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中国生物工程杂志

China Biotechnology
China Biotechnology  2011, Vol. 31 Issue (12): 57-62    DOI:
    
Ectopic Expression of Trim22 Down-regulates Pro-inflammatory Cytokines Production in LPS-stimulated Macrophage-like Cells
SUN Da-kang, AN Xin-ye, ZHOU Xiao-sheng, LI Meng, XUE Yan
Experiment Center of Clinical Medicine, Affiliated Hospital of Binzhou Medical University, Binzhou 256603, China
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Abstract  

Objective: To construct recombinant retrovirus vectors carrying human Trim22 gene, which could be stably expressed in U937 cell line, and to study the effects of Trim22 on pro-inflammatory cytokines production in LPS-stimulated macrophage-like cells. Methods: Trim22 gene was amplified with PCR and subcloned into retroviral vector (MSCV2.2 IRES-GFP). The recombinant vector was confirmed by restriction enzyme digestion, colony PCR and sequencing. The retroviral vector together with GAG-POL and VSV-G helper vectors were co-transfected into the packaging cells 293T by Lipofectamine. The supernatant of 293T cells was used to infect U937 cells and GFP positive U937 cells stably expressing Trim22 were sorted by flow cytometry. Eventually, PMA was used to differentiate U937 cells into macrophages and then LPS-induced pro-inflammatory cytokine levels were measured by ELISA. Results: Trim22 gene was successfully constructed into retrovirus vectors MSCV2.2 IRES-GFP, and the cloning site and reading frame were confirmed by enzyme digestion, colony PCR and sequencing. U937 cells stably expressing Trim22 and GFP were obtained by flow cytometry and were revealed by RT-PCR. Ectopic expression of Trim22 severely attenuated TNFα, IL6 production in LPS-stimulated macrophages derived from U937 cells. Conclusion: Trim22 gene retroviral vector was successfully constructed, and Trim22 could down-regulate LPS-mediated TNF-α, IL6 expression in macrophages-like cells.



Key wordsTrim22      Retrovirus vectors      Pro-inflammatory cytokines     
Received: 10 August 2011      Published: 25 December 2011
ZTFLH:  R392.11  
Cite this article:

SUN Da-kang, AN Xin-ye, ZHOU Xiao-sheng, LI Meng, XUE Yan. Ectopic Expression of Trim22 Down-regulates Pro-inflammatory Cytokines Production in LPS-stimulated Macrophage-like Cells. China Biotechnology, 2011, 31(12): 57-62.

URL:

https://manu60.magtech.com.cn/biotech/     OR     https://manu60.magtech.com.cn/biotech/Y2011/V31/I12/57


[1] McNab F W, Rajsbaum R, Stoye J P, et al. Tripartite-motif proteins and innate immune regulation. Curr Opin Immunol, 2011, 23(1):46-56.

[2] Niida M, Tanaka M, Kamitani T. Downregulation of active IKK beta by Ro52-mediated autophagy. Mol Immunol, 2010, 47(14):2378-2387.

[3] Shi M, Deng W, Bi E, et al. TRIM30 alpha negatively regulates TLR-mediated NF-kappa B activation by targeting TAB2 and TAB3 for degradation. Nat Immunol, 2008, 9(4):369-377.

[4] 孙大康, 安新业, 郑静, 等. Trim5α对TAB2诱导的NFκB启动子荧光素酶报告基因活性的影响. 免疫学杂志, 2011, (07):580-584. Sun D K, An X Y, Zhen J, et al. Immunology, 2011, (07):580-584.

[5] Singh R, Gaiha G, Werner L, et al. Association of TRIM22 with the type 1 interferon response and viral control during primary HIV-1 infection. J Virol, 2011, 85(1):208-216.

[6] Ohmine S, Sakuma R, Sakuma T, et al. The antiviral spectra of TRIM5alpha orthologues and human TRIM family proteins against lentiviral production. PLoS One, 2011, 6(1):e16121.

[7] Tachado S D, Li X, Swan K, et al. Constitutive activation of phosphatidylinositol 3-kinase signaling pathway down-regulates TLR4-mediated tumor necrosis factor-alpha release in alveolar macrophages from asymptomatic HIV-positive persons in vitro. J Biol Chem, 2008, 283(48):33191-33198.

[8] Yarilina A, DiCarlo E, Ivashkiv L B. Suppression of the effector phase of inflammatory arthritis by double-stranded RNA is mediated by type I IFNs. J Immunol, 2007, 178(4):2204-2211.

[9] Mirandola S R, Hallal D E, Farias A S, et al. Interferon-beta modifies the peripheral blood cell cytokine secretion in patients with multiple sclerosis. Int Immunopharmacol, 2009, 9(7-8):824-830.

[10] Carthagena L, Bergamaschi A, Luna J M, et al. Human TRIM gene expression in response to interferons. PLoS One, 2009, 4(3):e4894.

[11] Rajsbaum R, Stoye J P, O'Garra A. Type I interferon-dependent and-independent expression of tripartite motif proteins in immune cells. Eur J Immunol, 2008, 38(3):619-630.

[12] Yoshimi R, Chang T H, Wang H, et al. Gene disruption study reveals a nonredundant role for TRIM21/Ro52 in NF-kappaB-dependent cytokine expression in fibroblasts. J Immunol, 2009, 182(12):7527-7538.

[13] Eldin P, Papon L, Oteiza A, et al. TRIM22 E3 ubiquitin ligase activity is required to mediate antiviral activity against encephalomyocarditis virus. J Gen Virol, 2009, 90(Pt 3):536-545.

[14] Gao B, Duan Z, Xu W, et al. Tripartite motif-containing 22 inhibits the activity of hepatitis B virus core promoter, which is dependent on nuclear-located RING domain. Hepatology, 2009, 50(2):424-433.

[15] Deng Y J, Huang Z X, Zhou C J, et al. Gene profiling involved in immature CD4+ T lymphocyte responsible for systemic lupus erythematosus. Mol Immunol, 2006, 43(9):1497-1507.

[16] Kim J Y, Ozato K. The sequestosome 1/p62 attenuates cytokine gene expression in activated macrophages by inhibiting IFN regulatory factor 8 and TNF receptor-associated factor 6/NF-kappaB activity. J Immunol, 2009, 182(4):2131-2140.

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