En la Evaluación in vivo de roedores Plasmodium parasitemia y Merozoite Invasión por Citometría de Flujo
Summary
Los invade parásito de la malaria y se replica dentro de las células rojas de la sangre. La evaluación precisa de la invasión merozoite y parasitemia es por lo tanto crucial para evaluar el curso de la infección por malaria. Aquí se describe un protocolo basado en citometría de flujo para la medición de estos parámetros en un modelo de ratón de la malaria.
Abstract
During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.
Introduction
The clinical symptoms associated with malaria occur during the Plasmodium parasite’s asexual replicative cycle within red blood cells (RBCs). Merozoites, released during the liver stage of infection, quickly attach to and invade RBCs. After gaining entry into the cell, the parasite grows and matures, eventually undergoing schizogony, splitting open the cell, and releasing a cluster of newly formed merozoites which go on to repeat this cycle. As such, an assessment of malaria infection often involves monitoring both parasitemia, which is the percentage of RBCs appropriated by one or more parasites, and the rate of merozoite invasion into uninfected RBCs.
Flow cytometry is a powerful tool which can be used to record the properties of vast numbers of cells in a short period of time. This technique has clear applicability for the measurement of malaria parasitemia and invasion, and offers several advantages over traditional microscopy techniques. These include the accurate measurement of very low parasitemia, which would be prohibitively time consuming by microscopy, the unbiased nature of the measurement, and the ability to measure multiple cell parameters simultaneously. Flow cytometry is widely used to determine both parasitemia and merozoite invasion in in vitro culture1-9, however, techniques for measuring these parameters in vivo are less well developed, and can be complicated by the presence of additional cell types which interfere with analysis. No assays have been described for measurement of in vivo invasion, and while some assays exist for the analysis of in vivo parasitemia, these lack the ability to distinguish between parasitized RBCs (pRBCs) and RBCs containing Howell-Jolly bodies (HJ-RBCs)10-13. The later issue is particularly important as in mice HJ-RBCs may account for up to 0.9% of mature RBCs14-16, thereby preventing the accurate measurement of low parasitemia.
We have previously demonstrated an approach for the measurement of parasitemia and merozoite invasion in a rodent model of malaria infection14. Here, we provide a more detailed protocol and accompanying video. This approach builds on previous methodologies and allows for the accurate identification of parasitized RBCs, as distinct from leukocytes, RBC progenitors, and HJ-RBCs. Additionally, this assay allows the simultaneous measurement of merozoite invasion into two labeled RBC populations, a treated, or target, population, and a control population, thereby providing a robust platform for the assessment of invasion into different cell types.
Protocol
Representative Results
Discussion
Hemos descrito un método para la medición tanto de parasitemia y merozoite invasión de muestras in vivo. En términos de medición parasitemia, este método ofrece una ventaja sobre los métodos anteriores 10-13 en que HJ-glóbulos rojos pueden distinguirse de pRBCs, reduciendo así el número de eventos positivos falsos. Mientras HJ-glóbulos rojos son generalmente raros en los seres humanos, algunos estudios informan altos niveles en los ratones 15,16 hacer la distinción entre estas …
Divulgations
The authors have nothing to disclose.
Acknowledgements
Reconocemos el apoyo financiero de la Salud y medicina Consejo Nacional de Investigación (subvención APP605524, 490.037 y 1.047.082), el Consejo Australiano de Investigación (subvención DP12010061), la Estrategia de Infraestructuras de Investigación Cooperativa Nacional de Australia y el fondo de inversión de Educación del Departamento de Innovación, Industria , Ciencia e Investigación. PML es un receptor de un premio de postgrado australiano.
Materials
| bisBenzimide H 33342 trihydrochloride | Sigma-Aldrich | B2261 | Hoechst 33342. Store a 4mM stock solution at -20 °C in distilled water |
| Hoechst 34580 | Sigma-Aldrich | 63493 | Store a 2mM stock solution at -20 °C in distilled water |
| JC-1 Dye | Life Technologies | T-3168 | Store small aliquots of 6mM stock solution at -20 °C in DMSO |
| Anti-Mouse CD45 APC-eFluor 780 | eBioscience | 47-0451-80 | Clone 30-F11 |
| Anti-Mouse CD71 PerCP-eFluor 710 | eBioscience | 46-0711-80 | Clone R17217 |
| Atto 633 NHS ester | Sigma-Aldrich | 1464 | Atto 633-NHS. Store a 2mg/ml stock solution at -20 °C in DMF |
| EZ-Link Sulfo-NHS-LC-Biotin | Thermo Fisher Scientific | 21335 | Biotin-NHS. Store a 25mg/ml stock solution at -20 °C in DMF |
| Streptavidin PE-Cyanine7 | eBioscience | 25-4317-82 | Streptavidin PE-Cy7 |
| Heparin | Sigma-Aldrich | H478 | |
| 35µM filter cap tubes | Becton Dickinson | 352235 | |
| Flow cytometer: BD LSRFortessa | Becton Dickinson | ||
| Flow cytometer: BD FACSAria II | Becton Dickinson | ||
| Flow cytometer: BD Influx | Becton Dickinson | ||
| Flow cytometer: CyAn ADP Analyzer | Beckman Coulter |
References
- Jacobberger, J. W., Horan, P. K., Hare, J. D. Analysis of malaria parasite-infected blood by flow cytometry. Cytometry. 4 (3), 228-237 (1983).
- Bianco, A. E., Battye, F. L., Brown, G. V. Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry. Exp Parasitol. 62 (2), 275-282 (1986).
- Makler, M. T., Lee, L. G., Recktenwald, D. Thiazole orange: a new dye for Plasmodium species analysis. Cytometry. 8 (6), 568-570 (1987).
- Heyde, H. C., Elloso, M. M., van de Waa, J., Schell, K., Weidanz, W. P. Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum. Clin Diagn Lab Immunol. 2 (4), 417-425 (1995).
- Pattanapanyasat, K., et al. Culture of malaria parasites in two different red blood cell populations using biotin and flow cytometry. Cytometry. 25 (3), 287-294 (1996).
- Grimberg, B. T., Erickson, J. J., Sramkoski, R. M., Jacobberger, J. W., Zimmerman, P. A. Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry A. 73 (6), 546-554 (2008).
- Theron, M., Hesketh, R. L., Subramanian, S., Rayner, J. C. An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites. Cytometry A. 77 (11), 1067-1074 (2010).
- Bei, A. K., et al. A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum. Am J Hematol. 85 (4), 234-237 (2010).
- Clark, M. A., et al. RBC barcoding allows for the study of erythrocyte population dynamics and P. falciparum merozoite invasion. PLoS One. 9 (7), e101041 (2014).
- Malleret, B., et al. A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development. Sci Rep. 1 (118), (2011).
- Jimenez-Diaz, M. B., et al. Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Cytometry A. 75 (3), 225-235 (2009).
- Jimenez-Diaz, M. B., et al. Improvement of detection specificity of Plasmodium-infected murine erythrocytes by flow cytometry using autofluorescence and YOYO-1. Cytometry A. 67 (1), 27-36 (2005).
- Jun, G., Lee, J. S., Jung, Y. J., Park, J. W. Quantitative determination of Plasmodium parasitemia by flow cytometry and microscopy. J Korean Med Sci. 27 (10), 1137-1142 (2012).
- Lelliott, P. M., Lampkin, S., McMorran, B. J., Foote, S. J., Burgio, G. A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo. Malar J. 13 (1), 100 (2014).
- Morohashi, K., et al. Structural and functional abnormalities in the spleen of an mFtz-F1 gene-disrupted mouse. Blood. 93 (5), 1586-1594 (1999).
- Shet, A. S., et al. Morphological and functional platelet abnormalities in Berkeley sickle cell mice. Blood Cells Mol Dis. 41 (1), 109-118 (2008).
- Duraisingh, M. T., et al. Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes. EMBO J. 22 (5), 1047-1057 (2003).
- Okoyeh, J. N., Pillai, C. R., Chitnis, C. E. Plasmodium falciparum field isolates commonly use erythrocyte invasion pathways that are independent of sialic acid residues of glycophorin A. Infect Immun. 67 (11), 5784-5791 (1999).
- Dolan, S. A., Miller, L. H., Wellems, T. E. Evidence for a switching mechanism in the invasion of erythrocytes by Plasmodium falciparum. J Clin Invest. 86 (2), 618-624 (1990).
