Background
Malaria remains a major health problem worldwide, with an estimated 212 million disease cases and hundreds of thousands of related deaths in 2015 alone [
1]. Malarial parasites are transmitted to humans via bites of infected female
Anopheles mosquitoes; specifically, these parasites reside in the sporozoite form in mosquito salivary glands and are injected into human skin. The sporozoites rapidly leave the injection site and migrate through the bloodstream to the liver, where they invade hepatocytes and develop into liver-stage schizonts. A mature schizont contains thousands of daughter forms, termed merozoites, which are eventually released into the bloodstream where they invade erythrocytes and trigger the clinical symptoms of the disease. Of the five
Plasmodium species that infect humans,
Plasmodium falciparum is thought to cause the majority deaths, of which most cases (70%) involve children aged < 5 years in sub-Saharan Africa [
1].
Plasmodium vivax is the most widespread
Plasmodium species outside Africa. This organism is recognized as a major obstacle in malaria eradication campaigns because it can hide in a form called a hypnozoite [
2,
3] in the human liver, and can reactivate weeks, months or years after the primary infection to cause a relapse [
4‐
8].
The
Plasmodium liver stage has become an attractive target for the development of anti-malarial drugs and vaccines because it is the precursor to blood-stage disease and also serves as a reservoir of the hypnozoites [
9]. To date, only one approved drug, primaquine (PQ), has been shown to effectively eliminate liver-stage parasites, including
P. vivax hypnozoites [
10,
11]; however, this drug induces haemolytic toxicity in glucose-6-phosphate dehydrogenase-deficient individuals [
12] and drug tolerance and therapeutic failures have compromised its clinical use [
13‐
16]. Avoidance of these toxic effects have been considered in the development of an alternative drug, tafenoquine (TQ) [
17,
18], which targets hypnozoites and is currently in Phase III clinical trials [
19]. However, the development of more safe and efficacious anti-liver stage drugs, particularly those that target hypnozoites, remains essential [
20].
The lack of a robust, reliable in vitro model of the
Plasmodium liver stage has limited understanding of the biology of
Plasmodium liver stage and consequently, hampered development of drug and discovery programmes [
19]. Current in vitro models of human liver-stage parasites (mainly
P. falciparum and P. vivax) use both hepatoma cell lines, such as HepG2-A16 and HC-04 [
21‐
24], and primary human hepatocytes [
25‐
28]. The major advantages of the hepatoma cell lines include reliability and reduced variability between infection batches. Nonetheless, these cells exhibit abnormal cell regulation and proliferation and thus do not accurately recapitulate the biology of a liver-stage infection. Primary human hepatocytes are considered an ideal model for liver-stage cultures because these are the natural hosts. Nevertheless, primary hepatocytes are rarely used because of their limited availability and gradual loss of hepatic functions over time under conventional culture conditions [
29]. Several groups have attempted to establish models that could prolong functional phenotypes of primary hepatocytes in culture. The primary hepatocyte and human hepatoma HepaRG cell co-culture model has been shown to not only retain hepatic functions but also to help maintain the susceptibility of primary hepatocytes to
P. falciparum infection [
30]. Another advanced micropatterned co-culture (MPCC) model, in which primary hepatocytes are organized among supportive stromal cells (3T3-J2 fibroblast cells), has been shown to retain functional hepatocytes for up to 4–6 weeks [
28,
31‐
34] and thus, allow the establishment of
P. falciparum and
P. vivax liver stage forms [
28]. Recently, a microfluidic bilayer device was developed to promote long-term stability of primary hepatocytes and offer another platform for human liver-stage culture in vitro [
35].
A ‘continuous non-tumorous cell line’ in which hepatocyte phenotypes are maintained could be a necessary substitute for primary hepatocytes. Some alternative sources of human cells that mimic the phenotypes of hepatocytes have been developed. In recent advances, induced pluripotent stem cell-derived hepatocyte-like cells (iHLCs) have been shown to support
Plasmodium liver stages [
36]. Nevertheless, these iHLCs are functionally immature and their hepatic functions must be induced and extensively characterized before each use [
37]. Moreover, iHLCs that have been induced to differentiate usually exhibit limited life spans. Accordingly, iHLCs do not seem to be a simple or robust option for an in vitro model.
A novel ‘immortalized’ hepatocyte-like cell line (imHC) derived from human bone marrow mesenchymal stem cells (hMSCs) has been established [
38]. These imHCs maintain the production of hepatocyte-specific markers, including albumin (ALB), urea, glycogen, alpha-fetoprotein (AFP), tyrosine aminotransferase, hepatocyte nuclear factor-4-alpha (HNF-4ɑ), glucose-6-phosphate dehydrogenase and all major cytochrome P450 isotypes (CYP450s). In this report, the feasibility of imHCs as a model for establishing a malaria infection was demonstrated. imHCs support the growth of
P. vivax liver stages. This study also highlights the potential use of imHCs as a model for drug screening applications.
Methods
Ethical approval
In this study, human blood was collected and patient samples were used in strict accordance with the human use protocol TMEC 11-033, approved by the Institute Ethical Review Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand. Written informed consent was obtained from the patient for the publication of this report.
Plasmodium sporozoite preparation
Laboratory-reared female
Anopheles dirus mosquitoes were maintained in a colony at the Mahidol Vivax Research Unit in Bangkok, Thailand. The mosquitoes were membrane-fed with blood samples collected from
Plasmodium-infected patients in Kanchanaburi and Tak Provinces, Thailand. Briefly, 5 ml of patient blood was collected in a heparinized tube and centrifuged at 1500×
g for 10 min to remove plasma. The resulting pellet was washed with 10 ml of phosphate-buffered saline (PBS) and reconstituted to the original volume with naïve-type AB serum for mosquito feeding [
39]. Each feeder contained approximately 0.5 ml of infected blood and was used to feed approximately 100 5–7-day-old mosquitoes during a 30-min period. Mosquitoes were subsequently maintained on a 10% sucrose solution under a controlled environment at 26 °C and 75% humidity with a 12-h light/dark cycle. Midgut oocysts of
P. vivax were monitored on dissected mosquitoes using a mercurochrome staining method on day 7 post feeding [
40]. The salivary gland sporozoites were examined on day 14 post feeding and dissected from the infected mosquitoes using a standard protocol [
41]. In brief, salivary glands of 50 infected mosquitoes were dissected, placed in 50 μl of ice-cold RPMI 1640 medium (Gibco, Grand Island, NY, USA), pH 8.2 [
42], supplemented with 200 U/ml penicillin (Invitrogen, Carlsbad, CA, USA), 200 µg/ml streptomycin (Invitrogen), and 0.25 µg/ml amphotericin B (Invitrogen), and ground with a sterile pestle. The released sporozoites were counted in a hemocytometer and kept on ice until used, but for no more than 1 h to avoid a reduction in parasite infectivity (Patrapuvich R, unpublished data and [
43]).
Hepatocyte culture
imHCs were cultured in 1:1 DMEM:Ham’s F12 media (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin. The HC-04 hepatoma cell line (ATCC patent deposit no. PTA-3441: ATCC, Manassas, VA, USA) was cultured in 1:1 minimal essential medium (MEM):Ham’s F12 media (Invitrogen) supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Both cell lines were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Cells were subcultured every 2–4 days or once they had reached approximately 80% confluence, after detachment with 0.125% trypsin–EDTA (Invitrogen).
Growth curve
Hepatocytes were seeded at a density of 2 × 104 cells per well in a six-well plate (Thermo Scientific™, Nunc™; Waltham, MA, USA) and maintained as described above for 2 weeks with daily changes in medium. Viable cells were monitored and counted daily using a hemocytometer and an Olympus IX71 inverted phase/fluorescence microscope (Olympus, Tokyo, Japan). Growth curves were generated using GraphPad Prism software, version 7.0 (GraphPad Inc, La Jolla, CA, USA).
In vitro liver-stage infection
Hepatocytes were seeded at a density of 3 × 10
5 cells per well in a Matrigel (Corning Corp, Corning, NY, USA)-coated Millicell EZ SLIDE eight-well glass slide (Millipore, Billerica, MA, USA) and maintained in complete medium (1:1 MEM:Ham F12 supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin) at 37 °C. After incubation for 18–20 h, the medium was aspirated, and an aliquot of sporozoite suspension (3 × 10
5 in 200 µl of complete medium) was added to each well. After a 4-h inoculation at 37 °C, free sporozoites were removed by aspiration and a 400-µl aliquot of fresh infection medium (1:1 MEM:Ham F12 supplemented with 10% FBS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml amphotericin B) was added. The infected hepatocyte culture was maintained at 37 °C with daily changes in medium until the liver-stage parasites or exoerythocytic forms (EEs) were established and visualized using an indirect immunofluorescence assay. The total number of EEs in each well was manually quantified. Per cent sporozoite infectivity was determined by comparing the number of infected hepatocytes to the total number of inoculated sporozoites [
42].
Indirect immunofluorescence assay
After removing the culture medium, the infected hepatocyte cells were washed with PBS buffer thrice, fixed with 4% paraformaldehyde for 20 min, and permeabilized by exposure to 0.1% Triton X-100 for 3–5 min at room temperature (20–25 °C). The cells were then treated with 3% BSA (in PBS) solution, followed by incubation with mouse primary monoclonal anti-PbHSP70 antibodies (clone 4C9, kindly provided by Dr. Fidel P. Zavala [
44]) and rabbit primary polyclonal anti-PvUIS4 antibodies (1:500 dilution, kindly provided by Dr. Sebastian A. Mikolajczak [
45]). Subsequently, goat secondary IgG Alexa Fluor
® 488-conjugated anti-mouse antibodies (1:500 dilution, Invitrogen), goat secondary IgG Alexa Fluor
® 568-conjugated anti-rabbit 2nd antibodies (1:500 dilution, Invitrogen) and 0.1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) were added to the cells. Incubation with primary and secondary antibodies was performed at 37 °C for 1 h and 30 min, respectively. In some experiments, rabbit anti-acetylated histone H3K9 antibodies (1:200 dilution, Millipore) and mouse anti-acyl carrier protein (ACP) antibodies (1:200 dilution, kindly provided by Dr. Sebastian A. Mikolajczak [
45]) were used for detecting nuclear histones and apicoplasts of
P. vivax EEs, respectively. Finally, samples were covered with ProLong Gold antifade reagent (Invitrogen), sealed under coverslips, and viewed under a fluorescent microscope (400× magnification; AXIO Scope. A1 equipped with AxioVision Rel 4.8 Software; Carl Zeiss AG, Oberkochen, Germany). Fluorescence images were acquired using an Olympus FluoView™ FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan) equipped with a 60× oil objective. Images were captured using FV10-ASW 3.0 viewer software and prepared for publication using Adobe Illustrator CC (Adobe Systems, San Jose, CA, USA).
In some experiments, uninfected hepatocyte cells were grown in 96-well CellCarrier-96 optic black plates (PerkinElmer, Waltham, MA, USA) and stained with antibodies against the following hepatocyte markers: ALB (1:100 dilution, ab10241, Abcam, Cambridge, UK), AFP (1:100 dilution, SC8399, Santa Cruz Biotechnology, Dallas, TX, USA), low-density lipoprotein receptor (LDLR; 1:100 dilution, SC373830, Santa Cruz Biotechnology), Na+-taurocholate cotransporting polypeptide (NTCP; 1:100 dilution, ab131084, Abcam, Cambridge, UK), and HNF-4ɑ (1:100 dilution, SC6556, Santa Cruz Biotechnology). Cells were also stained with antibodies against the following cell surface receptors: CD81 (1:100 dilution, ab79559, Abcam), scavenger receptor type B class I (SR-BI) (1:100 dilution, NB400-104, Novus Biologicals, Littleton, CO, USA), and EphA2 (1:100 dilution, 37-4400, Thermo Fisher Scientific). Samples were then incubated with a goat anti-mouse Alexa Fluor® 488-conjugated (1:500 dilution, Invitrogen), goat anti-rabbit Alexa Fluor® 488-conjugated (1:500 dilution, Invitrogen), or donkey anti-goat Cy3-conjugated secondary antibody (1:500 dilution, BioLegend, San Diego, CA, USA), as appropriate; hepatocyte nuclei were stained with 2 µM Hoechst 33342 (Thermo Fisher Scientific). Mouse IgG2a, mouse IgG1, rabbit IgG and goat IgG were used as negative control for staining. Fluorescence images were captured and analysed using an Operetta High-Content Imaging System (PerkinElmer) with a 40× objective lens.
Primaquine treatment
PQ (Sigma-Aldrich, St Louis, MO, USA) was added to infected hepatocytes at concentrations ranging from 0.1 to 10 μM 2 h post sporozoite infection. The cultures were maintained for up to 5 days with daily changes in medium (including drug supplementation) and harvested on day 6 to determine sporozoite infectivity by immunofluorescence assay, as described above. Numbers and sizes of EEs were manually examined using a fluorescent microscope (ZEISS AXIO Scope.A1). The relative proportions of small EEs (diameter ≤ 5 µm) and schizonts (diameter > 5 µm) were determined relative to the 0.1% dimethyl sulfoxide untreated controls. Plasmodium vivax assays were conducted over three independent experiments using three batches of sporozoites generated from different P. vivax isolates.
Quantitative real-time PCR
Total RNA was extracted from the hepatocytes using a RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). The isolated total RNA was then treated with DNase I (Thermo Scientific, USA), according to the manufacturer’s instructions. The quantity and quality of total RNA were determined using a NanoDrop spectrophotometer (Thermo Scientific). For the CYP450 qRT-PCR analysis, 500 ng of total RNA was converted to cDNA using the SuperScript
® III First-Strand Synthesis System (Invitrogen). The primer sets and conditions used for CYP450 amplification have been described previously [
38]. Briefly, 2 μl of cDNA was diluted in a 10-μl reaction mixture containing 0.4 μM of each primer and 5 μl of Luminaris Color HiGreen qPCR Master Mix (Thermo Scientific, USA). Each reaction was run in a CFX96 Touch™ Real-Time PCR Detector (Bio-Rad, Hercules, CA, USA) with the following conditions: 50 °C for 2 min; 95 °C for 10 min; and 40 cycles of amplification at 95 °C for 15 s and 60 °C for 45 s. Reverse transcriptase- and template-negative controls were used for each gene-specific primer pair. The number of cycles required for the fluorescent signal to cross the threshold (Ct value) was determined for each CYP450 isotype. Obtained Ct values were subtracted from Ct of the respective house-keeping gene (GAPDH) obtained from the same cells to generate a relative gene expression value ΔCt. In some experiments, hepatocytes were treated with PQ for 6 days with daily changes in medium prior to RNA extraction.
Statistical analysis
At least three independent experiments were conducted in triplicate. Each individual sporozoite infection experiment was performed using a different parasite isolate. Differences in the results were determined using a standard Student’s t test, and a p value of < 0.05 was considered to indicate statistical significance. For non-normally distributed data, the Mann–Whitney U test was used in place of the paired t test.
Discussion
Urgent strategies aimed at achieving the current worldwide goal of eradication of malaria have included approaches for discovering drugs that target the
Plasmodium liver stage, particularly dormant hypnozoites [
20]. To date, however, biological features of
P. vivax hypnozoites remain largely unknown, mainly because of the difficulty of producing large amounts of
P. vivax sporozoites [
42] and the lack of a robust and reliable culture system for this purpose [
19]. Currently, humanized liver mouse models appear to be a powerful tool for obtaining human liver-stage parasites in vivo [
45,
54‐
56]. In the context of drug discovery, the cost and technical challenges associated with this animal model render it largely infeasible for early-stage drug screening, although it remains valuable during preclinical testing [
56]. Although in vitro primary human hepatocyte models have advanced the ability to identify potential molecules for liver-stage targeting [
28,
30,
34], the loss of hepatic functions over time and donor-to-donor variability [
28] remains a major challenge when using primary hepatocytes for in vitro assays. Therefore, most studies on liver-stage parasites heavily rely on human hepatoma cell lines as hosts for sporozoite infection [
57‐
59]. However, these hepatoma cells may not be suitable for assessment of drug metabolism and drug interactions because they usually lack various functional CYP450s and other phase I, II, and III drug-metabolizing enzymes. HC-04 cells tend to overgrow in culture, leading to the detachment of host cells, and thus, limiting long-term monitoring of EEs and hindering the establishment of hypnozoites. The reduction in HC-04 cell growth upon reaching confluency might also affect the development of EEs. In addition, infection rates among HC-04 cells alone are relatively low [
23] but, as in the present study, a Matrigel co-culture improved this parameter [
30]. Infection rates were 4–6 times higher in the Matrgel-HC-04 co-culture than in the original untreated HC-04 model (Patrapuvich R, unpublished data).
To overcome the noted deficiencies of HC-04 cells with respect to supporting liver-stage culture of malaria parasites, a novel hepatocyte cell line, imHC, was therefore tested the ability to support the development of P. vivax liver-stage.
imHCs displayed essential liver functions comparable with those of mature hepatocytes [
38], ceased proliferation upon reaching a high cell density, and remained in a monolayer even after months of culture. In addition, imHCs expressed the host factors CD81 [
48,
60], EphA2 [
49] and SR-BI [
50], which may be important for sporozoite invasion. No significant difference in infectivity between imHCs (CD81++) and HC-04 (CD81+) cells was observed, suggesting that CD81 was not the primary determinant of
P. vivax infection. This observation is consistent with the recent study from Manzoni et al. showing that antibodies against SR-BI but not against CD81 inhibit
P. vivax infection of primary human hepatocyte cultures [
61]. The results affirm the potential role of SR-BI during hepatocyte infection by
P. vivax sporozoites. Manzoni et al. also reported that
P. falciparum, another major human malarial parasite, relies on CD81 but not SR-BI for host entry [
61]. Given these data, it will be interesting to examine whether imHCs can better support
P. falciparum infection than HC-04 cells. The same study also showed that parasite protein P36 is essential for both SR-BI- and CD81-dependent sporozoite invasion [
61]. No direct interaction between P36-dependent invasion and EphA2 has been demonstrated [
49,
61,
62]. The role of EphA2 in
P. vivax infection remains to be further investigated.
In the present study,
P. vivax infection rate in imHCs (0.14%) was comparable or even higher than that observed in HC-04 cells [
23] and in a primary hepatocyte MPCC model [
28]. Nevertheless, these infection rates were relatively low in comparison to those observed in in vivo [
63,
64]. No significant difference in the development rates of
P. vivax EEs in imHCs versus those in HC-04 cells based on the percentages of EEs that developed into schizonts (> 10 μm) was observed. However, on day 7, there were many larger EEs among the schizonts growing in imHCs (> 20–45 μm); whereas most EEs in HC-04 cells were smaller (approximately 20–25 μm). The sizes of these day-7
P. vivax schizonts in imHCs were similar to those recently reported in MPCC on day 6 (approximately 30 μm) [
28] and in a humanized mice model (approximately 40–50 μm) [
45]. Larger mature EEs were clearly visible on day 10 (up to approximately 50 μm), but remained smaller than those (60–80 μm) observed in vivo [
45]. In imHCs, antibodies specific for PVMs and apicoplasts of EEs revealed a complex cellular structure similar to that observed in vivo [
45]. In this study, while MSP-1 expression was not used as a late liver-stage marker for determining parasite maturation, the presence of large multi-nuclei parasites on day 10 confirmed the establishment of mature
P. vivax schizonts in the imHC culture. Importantly, the long-term culture of
P. vivax-infected imHCs yielded a population of small (< 10 μm in diameter) forms, presumably hypnozoites, that maintained a single-nuclear structure as detected by histone-acetylation staining of the parasite nuclei [
45]. This finding demonstrates the capacity of imHCs to establish
P. vivax hypnozoites. These presumed hypnozoites in imHCs will continue to require further characterization. It was very interesting to note that a small form observed on day 14 did not exhibit the ‘UIS4-positive prominence’, previously considered as a unique feature of
P. vivax hypnozoites described by Mikolajczak et al. [
45]. In contrast, the young parasite showed on day 4 clearly displayed the prominence pattern. The reliability of this marker as a hypnozoite-specific trait should be considered with care. Here, CSP-VK247 sporozoites produced larger numbers of small forms (77% of EEs) than those of the CSP-VK210 genotype (36% of EEs). This result is in agreement with the recent report in humanized mice model showing that a greater proportion of CSP-VK247 sporozoites (~ 40% of EEs) form hypnozoites compared with CSP-VK210 sporozoites (~ 5% of EEs) [
45]. The higher percentage of small EEs obtained in the cultured imHCs compared to infections in in vivo mice may reflect the presence of a sub-population of slow-growing parasites in culture.
The responses of malaria parasites in imHCs to PQ, a drug known to target
Plasmodium liver stages, were tested to demonstrate the potential use of imHCs as a platform for drug screening. imHCs, which exhibit a cellular physiology closer to that of human hepatocytes compared to HC-04 cells (including CYP450 drug metabolic activity), provide a physiologically relevant platform for drug screening and, thus, would be expected to catch agents that cause drug-induced liver injury [
65], particularly during the initial stage of drug discovery. PQ more potently affected EEs in imHCs than in HC-04 cells. Increased PQ sensitivity was associated with higher levels of CYP450 activity in imHCs. CYP2C19, CYP3A4 and CYP2D6 have been identified as the three major enzymes involved in PQ metabolism [
53]. The observation that CYP2C19 and CYP3A4 were not induced in imHCs after PQ exposure suggests the essentiality of CYP2D6 in PQ metabolism [
52,
66].
Taken together, the findings suggest that imHCs could be an improved hepatoma cell line for study P. vivax liver stage parasites. This finding is expected to lead to an array of imHC applications that will facilitate understanding the biology of P. vivax hypnozoites and help discover anti-relapse drug, particularly in high-throughput screening formats.
Authors’ contributions
SH provided imHCs. JS provided P. vivax sporozoites. AD prepared and dissected the mosquitoes. YP, SR and KL maintained hepatocyte cultures, prepared sporozoites, and conducted the sporozoite infection experiments. YP, RP, SR and KL performed the immunofluorescence assay and identified liver-stage parasites. YP, PS and PK participated in fluorescence imaging and analysis. KS evaluated the expression of CYP450s. YP, RP and SB conceived the study, analysed data, and drafted the manuscript. JS and SH supervised the research. All authors read and approved the final manuscript.