Hostname: page-component-cd9895bd7-hc48f Total loading time: 0 Render date: 2024-12-18T19:54:47.341Z Has data issue: false hasContentIssue false
  • English
  • Français

Functional evaluation of gene silencing on macrophages derived from U937 cells using interference RNA (shRNA) in a model of macrophages infected with Leishmania (Viannia) braziliensis

Published online by Cambridge University Press:  07 October 2015

CLEMENCIA OVALLE-BRACHO ,
DIANA A. LONDOÑO-BARBOSA ,
CARLOS FRANCO-MUÑOZ  and
CARLOS CLAVIJO-RAMÍREZ
CLEMENCIA OVALLE-BRACHO*
Affiliation:
Centro Dermatológico Federico Lleras Acosta, Avenida 1 # 13ª-61, Bogotá D.C., Colombia Facultad de Medicina, Universidad Nacional de Colombia, Carrera 45 # 26-85, Bogotá D.C., Colombia
DIANA A. LONDOÑO-BARBOSA
Affiliation:
Centro Dermatológico Federico Lleras Acosta, Avenida 1 # 13ª-61, Bogotá D.C., Colombia
CARLOS FRANCO-MUÑOZ
Affiliation:
Centro Dermatológico Federico Lleras Acosta, Avenida 1 # 13ª-61, Bogotá D.C., Colombia
CARLOS CLAVIJO-RAMÍREZ
Affiliation:
Departamento de Biología, Universidad Nacional de Colombia, Carrera 45 # 26-85, Bogotá D.C., Colombia
*
*Corresponding Author: Clemencia Ovalle-Bracho, Centro Dermatológico Federico Lleras Acosta, Avenida 1 # 13ª-61, Bogotá D.C., Colombia. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Leishmaniasis development is multifactorial; nonetheless, the establishment of the infection, which occurs by the survival and replication of the parasite inside its main host cell, the macrophage, is mandatory. Thus, the importance of studying the molecular mechanisms involved in the Leishmania–macrophage interaction is highlighted. The aim of this study was to characterize a cellular model of macrophages derived from U937 cells that would allow for the identification of infection phenotypes induced by genetic silencing with interference RNA in the context of macrophages infected with Leishmania (Viannia) braziliensis. The model was standardized by silencing an exogenous gene (gfp), an endogenous gene (lmna) and a differentially expressed gene between infected and non-infected macrophages (gro-β). The silencing process was successful for the three genes studied, obtaining reductions of 88·9% in the GFP levels, 87·5% in LMNA levels and 74·4% for Gro-β with respect to the corresponding control cell lines. The cell model revealed changes in the infection phenotype of the macrophages in terms of number of amastigotes per infected macrophage, number of amastigotes per sampled macrophage and percentage of infected macrophages as a result of gene silencing. Thus, this cell model constitutes a research platform for the study of parasite–host interactions and for the identification of potentially therapeutic targets.

Keywords

Leishmaniamonocyte-derived macrophagesgene silencingRNAsmall interferingU937 cells
Type
Research Article
Information
Parasitology , Volume 142 , Issue 14 , December 2015 , pp. 1682 - 1692
Copyright
Copyright © Cambridge University Press 2015 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Alvar, J., Velez, I. D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J. and den Boer, M. (2012). Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 7, e35671.CrossRefGoogle ScholarPubMed
Biosciences, L.-C. (2013). Normalization Accuracy for Western Blotting. 1–10.Google Scholar
Carpenter, A. E., Jones, T. R., Lamprecht, M. R., Clarke, C., Kang, I. H., Friman, O., Guertin, D. A., Chang, J. H., Lindquist, R. A., Moffat, J., Golland, P. and Sabatini, D. M. (2006). CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology 7, R100.Google Scholar
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.Google Scholar
Elbashir, S. M., Harborth, J., Weber, K. and Tuschl, T. (2002). Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26, 199–213.Google Scholar
Fellmann, C. and Lowe, S. W. (2014). Stable RNA interference rules for silencing. Nature Cell Biology 16, 10–18.Google Scholar
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. and Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. Journal of Cell Science 114, 4557–4565.Google Scholar
Harborth, J., Elbashir, S. M., Vandenburgh, K., Manninga, H., Scaringe, S. A., Weber, K. and Tuschl, T. (2003). Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense and Nucleic Acid Drug Development 13, 83–105.Google Scholar
Hsiao, C. H., Ueno, N., Shao, J. Q., Schroeder, K. R., Moore, K. C., Donelson, J. E. and Wilson, M. E. (2011). The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania . Microbes and Infection 13, 1033–1044.CrossRefGoogle ScholarPubMed
Loria-Cervera, E. N. and Andrade-Narvaez, F. J. (2014). Animal models for the study of leishmaniasis immunology. Revista Do Instituto De Medicina Tropical De Sao Paulo 56, 1–11.CrossRefGoogle Scholar
Maia, C., Rolao, N., Nunes, M., Goncalves, L. and Campino, L. (2007). Infectivity of five different types of macrophages by Leishmania infantum . Acta Tropica 103, 150–155.Google Scholar
Martinet, W., Schrijvers, D. M. and Kockx, M. M. (2003). Nucleofection as an efficient nonviral transfection method for human monocytic cells. Biotechnology Letters 25, 1025–1029.Google Scholar
McManus, M. T. and Sharp, P. A. (2002). Gene silencing in mammals by small interfering RNAs. Nature Reviews Genetics 3, 737–747.Google Scholar
Mookerjee Basu, J., Mookerjee, A., Banerjee, R., Saha, M., Singh, S., Naskar, K., Tripathy, G., Sinha, P. K., Pandey, K., Sundar, S., Bimal, S., Das, P. K., Choudhuri, S. K. and Roy, S. (2008). Inhibition of ABC transporters abolishes antimony resistance in Leishmania infection. Antimicrobial Agents and Chemotherapy 52, 1080–1093.Google Scholar
Ovalle-Bracho, C., Franco-Munoz, C., Londono-Barbosa, D., Restrepo-Montoya, D. and Clavijo-Ramirez, C. (2015). Changes in macrophage gene expression associated with Leishmania (Viannia) braziliensis infection. PLoS ONE 10, e0128934.Google Scholar
Paul, C. P., Good, P. D., Winer, I. and Engelke, D. R. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnology 20, 505–508.Google Scholar
Rao, D. D., Vorhies, J. S., Senzer, N. and Nemunaitis, J. (2009). siRNA vs. shRNA: similarities and differences. Advance Drug Delivery Reviews 61, 746–759.Google Scholar
Reynolds, A., Anderson, E. M., Vermeulen, A., Fedorov, Y., Robinson, K., Leake, D., Karpilow, J., Marshall, W. S. and Khvorova, A. (2006). Induction of the interferon response by siRNA is cell type- and duplex length-dependent. RNA 12, 988–993.CrossRefGoogle ScholarPubMed
Rosas, L. E., Keiser, T., Pyles, R., Durbin, J. and Satoskar, A. R. (2003). Development of protective immunity against cutaneous leishmaniasis is dependent on STAT1-mediated IFN signaling pathway. European Journal of Immunology 33, 1799–1805.Google Scholar
Santhosh, C., Tamhane, M. C., Kamat, R. H., Patel, V. V. and Mukhopadhyaya, R. (2008). A lentiviral vector with novel multiple cloning sites: stable transgene expression in vitro and in vivo . Biochemical and Biophysical Research Communications 371, 546–550.Google Scholar
Tietz, S. M. and Berghoff, M. (2012). Gene silencing of MK2 in hard-to-transfect human U937 cells. Journal of Biomolecular Techniques 23, 47–50.Google Scholar
Tulac, S., Dosiou, C., Suchanek, E. and Giudice, L. C. (2004). Silencing lamin A/C in human endometrial stromal cells: a model to investigate endometrial gene function and regulation. Molecular Human Reproduction 10, 705–711.Google Scholar
Van De Parre, T. J., Martinet, W. and De Meyer, G. R. (2007). mRNA transfection in monocytes and macrophages. In New Messenger RNA Research Communications (ed. Kwang, L. B.). Nova Science Publishers, Inc., Hauppauge, NY, pp. 181–190.Google Scholar
Supplementary material: File

Ovalle-Bracho supplementary material S1

Ovalle-Bracho supplementary material

Download Ovalle-Bracho supplementary material S1(File)
File 429.1 KB