In the early immune response to Plasmodium falciparum-infected erythrocytes (iRBC), Natural Killer (NK) cells are activated, which suggests an important role in innate anti-parasitic immunity. However, it is not well understood whether NK cells directly recognize iRBC or whether stimulation of NK cells depends mainly on activating signals from accessory cells through cell-to-cell contact or soluble factors. In the present study, we investigated the influence of membrane-bound host Heat shock protein (Hsp) 70 in triggering cytotoxicity of NK cells from malaria-naïve donors or the cell line NK92 against iRBC. Hsp70 and HLA-E membrane expression on iRBC and potential activatory NK cell receptors (NKG2C, CD94) were assessed by flow cytometry and immunoblot. Upon contact with iRBC, Granzyme B (GzmB) production and release was initiated by unstimulated and Hsp70-peptide (TKD) pre-stimulated NK cells, as determined by Western blot, RT-PCR and ELISPOT analysis. Eryptosis of iRBC was determined by Annexin V-staining. Our results suggest that presence of Hsp70 and absence of HLA-E on the membrane of iRBC prompt the infected host cells to become targets for NK cell-mediated cytotoxicity, as evidenced by impaired parasite development. Contact of iRBC with NK cells induced release of GzmB. We propose that following GzmB uptake, iRBC undergo eryptosis via a perforin-independent, GzmB-mediated mechanism. Since NK activity toward iRBC could be specifically enhanced by TKD peptide and abrogated to baseline levels by blocking Hsp70 exposure, we propose TKD as an innovative immunostimulatory agent to be tested as an adjunct to anti-parasitic treatments in vivo.

A balanced proinflammatory cytokine response to Plasmodium ssp. infection is crucial to control the disease outcome. To elucidate the effect of cytokines and Plasmodium falciparum-infected erythrocytes on the regulation of interleukin (IL)-6 receptor (IL-6R), ciliary neurotrophic factor alpha (CNTFR-α) and glycoprotein (gp)130 in natural killer (NK) cells in the context of malaria, we assessed their gene expression and surface expression in NK92 cells. P. falciparum alone did not alter gene expression of the investigated receptors in NK92 cells. Analysis revealed a low effect of IL-6 on IL-6R surface expression in NK92 cells. However, at transcriptional level, a downregulation of IL-6R was observed following IL-6 stimulation. Thus, IL-6 might act within a negative feedback loop to terminate signal transduction by downregulating IL-6R expression. Additionally, we observed that IL-6R and CNTFR-α surface expression were regulated by a combination of IL-2, 12, and 18, and gp130 was influenced by interferon-α. Our results show that the IL-6 family receptors in NK92 cells are not directly influenced by P. falciparum. However, cytokines usually derived from accessory cells during malaria episodes may regulate IL-6 receptor signaling pathways. This finding encourages future studies in a more physiological context and with primary cells isolated from humans with and without malaria.

BackgroundMechanisms by which anti-malarial immune responses occur are still not fully clear. Natural killer (NK) cells are thought to play a pivotal role in innate responses against Plasmodium falciparum. In this study, the suitability of NK92 cells as models for the NK mechanisms involved in the immune response against malaria was investigated.MethodsNK92 cells were assessed for several signs of activation and cytotoxicity due to contact to parasites and were as well examined by oligonucleotide microarrays for an insight on the impact P. falciparum-infected erythrocytes have on their transcriptome. To address the parasite side of such interaction, growth inhibition assays were performed including non-NK cells as controls.ResultsBy performing microarrays with NK92 cells, the impact of parasites on a transcriptional level was observed. The findings show that, although not evidently activated by iRBCs, NK92 cells show transcriptional signs of priming and proliferation. In addition, decreased parasitaemia was observed due to co-incubation with NK92 cells. However, such effect might not be NK-specific since irrelevant cells also affected parasite growth in vitro.ConclusionsAlthough NK92 cells are here shown to behave as poor models for the NK immune response against parasites, the results obtained in this study may be of use for future investigations regarding host-parasites interactions in malaria.

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