Een<em> In Vitro</em> Model voor het meten van immuunrespons op Malaria in de context van HIV Co-infectie
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-infectie, infectie met meerdere gelijktijdige infecties, is de norm in de natuurlijke omgeving. Co-infectie kan een grote impact hebben op de ziekte pathologie en op de klinische behandeling van iedere infectie. In de context van co-infectie, vaccins en werkzaamheid van het geneesmiddel, alsmede diagnostische tests kunnen negatief beïnvloed (besproken in 1). Ondanks het belang, de meeste pathogeen onderzoek beschouwt eenmalig infecties.
Malaria en HIV-1 (HIV) zijn belangrijke oorzaken van morbiditeit en mortaliteit wereldwijd. Gebieden van malaria en HIV endemiciteit delen een brede geografische overlap, waardoor miljoenen mensen het risico lopen van co-infectie en dus een risico op meer ernstige klinische ziekte 2-10. De twee ziekten negatief interageren. Bij HIV-geïnfecteerde individuen, hogere HIV virale lading en tijdelijke dalingen in CD4 + T-cel aantallen te zien tijdens een malaria infectie, terwijlmalariaparasiet lasten en risico's van de klinische en ernstige malaria zijn hoger in co-geïnfecteerde individuen 2,3,5,7,8,10. De mechanismen die HIV verhoogt malaria ernst worden niet volledig begrepen en garandeert verder onderzoek.
Hier beschrijven we een werkwijze waarmee malaria en HIV-infectie kan worden bestudeerd in vitro. Specifiek, maakt deze werkwijze voor de behandeling van malaria-specifieke immuunresponsen in de context van HIV-infectie. Onze protocol beschrijft een veelzijdige co-kweeksysteem met vers geïsoleerde perifere mononucleaire cellen (PBMCs) geïsoleerd uit chronisch HIV geïnfecteerde donoren en in vitro gekweekte P. falciparum geparasiteerde erytrocyten (PfRBC). Het effect van HIV antiretrovirale therapie volgende reacties kunnen worden onderzocht met behulp prospectief PBMC uit HIV (+) donors en na de behandeling.
We hebben dit systeem gebruikt om de impact van HIV-infectie te onderzoekenmalaria-specifieke aangeboren immuunrespons 11,12 en konden vaststellen dat malaria-specifieke IFNy en TNF responsen geremd in NK-cellen, NKT-cellen, γδ T-cellen van HIV (+) donoren pre- en post-HIV antiretrovirale therapie . Bovendien, waren we in staat om dit systeem te gebruiken om te bepalen dat monocytische functies ook worden aangetast bij HIV (+) donoren, maar herstellen na HIV antiretrovirale therapie.
Protocol
Representative Results
Discussion
Ons protocol is geoptimaliseerd om zo realistisch studie HIV-malaria co-infectie in vitro. Eerst verse menselijke RBCs en serum zijn vereist voor malariaparasiet cultuur. Dit is essentieel voor een gezonde populatie van malariaparasieten verkrijgen. Parasiet lysaten kan niet worden vervangen voor levende parasieten als cytokine productie is veel sneller en intenser bij het gebruik van live-P. falciparum geïnfecteerde RBC (PfRBC) 17,18. Bovendien activeren van celtypen zoals NK-cellen,…
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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