G·A·B·B·A

Malaria is caused by the protozoan parasite Plasmodium and is one of the most prevalent infectious diseases worldwide. Plasmodium sporozoites invade hepatocytes, initiating the hepatic stage of malaria within minutes after inoculation into mammals by a blood-feeding Anopheles mosquito. This stage, although asymptomatic, is crucial for the establishment of infection. Thus, it constitutes an ideal target for prophylaxis. However, the infection mechanisms as well as the alterations induced by the parasite in the host liver cells remain to be clarified.

In the present work, we aimed at unravelling the alterations induced in host cells as a result of Plasmodium infection employing a genome-wide molecular microarray-based approach. Using an in vitro model of murine malaria infection, total RNA was extracted from P. berghei-infected Hepa1-6 hepatoma cells at 6, 12, 18 and 24 hours post-infection. The corresponding cRNAs were hybridized with Affymetrix oligonucleotide microarrays (GeneChip® Mouse Genome 430 2.0), and the transcriptome of infected cells was compared to that of non-infected ones, used as control. The transcriptional profile of infected cells differed substantially from that of non-infected ones, portraying the host response to Plasmodium infection. We identified a total of 611 host genes differentially expressed in Plasmodium-infected cells. Microarray-based data for a subset of these genes was validated using quantitative RT-PCR analysis. A higher number of modulated genes (38%), and with more pronounced modulation, was identified at 6 hours post-infection than at later time points (13%).

A comparison of these results with published data on host responses to Toxoplasma and Trypanosoma suggested that the modulations of the host transcriptional profiles are highly pathogen-specific. Whether the unique pattern of the host response to Plasmodium is due to changes induced by the parasite’s migration through hepatocytes prior to infection remains to be elucidated. A time-course analysis of the host transcriptional response to Plasmodium sporozoites infection can provide novel insights and a greatly expanded framework for further investigations. This approach alone does not discern which host genes are, in fact, potentially able to impair Plasmodium infection and, therefore, constitute possible targets for drug treatment. Hence, we decided to perform a functional analysis for 50 genes selected from the analysis of the host transcriptome. These were modulated by RNA interference in order to assess their possible role in P. berghei infection.

One of those genes whose knock-down led to a significant infection increase was the Vascular Endothelial Growth Factor (VEGF). VEGF levels were increased in cell supernatants 2 hours post P. berghei infection, probably due to cytosol leakage during sporozoite migration. Moreover, VEGF significantly decreases infection in a dose dependent manner, with a minimum effective concentration of 100 ng mL-1 and only if present in the first 2 hours of infection. Sporozoites incubated with VEGF retained their normal infection ability, indicating that this cytokine acts exclusively on the host cell. Flt-1 was the only VEGF receptor found in Hepa1-6 cells. Additionally, Placental Growth Factor (PlGF), another ligand for Flt-1, induced a similar infection decrease as VEGF, both in vitro and ex vivo. Inhibition of the tyrosine kinase activity of Flt-1 did not influence infection nor did it reverse the effect of VEGF, indicating that the latter does not occur through signaling. These findings suggest that Flt-1 is able to restrain Plasmodium infection at early time points. Therefore, it will be of considerable interest to further examine the mechanisms by which interactions of VEGF or PlGF molecules with Flt-1 modulate Plasmodium infection in hepatocytes.

Altogether, this work provides for the first time a full chronological picture of the transcriptional modifications occurring in the mammalian host cell as a result of Plasmodium sporozoite infection. Furthermore, it identifies a host cytokine whose receptor may influence the fate of malaria liver infection.