Brug af RNA-interferens Undersøge Innate immunresponset i musemakrofager
Summary
In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.
Abstract
Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.
Introduction
I denne protokol, beskriver vi effektive metoder til at hæmme gener i mus makrofagcellelinier anvender siRNAs og overvåge virkningerne af disse behandlinger på medfødte immunreaktion. Disse procedurer udføres i 96-brønds format, hvilket muliggør RNAi skærme i mellem- eller høj-throughput fashion.
Som reaktion på infektion, mennesker montere en øjeblikkelig medfødte immunreaktion og en reaktion langsommere, men mere specifikt adaptive immunrespons. Denne respons hurtig medfødte immunforsvar involverer rekruttering og aktivering af fagocytiske medfødte immunceller, herunder makrofager 1. Klassisk aktiverede makrofager er involveret i akutte inflammatoriske reaktioner og producere antimikrobielle proteiner og forbindelser, cytokiner og chemokiner, og præsentere antigener 2,3. Alternativt aktiverede makrofager spiller en rolle i reguleringen af immunitet, opretholde tolerance, vævsreparation og sårheling 4-8. På grund af deres brede vifte af funktionerKan makrofager spille en rolle i mange sygdomme, herunder atherosclerose, arthritis og cancer 9. Således har studiet af makrofager været et centralt område for forskning i nogen tid i en lang række sygdomstilstande områder.
På trods af deres betydning i medfødte immunreaktion, kan makrofager være udfordrende celler til at arbejde med. Især er det vanskeligt at opnå effektiv transfektion ved hjælp af lipid reagenser i makrofager uden associeret toksicitet 10,11. Selv når siRNA effektivt leveret til makrofager kan robustheden af RNAi-induceret gen knockdown ofte være temmelig moderat og kan variere fra gen til genet.
For at overvinde disse tekniske udfordringer har vi optimeret transfektion og knockdown teknikker 12-16 i to mus makrofagcellelinier, RAW264.7 17 og J774A.1 18. Denne fremgangsmåde bruger Amaxa Nucleofector 96 brønde Shuttle System for transfektion; dette systemanvender en kombination af specialiserede reagenser og elektroporering til at transficere celler i 96-brønds format 19. Efter transfektion beskriver vi metoder til at maksimere celle genopretning og levedygtighed og for at maksimere efterfølgende siRNA-induceret gen knockdown. For at illustrere anvendeligheden af denne tilgang, beskriver vi en protokol for siRNA levering til disse makrofagcellelinier, stimulere disse celler med lipopolysaccharid (LPS), og overvåge respons på niveau med produktion af flere pro-inflammatoriske cytokiner medfødte immunforsvar. Vi leverer prøve data, hvor vi målretter Toll-lignende receptor (TLR) familie, hvis medlemmer fornemmer LPS og andre patogen forbundet molekylære mønstre (PAMPs), at regulere medfødte immunitet.
Protocol
Representative Results
Discussion
Talrige undersøgelser er blevet offentliggjort i de enkelte gener er blevet rettet af siRNA i murine makrofager. Mens lipidmedieret transfektion er blevet brugt til at levere siRNA til makrofagcellelinier på et individuelt grundlag, disse metoder lider potentielle virkninger på levedygtighed, begrænset gen knockdown, og variabilitet fra gen til gen. At udvikle mere robuste analyser, der kunne bruges til at målrette gener i mellem- eller high-throughput fashion, vi optimerede teknikker, der anvender den Amaxa nucleo…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.
Materials
| Amaxa nucleofector 96-well shuttle system | Lonza | AAM-1001S | |
| Amaxa SF cell line 96-well nucleofector kit | Lonza | V4SC-2096 | |
| RAW264.7 mouse macrophage cell line | ATCC | TIB-71 | |
| J774A.1 mouse macrophage cell line | ATCC | TIB-67 | |
| siGenome smartpool siRNA | Dharmacon | varies depending on gene | |
| Non-targeting control siRNA pool | Dharmacon | D-001206-13-20 | |
| Block-iT fluorescent oligo for electrooration | Invitrogen | 13750062 | |
| Ultrapure E. coli O111:B4 LPS | List Biological Laboratories | 421 | |
| DMEM, high glucose | Invitrogen | 11995-065 | |
| RPM1-1640 | Invitrogen | 11875-093 | |
| Penicillin-Streptomycin Solution (Pen/Strep) | Fisher | SV30010 | |
| 0.25% Trypsin-EDTA | Invitrogen | 25200072 | |
| 96 well tissue culture plates | Fisher | 07-200-89 | |
| 96 well round bottom sterile plates (not coated) | Fisher | 07-200-745 | |
| Mouse IL-6 Duoset ELISA kit | R&D Biosystems | DY406 | |
| Mouse TNFa Duoset ELISA kit | R&D Biosystems | DY410 | |
| Fluorescein diacetate | Sigma-Aldrich | F7378 | |
| RLT Bufer | Qiagen | 79216 | |
| Rneasy mini kit | Qiagen | 74134 | |
| Vybrant Phagocytosis Assay Kit | Invitrogen | V-6694 |
References
- Kaufmann, S. H. E., Medzhitov, R., Gordon, S. . The innate immune response to infection. , (2004).
- Adams, D. O., Hamilton, T. A. The cell biology of macrophage activation. Annual review of immunology. 2, 283-318 (1984).
- Fujiwara, N., Kobayashi, K. Macrophages in inflammation. Current drug targets. Inflammation and allergy. 4, 281-286 (2005).
- Gordon, S., Martinez, F. O. Alternative activation of macrophages: mechanism and functions. Immunity. 32, 593-604 (2010).
- Martinez, F. O., Helming, L., Gordon, S. Alternative activation of macrophages: an immunologic functional perspective. Annual review of immunology. 27, 451-483 (2009).
- Fairweather, D., Cihakova, D. Alternatively activated macrophages in infection and autoimmunity. Journal of autoimmunity. 33, 222-230 (2009).
- Benoit, M., Desnues, B., Mege, J. L. Macrophage polarization in bacterial infections. Journal of immunology. 181, 3733-3739 (2008).
- Mantovani, A., Sica, A., Locati, M. Macrophage polarization comes of age. Immunity. 23, 344-346 (2005).
- Burke, B., Lewis, C. E. . The macrophage. , (2002).
- Lee, G., Santat, L. A., Chang, M. S., Choi, S. RNAi methodologies for the functional study of signaling molecules. PloS one. 4, (2009).
- Carralot, J. P., et al. Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure. Journal of biomolecular screening. 14, 151-160 (2009).
- Alper, S., et al. Identification of innate immunity genes and pathways using a comparative genomics approach. ProcNatl Acad Sci USA. 105, 7016-7021 (2008).
- De Arras, L., et al. An evolutionarily conserved innate immunity protein interaction network. J. Bio. Chem. , 1967-1978 (2013).
- Yang, I. V., et al. Novel regulators of the systemic response to lipopolysaccharide. Am J Respir Cell Mol Biol. 45, 393-402 (2011).
- Yang, I. V., et al. Identification of novel innate immune genes by transcriptional profiling of macrophages stimulated with TLR ligands. Molecular immunology. 48, 1886-1895 (2011).
- Yang, I. V., et al. Identification of novel genes that mediate innate immunity using inbred mice. Génétique. 183, 1535-1544 (2009).
- Raschke, W. C., Baird, S., Ralph, P., Nakoinz, I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell. 15, 261-267 (1978).
- Ralph, P., Moore, M. A., Nilsson, K. Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. The Journal of experimental medicine. , 1528-1533 (1976).
- Zumbansen, M., et al. First siRNA library screening in hard-to-transfect HUVEC cells. Journal of RNAi and gene silencing. 6, 354-360 (2010).
- Lu, Y. C., Yeh, W. C., Ohashi, P. S. LPS/TLR4 signal transduction pathway. Cytokine. 42, 145-151 (2008).
- Aliprantis, A. O., et al. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science. 285, 736-739 (1999).
- Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature. 413, 732 (2001).
- Conforti, R., et al. Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands. Cancer Res. 70, 490-500 (2010).
- Fernandez-Botran, R., Větvička, V. . Methods in Cellular Immunology. 8, 8 (2001).
