Limited response of NK92 cells to Plasmodium falciparum-infected erythrocytes

<p>Abstract</p> <p>Background</p> <p>Mechanisms 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 <it>Plasmodium falciparum</it>. In this study, the suitability of NK92 cells as models for the NK mechanisms involved in the immune response against malaria was investigated.</p> <p>Methods</p> <p>NK92 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 <it>P. falciparum</it>-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.</p> <p>Results</p> <p>By 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 <it>in vitro</it>.</p> <p>Conclusions</p> <p>Although 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.</p

Plasmodium falciparum-Infected Erythrocytes Induce Granzyme B by NK Cells through Expression of Host-Hsp70

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

Investigation of the effector mechanisms of Natural Killer Cells against Plasmodium falciparum-infected erythrocytes

Natürliche Killerzellen (NK-Zellen) sind wichtige Effektorzellen der angeborenen Immunantwort, die entscheidend sind für die initiale Reaktion auf Plasmodium falciparum-Infektion sowie die Regulation nachfolgender adaptiver Prozesse. In der vorliegenden Arbeit sollte ihr Einfluss auf P. falciparum infizierte Erythrozyten (iRBZ) in vitro untersucht werden. Insbesondere sollten Mechanismen der direkten Aktivierung von NK-Zellen und deren Einflussnahme auf die Parasitenentwicklung durch Ausübung von Effektorfunktionen analysiert werden. Zunächst wurde ermittelt, inwiefern die NK92-Zelllinie als Modell bei der untersuchten Interaktion geeignet ist. Die Transkriptionsanalyse von NK92-Zellen zeigte, dass die Mehrheit der regulierten Gene nach 24h Kokultur mit iRBZ im Zusammenhang mit Zellzyklusprogression, Antiapoptose und Zellwachstum stand. Weiterhin waren einige Gene aktiviert, die Immunantwort und Aktivierung der NK-Zellen regulieren. Allerdings sollten diese Ergebnisse unter dem Aspekt der offensichtlichen Voraktivierung der verwendeten Zelllinie betrachtet werden, die sich als konstitutive Expression von Aktivierungsmarkern und Interferon-gamma-Produktion äußerte. Obwohl die NK92-Zellen einen wachstumshemmenden Einfluss auf P. falciparum auszuüben schienen, war dieser nicht spezifisch und die Zelllinie erwies sich unter den beobachteten Bedingungen als ungeeignetes Modellsystem. Nachfolgende Analyse von Rezeptoren der IL-6 Familie machte deutlich, dass diese durch unterschiedliche Zytokine (IFN-alpha, IL-6 und IL-2/-12/-18), aber nicht die parasitierten Erythrozyten beeinflusst werden konnten. IL-6 scheint in einem negativen Feedback-Loop die Beendigung der Signalkette durch verminderte Expression der untersuchten Rezeptoren zu bewirken, während die anderen Zytokine in unterschiedlichem Maße eine verstärkte Rezeptorexpression induzierten. Weitere Untersuchungen von Rezeptoren und Liganden ergaben einerseits keinen Einfluss auf den aktivierenden Rezeptor NKG2C und andererseits auf MICA/B sowie HLA-E. Jedoch könnte der Korezeptor CD94 von sowohl aktivierenden als auch inhibierenden NKG2-Rezeptoren bei der Bindung von Hitzeschockprotein 70 (Hsp70) auf der Erythrozytenmembran eine Rolle spielen, da er immer auf der NK-Zelloberfläche exprimiert war. Aufgrund der durchgeführten Untersuchungen wurde eine Hypothese zur Erkennung und Eliminierung der iRBZ erstellt. Diese beinhaltet, dass NK-Zellen über Hsp70 auf der iRBZ-Membran zur erhöhten Granzym B-Freisetzung stimuliert werden, was in der Eryptose der parasitierten Erythrozyten resultiert. In der vorliegenden Arbeit konnte diese Hypothese bestätigt werden. Des Weiteren konnte der Effekt durch das Hsp70-Epitop TKD-Peptid verstärkt werden. Es ist daher denkbar, TKD oder Hsp70 selbst als Immunostimulans oder als Adjuvans in der Impfstoffentwicklung zu nutzen, wodurch sich neue Alternativen für die Prävention oder Behandlung von Malaria eröffnen.Natural Killer (NK) cells are important effector cells of the innate immune system and are thought to play a crucial role in early responses to Plasmodium falciparum-infection and by regulating adaptive immunity. The aim of this thesis was to investigate the interaction between NK cells and P. falciparum-infected erythrocytes in vitro. Particular interest was on elucidation of mechanisms occurring during such interaction and to rule out whether the intracellular parasite has a direct impact on activation of NK cell effector functions. First, assessment of suitability of the NK92 cell line as a model to study NK cell-iRBC-interaction was addressed. Transcription analysis of NK92 cells showed that the majority of regulated genes after 24h co-culture were linked to cell cycle progression, anti-apoptosis and cell growth. Additionally, genes important for regulation of immune responses and NK cell activation were altered. However, those results have to be interpreted in the context of obvious pre-activation of the used cell line. Although NK92 seemed to exert a growth-limiting influence on P. falciparum, this effect was not specific. Given these circumstances, NK92 cells cannot be considered as a good model to study NK cell-parasite-interactions in malaria in vitro. Subsequent analysis of IL-6-family receptors revealed a differential regulation by several cytokines (IFN-alpha, IL-6 und IL-2/-12/-18-mix), but not the parasitized erythrocytes themselves. IL-6 seemed to stop signal transduction via a negative feed-back loop, while the other cytokines resulted in increased receptor expression. Further investigation of receptors and ligands demonstrated no involvement of the activating receptor NKG2C or ligands like MICA/B and HLA-E. However, CD94 could serve as a co-receptor of erythrocyte membrane-associated heat shock protein (Hsp) 70, since it was constitutively expressed on the NK cell surface. Due to Hsp70 on iRBC, NK cells were stimulated to elevated granzyme B release leading to subsequent eryptosis of the parasitized erythrocytes. This proposed mechanism is promising since the recognised Hsp70 epitope TKD-peptide could serve as an immunostimulants or Hsp70 itself as an adjuvants in vaccine development which could open the way for new alternatives of malaria prevention or treatment

Eryptosis of iRBC after co-culture with PBMCs or purified NK cells.

<p>iRBC were co-cultured 3∶1 for 24 hours with NK cells, NK cells+TKD (5 day stimulation), PBMC, PBMC+TKD (5 day stimulation) of the same donor. 0.5×10⁶ erythrocytes were washed in 5 mM Ringer solution. Afterwards, cells were stained for 15 minutes with Annexin-V (1∶500) and propidium iodide (1∶50). Eryptotic cells were determined as Annexin-V-positive (AV⁺) and propidium iodide-negative (PI⁻). A: Baseline levels of eryptotic uRBC and iRBC at the start of the experiment and after 24 h of culture in growth medium without leukocytes. RBC were stained with AV+PI or left unstained. Gating was done based on FSC/SSC properties and the unstained control. AV was measured in the FL1 channel and PI in the FL2 channel. B: Percentage of AV⁺/PI⁻-iRBC after co-culture in growth medium (GM) or with different effector cells in relation to starting parasitemia (** p≤0.01, student's <i>t</i> test, n = 6). C: Normalized FSC of iRBC after co-culture in growth medium (GM) or with different effector cells (* p≤0.05, student's <i>t</i> test, n = 6). FSC values of co-cultured iRBC were normalized to untreated iRBC (GM). D: PI⁺ necrotic iRBC after culture in growth medium (GM) or with various effector cells. Numbers of necrotic cells were normalized to untreated iRBC (GM).</p

Growth delay in <i>Plasmodium falciparum</i> development after NK cell contact.

<p>A: Representative figure of 3D7-iRBC before the start of co-cultures. Parasites were synchronized by magnetic cell sorting columns for late stages. B: Representative figure of 3D7-iRBC after 24 h of incubation. The parasites have developed into normal ring-stage forms that are expected for this time point. Infected RBC were either co-cultured 3∶1 with autologous NK cells (C), NK cells+TKD (pre-stimulated for 5 days before co-culture with 2 µg/ml TKD peptide) (D), PBMCs (E), or PBMCs stimulated with TKD peptide (F). Normal parasite development of a control culture of parasites without leukocytes was observed in parallel. A blood smear was prepared before and after 24 hours of incubation and stained with 10% Giemsa. Experiments were repeated with RBC from 3 different donors.</p

Transcriptional and translational changes of GzmA, GzmB and Perforin in NK92 cells after 24 hours stimulation.

<p>NK92 cells were left untreated (ns) or cultured with IL-12/-18 (IL), IFN-α, uRBC or iRBC (1∶3) for 24 h. A: Changes on transcriptional level of stimulated cells in comparison to untreated cells were analyzed for GzmA, GzmB and Perforin. β-Actin expression served as house-keeping gene normalizer. Data are represented as mean ± SD (student's <i>t</i> test * p<0.05; ** p<0.01) and are representative of three independent experiments each performed in duplicate. B: After 24 h, 1×10⁷ NK92 cells were lysed, incubated 30 min on ice and subsequently centrifuged at 13000× <i>g</i> for 15 min at 4°C. 10 µg of total supernatant protein were separated by 7.5% SDS-PAGE and blotted onto a nitrocellulose membrane. After blocking, membranes were incubated for 1 h with anti-β-Actin (lane 1), anti-hGzmA (lane 2), anti-hGzmB (lane 3), or anti-hPrf (lane 4).</p

Granzyme B-Elispot of NK92 cells (A) or isolated NK cells (B,C) after 24 hours of co-culture with i/uRBC.

<p>Some cultures were pre-activated 5 days with TKD peptide or the scrambled NGL peptide and/or pre-incubated with blocking Hsp70 antibody (cmHsp70.2) or a blocking antibody IgM-isotype control for 20 minutes before the start of the experiment. After stimulation, 2000 NK cells were cultured either alone, with iRBC or uRBC (1∶3 or 10∶1) on a GzmB antibody-coated 96-well plate. GzmB-releasing cells were counted after 24 hours of incubation (* p≤0.05, ** p≤0.01, *** p≤0.001, student's <i>t</i> test, n = 6).</p

Flow cytometry analysis of erythrocytes for possible NK cell ligands.

<p>0.5×10⁶ i/uRBC or Hela cells were stained with anti-hMICA/B-PE (A), anti-hHLA-E-PE (B) or the respective isotype control. As a control, 0.5×10⁶ iRBC, uRBC or NK92 cells were also stained with anti-hHLA-E-PE (B) or a PE-isotype control. The presence of Hsp70 on uRBC (C) and iRBC (D, E) was determined with anti-Hsp70 mAb cmHsp70.1-FITC compared to FITC isotype. Parasite DNA was stained with Hoechst dye (schizonts, D) that is detected in the Pacific blue channel or Hydroethidine (rings, E) that is detected in the PE channel. Experiments were repeated 3 times.</p