The liver stages of malaria parasites are inhibited by cytokines such as interferon-γ or Interleukin (IL)-6. Binding of these cytokines to their receptors at the surface of the infected hepatocytes leads to the production of nitric oxide (NO) and radical oxygen intermediates (ROI), which kill hepatic parasites. However, conflicting results were obtained with TNF-α possibly because of differences in the models used. We have reassessed the role of TNF-α in the different cellular systems used to study the Plasmodium pre-erythrocytic stages.
Methods and Findings
Human or mouse TNF-α were tested against human and rodent malaria parasites grown in vitro in human or rodent primary hepatocytes, or in hepatoma cell lines. Our data demonstrated that TNF-α treatment prevents the development of malaria pre-erythrocytic stages. This inhibitory effect however varies with the infecting parasite species and with the nature and origin of the cytokine and hepatocytes. Inhibition was only observed for all parasite species tested when hepatocytes were pre-incubated 24 or 48 hrs before infection and activity was directed only against early hepatic parasite. We further showed that TNF-α inhibition was mediated by a soluble factor present in the supernatant of TNF-α stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified.
Treatment TNF-α prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. However, the nature of the cytokine-host cell-parasite combination must be carefully considered for extrapolation to the human infection.
Citation: Depinay N, Franetich JF, Grüner AC, Mauduit M, Chavatte J-M, Luty AJF, et al. (2011) Inhibitory Effect of TNF-α on Malaria Pre-Erythrocytic Stage Development: Influence of Host Hepatocyte/Parasite Combinations. PLoS ONE6(3): e17464. https://doi.org/10.1371/journal.pone.0017464
Editor: Fabio Costa, State University of Campinas, Brazil
Received: November 3, 2010; Accepted: January 25, 2011; Published: March 4, 2011
Copyright: © 2011 Depinay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by intramural grants from the Agency for Science, Technology and Research (A*STAR) and Institut National de la Santé et de la Recherche Médicale (INSERM), and by a grant from the European community (MALINV contract number LSH-CT-2005-01299) (L.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Laurent Rénia, Georges Snounou and Anne Charlotte Grüner are editors of PLoS ONE. However, this does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Tumour necrosis factor (TNF)-α is a cytokine with pleiotropic effects including anti-microbial activities . In malaria infections, it has been shown that TNF-α could have both beneficial and detrimental effects. TNF-α is detected in the circulation during the erythrocytic phase of the infection in humans – and in mice , ). In both hosts, high levels of this cytokine have been associated with malarial pathology such as fever , and cerebral malaria , . On the other hand, TNF-α has also been shown to have a potent anti-parasitic activity. Administration of recombinant TNF-α protected against blood stage infection with Plasmodium chabaudi in mice , while mice deficient for TNF-α controlled P. chabaudi adami blood infections less efficiently . In humans, sustained high levels of TNF-α were associated with rapid clearance of fever and parasites . There are controversial observations concerning the role of TNF-α against the pre-erythrocytic (PE) stages of the malarial infection. Schofield et al. reported that recombinant human TNF-α directly inhibited P. berghei grown in vitro in the HepG2 human hepatoma cell line, and in vivo in rats and mice . However, we have previously found that P. yoelii parasites grown in cultured purified mouse primary hepatocytes were unaffected by TNF-α, whereas this cytokine inhibited the hepatic development of this parasite in vivo . We then showed for this rodent malaria model that the inhibitory effect observed in vivo was actually mediated by the IL-6 secreted by non-parenchymal liver cells in response to TNF-α stimulation . Thus depending on the host/parasite combination, different effects of TNF-α on PE parasites were reported. In this study, we wished to reassess the role of some of the biological and experimental parameters on the inhibition of Plasmodium hepatic stages observed in in vitro assays of TNF-α activity.
Materials and Methods
All experiments and procedures involving mice were approved by the “Direction Départementale des Service Vétérinaires de Paris, France” (Authorisation No 75–129) and performed in compliance with regulations of the French Ministry of Agriculture for animal experimentation (1987). Human liver fragments used to prepare primary hepatocyte cultures were collected after written informed consent from patients undergoing a partial hepatectomy. The collection and use of these tissues were undertaken in accordance with French national ethical regulations and have been approved by the Ethic Committee of the Centre Hospitalo-Universitaire Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.
Cytokines and chemicals
Different batches of human and murine TNF-α with similar specific activity were obtained from R&D systems. Endotoxin levels in the different batches used in this study were always below 1.0 EU per 1 µg as reported by the manufacturer. S-methyl-thiourea (SMT, Sigma), a potent inhibitor of iNOS  and N-acetyl-cysteine (NAC), which prevents oxygen free radical , were obtained from Sigma.
Mice and parasites
BALB/cJ mice were purchased from Harlan Laboratories (Gannat, France), and were housed in a pathogen-free rodent barrier facility. Sporozoites of the uncloned line of Plasmodium yoelii yoelii 265BY strain (Pyy265BY), of P. yoelii yoelii 17X strain clone 1.1 (Py17X), and of P. berghei ANKA cloned line transfected with GFP (PbA)  were obtained from infected salivary glands of Anopheles stephensi mosquitoes, 16 to 21 days after an infective mouse blood meal. After aseptic dissection, salivary glands were homogenized in a glass grinder and diluted in culture medium . Sporozoites of the NF54 strain of Plasmodium falciparum were obtained from infected salivary glands of A. gambiae mosquitoes which have been fed two weeks before on infected human blood cultures using a membrane-based feeder system . After aseptic dissection, salivary glands were disrupted by trituration in a glass tissue grinder, diluted in culture medium and the sporozoites were counted using a KovaSlide® chamber.
Mouse hepatoma cells Hepa1–6 (ATCC CRL-1830) (105 per well), were cultured in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen), 2 mM glutamine, 1% penicillin-streptomycin of a 100 X stock solution (Invitrogen). Human hepatocarcinoma cells HepG2/CD81+ (HepG2 stably expressing CD81) (105 per well)  were cultured in supplemented DMEM as above, in culture dishes coated with rat tail collagen I (Becton Dickinson. Mouse primary hepatocytes were prepared as described, with minor modifications . Cells were isolated by collagenase perfusion (Boehringer Mannheim) of liver fragments and were further purified over a 60% Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). Mouse hepatocyte purity and viability were >95% as assessed by Trypan blue dye exclusion. Human primary hepatocyte cultures were prepared as described with minor modifications . Briefly, cells were isolated by collagenase (Roche) perfusion of human liver fragments, which were collected and used in agreement with the French ethical regulations, and were further purified over a 40% Percoll gradient. Human hepatocyte purity and viability were >99% as assessed by Trypan blue dye exclusion. Mouse and human hepatocytes were seeded in eight-chamber plastic Lab-Tek slides (Nunc) coated with rat tail collagen I (Becton Dickinson) at a density of 1×105 cells per well for primary murine hepatocytes, and of 2×105 cells per well for primary human hepatocytes cells, and cultured at 37°C, 5% CO2, in DMEM medium as above supplemented with 10−7 M dexamethasone (Sigma) after complete cell adherence (12–24 hours).
Evaluation of TNF-α cytotoxicity
Toxicity of the cytokine to primary culture of hepatocytes or hepatoma cell lines in flat-bottom 96 wells (20×103 cells/well) was evaluated using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Briefly, cytokines were added in triplicate at decreasing concentrations. Medium was replaced 3 hours after sporozoite infection and every day thereafter up to day 4 (for rodent parasites) or 7 (for P. falciparum) with fresh medium containing the cytokine at the same concentration. Twenty-four hours after the last medium change, 110 µl of a solution containing 10 µl of MTT (Sigma) solution (5 mg/ml) and 100 µl of medium were added and the cultures incubated for a further 4 hours. The formazan crystals that were formed were dissolved using 100 µl of a 1∶1 DMSO: ethanol solution. Optical density was read immediately at 570 nm with a reference wavelength at 630 nm .
In vitro assay of sporozoite invasion of and development in hepatocytes and HepG2 cells
After removal of medium from the culture chambers, ten thousands sporozoites were added in 100 µl of fresh supplemented medium with various quantities of TNF-α tested, and at different times during cultivation. Medium was replaced at −24, 0, 3, and 24 h after rodent malaria sporozoite inoculation, and at −24, 0, 3, 24 h and then every day up to day 5 after P. falciparum sporozoite inoculation of primary human hepatocytes with fresh medium containing or not the cytokine. In one experiment, supernatant was collected from wells containing human hepatocytes treated with TNF-α (100 ng/m) of for 2 days. Plasmodium sporozoites (104 in a 50 µl) were added to this supernatant (50 µl) or to fresh medium (50 µl). The final solution (100 µl) containing the sporozoite was added to the culture. Medium was replaced after 3 h, 24 h, and then every day up to day 5 after sporozoite inoculation. Experimental determination of the number of liver stage parasites was performed in triplicate or quadruplicate. Cultures were stopped 48 h (for rodent malaria species) or 5 days (for P. falciparum) after sporozoite infection, fixed with cold methanol and schizont numbers were assessed by immunofluorescence using antibodies recognizing Plasmodium liver stages as previously described ,  and were quantified by microscopic examination or using the Odyssey infra red imaging system (Li-COR Biosciences) . Percentage of inhibition of the development is calculated by comparing the numbers of parasitic forms in the experimental wells versus control wells.
Effect of human TNF-α on HepG2-CD81 cells infected with murine Plasmodium species
HepG2 is a hepatoma cell line easily propagated in vitro  that has been shown to sustain the development of the P. berghei  but not the P. yoelii  liver stages. Recently Silvie et al.  showed that HepG2 cells transduced with CD81 are thereby made permissive to P. yoelii development, providing a good system to study P. yoelii liver stage biology, thus reducing the need for the more tedious primary murine hepatocyte cultures. We employed this cell line to assess the role of TNF-α against the hepatic stages of malaria parasites.
When human TNF-α was added to HepG2-CD81 cultures over a four-day period centred on the time of sporozoite inoculation, Pyy265BY hepatic parasite development was inhibited in a dose dependent manner (Figure 1A). Significant inhibition was observed even at a low dose of 10 ng/ml. Only minor increases in the level of inhibition were obtained at doses above 100 ng/ml (Figure 1A). We then tested the influence of the timing of TNF-α addition on the level of inhibition observed. HepG2-CD81 cultures where the cytokine was added only 1 day before sporozoite inoculation were also significantly inhibited by 100 ng/ml of TNF-α (Figure 1B). However, no inhibition of parasite development could be observed when TNF-α was first added starting at the time of sporozoite inoculation or thereafter (Figure 1B). The inhibitory activity of TNF-α was specific to the parasites because TNF-α had no cytotoxic effect on infected or normal HepG2 cells as measured by the MTT assay, even when added at the highest doses used. Indeed, the optical density values obtained in wells containing untreated cells [(0.953±0.2 arbitrary units (A.U.)] or cells treated for 4 days with 100 ng/ml TNF-α and infected with P. yoelii sporozoites (1.29±0.0.05 A.U.) did not differ significantly.
A. HepG2/CD81 hepatoma cell cultures were treated with various concentrations of recombinant human TNF-α, 48 h prior to, at the time of, and then 3 h and 24 h after sporozoite inoculation. Cultures were stopped 45 h after infection. Data are presented as the mean (± SD) reduction in Pyy265 liver schizonts numbers and represent data from two independent experiments (one represented by filled circles and the other by open circles). Parasite number reduction was calculated by enumerating 48 h liver schizonts in triplicate cultures exposed or not to TNF-α. Data are presented as the mean (± SD) reduction in liver schizont numbers in triplicate wells compared to the mean number in 6 control wells. The number of liver schizonts in control wells was 67±9.9. B. Timing of the antiparasitic effect of human TNF-α. HepG2/CD81 cells were incubated with 100 ng/ml of human TNF-α for different times. Cultures were stopped 48 h after sporozoite inoculation. Data are presented as the mean (± SD) numbers of liver schizonts in triplicate experimental wells and in six control wells. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test). The rate of infection Pyy265 sporozoites for HepG2/CD81 hepatoma cell was between 0.5 to 2% depending on the experiment. Data are representative of three independent experiments with similar results.
We next tested the effect of human TNF-α on PbA parasites, thus providing a reasonable comparison with the study of Schofield et al. where an inhibitory effect of human TNF-α against P. berghei NK65 strain was reported . When human TNF-α at an inhibiting dose of 100 ng/ml was added to HepG2-CD81 cultures starting 48 h before sporozoite inoculation and over a four-day period until the end of the parasite cultivation period 48 h thereafter, PbA liver stage development was also inhibited in a dose dependent manner (Figure 2), though the magnitude of the inhibition was lesser than that observed for Pyy265BY.
HepG2/CD81 hepatoma cell cultures were treated with various concentrations of recombinant human TNF-α, 48 h prior to, at the time of, and then 3 h and 24 h after sporozoite inoculation. Cultures were stopped 48 h after sporozoite inoculation. Data are presented as the mean (± SD) reduction in 48 h PbA liver schizont numbers from triplicate experimental wells compared to those from 8 control wells. The number of liver schizonts in the control wells was 357±31. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test). The rate of infection PbA sporozoites for HepG2/CD81 hepatoma cell was between 0.3 to 2% depending on the experiment. Data are representative of two independent experiments with similar results.
Effect of TNF-α on mouse hepatoma cell line Hepa 1–6 and primary mouse hepatocytes infected with murine Plasmodium species
In order to test whether the findings obtained using HepG2-CD81 cells can be extended to other host cell/parasite combinations, we repeated the TNF-α treatment (100 ng/ml initiated at 48 h before sporozoite inoculation and maintained for the subsequent 48 h) with mouse hepatoma cells Hepa 1–6 and primary cultures of purified mouse hepatocytes, both of which sustain the growth of Plasmodium species that infect rodents , . Mouse TNFα significantly inhibited PbA in Hepa1-6 cells (Figure 3A) or in primary hepatocyte cultures (Figure 3B), whereas for Pyy265BY, inhibition was only observed in primary hepatocytes (Figure 3).
(A) Hepa 1–6 cells and (B) primary hepatocyte cultures were treated with 100 ng/ml of recombinant human or mouse TNF-α, 48 h prior to, at the time of, and then 3 h and 24 h after sporozoite inoculation. Cultures were stopped 48 h later. Data are presented as the mean (± SD) reduction in PbA liver schizont numbers at 48 h in triplicate experimental wells as compared to those enumerated in 6 control wells. In the control wells, there were 110.3±13.1 liver schizonts in Hepa1-6 and 602±83.4 in the primary mouse hepatocytes infected with PbA, and 25.5±3.6 liver schizonts in Hepa1-6 and 443.5±133.5 in primary hepatocytes infected with Pyy265 BY. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test). The rate of infection for Pyy265 sporozoites was 0.02-.004% for Hepa1.6 cells and 0.1–2% for primary mouse hepatocytes. For PbA sporozoites, it was of 0.1–0.5% for Hepa1.6 cells and 0.5–1% for primary mouse hepatocytes. Data are representative of two independent experiments with similar results. ND, not done.
We next tested the effect of human TNF-α on mouse cells since it was shown previously that in vivo treatment with this cytokine inhibited P. berghei NK65 liver stage development . However, human TNF-α may have an indirect inhibitory effect on the liver stage in situ, for e.g. by inducing the production of another anti-liver stage cytokine such as IL-6 by nonparenchymal liver cells. We wished to ascertain whether the inhibition noted above was due to direct interaction with hepatocytes . The liver stage development of parasites (Pyy265BY or PbA) grown in primary mouse hepatocytes was not inhibited by treatment with human TNF-α (Figure 3B). These results indicate that parasite species are differentially susceptible to the inhibitory activity of TNF-α, and that this is influenced by the origin of the TNF-α as well as the type and origin of the host cells in which the parasites develop. It is worth noting that treatment with TNF-α had no cytotoxic effect on infected or non-infected primary mouse cells as measured by the MTT assay. The difference in the optical density values obtained for wells containing untreated cells (0.47±0.09 A.U.) or cells treated for 4 days with 100 ng/ml TNF-α and infected with P. yoelii sporozoites (0.41±0.05 A.U.) were not significant.
Effect of TNF-α on primary human hepatocytes infected with P. falciparum
We then tested the effect of TNF-α on the development of P. falciparum in human primary hepatocytes, a host/parasite combination of direct clinical relevance. We used highly purified human hepatocytes to prevent indirect effect of TNF-α on contaminating nonparenchymal cells as shown previously . P. falciparum liver stage development was inhibited when the cultures were treated with human TNF-α at 100 ng/ml, but not 1 ng/ml, 24 h or 48 h prior to sporozoite inoculation and until day 5 thereafter (Figure 4). Inhibition was more pronounced when the hepatocytes were pre-incubated with human TNF-α 48 h as compared to 24 h before sporozoite inoculation. As for the other hepatocyte cells, treatment with TNF-α had no cytotoxic effect on primary human hepatocytes as measured by the MTT assay. The optical density values obtained for wells containing untreated cells (0.78±0.005 A.U.) or cells treated with for 4 days with 100 ng/ml TNF-α and infected with P. yoelii sporozoites (0.793±0.03 A.U.) did not differ significantly.
Primary human hepatocyte cell cultures were treated with various concentrations of recombinant human TNF-α, 24 h (A) or 48 h (B) before, at the time of sporozoite inoculation, and then every day for day 1 to day 5. Cultures were stopped 5 days later after sporozoite inoculation. The data presented are mean numbers (± SD) of 5 days P. falciparum liver stages from triplicate experimental wells and from 8 control wells. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test). The rate of infection for P. falciparum sporozoites was 0.2–0.5% for primary human hepatocytes depending on the experiment. Data are representative of two independent experiments with similar results.
Absence of effect of nitric oxide derivatives and radical oxygen intermediates inhibitors on TNF-α mediated pre-erythrocytic stage inhibition
In order to determine how TNF-α inhibits liver stage parasite development, we used inhibitors that block the NO or the ROI pathways, both of which have been implicated previously in PE killing. First, we used S-methyl-thiourea (SMT), a competitive inhibitor of the inducible nitric oxide synthase . Addition of SMT to cultures treated with 100 ng/ml of human TNF-α did not reverse the effect of the cytokine against Pyy265BY HepG2/CD81 (Figure 5A) or against P. falciparum in primary human hepatocytes (Figure 6A). We next tested whether inhibition was mediated via the ROI pathway by using N-acetyl-cysteine (NAC), the precursor of glutathione and a potent endogenous antioxidant . Addition of NAC did not reverse TNF-α mediated inhibition of Pyy265BY in HepG2/CD81 (Figure 5B), or that of P. falciparum in primary human hepatocytes (Figure 6A). We also observed that SMT and NAC did not reverse the inhibitory effect of TNF-α on PbA-infected HepG2-CD81 hepatoma cells (data not shown).
HepG2/CD81 hepatoma cells treated or not with 100 ng/ml of human TNF-α together with or without SMT (A) or NAC (B) 48 h before, at the time of, and then 3 h and 24 h after sporozoite inoculation. Cultures were stopped 48 h later. Data are presented as the mean (± SD) reduction in liver schizont numbers in triplicate wells compared to the mean number in 6 control wells. The numbers of Pyy265BY liver schizonts in the 6 control wells were 145.5±9 (A), and 148±9 (B). The results are representative of three independent experiments. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test).
A. Primary human hepatocytes were treated or not with 100 ng/ml of human TNF-α together with or without SMT or NAC at 48 h before, at the time and every day for day 1 to day 5 after sporozoite inoculation. B. In the same experiment, supernatants from cells treated previously for 48 h with human TNF-α were added together with P. falciparum sporozoites to fresh human primary hepatocytes. Medium was changed after 3 hr and every day after sporozoite inoculation. In both experimental settings, cultures were stopped 5 days later. Data are presented are the mean (± SD) reduction in liver schizont numbers in triplicate wells to the mean number in 6 control wells and are derived from one of two experiments. The numbers of P. falciparum 5 day-liver schizonts in the 6 control wells were 179.2±26.1. * p<0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test).
A soluble mediator synthesized by human TNF-α-stimulated human hepatocytes prevents P. falciparum development
Since inhibition was observed only when cells were treated 48 h or 24 h before sporozoite infection but not on the time of sporozoite inoculation, we hypothesized that TNF-α-treated hepatocytes might release a parasite-inhibitory soluble mediator as previously shown for IL-1 or other inflammatory stimuli , . P. falciparum sporozoites in normal medium were added to supernatant medium obtained from human hepatocytes treated for 48 h with TNF-α (1/1 volume). The mixture was then added to fresh human hepatocytes to initiate the infection. A strong significant inhibition of sporozoite development was observed, and it reached levels similar to those induced by direct TNF-α pre-treatment of the cultures (Figure 6B). This showed that an inhibitory soluble mediator was produced by TNF-α-stimulated hepatocytes.
The in vitro experiments presented here were designed to assess parameters, such as the origin of the cytokine, the origin of the host cells, the parasite species, or the schedule of application that might affect the anti-liver stage activity of TNF-α. Different types of hepatocytes and hepatoma cell lines together with 3 Plasmodium species were used. Our main finding is that human TNF-α was able to inhibit the hepatic development of two rodent malaria species, P. yoelii and to a lesser extent P. berghei (Figure 1), and most importantly that of P. falciparum (Figure 4). We showed that this activity of TNF-α was dependent on the host cell type and on the schedule of this cytokine's administration. Maximal inhibition could be obtained when human TNF-α was administered 48 h before sporozoite inoculation to HepG2-CD81 cells (Figure 1). Significant inhibition was still observed when cells were treated 24 h before sporozoite inoculation both in HepG2-CD81 infected with P. yoelii and in highly purified primary human hepatocytes infected with P. falciparum. The good concordance between the data derived from P. yoelii/HepG2-CD81 combination and that from the P. falciparum/human primary hepatocyte combination makes it a good surrogate in future studies of the role of non-specific immune components against the malaria hepatic stages. These results differed from those obtained using primary mouse hepatocytes where an effect was observed only when the cultures were treated 48 h (Figure 3) but not 24 h  before P. yoelii sporozoite inoculation. Of the different parameters that might account for this difference we favour those related to the TNF-αmechanism of action. Murine TNF-α and human TNF-α differ in their affinity to the various host TNF receptors. Human TNF-α signals only through TNFR1 in mouse cells  and, as shown here, it had no effect on primary mouse hepatocytes infected with P. yoelii or with P. berghei (Figure 3). Signalling by TNF-R1 signals is effected through the TRAD/NEMO pathway to NF-kB or through FAD to activate caspase for apoptosis. TNF-R2 also mediates NF-kB activation through the TRAF pathway , however, in hepatocytes only TNFR1 mediates activation of NF-kB . The NF-kB pathway is necessary for the induction of the NO or ROI in hepatocytes . These two mediators have been shown to inhibit the Plasmodium liver stage , . We did not observe the induction or NO and ROI, which strongly suggests that it was TNFR2 but not TNFR1 that was involved in TNF-α signalling in infected hepatocytes. It has been proposed recently that malaria parasites manipulate their host hepatocytes to make them resistant to the apoptosis induced by TNF-α in vivo or in vitro  through interference with the NF-kB pathway  and consequently allowing them to escape the TNFR1-signaled cytotoxic effect of TNF-α. In addition, since signalling through TNF-R2 has also been involved in the necrotic effect of TNF-α , we tested whether treatment with TFN-α induced infected hepatocyte necrosis. Such an effect was ruled out because cell cytotoxicity was not observed in the MTT assays conducted after treatment with TNF-α.
The fact that the inhibitory effect of TNF-α was observed only when cultures were pre-incubated with the cytokine suggested that stimulated hepatocytes secrete an inhibitory factor and/or that the TNF-α treatment makes them refractory to infection. Host cell refractoriness is unlikely because addition of the supernatant from TNF-α-stimulated hepatocytes to the cultures was sufficient to obtain hepatic parasite inhibition. TNF-α alone or together with IL-6 and IL-1, is known to induce the synthesis of acute phase response proteins by hepatocytes. Although the acute phase response to inflammatory stimuli is evolutionary conserved, species-specific differences exist , . IL-1 was previously shown to prevent sporozoite development in human or rat primary hepatocytes in vitro through the action of an acute phase protein, the C-reactive protein (CRP) , . Human or rat C-reactive proteins can bind sporozoite and prevent their invasion and further development in hepatocytes , . However, Yap et al.  have shown that CRP is not produced by human hepatocytes after TNF-α stimulation. They also showed that TNF-α treatment blocks the induction of CRP stimulated by IL-1 or IL-6 treatment of human hepatocytes. This suggest strongly that this acute phase protein does not mediate the TNF-α effect. It has been reported previously that two other acute phase proteins, the protease inhibitors α1-antitrypsin and α2 macroglobulin, were also able to prevent sporozoite infection and development . Parasite proteases are necessary for sporozoite invasion in hepatocytes  and thus may be targeted by these two protease inhibitors. However, although TNF-α has been shown to increase the synthesis of α1-antitrypsin  or α2 macroglobulin  in HepG2 cells, it does not induce these molecules in human hepatocytes . Thus, the nature of the inhibitory mediator secreted by human hepatocytes is still unknown and deserves further study. In the mouse liver the profile of acute phase proteins induced by inflammatory stimuli is different, for example mouse hepatocytes do not synthesize CRP. Serum Amyloid A is induced by TNF-α in mouse hepatocytes  and it might be responsible for the inhibition that is consequent to TNF-α-stimulation of mouse primary hepatocytes or mouse hepatoma cell lines. However, Serum Amyloid A is not induced in human hepatocytes by TNF-α stimulation  suggesting that other mediators might be involved.
During malaria blood stage infection, the production of TNF-α is increased , , and these cytokine might modulate new liver stage infections , . By extension, any systemic inflammations or infections or more localized liver infections, of viral or bacterial origin, that induces high level of TNF-α might also have an inhibitory effect on the liver stages, which could consequently influence the outcome of a subsequent blood infection and its associated pathology , .
LR, DM and GS are currently part of an official collaboration between SIgN/A*STAR and INSERM (Laboratoire International Associé, INSERM).
Conceived and designed the experiments: ND JFF LR. Performed the experiments: ND JFF ACG MM GS LR. Analyzed the data: ND JFF DM GS LR. Contributed reagents/materials/analysis tools: JMC AJFL GJvG RWS JMS LH. Wrote the paper: ND GS LR.
- 1. Grau GE, Taylor TE, Molyneux ME, Wirima JJ, Vassalli P, et al. (1989) Tumor necrosis factor and disease severity in children with falciparum Malaria. N Engl J Med 320: 1586–1591.GE GrauTE TaylorME MolyneuxJJ WirimaP. Vassalli1989Tumor necrosis factor and disease severity in children with falciparum Malaria.N Engl J Med32015861591
- 2. Kern P, Hemmer CJ, Vandamme J, Gruss HJ, Dietrich M (1989) Elevated tumor necrosis factor-alpha and interleukin-6 serum levels as markers for complicated plasmodium-falciparum malaria. Am J Med 87: 139.P. KernCJ HemmerJ. VandammeHJ GrussM. Dietrich1989Elevated tumor necrosis factor-alpha and interleukin-6 serum levels as markers for complicated plasmodium-falciparum malaria.Am J Med87139
- 3. Kwiatkowski DP, Hill AVS, Sambou I, Twumasi PM, Castracane J, et al. (1990) TNF concentration in fatal cerebral, non fatal cerebral, and uncomplicated Plasmodium falciparum malaria. Lancet 336: 1201–1205.DP KwiatkowskiAVS HillI. SambouPM TwumasiJ. Castracane1990TNF concentration in fatal cerebral, non fatal cerebral, and uncomplicated Plasmodium falciparum malaria.Lancet33612011205
- 4. Amani V, Vigario AM, Belnoue E, Marussig M, Fonseca L, et al. (2000) Involvement of IFN-gamma receptor-mediated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection. Eur J Immunol 30: 1646–1655.V. AmaniAM VigarioE. BelnoueM. MarussigL. Fonseca2000Involvement of IFN-gamma receptor-mediated signaling in pathology and anti-malarial immunity induced by Plasmodium berghei infection.Eur J Immunol3016461655
- 5. Grau GE, Fajardo LF, Piguet PF, Allet B, et al. (1987) Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria. Science 237: 1210–1212.GE GrauLF FajardoPF PiguetB. Allet1987Tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria.Science23712101212
- 6. Karunaweera ND, Grau GE, Gamage P, Carter RL, Mendis KN (1992) Dynamics of fever and serum levels of tumor necrosis factor are closely associated during clinical paroxysms in Plasmodium vivax malaria. Proc Natl Acad Sci USA 89: 3200–3203.ND KarunaweeraGE GrauP. GamageRL CarterKN Mendis1992Dynamics of fever and serum levels of tumor necrosis factor are closely associated during clinical paroxysms in Plasmodium vivax malaria.Proc Natl Acad Sci USA8932003203
- 7. Clark IA, Hunt NH, Butcher GA, Cowden WB (1987) Inhibition of murine malaria Plasmodium chabaudi in vivo by recombinant interferon gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole. J Immunol 139: 3493–3496.IA ClarkNH HuntGA ButcherWB Cowden1987Inhibition of murine malaria Plasmodium chabaudi in vivo by recombinant interferon gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole.J Immunol13934933496
- 8. Hernandez-Valladares M, Naessens J, Musoke AJ, Sekikawa K, Rihet P, et al. (2006) Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ. Exp Parasitol 114: 271–278.M. Hernandez-ValladaresJ. NaessensAJ MusokeK. SekikawaP. Rihet2006Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ.Exp Parasitol114271278
- 9. Mordmuller BG, Metzger WG, Juillard P, Brinkman BM, Verweij CL, et al. (1997) Tumor necrosis factor in Plasmodium falciparum malaria: high plasma level is associated with fever, but high production capacity is associated with rapid fever clearance. Eur Cytokine Netw 8: 29–35.BG MordmullerWG MetzgerP. JuillardBM BrinkmanCL Verweij1997Tumor necrosis factor in Plasmodium falciparum malaria: high plasma level is associated with fever, but high production capacity is associated with rapid fever clearance.Eur Cytokine Netw82935
- 10. Schofield L, Nussenzweig RS, Nussenzweig V (1988) CD8+ T cells and gamma-interferon required for immunity to sporozoite challenge. Report of the tenth meeting of the scientific working group on the immunology of Malaria 17–19.L. SchofieldRS NussenzweigV. Nussenzweig1988CD8+ T cells and gamma-interferon required for immunity to sporozoite challenge.Report of the tenth meeting of the scientific working group on the immunology of Malaria1719
- 11. Nussler AK, Pied S, Goma J, Renia L, Miltgen F, et al. (1991) TNF inhibits malaria hepatic stages in vitro via IL-6 liver synthesis. Int Immunol 3: 317–321.AK NusslerS. PiedJ. GomaL. ReniaF. Miltgen1991TNF inhibits malaria hepatic stages in vitro via IL-6 liver synthesis.Int Immunol3317321
- 12. Southan GJ, Szabo C, Thiemermann C (1995) Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity. Br J Pharmacol 114: 510–516.GJ SouthanC. SzaboC. Thiemermann1995Isothioureas: potent inhibitors of nitric oxide synthases with variable isoform selectivity.Br J Pharmacol114510516
- 13. Cotgreave IA (1997) N-acetylcysteine: pharmacological considerations and experimental and clinical applications. Adv Pharmacol 38: 205–227.IA Cotgreave1997N-acetylcysteine: pharmacological considerations and experimental and clinical applications.Adv Pharmacol38205227
- 14. Franke-Fayard B, Trueman H, Ramesar J, Mendoza J, van der Keur M, et al. (2004) A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle. Mol Biochem Parasitol 137: 23–33.B. Franke-FayardH. TruemanJ. RamesarJ. MendozaM. van der Keur2004A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.Mol Biochem Parasitol1372333
- 15. Nudelman S, Renia L, Charoenvit Y, Yuan L, Miltgen F, et al. (1989) Dual action of anti-sporozoite antibodies in vitro. J Immunol 143: 996–1000.S. NudelmanL. ReniaY. CharoenvitL. YuanF. Miltgen1989Dual action of anti-sporozoite antibodies in vitro.J Immunol1439961000
- 16. Ponnudurai T, Lensen AHW, Meuwissen JHET (1983) An automated large-scale culture system of Plasmodium falciparum using tangential flow filtration for medium change. Parasitology 87: 439–445.T. PonnuduraiAHW LensenJHET Meuwissen1983An automated large-scale culture system of Plasmodium falciparum using tangential flow filtration for medium change.Parasitology87439445
- 17. Silvie O, Greco C, Franetich JF, Dubart-Kupperschmitt A, Hannoun L, et al. (2006) Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species. Cell Microbiol 8: 1134–1146.O. SilvieC. GrecoJF FranetichA. Dubart-KupperschmittL. Hannoun2006Expression of human CD81 differently affects host cell susceptibility to malaria sporozoites depending on the Plasmodium species.Cell Microbiol811341146
- 18. Marussig M, Renia L, Motard A, Miltgen F, Petour P, et al. (1997) Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection. Int Immunol 9: 1817–1824.M. MarussigL. ReniaA. MotardF. MiltgenP. Petour1997Linear and multiple antigen peptides containing defined T and B epitopes of the Plasmodium yoelii circumsporozoite protein: antibody-mediated protection and boosting by sporozoite infection.Int Immunol918171824
- 19. Silvie O, Franetich JF, Charrin S, Mueller MS, Siau A, et al. (2004) A Role for Apical Membrane Antigen 1 during Invasion of Hepatocytes by Plasmodium falciparum Sporozoites. J Biol Chem 279: 9490–9496.O. SilvieJF FranetichS. CharrinMS MuellerA. Siau2004A Role for Apical Membrane Antigen 1 during Invasion of Hepatocytes by Plasmodium falciparum Sporozoites.J Biol Chem27994909496
- 20. Mosmann TR (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63.TR Mosmann1983Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays.J Immunol Methods655563
- 21. Renia L, Mattei DM, Goma J, Pied S, Dubois P, et al. (1990) A malaria heat shock like protein epitope expressed on the infected hepatocyte surface is the target of antibody-dependent cell-mediated cytotoxic mechanisms by non-parechymal liver cells. Eur J Immunol 20: 1445–1449.L. ReniaDM MatteiJ. GomaS. PiedP. Dubois1990A malaria heat shock like protein epitope expressed on the infected hepatocyte surface is the target of antibody-dependent cell-mediated cytotoxic mechanisms by non-parechymal liver cells.Eur J Immunol2014451449
- 22. Tsuji M, Mattei D, Nussenzweig RS, Eichinger D, Zavala F (1994) Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites. Parasitol Res 80: 16–21.M. TsujiD. MatteiRS NussenzweigD. EichingerF. Zavala1994Demonstration of heat-shock protein 70 in the sporozoite stage of malaria parasites.Parasitol Res801621
- 23. Gego A, Silvie O, Franetich JF, Farhati K, Hannoun L, et al. (2006) New approach for high-throughput screening of drug activity on Plasmodium liver stages. Antimicrob Agents Chemother 50: 1586–1589.A. GegoO. SilvieJF FranetichK. FarhatiL. Hannoun2006New approach for high-throughput screening of drug activity on Plasmodium liver stages.Antimicrob Agents Chemother5015861589
- 24. Knowles BB, Howe C, Aden D (1980) Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science 209: 497–499.BB KnowlesC. HoweD. Aden1980Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen.Science209497499
- 25. Hollingdale MR, Leland P, Schwartz AL (1983) In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei in a hepatoma cell line. Am J Trop Med Hyg 32: 682–684.MR HollingdaleP. LelandAL Schwartz1983In vitro cultivation of the exoerythrocytic stage of Plasmodium berghei in a hepatoma cell line.Am J Trop Med Hyg32682684
- 26. Calvo-Calle JM, Moreno A, Eling WMC, Nardin EH (1994) In vitro development of infectious liver stages of Plasmodium yoelii and P. berghei malaria in human cell lines. Exp Parasitol 79: 362–373.JM Calvo-CalleA. MorenoWMC ElingEH Nardin1994In vitro development of infectious liver stages of Plasmodium yoelii and P. berghei malaria in human cell lines.Exp Parasitol79362373
- 27. Mota MM, Rodriguez A (2000) Plasmodium yoelii: Efficient in Vitro invasion and complete development of sporozoites in mouse hepatic cell lines. Exp Parasitol 96: 257–259.MM MotaA. Rodriguez2000Plasmodium yoelii: Efficient in Vitro invasion and complete development of sporozoites in mouse hepatic cell lines.Exp Parasitol96257259
- 28. Nussler AK, Pied S, Pontet M, Miltgen F, Renia L, et al. (1991) Inflammatory status and pre-erythrocytic stages of malaria. Role of the C-reactive protein. Exp Parasitol 72: 1–7.AK NusslerS. PiedM. PontetF. MiltgenL. Renia1991Inflammatory status and pre-erythrocytic stages of malaria. Role of the C-reactive protein.Exp Parasitol7217
- 29. Pied S, Nussler AK, Pontet M, Miltgen F, Matile H, et al. (1989) C-reactive protein protects against pre-erythrocytic stages of malaria. Infect Immun 57: 278–282.S. PiedAK NusslerM. PontetF. MiltgenH. Matile1989C-reactive protein protects against pre-erythrocytic stages of malaria.Infect Immun57278282
- 30. Tartaglia LA, Weber RF, Figari IS, Reynolds C, Palladino MA Jr, et al. (1991) The two different receptors for tumor necrosis factor mediate distinct cellular responses. Proc Natl Acad Sci USA 88: 9292–9296.LA TartagliaRF WeberIS FigariC. ReynoldsMA Palladino Jr1991The two different receptors for tumor necrosis factor mediate distinct cellular responses.Proc Natl Acad Sci USA8892929296
- 31. Bradley JR, Pober JS (2001) Tumor necrosis factor receptor-associated factors (TRAFs). Oncogene 20: 6482–6491.JR BradleyJS Pober2001Tumor necrosis factor receptor-associated factors (TRAFs).Oncogene2064826491
- 32. Yamada Y, Webber EM, Kirillova I, Peschon JJ, Fausto N (1998) Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor. Hepatology 28: 959–970.Y. YamadaEM WebberI. KirillovaJJ PeschonN. Fausto1998Analysis of liver regeneration in mice lacking type 1 or type 2 tumor necrosis factor receptor: requirement for type 1 but not type 2 receptor.Hepatology28959970
- 33. Lee HJ, Oh YK, Rhee M, Lim JY, Hwang JY, et al. (2007) The role of STAT1/IRF-1 on synergistic ROS production and loss of mitochondrial transmembrane potential during hepatic cell death induced by LPS/d-GalN. J Mol Biol 369: 967–984.HJ LeeYK OhM. RheeJY LimJY Hwang2007The role of STAT1/IRF-1 on synergistic ROS production and loss of mitochondrial transmembrane potential during hepatic cell death induced by LPS/d-GalN.J Mol Biol369967984
- 34. Nussler AK, Drapier JC, Renia L, Pied S, Miltgen F, et al. (1991) L-arginine dependent destruction of intrahepatic malaria parasite in response to tumor necrosis factor and/or interleukin 6 stimulation. Eur J Immunol 21: 227–230.AK NusslerJC DrapierL. ReniaS. PiedF. Miltgen1991L-arginine dependent destruction of intrahepatic malaria parasite in response to tumor necrosis factor and/or interleukin 6 stimulation.Eur J Immunol21227230
- 35. Pied S, Renia L, Nussler AK, Miltgen F, Mazier D (1991) Inhibitory activity of IL-6 on malaria hepatic stages. Parasite Immunol 13: 211–217.S. PiedL. ReniaAK NusslerF. MiltgenD. Mazier1991Inhibitory activity of IL-6 on malaria hepatic stages.Parasite Immunol13211217
- 36. Van De Sand C, Horstmann S, Schmidt A, Sturm A, Bolte S, et al. (2005) The liver stage of Plasmodium berghei inhibits host cell apoptosis. Mol Microbiol 58: 731–742.C. Van De SandS. HorstmannA. SchmidtA. SturmS. Bolte2005The liver stage of Plasmodium berghei inhibits host cell apoptosis.Mol Microbiol58731742
- 37. Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, et al. (2007) Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131: 492–504.AP SinghCA BuscagliaQ. WangA. LevayDR Nussenzweig2007Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite.Cell131492504
- 38. Erickson SL, de Sauvage FJ, Kikly K, Carver-Moore K, Pitts-Meek S, et al. (1994) Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice. Nature 372: 560–563.SL EricksonFJ de SauvageK. KiklyK. Carver-MooreS. Pitts-Meek1994Decreased sensitivity to tumour-necrosis factor but normal T-cell development in TNF receptor-2-deficient mice.Nature372560563
- 39. Fey GH, Gauldie J (1990) The acute phase response of the liver in inflammation. Prog Liver Dis 9: 89–116.GH FeyJ. Gauldie1990The acute phase response of the liver in inflammation.Prog Liver Dis989116
- 40. Heinrich PC, Castell JV, Andus T (1990) Interleukin-6 and the acute phase response. Biochem J 265: 621–636.PC HeinrichJV CastellT. Andus1990Interleukin-6 and the acute phase response.Biochem J265621636
- 41. Yap SH, Moshage HJ, Hazenberg BPC, Roelofs HMJ, Bijzet J, et al. (1991) Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes. Biochim Biophys Acta 1091: 405–408.SH YapHJ MoshageBPC HazenbergHMJ RoelofsJ. Bijzet1991Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes.Biochim Biophys Acta1091405408
- 42. Pied S, Tabone MD, Chatellier G, Marussig M, Jardel C, et al. (1995) Non specific resistance against malaria pre-erythrocytic stages: involvement of acute phase proteins. Parasite 2: 263–268.S. PiedMD TaboneG. ChatellierM. MarussigC. Jardel1995Non specific resistance against malaria pre-erythrocytic stages: involvement of acute phase proteins.Parasite2263268
- 43. Coppi A, Pinzon-Ortiz C, Hutter C, Sinnis P (2005) The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion. J Exp Med 201: 27–33.A. CoppiC. Pinzon-OrtizC. HutterP. Sinnis2005The Plasmodium circumsporozoite protein is proteolytically processed during cell invasion.J Exp Med2012733
- 44. Baumann H, Richards C, Gauldie J (1987) Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells. J Immunol 139: 4122–4128.H. BaumannC. RichardsJ. Gauldie1987Interaction among hepatocyte-stimulating factors, interleukin 1, and glucocorticoids for regulation of acute phase plasma proteins in human hepatoma (HepG2) cells.J Immunol13941224128
- 45. Magielska-Zero D, Bereta J, Czuba-Pelech B, Pajdak W, Gauldie J, et al. (1988) Inhibitory effect of human recombinant interferon gamma on synthesis of acute phase proteins in human hepatoma Hep G2 cells stimulated by leukocyte cytokines, TNF alpha and IFN-beta 2/BSF-2/IL-6. Biochem Int 17: 17–23.D. Magielska-ZeroJ. BeretaB. Czuba-PelechW. PajdakJ. Gauldie1988Inhibitory effect of human recombinant interferon gamma on synthesis of acute phase proteins in human hepatoma Hep G2 cells stimulated by leukocyte cytokines, TNF alpha and IFN-beta 2/BSF-2/IL-6.Biochem Int171723
- 46. Ghezzi P, Sipe JD (1988) Dexamethasone modulation of LPS, IL-1, and TNF stimulated serum amyloid A synthesis in mice. Lymphokine Res 7: 157–166.P. GhezziJD Sipe1988Dexamethasone modulation of LPS, IL-1, and TNF stimulated serum amyloid A synthesis in mice.Lymphokine Res7157166
- 47. Castell JV, Gomez-Lechon MJ, David M, Andus T, Geiger T, et al. (1989) Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes. FEBS Lett 242: 237–239.JV CastellMJ Gomez-LechonM. DavidT. AndusT. Geiger1989Interleukin-6 is the major regulator of acute phase protein synthesis in adult human hepatocytes.FEBS Lett242237239
- 48. Mazier D, Goma J, Pied S, Renia L, Nussler AK, et al. (1990) Hepatic phase of Malaria. A crucial role as “go-between” with other stages. Bull World Health Organ 68: 126–131.D. MazierJ. GomaS. PiedL. ReniaAK Nussler1990Hepatic phase of Malaria. A crucial role as “go-between” with other stages.Bull World Health Organ68126131
- 49. Mazier D, Renia L, Nussler AK, Pied S, Goma J, et al. (1990) Hepatic phase of malaria parasite is the target of cellular mechanisms induced by the previous and the subsequent stage. A crucial role for the liver nonparenchymal cells. Immunol Lett 25: 65–70.D. MazierL. ReniaAK NusslerS. PiedJ. Goma1990Hepatic phase of malaria parasite is the target of cellular mechanisms induced by the previous and the subsequent stage. A crucial role for the liver nonparenchymal cells.Immunol Lett256570