regulated by individual SLA genes but could rather be under the influence of non-classical MHC genes or controlled by downstream pathways yet to be described. The involvement of other closely linked genes, whose variants are in linkage disequilibrium (LD) can not be discarded [20, 21]. For example, it has been found that differential expression of LTB (also known as TNF beta) in MHC class II-defined B cell lines is associated with certain MHC class II haplotypes but not others. This association could be explained by LD between LTB and MHC haplotypes or by the influence of polymorphism in the MHC class II molecules and their interactions on the control of gene expression . Another example is represented by BRD2 in humans. This transcription factor, without an established immune function and located in the MHC class II region, is strongly linked to the MHC in most vertebrates .
Although it is not possible from the results in this study to establish a direct causal relationship between particular SLA-DRB1 alleles and differential transcription of inflammatory genes observed, it is undeniable that there seems to be an association. These observations will be better explained by the characterization of the haplotypes linked to these alleles and further exploration of the immune response in animals with defined MHC haplotypes.
Gene-specific primers and PCR conditions for relative quantification in the Light Cycler system
Primers (5'-> 3') b
Prod. size (bp) c
Ann. temp. (°C) d
Acq. temp. (°C) e
List of abbreviations
- LTB :
Lymphotoxin beta (TNF superfamily, member 3)
- BRD2 :
Bromodomain containing 2
- CCL4 :
Chemokine (C-C motif) ligand 4
- IL1B :
Interleukin 1 beta
- SLA-DQA :
SLA class II DQ alpha
- TLR2 :
Toll-like receptor 2
- CASP1 :
- RPL19 :
Ribosomal protein L19, EBI IPD, European Bioinformatics Institute Immuno-Polymorphism Database
Basic Local Alignment Search Tool
Reverse transcription – polymerase chain reaction
Swine leukocyte antigen
Major histocompatibility complex
Blood mononuclear cells
Gene Expression Omnibus
Minimum information about a microarray experiment
Locally weighted scatter plot smoothing algorithm
False Discovery Rate.
We wish to acknowledge the financial support of Ontario Pork Producers to BAM and the Government of Iran to RJJ. The assistance of the personnel at Arkell Research Station (University of Guelph), technical support of Sophia Lim, and assistance in statistical analysis of William Sears are greatly appreciated.
- Ando A, Chardon P: Gene organization and polymorphism of the swine major histocompatibility complex. Anim Sci J. 2006, 77: 127-133. 10.1111/j.1740-0929.2006.00331.x.View ArticleGoogle Scholar
- Kumanovics A, Takada T, Lindahl KF: Genomic organization of the mammalian MHC. Annu Rev Immunol. 2003, 21: 629-657. 10.1146/annurev.immunol.21.090501.080116.View ArticlePubMedGoogle Scholar
- Smith DM, Lunney JK, Ho CS, Martens GW, Ando A, Lee JH, Schook L, Renard C, Chardon P: Nomenclature for factors of the swine leukocyte antigen class II system, 2005. Tissue Antigens. 2005, 66: 623-639. 10.1111/j.1399-0039.2005.00492.x.View ArticlePubMedGoogle Scholar
- Vaiman M, Chardon P, Rothschild MF: Porcine major histocompatibility complex. Rev Sci Tech. 1998, 17: 95-107.PubMedGoogle Scholar
- Ho CS, Rochelle ES, Martens GW, Schook LB, Smith DM: Characterization of swine leukocyte antigen polymorphism by sequence-based and PCR-SSP methods in Meishan pigs. Immunogenetics. 2006, 58: 873-882. 10.1007/s00251-006-0145-y.View ArticlePubMedGoogle Scholar
- Lunney JK: Advances in swine biomedical model genomics. Int J Biol Sci. 2007, 3: 179-184.PubMed CentralView ArticlePubMedGoogle Scholar
- Bridle BW, Wilkie BN, Jevnikar AM, Mallard BA: Rat genotype influences quantitative and qualitative aspects of xenogeneic immune response to pig blood mononuclear cells. Xenotransplantation. 2006, 13: 299-307. 10.1111/j.1399-3089.2006.00306.x.View ArticlePubMedGoogle Scholar
- Bridle BW, Wilkie BN, Jevnikar AM, Mallard BA: Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells. Transpl Immunol. 2007, 17: 262-270. 10.1016/j.trim.2007.01.010.View ArticlePubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410.View ArticlePubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralView ArticlePubMedGoogle Scholar
- Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J, Ansorge W, Ball CA, Causton HC, Gaasterland T, Glenisson P, Holstege FC, Kim IF, Markowitz V, Matese JC, Parkinson H, Robinson A, Sarkans U, Schulze-Kremer S, Stewart J, Taylor R, Vilo J, Vingron M: Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001, 29: 365-371. 10.1038/ng1201-365.View ArticlePubMedGoogle Scholar
- Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP: Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002, 30: e15-10.1093/nar/30.4.e15.PubMed CentralView ArticlePubMedGoogle Scholar
- Benjamini Y, Hochberg Y: Controlling the false discovery rate: a practical and powerful approach to multiple testing. J Roy Statistic Soc Ser B. 1995, 57: 289-300.Google Scholar
- Tao W, Mallard B, Karrow N, Bridle B: Construction and application of a bovine immune-endocrine cDNA microarray. Vet Immunol Immunopathol. 2004, 101: 1-17. 10.1016/j.vetimm.2003.10.011.View ArticlePubMedGoogle Scholar
- Buckley BA: Comparative environmental genomics in non-model species: using heterologous hybridization to DNA-based microarrays. J Exp Biol. 2007, 210: 1602-1606. 10.1242/jeb.002402.View ArticlePubMedGoogle Scholar
- Lumsden JS, Kennedy BW, Mallard BA, Wilkie BN: The influence of the swine major histocompatibility genes on antibody and cell-mediated immune responses to immunization with an aromatic-dependent mutant of Salmonella typhimurium. Can J Vet Res. 1993, 57: 14-18.PubMed CentralPubMedGoogle Scholar
- Madden KB, Moeller RF, Douglass LW, Goldman T, Lunney JK: Trichinella spiralis: genetic basis and kinetics of the anti-encysted muscle larval response in miniature swine. Exp Parasitol. 1993, 77: 23-35. 10.1006/expr.1993.1057.View ArticlePubMedGoogle Scholar
- Renard C, Vaiman M: Possible relationships between SLA and porcine reproduction. Reprod Nutr Dev. 1989, 29: 569-576. 10.1051/rnd:19890506.View ArticlePubMedGoogle Scholar
- Mallard BA, Kennedy BW, Wilkie BN: The effect of swine leukocyte antigen haplotype on birth and weaning weights in miniature pigs and the role of statistical analysis in this estimation. J Anim Sci. 1991, 69: 559-564.PubMedGoogle Scholar
- Dorak MT, Shao W, Machulla HK, Lobashevsky ES, Tang J, Park MH, Kaslow RA: Conserved extended haplotypes of the major histocompatibility complex: further characterization. Genes Immun. 2006, 7: 450-467. 10.1038/sj.gene.6364315.View ArticlePubMedGoogle Scholar
- Trowsdale J: HLA genomics in the third millennium. Curr Opin Immunol. 2005, 17: 498-504.View ArticlePubMedGoogle Scholar
- Guo W, Mourad W, Charron D, Al Daccak R: Ligation of MHC class II molecules differentially upregulates TNF beta gene expression in B cell lines of different MHC class II haplotypes. Hum Immunol. 1999, 60: 312-322. 10.1016/S0198-8859(98)00131-1.View ArticlePubMedGoogle Scholar
- Shiina T, Inoko H, Kulski JK: An update of the HLA genomic region, locus information and disease associations: 2004. Tissue Antigens. 2004, 64: 631-649. 10.1111/j.1399-0039.2004.00327.x.View ArticlePubMedGoogle Scholar
- Littell RC, Milliken GA, Stroup WW, Wolfinger RD: SAS System for Mixed Models. Edited by: Inc. SASI. 1996, Cary, NC, USA, 656.Google Scholar