En<em> In Vitro</em> Modell for måling av immunresponser mot Malaria i sammenheng med HIV Samtidig infeksjon
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-infeksjon, infeksjon med flere samtidige infeksjoner, er normen i naturlige omgivelser. Co-infeksjon kan ha en stor innvirkning på sykdommen patologi og på den kliniske behandlingen av hver infeksjon. I sammenheng med ko-infeksjon, vaksine og legemiddel effekt, så vel som diagnostiske tester kan bli negativt påvirket (oversikt i 1). Imidlertid, til tross for sin betydning, de fleste av patogenet forskning vurderer bare én infeksjoner.
Malaria og HIV-1 (HIV) er ledende årsaker til sykelighet og dødelighet globalt. Områder av malaria og HIV endemicity dele en bred geografisk overlapp, sette millioner av mennesker i fare for co-infeksjon og dermed i fare for alvorligere klinisk sykdom 2-10. De to sykdommene negativt samhandle. Hos HIV-infiserte individer, høyere HIV-virusmengde og midlertidige reduksjoner i CD4 + T-celletall kan sees under en malariainfeksjon, mensmalariaparasitten byrder og risiko for klinisk og alvorlig malaria er høyere i co-infiserte individer 2,3,5,7,8,10. Mekanismer som HIV øker malaria alvorlighetsgrad er ikke fullt ut forstått og garanterer videre etterforskning.
Her beskriver vi en metode som malaria og HIV-infeksjon kan studeres in vitro. Konkret gir denne metoden for undersøkelse av malaria-spesifikke immunresponser i sammenheng med HIV-infeksjon. Vår protokollen beskriver en allsidig ko-kultur-system ved bruk av nylig isolerte perifere mononukleære blodceller (PBMC) isolert fra kronisk HIV-infiserte givere og in vitro dyrkede P. falciparum parasitized erytrocytter (PfRBC). Virkningen av HIV antiretroviral terapi på disse svarene kan også bli undersøkt ved hjelp prospektivt innsamlede PBMC fra HIV (+) givere før og etter behandling.
Vi har brukt dette systemet for å undersøke effekten av HIV-smittepå malaria-spesifikke medfødte immunresponser 11,12, og var i stand til å bestemme at malariaspesifikke IFNy og TNF responser er svekket i NK-celler, NKT celler, γδ T-celler fra HIV (+) givere før og etter HIV antiretroviral behandling . I tillegg var vi i stand til å bruke dette systemet for å fastslå at monocyttiske funksjoner er også svekket i HIV (+) givere, men gjenopprette post-HIV antiretroviral behandling.
Protocol
Representative Results
Discussion
Vår protokoll har blitt optimalisert for å mest mulig realistisk studere HIV-malaria-infeksjon in vitro. Først blir friske humane RBC-er og serum som kreves for malariaparasitten kultur. Dette er viktig for å oppnå en sunn befolkning på malariaparasitter. Parasitt lysatene kan ikke erstattes av levende parasitter som cytokinproduksjon er mye raskere og mer intens når du bruker levende P. falciparum smittet RBC (PfRBC) 17,18. I tillegg, aktivering av celletyper som NK-celler, krever he…
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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