Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (3): 583-593.doi: 10.3864/j.issn.0578-1752.2015.03.17

• ANIMAL SCIENCE·VETERINARY SCIENCERE • Previous Articles     Next Articles

Impact of Concentrations and Immune-stimulating Times of Poly I:C on Gene Expression of Porcine Peripheral Blood Mononuclear Cells

WANG Ji-ying1, WANG Yan-ping1, WANG Huai-zhong1, WANG Hai-fei2, LIU Jian-feng2, GUO Jian-feng1   

  1. 1Shandong Key Laboratory of Animal Disease Control and Breeding/Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100
    2Key Laboratory of Animal Genetics, Breeding and Reproduction, Ministry of Agriculture/College of Animal Science and Technology, China Agricultural University, Beijing 100193
  • Received:2014-03-21 Online:2015-01-31 Published:2015-01-31

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Abstract: 【Objective】 Polyinosinic:polycytidylic acid (Poly I:C) is a synthetic double-stranded polyribonucleotide. As an analogue of viral double-stranded RNA, Poly I:C is widely used as immunologic stimulant to study the immune regulation and inflammatory reaction of an organism to the RNA viruses. So far, no studies were reported on determination of the optional concentration and stimulating time of porcine peripheral blood mononuclear cells (PBMC) in response to Poly I:C stimulation. In the present study, we systematically analyzed the impact of Poly I:C concentrations and stimulating times on the expression of several cytokines and pattern recognition receptors and the optional concentration and stimulating time of Poly I:C in the PBMC were determined, which would provide an experimental foundation for the future use of Poly I:C and porcine PBMC to study the immune response of pigs to RNA viruses. 【Method】 Using isolated PBMC and 1:5 EDTA-anticoagulated diluted blood of three Landrace piglets, the in vitro immune stimulating experiment was performed with a series of Poly I:C concentrations (0, 10, 20 and 40 μg·mL-1) and immune-stimulating culture times (4 h, 8 h, 12 h and 24 h). The expression of several cytokines (IL6, IL8, TNFα, IL10, IRF3, IFNα and IFNγ) and pattern recognition receptors (TLR3 and TLR4) were determined by qPCR. According to the relative expression trends of these cytokines and receptors, it was analyzed to achieve the optimal Poly I:C concentration and immune-stimulating culture time. 【Result】 The expression of the cytokines and receptors were affected by Poly I:C concentrations as well as immune-stimulating times. Each gene had its characteristic expression trend, and varied in Poly I:C concentration and immune-stimulating time to attain the highest expression. For the two interferon genes, IFNα and IFNγ, the highest expressions were observed when the stimulating time was 4 h, and their expression decreased gradually with the stimulating time. For the other five cytokines (IL6, IL8, TNFα, IL10 and IRF3) and two receptor genes (TLR3 and TLR4), the expressions were gradually increased with the Poly I:C concentrations and stimulation times, and reached the highest when Poly I:C concentrations and stimulation times were 20-40 μg·mL-1 and 12-24 h, respectively. However, for the genes whose expressions were highest when Poly I:C concentration and stimulation time were 20 μg·mL-1 and 12 h, their expressions showed small decline when Poly I:C concentration and stimulation time were 40 μg·mL-1 and 24 h. Furthermore, in the study, it was also tested the expressions of the above genes of 1:5 EDTA-anticoagulated diluted blood in response to various Poly I:C concentrations and stimulation times. The results indicated that the whole expressions of these genes were lower than those in PBMC, especially for two interleukin genes, IL6 and IL8, whose expression in diluted whole blood was not only low but the variation trends with Poly I:C concentrations and stimulation time were reversed with those in PBMC. Compared with PBMC, though diluted blood preserved the integrity of the immune state of the pig’s blood, the major drawback is that the anticoagulant is kept in it. The anticoagulant used in the study was EDTAK2, which can combine with calcium ion to form chelate, so as to prevent blood clotting. However, calcium ion is an important signal molecule in cells, and the combination of calcium ion with EDTA may have a certain impact on the subsequent cell function. In addition, some of the compounds and plasma proteins in whole blood may be also involved in the regulation of immune cells to Poly I:C immune stimulation so as to reduce immune response. 【Conclusion】 Comprehensive analysis of the expression trends of the cytokines and receptors were detected, and it was concluded that, in the porcine PBMC stimulation experiment using Poly I:C, the optimal concentration and immune-stimulating time are 20 μg·mL-1 and 24 hours, respectively. EDTA- anticoagulated diluted blood has less immune response to Poly I:C and different variation trends with Poly I:C concentrations and stimulation times.

Key words: pig, polyinosinic-cytidylic acid (Poly I:C), porcine peripheral blood mononuclear cell (PBMC), gene expression

[1]    Reiner G, Eckert J, Peischl T, Bochert S, J kel T, Mackenstedt U, Joachim A, Daugschies A, Geldermann H. Variation in clinical and parasitological traits in Pietrain and Meishan pigs infected with Sarcocystis miescheriana. Veterinary Parasitology, 2002, 106(2): 99-113.
[2]    Flori L, Gao Y, Lalo D, Lemonnier G, Leplat J J, Teillaud A, Cossalter A M, Laffitte J, Pinton P, de Vaureix C. Immunity traits in pigs: Substantial genetic variation and limited covariation. PLoS One, 2011, 6(7): e22717.
[3]    Hunter D J. Gene-environment interactions in human diseases. Nature Reviews Genetics, 2005, 6(4): 287-298.
[4]    Meurens F, Summerfield A, Nauwynck H, Saif L, Gerdts V. The pig: a model for human infectious diseases. Trends in Microbiology, 2012, 20(1): 50-57.
[5]    Lion E, Anguille S, Berneman Z N, Smits E L, Van Tendeloo V F. Poly (I: C) enhances the susceptibility of leukemic cells to NK cell cytotoxicity and phagocytosis by DC. PLoS One, 2011, 6(6): e20952.
[6]    Trapp S, Derby N R, Singer R, Shaw A, Williams V G, Turville S G, Bess J W, Lifson J D, Robbiani M. Double-stranded RNA analog poly (I: C) inhibits human immunodeficiency virus amplification in dendritic cells via type I interferon-mediated activation of APOBEC3G. Journal of Virology, 2009, 83(2): 884-895.
[7]    Kato H, Takeuchi O, Sato S, Yoneyama M, Yamamoto M, Matsui K, Uematsu S, Jung A, Kawai T, Ishii K J, Yamaguchi O, Otsu K, Tsujimura T, Koh C S, Reis e Sousa C, Matsuura Y, Fujita T, Akira S. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature, 2006, 441(7089): 101-105.
[8]    Matsumoto M, Seya T. TLR3: interferon induction by double-stranded RNA including poly (I: C). Advanced Drug Delivery Reviews, 2008, 60(7): 805-812.
[9]    Gao Y, Flori L, Lecardonnel J, Esquerré D, Hu ZL, Teillaud A, Lemonnier G, Lefèvre F, Oswald I, Rogel-Gaillard C. Transcriptome analysis of porcine PBMCs after in vitro stimulation by LPS or PMA/ionomycin using an expression array targeting the pig immune response. BMC Genomics, 2010, 11: 292.
[10]   Mian M F, Stampfli M R, Mossman K L, Ashkar A A. Cigarette smoke attenuation of poly I: C-induced innate antiviral responses in human PBMC is mainly due to inhibition of IFN-beta production. Molecular Immunology, 2009, 46(5): 821-829.
[11]   王继英. 猪免疫性状的全基因组关联分析及拷贝数变异的检测[D]. 北京: 中国农业大学, 2012.
Wang J Y. Genome-wide association studies for immune traits and detection of copy number variations in swine [D]. Beijing: China Agricultural University, 2009. (in Chinese)
[12]   Cao Y, Lu Z, Li Y, Sun P, Li D, Li P, Bai X, Fu Y, Bao H, Zhou C, Xie B, Chen Y, Liu Z. Poly (I: C) combined with multi-epitope protein vaccine completely protects against virulent foot-and-mouth disease virus challenge in pigs. Antiviral Research, 2013, 97(2): 145-153.
[13]   Wang X, Ao H, Zhai L, Bai L, He W, Yu Y, Wang C. Genome-wide effects of DNA methyltransferase inhibitor on gene expression in double-stranded RNA transfected porcine PK15 cells. Genomics, 2013, 103(5-6):371-379.
[14]   Liu X, Huang J, Yang S, Zhao Y, Xiang A, Cao J, Fan B, Wu Z, Zhao J, Zhao S, Zhu M. Whole blood transcriptome comparison of pigs with extreme production of in vivo dsRNA-induced serum IFN-a. Developmental and Comparative Immunology, 2014, 44(1): 35-43.
[15]   Sorensen N S, Skovgaard K, Heegaard P M. Porcine blood mononuclear cell cytokine responses to PAMP molecules: comparison of mRNA and protein production. Veterinary Immunology and Immunopathology, 2011, 139(2): 296-302.
[16]   Uddin M J, Nuro-Gyina P K, Islam M A, Tesfaye D, Tholen E, Looft C, Schellander K, Cinar M U. Expression dynamics of Toll-like receptors mRNA and cytokines in porcine peripheral blood mononuclear cells stimulated by bacterial lipopolysaccharide. Veterinary Immunology and Immunopathology, 2012, 147(3/4): 211-222.
[17]   Skovgaard K, Mortensen S, Poulsen K T, Angen Ø, Heegaard P M. Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae. Veterinary Immunology and Immunopathology, 2007, 118(1): 140-146.
[18]   Wikström F H, Fossum C, Fuxler L, Kruse R, Lövgren T. Cytokine induction by immunostimulatory DNA in porcine PBMC is impaired by a hairpin forming sequence motif from the genome of Porcine Circovirus type 2 (PCV2). Veterinary Immunology and Immunopathology, 2011, 139(2): 156-166.
[19]   Martino A, Cabiati M, Campan M, Prescimone T, Minocci D, Caselli C, Rossi A M, Giannessi D, Del Ry S. Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-α mRNA expression. Journal of Biotechnology, 2011, 153(3/4): 92-99.
[20]. Cinar M U, Islam M A, Uddin M J, Tholen E, Tesfaye D, Looft C, Schellander K. Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA. BMC Research Notes, 2012(5): 107.
[21]. Wang J, Wang Y, Wang H, Hao X, Wu Y, Guo J. Selection of reference genes for gene expression studies in porcine whole blood and PBMC under poly I:C stimulation. Asian-Australasian Journal of Animal Sciences, 2014, 27(4): 471-478.
[22]   Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta (CT)) method. Methods, 2001, 25(4): 402-408.
[23]   盛秀胜. 全血培养对体外培养 PBMC 细胞凋亡的影响. 温州医学院学报, 2005, 35(1): 66-68.
Sheng X S. Effects of whole blood to apoptosis of PBMC in vitro. Journal of Wenzhou Medical College, 2005, 35(1): 66-68. (in Chinese)
[24]   Yaqoob P, Newsholme E A, Calder P C. Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells. Cytokine, 1999, 11(8): 600-605.
[25]   Damsgaard C T, Lauritzen L, Calder P C, Kjær T M, Frøkiær H. Whole-blood culture is a valid low-cost method to measure monocytic cytokines-a comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes. Journal of Immunological Methods, 2009, 340(2): 95-101.
[26]   Dolmetsch R E, Lewis R S, Goodnow C C, Healy J I. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature, 1997, 386(6627): 855-858.
[27]   Feske S, Giltnane J, Dolmetsch R, Staudt L M, Rao A. Gene regulation mediated by calcium signals in T lymphocytes. Nature Immunology, 2001, 2(4): 316-324.
[28] Duvigneau J C, Sipos W, Hartl R T, Bayer M, Moldzio R, Stevenson L, Adair B, Gemeiner M. Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells. Journal of Immunological Methods, 2007, 324(1/2): 38-47.
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