Un<em> In Vitro</em> Modelo para medir la respuesta inmune a la malaria en el contexto del VIH Co-infección
Summary
Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.
Abstract
Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood.
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility.
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.
Introduction
Co-infección, la infección con múltiples infecciones concurrentes, es la norma en los ambientes naturales. Co-infección puede tener un impacto importante en la patología de la enfermedad y en el manejo clínico de cada infección. En el contexto de la co-infección, la vacuna y la eficacia del medicamento, así como las pruebas de diagnóstico, puede ser afectado negativamente (revisado en 1). Sin embargo, a pesar de su importancia, la mayoría de las investigaciones de patógenos considera infecciones solamente individuales.
La malaria y el VIH-1 (VIH) son las principales causas de morbilidad y mortalidad a nivel mundial. Áreas de la malaria y el VIH endemicidad comparten una amplia superposición geográfica, poniendo a millones de personas en riesgo de coinfección y consecuentemente en riesgo de enfermedad clínica más grave 2-10. Las dos enfermedades interactúan negativamente. En las personas infectadas por el VIH, las cargas virales más altas de VIH y disminuciones temporales en los recuentos de células T CD4 + se puede ver durante una infección de la malaria, mientras quecargas de parásitos de la malaria y el riesgo de malaria clínica y grave son más altos en los individuos coinfectados 2,3,5,7,8,10. Los mecanismos por los que el VIH aumenta la gravedad de la malaria no se entienden completamente y garantiza una mayor investigación.
Aquí se describe un método por el cual la malaria y la co-infección con el VIH puede ser estudiado in vitro. En concreto, este método permite el examen de las respuestas inmunitarias donde la malaria es específica en el contexto de la infección por el VIH. Nuestro protocolo describe un sistema versátil co-cultivo utilizando recién aisladas células mononucleares de sangre periférica (PBMCs) aisladas de donantes crónicamente infectados por el VIH in vitro y se cultivaron P. falciparum parasitada eritrocitos (PfRBC). El impacto de la terapia antirretroviral contra el VIH en estas respuestas también puede ser examinada utilizando PBMCs recogidos prospectivamente del VIH (+) donantes antes y después de la terapia.
Hemos utilizado este sistema para investigar el impacto de la infección por VIHen la respuesta inmune innata la malaria específico 11,12, y fueron capaces de determinar que IFN y TNF respuestas donde la malaria es específica se deterioran en las células NK, células NKT, delta gamma células T de VIH (+) donantes pre y post tratamiento antirretroviral del VIH . Además, hemos sido capaces de utilizar este sistema para determinar que las funciones de monocitos también se vean afectados en donantes VIH (+), pero se recuperan después de VIH el tratamiento antirretroviral.
Protocol
Representative Results
Discussion
Nuestro protocolo se ha optimizado con el fin de estudiar más realista co-infección por el VIH in vitro de la malaria. En primer lugar, se requiere que los eritrocitos humanos frescos y suero para la cultura parásito de la malaria. Esto es vital para obtener una población saludable de parásitos de la malaria. Lisados de parásitos no pueden ser sustituidos por parásitos vivos como la producción de citoquinas es mucho más rápida e intensa al utilizar P. en vivo falciparum infecta…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
C.A.M.F. and L.S. participated in protocol design, acquisition and analysis of data, and drafting of the article.
The authors wish to thank Dr. Kain, Dr. Loutfy, Dr. Wasmuth, and Dr. Ayi for their contributions.
C.F. was supported by a CTN/Ontario HIV Treatment Network (OHTN) postdoctoral fellowship. L.S. is supported by an OHTN Junior Investigator Development Award. The present work was supported by a Canadian Institutes of Health Research (CIHR) operating grant (MOP-13721 and 115160), and a CIHR New Investigator Catalyst grant.
Materials
| alanine | Sigma | A7377 | |
| antibodies (see other table) | |||
| BD Cytofix/Cytoperm with BD GolgiPlug | BD | 555028 | includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer |
| BD Vacutainer ACD Solution A | BD | 364506 | |
| BD Vacutainer Sodium Heparin | BD | 17-1440-02 | |
| DPBS (no calcium, no magnesium) | Corning | 21-031-CV | |
| Fetal Bovine Serum | Sigma | F1051 | heat inactivate before use |
| Ficoll-Paque PLUS | GE Healthcare | 17-1440-02 | |
| gentamycin (10mg/ml) | Gibco | 15710-064 | |
| Hema3 Staining Set | Fisher | 122-911 | |
| HEPES | Fisher | BP310-500 | |
| hypoxanthine | Sigma | H9636 | |
| Ionomycin | Sigma | I3909 | |
| MEM non-essential amino acids (10mM) | Gibco | 11140 | |
| PMA | Sigma | P8139 | |
| RPMI-1640 powder | Life-Technologies | 31800-022 | |
| RPMI-1640 with L-glutamine and HEPES | Thermo Scientific | SH30255.01 | |
| sodium bicarbonate (powder, cell culture) | Sigma | S5761 | |
| Sodium Pyruvate (100mM) | Gibco | 11360 | |
| Tris (Trizma base) | Sigma | T6066 | |
| Trizol | Ambion | 15596018 | |
| Trypan Blue (0.4%) | Gibco | 15250-061 | |
| BD CompBead | BD | 552843, 552845 | depends on antibodies used |
| Parasite Gas Mixture | By special order | 3% CO2, 1% O2, balance N2 |
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