Inhibitory Effect of TNF-α on Malaria Pre-Erythrocytic Stage Development: Influence of Host Hepatocyte/Parasite Combinations

BACKGROUND: The liver stages of malaria parasites are inhibited by cytokines such as interferon-gamma 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-alpha possibly because of differences in the models used. We have reassessed the role of TNF-alpha in the different cellular systems used to study the Plasmodium pre-erythrocytic stages. METHODS AND FINDINGS: Human or mouse TNF-alpha 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-alpha 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-alpha inhibition was mediated by a soluble factor present in the supernatant of TNF-alpha stimulated hepatocytes but it was not related to NO or ROI. Treatment TNF-alpha prevents the development of human and rodent malaria pre-erythrocytic stages through the activity of a mediator that remains to be identified. CONCLUSIONS: Treatment TNF-alpha 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

Gene Disruption of Plasmodium falciparum p52 Results in Attenuation of Malaria Liver Stage Development in Cultured Primary Human Hepatocytes

Difficulties with inducing sterile and long lasting protective immunity against malaria with subunit vaccines has renewed interest in vaccinations with attenuated Plasmodium parasites. Immunizations with sporozoites that are attenuated by radiation (RAS) can induce strong protective immunity both in humans and rodent models of malaria. Recently, in rodent parasites it has been shown that through the deletion of a single gene, sporozoites can also become attenuated in liver stage development and, importantly, immunization with these sporozoites results in immune responses identical to RAS. The promise of vaccination using these genetically attenuated sporozoites (GAS) depends on translating the results in rodent malaria models to human malaria. In this study, we perform the first essential step in this transition by disrupting, p52, in P. falciparum an ortholog of the rodent parasite gene, p36p, which we had previously shown can confer long lasting protective immunity in mice. These P. falciparum P52 deficient sporozoites demonstrate gliding motility, cell traversal and an invasion rate into primary human hepatocytes in vitro that is comparable to wild type sporozoites. However, inside the host hepatocyte development is arrested very soon after invasion. This study reveals, for the first time, that disrupting the equivalent gene in both P. falciparum and rodent malaria Plasmodium species generates parasites that become similarly arrested during liver stage development and these results pave the way for further development of GAS for human use

Chloroquine Potentiates Primaquine Activity against Active and Latent Hepatic Plasmodia Ex Vivo: Potentials and Pitfalls

Contains fulltext : 228687.pdf (Publisher’s version ) (Open Access

Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites

Contains fulltext : 241912.pdf (Publisher’s version ) (Open Access

The Host Protein Aquaporin-9 is Required for Efficient Plasmodium falciparum Sporozoite Entry into Human Hepatocytes

Contains fulltext : 235497.pdf (Publisher’s version ) (Open Access

Laser capture microdissection enables transcriptomic analysis of dividing and quiescent liver stages of Plasmodium relapsing species

International audienceDormant liver stage forms (hypnozoites) of the malaria parasite Plasmodium vivax present major hurdles to control and eradicate infection. Despite major research efforts, the molecular composition of hypnozoites remains ill defined. Here, we applied a combination of state-of-the-art technologies to generate the first transcriptome of hypnozoites. We developed a robust laser dissection microscopy protocol to isolate individual Plasmodium cynomolgi hypnozoites and schizonts from infected monkey hepatocytes and optimized RNA-seq analysis to obtain the first transcriptomes of these stages. Comparative transcriptomic analysis identified 120 transcripts as being differentially expressed in the hypnozoite stage relative to the dividing liver schizont, with 69 and 51 mRNAs being up- or down-regulated, respectively, in the hypnozoites. This lead to the identification of potential markers of commitment to and maintenance of the dormant state of the hypnozoite including three transcriptional regulators of the ApiAP2 family, one of which is unique to P. cynomolgi and P. vivax, and the global translational repressor, eIF2a kinase eIK2, all of which are upregulated in the hypnozoite. Together, this work not only provides a primary experimentally-derived list of molecular markers of hypnozoites but also identifies transcriptional and posttranscriptional regulation of gene expression as potentially being key to establishing and maintaining quiescence

Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity

Fully effective vaccines for complex infections must elicit a diverse repertoire of antibodies (humoral immunity) and CD8+ T-cell responses (cellular immunity). Here, we present a synthetic glyco-adjuvant named p(Man-TLR7), which, when conjugated to antigens, elicits robust humoral and cellular immunity. p(Man-TLR7) is a random copolymer composed of monomers that either target dendritic cells (DCs) via mannose-binding receptors or activate DCs via Toll-like receptor 7 (TLR7). Protein antigens are conjugated to p(Man-TLR7) via a self-immolative linkage that releases chemically unmodified antigen after endocytosis, thus amplifying antigen presentation to T cells. Studies with ovalbumin (OVA)-p(Man-TLR7) conjugates demonstrate that OVA-p(Man-TLR7) generates greater humoral and cellular immunity than OVA conjugated to polymers lacking either mannose targeting or TLR7 ligand. We show significant enhancement of Plasmodium falciparum-derived circumsporozoite protein (CSP)-specific T-cell responses, expansion in the breadth of the alpha CSP IgG response and increased inhibition of sporozoite invasion into hepatocytes with CSP-p(Man-TLR7) when compared with CSP formulated with MPLA/QS-21-loaded liposomes-the adjuvant used in the most clinically advanced malaria vaccine. We conclude that our antigen-p(Man-TLR7) platform offers a strategy to enhance the immunogenicity of protein subunit vaccines

Human TNF-α inhibits the pre-erythrocytic stage of Pyy265BY.

<p>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. * <i>p</i><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.</p

A soluble mediator but not NO or RO intermediates synthesized by human TNF-α-stimulated human hepatocytes inhibits <i>P. falciparum</i> development.

<p>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 <i>P. falciparum</i> 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 <i>P. falciparum</i> 5 day-liver schizonts in the 6 control wells were 179.2±26.1. * <i>p</i><0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test).</p

Human TNF-α inhibits the pre-erythrocytic stage of <i>P. falciparum</i>.

<p>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 <i>P. falciparum</i> liver stages from triplicate experimental wells and from 8 control wells. * <i>p</i><0.05 versus control non-treated cultures (Kruskal-Wallis test, followed by Dunn test). The rate of infection for <i>P. falciparum</i> sporozoites was 0.2–0.5% for primary human hepatocytes depending on the experiment. Data are representative of two independent experiments with similar results.</p