The online version of this article (https://doi.org/10.1186/s12936-018-2198-4) contains supplementary material, which is available to authorized users.
Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay.
A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay.
imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening.
imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.
Additional file 1: Figure S1. Long-term immortalized hepatocyte-like cell (imHC) cultures. Upon achieving confluence, imHCs were maintained in MEM/F12 medium containing 10% fetal bovine serum for more than 6 weeks. Cells were routinely observed, and brightfield images were taken weekly. imHCs remained in a cell monolayer after several weeks of subculture. Scale bar = 50 μm.
Additional file 2: Figure S2. Immortalized hepatocyte-like cells (imHCs) were susceptible to Plasmodium vivax liver-stage infection. Representative immunofluorescence images depict P. vivax exoerythocytic forms (EEs) in imHCs on days 4 and 7 post infection. Anti-UIS4 (red) was used to identify EEs. The hepatocyte nuclei were stained using DAPI. Schizonts and small EEs were observed on day 7 post-infection. Scale bar = 5 μm.
Additional file 3: Figure S3. Immortalized hepatocyte-like cells (imHCs) supported Plasmodium vivax liver-stage development. Representative immunofluorescence images of P. vivax exoerythocytic forms (EEs) in imHCs on day 10 post infection. EEs were visualized using antibodies against parasite UIS4 (red). The hepatocyte nuclei were stained using DAPI. Mature schizonts containing numerous merozoites were observed on day 10 post-infection. Scale bar = 10 μm.
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- A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax
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