Ein<em> In-vitro-</em> Modell zur Messung der Immunantwort auf Malaria im Kontext der HIV-Koinfektion
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-Infektion, Infektion mit mehreren gleichzeitigen Infektionen, ist die Norm in natürlichen Umgebungen. Co-Infektion kann einen großen Einfluss auf die Krankheitspathologie und der klinischen Behandlung von jeder Infektion. Im Zusammenhang mit der Ko-Infektion, Impfstoff und Wirksamkeit von Medikamenten sowie diagnostische Tests können negativ beeinflusst werden (Übersicht in 1). Trotz ihrer Bedeutung ist die Mehrheit der Erreger der Forschung ist jedoch der Auffassung nur einzelne Infektionen.
Malaria und HIV-1 (HIV) sind die führenden Ursachen von Morbidität und Mortalität weltweit. Bereiche von Malaria und HIV Endemizität teilen eine breite geografische Überschneidungen, indem Millionen von Menschen, die von Co-Infektion und somit ein Risiko für schwerere klinische Erkrankung 2-10. Die beiden Krankheiten negativ interagieren. In HIV-infizierten Personen, höhere HIV-Viruslast und vorübergehende Abnahmen der CD4 + T-Zellzahlen während einer Malaria-Infektion zu sehen, währendMalariaparasiten Lasten und Risiko von klinischen und schwerer Malaria sind in co-infizierten Personen 2,3,5,7,8,10 höher. Die Mechanismen, durch die HIV erhöht Malaria Schweregrad sind nicht vollständig verstanden und rechtfertigen weitere Untersuchungen.
Hier beschreiben wir ein Verfahren, mit dem Malaria und HIV-Koinfektion kann in vitro untersucht werden. Insbesondere ermöglicht dieses Verfahren für die Untersuchung von Malaria-spezifischen Immunantworten im Zusammenhang mit HIV-Infektion. Unser Protokoll beschreibt ein vielseitiges Kokultursystem frisch isolierten peripheren mononukleären Blutzellen (PBMCs) von chronisch HIV-infizierten Spendern und in vitro kultivierten P. isoliert falciparum parasitierten Erythrozyten (PfRBC). Die Auswirkungen von HIV antiretroviralen Therapie auf diesen Reaktionen können auch unter Verwendung prospektiv erfasst PBMCs von HIV (+) Spender vor und nach der Behandlung untersucht werden.
Hat dieses System verwendet, um den Einfluss der HIV-Infektion zu untersuchenMalaria-spezifischen angeborenen Immunantwort 11,12 und waren in der Lage, um festzustellen, dass die Malaria-spezifische IFNy und TNF-Antworten werden in NK-Zellen beeinträchtigt, NKT-Zellen, γδ T-Zellen von HIV (+) Spender vor und nach der HIV antiretroviralen Therapie . Darüber hinaus konnten wir dieses System verwenden, um festzustellen, dass Monozyten-Funktionen werden auch in HIV (+) Spendern beeinträchtigt, aber erholen post HIV antiretroviralen Therapie.
Protocol
Representative Results
Discussion
Unser Protokoll wurde entwickelt, um HIV-Malaria-Koinfektion meisten realistisch studieren in vitro optimiert. Zunächst werden frische menschliche Erythrozyten und Serum für die Malaria-Parasiten Kultur erforderlich. Dies ist entscheidend, um eine gesunde Population von Malaria-Parasiten zu erhalten. Parasite Lysaten kann nicht für Live-Parasiten substituiert sein, wie Zytokin-Produktion viel schneller und intensiver bei der Verwendung von Live-P. falciparum infizierten RBC (PfRBC) 17,18.…
Disclosures
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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