The effects of anti-Fas ribozyme on T lymphocyte apoptosis in mice model with chronic obstructive pulmonary disease

Document Type : Original Article

Authors

1 Department of Respiratory Medicine, the Affiliated Shenzhen Longgang Center Hospital, Zunyi Medical University, Shenzhen City, Guangdong Province, China

2 Zhuhai Campus of Zunyi Medical University, Zhuhai City, Guangdong Province, China

Abstract

Objective(s): In this study, we aimed to investigate the effects of anti-Fas ribozyme on the apoptosis of T lymphocytes (T cells) in mice model with chronic obstructive pulmonary disease (COPD).
Materials and Methods: Male 6-week-old C57BL/6 mice were used to establish the COPD model by exposure to cigarette smoke. The COPD mice were sacrificed for spleen dissection and T cell isolation. T cells were randomly divided into four groups (n=10 per group). Group A was used as the control. B, C, and D groups were transfected with empty lentivirus, anti-Fas ribozyme, and an anti-Fas ribozyme mutant, respectively. The expression of Fas mRNA and protein in the T cells were evaluated using qPCR and Western blot, respectively. Flow cytometry was used to evaluate the apoptosis of CD4T cells and calculate the ratio of CD4+ to CD8+ T cells (CD4/CD8).
Results: Anti-Fas ribozyme significantly inhibited the expression of Fas in the T cells of COPD mice. In addition, the number of apoptotic CD4 T cells and CD4/CD8of the C and D groups were significantly lower and higher than those of group A, respectively (P<0.05). The apoptotic CD4T cells and CD4CD8of the C group were significantly lower and higher than those of group D, respectively (P<0.05).
Conclusion: Anti-Fas ribozyme significantly inhibited the expression of Fas, increased CD4+/ CD8+, and inhibited the apoptosis of T cells in COPD mice.

Keywords


1. Cazzola M, Page CP, Calzetta L, Matera MG. Emerging anti-inflammatory strategies for COPD. Eur Respir J 2012; 40:724-741.
2. Barnes PJ. Chronic obstructive pulmonary disease. N Engl J Med 2000; 343:269-280.
3. Schikowski T, Mills IC, Anderson HR, Cohen A, Hansell A, Kauffmann F, et al. Ambient air pollution: A cause of COPD. Eur Respir J 2014; 43:250-263.
4. Spurzem JR, Rennard SI. Pathogenesis of COPD. Semin Respir Crit Care Med 2005; 26:142-153.
5. Górska K, Krenke R, Kosciuch J, Korczynski P, Zukowska M, Domagalakulawik J, et al. Relationship between airway inflammation and remodeling in patients with asthma and chronic obstructive pulmonary disease. Eur J Med Res 2009; 14:1-7.
6. Saha S, Brightling CE. Eosinophilic airway inflammation in COPD. Int J Chron Obstruct Pulmon Dis 2006; 1:39-47.
7. Kurts C, Carbone FR, Barnden M, Blanas E, Allison J, Heath WR, et al. CD4+ T cell help impairs CD8+ T cell deletion induced by cross-presentation of self-antigens and favors autoimmunity. J Exp Med 1997; 186:2057-2062.
8. Roos-Engstrand E. Increased intraepithelial CD4+ and CD8+ T-Cells in COPD. Respir Med:COPD Update 2007; 3:124.
9. Yin YM, Qin J, Dai YP, Zeng FW, Pei H, Wang J. The CD4+/CD8+ ratio in pulmonary tuberculosis: systematic and meta-analysis article. Iran J Pub Health 2015; 44:185-193.
10. Sampson AP. The role of eosinophils and neutrophils in inflammation. Clin Exp Allergy 2000; 30:22–27.
11. Geering B, Stoeckle C, Conus S, Simon HU. Living and dying for inflammation: neutrophils, eosinophils, basophils. Trends Immunol 2013; 34:398-409.
12. Hasgall P, Bellehsen L, Hammel I, Amichai D, Levischaffer F. In Vitro exposure to cigarette smoke activates eosinophils: implications for lung inflammation. Internet J Asthma Allergy Immunol 2006; 5: 1-12.
13. Finkelstein R, Fraser RS, Ghezzo H, Cosio MG. Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med 1995; 152:1666-1672.
14. Siegel RM, Martin DA, Hornung F, Dudley EC, Zheng L, Lenardo MJ. Signaling Through Fas (CD95, APO-1) and Related Death Receptors: Humana Press; 2000.p. 135-152.
15. Nagata S. Fas-induced apoptosis. Inter Med 1998; 37:179-181.
16. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 1995; 270:1189-1192.
17. Gratas C, Tohma Y, Barnas C, Taniere P, Hainaut P, Ohgaki H. Up regulation of Fas (APO-1/CD95) Ligand and down-regulation of Fas expression in human esophageal cancer. Cancer Res 1998; 58:2057-2062.
18. Chio CC, Wang YS, Chen YL, Lin SJ, Yang BC. Down-regulation of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumour-infiltrating cells, and liver damage but accelerates tumour formation in nude mice. Br J Cancer 2001; 85:1185-1192.
19. Du Z, Ricordi C, Inverardi L, Podack E, Pastori RL. Efficient ex vivo inhibition of perforin and Fas ligand expression by chimeric tRNA-hammerhead ribozymes. Hum Gene Ther 1998; 9:1551-1560.
20. Bühling F, Wille A, Röcken C, Wiesner O, Baier A, Meinecke I, et al. Altered expression of membrane-bound and soluble CD95/Fas contributes to the resistance of fibrotic lung fibroblasts to FasL induced apoptosis. Respir Res 2005; 6:1-9.
21. Nielsen H. Group I intron ribozymes. Methods Mol Biol 2012; 848:873.
22. Enyeart PJ, Mohr G, Ellington AD, Lambowitz AM. Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis. Mobile DNA 2014; 5:1-19.
23. Trang P, Kim K, Liu F. Developing RNase P ribozymes for gene-targeting and antiviral therapy. Cell Microbiol 2004; 6:499–508.
24. Usman N, Beigelman L, Mcswiggen JA. Hammerhead ribozyme engineering. Curr Opin Struct Biol 1996; 6:527-533.
25. Yu M, Ojwang J, Yamada O, Hampel A, Rapapport J, Looney D, et al. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc Nat Acad Sci U S A 1993; 90:6340-6344.
26. Ghorani V, Boskabady MH, Khazdair MR, Kianmeher M. Experimental animal models for COPD: a methodological review. Tob Induc Dis 2017; 15:25.
27. Mallia P, Johnston SL. Mechanisms and experimental models of chronic obstructive pulmonary disease exacerbations. Proc Am Thorac Soc 2005; 2:371-372.
28. Demoor T, Bracke KR, Joos GF, Brusselle GG. Increased T-regulatory cells in lungs and draining lymph nodes in a murine model of COPD. Eur Respir J 2010; 35:688-689.
29. Wollin L, Pieper MP. Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD. Pulmon Pharmacol Ther 2010; 23:345-354.
30. Kacherovsky N, Liu GW, Jensen MC, Pun SH. Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection. Biotechnol Bioeng 2015; 112:1429-1436.
31. Churg A, Cosio M, Wright JL. Mechanisms of cigarette smoke-induced COPD: insights from animal models. Am J Physiol Lung Cell Mol Physiol 2008; 294:612-631.
32. Wright JL, Churg A. Animal models of cigarette smoke-induced COPD. Chest 2002; 122:301S-36S.
33. Curtis JL. At the Checkpoint: lung CD8(+) T Cells, respiratory viruses, and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2016; 193:600-602.
34. Zhuo S, Xu W, Liang S, Zhang C, Ye C, Yu H, et al. Relationship between the apoptosis of T lymphocyte subsets and the expression of Fas, FasL and Bcl-2 in patients with COPD. China J Clin  2011; 5:1554-1560.
35. Yang Y, Li Y. The study of oxidant, antioxidant status of patiants with chronic obstructive pulmonary disease (in Chinese). Pract J Cardiac Cereb Pneumal Vasc Dis 2007; 15:85-86.