Un<em> In Vitro</em> Modèle de mesure de réponses immunitaires contre le paludisme dans le contexte du VIH co-infection
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-infection, l'infection par de multiples infections simultanées, est la norme dans les milieux naturels. Co-infection peut avoir un impact majeur sur la pathologie de la maladie et sur la prise en charge clinique de chaque infection. Dans le cadre de la co-infection, le vaccin et l'efficacité des médicaments, ainsi que des tests de diagnostic, peut être eu une incidence négative (revue pt 1). Cependant, malgré son importance, la majorité des recherches de l'agent pathogène considère seulement les infections simples.
Le paludisme et le VIH-1 (VIH) sont les principales causes de morbidité et de mortalité dans le monde. Les zones de paludisme endémique et VIH partagent un large chevauchement géographique, en mettant des millions de personnes à risque de co-infection et par conséquent un risque de maladie clinique plus sévère 2 – 10. Les deux maladies interagissent négativement. Chez les personnes infectées par le VIH, la hausse des charges virales de VIH et des diminutions temporaires de CD4 + lymphocytes T peut être vu lors d'une infection par le paludisme, tandis quepaludisme charges parasitaires et risque de paludisme clinique et grave sont plus élevés dans les individus co-infectés 2,3,5,7,8,10. Les mécanismes par lesquels le VIH augmente la gravité du paludisme ne sont pas entièrement comprises et justifient une enquête plus approfondie.
Nous décrivons ici une méthode par laquelle le paludisme et la co-infection VIH peut être étudiée de vitro. Plus précisément, cette méthode permet à l'examen des réponses immunitaires spécifiques contre le paludisme dans le contexte de l'infection à VIH. Notre protocole décrit un système polyvalent de co-culture en utilisant des cellules mononucléaires de sang périphérique fraîchement isolées (PBMC) isolées de donneurs infectés chroniquement par le VIH in vitro et en culture P. falciparum parasité érythrocytes (PfRBC). L'impact du traitement antirétroviral sur ces réponses peut également être examiné en utilisant PBMC recueillies de façon prospective du VIH (+) donateurs pré- et post-traitement.
Nous avons utilisé ce système pour étudier l'impact de l'infection à VIHsur les réponses immunitaires innées spécifiques au paludisme 11,12, et ont pu déterminer que IFN-y et TNF réponses spécifiques au paludisme sont altérées dans les cellules NK, les cellules NKT, yô cellules du VIH (+) donateurs pré- et post-traitement antirétroviral VIH T . En outre, nous avons été en mesure d'utiliser ce système pour déterminer que les fonctions monocytaires sont également affaiblies par le VIH (+) les bailleurs de fonds, mais récupérer post-VIH thérapie antirétrovirale.
Protocol
Representative Results
Discussion
Notre protocole a été optimisé afin d'étudier plus réaliste la co-infection VIH-de paludisme in vitro. Tout d'abord, les globules rouges humains frais et le sérum sont nécessaires pour la culture du parasite du paludisme. Ceci est essentiel pour obtenir une population saine des parasites du paludisme. Lysats parasite ne peut pas être remplacé par des parasites vivants que la production de cytokines est beaucoup plus rapide et intense lors de l'utilisation en direct P. falciparum in…
Declarações
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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