Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (3): 574-581.doi: 10.3864/j.issn.0578-1752.2017.03.016

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Growth and Expression of NOD2 mRNA in Bovine Mammary Epithelial Cells Treated with Different Concentrations of MDP in Vitro

XU DanDan, WANG JianFa, ZHANG Xu, LIU DongYu, XU XiaoNan, WANG Le, CHEN Jia, SHAN XuFei, WANG XiaoYa, WU Rui, YANG Bin   

  1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, Heilongjiang
  • Received:2016-04-13 Online:2017-02-01 Published:2017-02-01

Abstract: 【Objective】 Dairy cow mastitis is one of the most common diseases causing serious economic losses in dairy-farming industry. Bacterial infection is the main cause of mastitis. Innate immunity is the first line of defense against the invasion of pathogenic bacteria in mammary gland. NOD2 is an important member of the innate immune pattern recognition receptor of nucleotide-binding oligomerization domain (NOD) family, which defenses against various microbial invasions by recognizing its specific ligand-muramyl dipeptide (MDP), a component widely existing in gram positive bacteria and gram negative bacteria cell wall. Bovine mammary epithelial cell (BMEC) is the immune barrier of dairy cow mammary gland other than secreting milk. Here, the effect of MDP on the in vitro growth state of BMEC and the expression ofNOD2 in the BMEC was explored in this experiment.【Method】 Mammary gland tissue of healthy and lactating Holstein cows was chosen as raw materials. Collagenase digestion method combined with concentration gradient of trypsin was used to separate BMEC. Cytokeratin-18 specific expression in epithelial cells and vimentin specific expression in fibroblasts were used to identify the obtained cells by immunofluorescent techniques. BMEC was set to 6 treatment groups, including MDP stimulating concentrations of 0 (control group), 1, 5, 10, 15 and 20 μg·mL-1. Twenty-four hours of poststimulation, BMEC status were observed under a microscope, meanwhile total RNA was extracted from BMEC and reverse transcribed to cDNA. Real time fluorescent quantitative PCR method was used to detect the expression of NOD2 in BMEC.【Result】Those cells separated by collagenase digestion method combined with concentration gradient of trypsin, immunofluorescence results of CK-18 reaction was positive and vimentin reaction was negative. All cells were in a good growth condition. In the control group and in the groups of MDP stimulating concentration at 1, 5 and 10 μg·mL-1, BMEC grew well without any visible abnormalities. There was a small amount of BMEC detached from bottom in the group of MDP stimulating concentration at 15 μg·mL-1. However, the group of MDP stimulating concentration at 20 μg·mL-1 showed a large number of BMEC detached and floated from bottom. Even though those BMEC were still attached to the bottom, their morphology had already changed. Compared with the control group, the expression of NOD2 mRNA in BMEC was positively correlated with the stimulating concentrations of MDP. In other words, 24 h of poststimulation, the expression of NOD2 mRNA in BMEC gradually increased along with the stimulating concentrations of MDP.【Conclusion】High purity BMEC was successfully obtained. The obtained cells grew well and could be used in the following experiments. Although the expression of NOD2 mRNA was positively correlated with the stimulating concentrations of MDP, the stimulating concentrations of MDP in vitro culture BMEC should be controlled below 10 μg·mL-1 in order to maintain the normal growth condition. These results suggested that BMEC could participate in the immune defense response of bovine mammary gland through the NOD2 receptor pathway. But this defense capability was influenced by the number of bacteria or the intensity of bacterial virulence. In a certain number or virulence of bacteria, the immune defense response of bovine mammary gland was enhanced along with the increasing number of bacteria or the enhancement of virulence to eliminate intramammary pathogens. While the number or virulence of bacteria exceeded to a certain range, bovine mammary gland tissue would be seriously damaged, so the immune defense barrier would be collapsed. Under this condition, the local bovine mammary gland or even all over the body would present obvious clinical symptoms.

Key words: bovine mammary epithelial cell, MDP, NOD2

[1]    CANNING P, RUAN Q, SCHWERD T, HRDINKA M, MAKI J L, SALEH D, SUEBSUWONG C, RAY S, BRENNAN P E, CUNY G D, UHLIG H H, GYRD H M, DEGTEREV A, BULLOCK A N. Inflammatory signaling by NOD-RIPK2 Is Inhibited by clinically relevant type II kinase inhibitors. Chemistry & Biology, 2015, 22(9): 1174-1184.
[2]    TAMAKI Y, SHOICHIRO K. Intracellular recognition of pathogens and autophagy as an innate immune host defence. Journal of Biochemistry, 2011, 150(2): 143-149.
[3]    TRAVASSOS L H, CARNEIRO L A, GIRARDIN S, PHILPOTT D J. Nod proteins link bacterial sensing and autophagy. Autophagy, 2010, 6(3): 409-411.
[4]    PORCHERIE A, CUNHA P, TROTEREAU A, ROUSSEL P, GILBERT FB, RAINARD P, GERMON P. Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine  udder and mammary epithelial cells. Veterinary Research, 2012, 43(1): 1-8.
[5]    ZHU Y H, LIU P Q, WENG X G, ZHUGE Z Y, ZHANG R, MA J L, QIU X Q, LI R Q, ZHANG X L,WANG J F. Short communication: Pheromonicin-SA affects mRNA expression of toll-like receptors, cytokines, and lactoferrin by Staphylococcus aureus-infected bovine mammary epithelial cells. Journal of Dairy Science, 2012, 95(2): 759-764.
[6]    WHELEHAN C J, MEADE K G, ECKERSALL P D. Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression. Veterinary Immunology and Immunopathology, 2011, 140(3/4): 181-189.
[7]    刘晓云, 郑家珍, 王艳青, 裴建秋, 樊爱萍. 牛乳腺上皮细胞的快速分离和培养. 河北大学学报(自然科学版), 2015, 35(4): 385-389.
LIU X Y, ZHENG J Z, WANG Y Q, PEI J Q, FAN A P. Isolation and culture of bovine mammary epithelial cell. Journal of Hebei University (Natural Science Edition), 2015, 35(4): 385-389. (in Chinese)
[8]    詹康, 贡笑笑, 左晓昕, 陈银银, 占今舜, 赵国琦. 奶牛乳腺上皮细胞系的培养与鉴定. 动物营养学报, 2015(8): 2544-2550.
ZHAN K, GONG X X, ZUO X X, CHEN Y Y, ZHAN J S, ZHAO G Q. Culture and identification of the bovine mammary epithelial cell line. Chinese Journal of Animal Nutrition, 2015(8): 2544-2550. (in Chinese)
[9]    WU Q, LIU M C, YANG J, WANG J F, ZHU Y H. Lactobacillus rhamnosus GR-1 ameliorates Escherichia coli-Induced inflammation and cell damage via attenuation of ASC-independent NLRP3 inflammasome activation. Applied and Environmental Microbiology, 2015, 82(4): 1173-1182.
[10]   NAOHIRO I, GABRIEL N. NODs: intracellular proteins involved in inflammation and apoptosis. Nature Reviews Immunology, 2003, 3(5): 371-382.
[11]   WARREN S, MURRAY P J, ATSUSHI K, TOMOHIRO W. Signalling pathways and molecular interactions of NOD1 and NOD2. Nature Reviews Immunology, 2006, 6(1): 9-20.
[12]   PETR H, ZINKERNAGEL A S, GABRIELA J, BOTWIN G J, JEAN-PIERRE H, MICHAEL K, VICTOR N, LARS E. NOD2 contributes to cutaneous defense against Staphylococcus aureus through α-toxin-dependent innate immune activation. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(31): 12873-12878.
[13]   CORREA R G, MILUTINOVIC S, REED J C. Roles of NOD1 (NLRC1) and NOD2 (NLRC2) in innate immunity and inflammatory diseases. Bioscience Reports, 2012, 32(6): 597-608.
[14]   KAORU G, RUBINO S J, MAGALHAES J G, CATHERINE S, LIONEL L B, CHO J H, ROBERTSON S J, KIM C J, RUPERT K, PHILPOTT D J, GIRARDIN S E. Identification of an innate T helper type 17 response to intestinal bacterial pathogens. Nature Medicine, 2011, 17(7): 837-844.
[15]   BASTIAN O, ANJA P, BERND S, ANDREAS C H, SIMONE R, SVEN H, RALF R S, NORBERT S, STEFAN H. Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized Streptococcus pneumoniae. Journal of Biological Chemistry, 2004, 279(35): 36426-36432.
[16]   ERAN E, TILL S, JORGE H M, RICHARD A F. Regulation of the antimicrobial response by NLR proteins. Immunity, 2011, 34(5): 665-679.
[17]   NIGRO G, FAZIO L L, MARTINO M C, ROSSI G, TATTOLI L, LIPAROTI V, CRISTINA D C, MOLINARO A, DANA J P, MARIA L B. Muramylpeptide shedding modulates cell sensing of Shigella flexneri. Cellular Microbiology, 2008, 10(3): 682–695.
[18]   KIM Y G, PARK J H, SHAW M H, FRANCHI L, INOHARA N, GABRIEL N. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recognition and host defense after exposure to toll-like receptor ligands. Immunity, 2008, 28(2): 246-257.
[19]   WARREN S E, MAO D P, RODRIGUEZ A E, MIAO E A, ALAN A. Multiple Nod-like receptors activate caspase 1 during Listeria monocytogenes infection. Journal of Immunology, 2008, 180(11): 7558-7564.
[20]   BERRINGTON W R, RAVI I, WELLS R D, SMITH K D, SKERRETT S J, HAWN T R. NOD1 and NOD2 regulation of pulmonary innate immunity to legionella pneumophila. European Journal of Immunology, 2010, 40(12): 3519-3527.
[21]   KENICHI S, SHUANG C, DEMPSEY P W, ROSALINDA S, RANDA A, SLEPENKIN A V, ELLENA P, DOHERTY T M, DAVID U, CROTHER T R, MOSHE A. The NOD/RIP2 pathway is essential for host defenses against Chlamydophila pneumoniae lung infection. Plos Pathogens, 2009, 5(4): 1243-1248.
[22]   TAKEUCHI O, AKIRA S. Pattern recognition receptors and inflammation. Cell, 2010, 140(6): 805-820.
[23]   SHAW M H, REIMER T, SÁNCHEZ-VALDEPEÑAS C, WARNER N, KIM Y G, FRESNO M, NUÑEZ C. T cell intrinsic role of Nod2 in promoting type 1 immunity to Toxoplasma gondii. Nature Immunology, 2009, 10(12): 1267-1274.
[24]   BRAIN O, ALLAN P, SIMMONS A. NOD2-mediated autophagy and Crohn disease. Autophagy, 2010, 6(3): 412-414.
[25]   YAMAMOTO-FURUSHO J K, KORZENIK J R. Crohn's disease: Innate immunodeficiency? World Journal of Gastroenterology, 2006, 12(42): 6751-6755.
[26]   HUGOT J P, CHAMAILLARD M, ZOUALI H, LESAGE S, CÉZARD J P, BELAICHE J, ALMER S, TYSK C, O'MORAIN C A, GASSULL M, BINDER V, FINKEL Y, CORTOT A, MODIGLIANI R, PIERRE L P, CORINE G R, MACRY J, COLOMBEL J F, SAHBATOU M, THOMAS G. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature, 2001, 411(6837): 599-603.
[27]   LAM C, MAGALHAES J G, TATTOLI I, PHILPOTT D J, TRAVASSOS L H. NOD-like proteins in inflammation and disease. Journal of Pathology, 2008, 214(2): 136-148.
[28]   FRANCHI L, PARK J H, SHAW M H, MARINA G N, CHEN G, KIM Y G, NUNEZ G. Intracellular NOD-like receptors in innate immunity, infection and disease. Cellular Microbiology, 2008, 10(1): 1-8.
[29]   崔新洁, 胡庆亮, 李奕平, 陶琳, 修磊, 刘秉春, 陈媛, 王潇. 金黄色葡萄球菌诱导牛原代乳腺上皮细胞的凋亡. 中国农业科学, 2013, 46(15): 3212-3219.
CUI X J, HU Q L, LI L P, TAO L, XIU L, LIU B C, CHEN Y, WANG X. The apoptosis of bovine primary mammary epithelial cells induced by Staphylococcus aureus. Scientia Agricultura Sinica, 2013, 46(15): 3212-3219. (in Chinese)
[30]   BOUGARN S, CUNHA P, HARMACHE A, FROMAGEAU A, GILBERT F B, RAINARD P. Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells. Clinical and Vaccine Immunology, 2010, 17(11): 1797-809.
[31]   GILBERT F B, CUNHA P, JENSEN K, GLASS E J, FOUCRAS G, ROBERT-GRANIÉ C, RUPP R, RAINARD P. Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. Veterinary Research, 2013, 44(1): 1-23.
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