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The Journal of Immunology, 2006, 177: 1229-1239.
Copyright © 2006 by The American Association of Immunologists

NK Cell Responses to Plasmodium Infection and Control of Intrahepatic Parasite Development1

Jacques Roland2,*, Valérie Soulard*, Christèle Sellier*, Anne-Marie Drapier*, James P. Di Santo{dagger}, Pierre-André Cazenave* and Sylviane Pied*

* Unité d’Immunophysiopathologie Infectieuse, Centre National de la Recherche Scientifique Unité de Recherche Associée 1961, Université Pierre et Marie Curie, Institut Pasteur, Paris, France; and {dagger} Unité des Cytokines et Développement Lymphoïde, Institut National de la Santé et de la Recherche Médicale U 668, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Various components of innate and adaptive immunity contribute to host defenses against Plasmodium infection. We investigated the contribution of NK cells to the immune response to primary infection with Plasmodium yoelii sporozoites in C57BL/6 mice. We found that hepatic and splenic NK cells were activated during infection and displayed different phenotypic and functional properties. The number of hepatic NK cells increased whereas the number of splenic NK cells decreased. Expression of the Ly49 repertoire was modified in the spleen but not in the liver. Splenic and hepatic NK cells have a different inflammatory cytokines profile production. In addition, liver NK cells were cytotoxic to YAC-1 cells and P. yoelii liver stages in vitro but not to erythrocytic stages. No such activity was observed with splenic NK cells from infected mice. These in vitro results were confirmed by the in vivo observation that Rag2–/– mice were more resistant to sporozoite infection than Rag2–/– {gamma} c–/– mice, whereas survival rates were similar for the two strains following blood-stage infection. Thus, NK cells are involved in early immune mechanisms controlling Plasmodium infection, mostly at the pre-erythrocytic stage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Malaria is one of the main causes of morbidity and mortality in tropical countries. The infection is initiated in the mammalian host when sporozoites, transmitted through the bite of an infected mosquito, invade the liver cells. The life cycle of Plasmodium within this host is characterized by two distinct developmental stages: the hepatic phase and the subsequent erythrocytic phase. During its development, the parasite exposes to the host immune system several waves of Ags specific to its extracellular forms (sporozoite and merozoite) and intracellular forms (within hepatocytes and erythrocytes). The immune response to Plasmodium is therefore complex and involves several components of the innate and adaptive immune systems. Protective immunity, limiting circulating parasite load and preventing severe disease, may develop after several exposures to infection. This immunity is mediated not only by Abs (1), but also by cellular effector mechanisms, including the CD4 and CD8 subsets of {alpha}beta T cells (2, 3, 4, 5, 6), {gamma}{delta} T cells (7, 8), NKT cells (9, 10, 11), dendritic cells (12), macrophages (13), and NK cells (14, 15). Most studies of protective immune responses during malaria have focused on the adaptive immune responses induced by immunization with irradiated sporozoites (16). Little is known about the establishment of protective immune responses during primary infection (17).

NK cells are essential effector cells of the innate immune system (18). They play an important role in the early phase of host defense against a variety of pathogens, including viruses, bacteria, and parasites. Recent advances in our understanding of NK cell receptors have provided new tools for the identification of subpopulations within the heterogeneous NK population. NK cells bear receptors from several families, with different physiological functions, illustrating the complexity of innate recognition by NK cells (19). Some of the murine NK receptors recognizing MHC molecules belong to the homodimeric Ly49 family (20). The members of this family interact with the classical class I molecules (21), and heterodimeric NKG2/CD94 molecules interact with nonclassical class I molecules such as Qa-1 (22). Each receptor family contains both inhibitory and activating receptors (23). The activity of NK cells depends on the balance between activating and inhibitory signals. As a result, NK cells may produce cytokines and/or display cytotoxic activity. NK cells have been shown to play a role in the early control of Plasmodium infection and in establishment of the adaptive immune response in experimental infection with erythrocytic stages of Plasmodium chabaudi (15), Plasmodium berghei (24, 25), or Plasmodium yoelii (8) in mice and in Plasmodium falciparum-infected patients (26). This protective role has been confirmed in beige mice and in NK cell-depleted mice, by treatment with NK cell-depleting Abs (8, 15, 27). IFN-{gamma} is one of the mediators of NK cell effector functions in malaria (15, 26). However, the anti-asialo-GM1 Abs used in these studies affect not only NK cells, but also other cell types, such as macrophages and activated T cells (28, 29, 30). Little is currently known about the NK cell response during primary infection with sporozoites.

We investigated the role of NK cells in establishment of the immune response during malaria, using an experimental model consisting of C57BL/6 mice infected with sporozoites of P. yoelii 265By, in which the complete developmental cycle of the parasite in a mammalian host is induced (as similar as possible to the natural infection). We performed an extensive phenotypic and functional analysis of NK cells during the course of infection. Both splenic and hepatic NK cells were activated following sporozoite injection. The number of NK cells decreased in the spleen but increased in the liver. The repertoire of Ly49 receptors expressed was found to be modified in splenic NK cells but not in hepatic NK cells. Both splenic and hepatic NK cells produced IFN-{gamma} and TNF-{alpha} during infection, although with different profiles of production. Functional analysis showed an increase in the cytotoxicity of NK cells in the liver, but not in the spleen. In particular, hepatic NK cells inhibited the development of P. yoelii in hepatocytes in vitro. Finally, comparison of the outcome of primary P. yoelii infection in RAG2–/– and RAG2–/– {gamma} c–/– mice, initiated with sporozoites or with parasitized RBC (pRBC),3 showed that NK cells acted on the pre-erythrocytic phase of parasite development in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6N (B6) mice were purchased from Charles River. Rag2–/– and alymphoid Rag2–/–{gamma} c–/– mice with a C57BL/6 genetic background (31, 32) were maintained at the animal facilities of the Institut Pasteur (Paris, France) in pathogen-free conditions. We used only female mice between 8 and 12 wk of age. Experiments were conducted in accordance with European guidelines for animal care and use.

Parasites and parasitemia

P. yoelii 265By sporozoites were obtained by dissecting the salivary glands of infected Anopheles stephensi mosquitoes as previously described (9). The mosquitoes were provided by the Centre de Production et Infection des Anophèles, Institut Pasteur. Mice were injected i.v. with 40, 400, or 4000 sporozoites diluted in sterile PBS. Some infections were initiated by i.p. injection of 106 pRBCs. The mice were either killed on the day after infection indicated or used for the follow-up of parasitemia, which was monitored on Giemsa-stained blood smears.

Cell preparation

Suspensions of cells were obtained from the spleen, peritoneal cavity, and bone marrow by homogenization in DMEM containing 3% FCS. The liver was perfused with RPMI 1640 medium, and lymphoid cells were obtained by homogenizing the organ with a Potter-Elvehjem homogenizer and centrifugation at 1200 x g on a 35% (v/v) Percoll (Pharmacia) solution. Erythrocytes were lysed in ACK lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). We determined the number of lymphoid cells by flow cytometry with latex beads (Beckman Coulter).

Flow cytometry

Anti-Ly49A Abs (JR9–318) were isolated and labeled with FITC as previously described (33). Biotin-conjugated anti-CD4 (GK1.5) and anti-CD8 (53–6.7), PE-conjugated anti-NK1.1 (PK136), FITC-conjugated anti-Ly49C/I (SW5E6), anti-Ly49G2 (4D11), anti-Ly49D (4E5), anti-CD95 (Jo2), allophycocyanin-conjugated streptavidin, anti-CD3{epsilon} (145-2C11), anti-TCRbeta (H57-597), and biotin-conjugated anti-CD69 (H1.2F3) Abs were purchased from BD Pharmingen. FACS analysis was conducted with a FACSCalibur flow cytometer (BD Biosciences) and CellQuest 3.3 software. More than 50,000 events were collected in a live leukocyte acquisition gate defined on the basis of forward and side light scatter and the exclusion of propidium iodide-labeled (dead) cells.

Cytokine production

Splenocytes or hepatic lymphoid cells were incubated at a concentration of 106 cells/ml in 24-well plates for 1 h at 37°C in an atmosphere containing 5% CO2. They were cultured in complete RPMI 1640 medium supplemented with 10% FCS, 5 x 10–5 M 2-ME, 2 mM glutamine, 100 IU/ml penicillin-streptomycin (Invitrogen Life Technologies), and brefeldin A (Sigma-Aldrich) at a final concentration of 10 µg/ml. Cells were washed, and fluorescent Abs were used for surface staining. Cells were then washed in 3% FCS in PBS, fixed by incubation for 45 min in 4% paraformaldehyde at room temperature, permeabilized by incubation with Permwash solution (BD Pharmingen), and stained by incubation for 30 min with allophycocyanin-conjugated anti-IFN-{gamma} (XMG1.2), anti-TNF{alpha} (MP6-XT22), and anti-IL-4 (11B11) Abs (BD Pharmingen).

Apoptosis assay

Spleen cells (1 x 106/ml) were incubated at 37°C in complete medium (RPMI 1640, 10% FCS) in the presence or absence of 1 µg/ml staurosporine (Sigma-Aldrich). Cells were stained 2 h later with anti-NK1.1-PE, biotin-conjugated anti-CD3 Abs and annexin V-FITC (BD Biosciences), then with PE-Cy7-conjugated streptavidin (BD Biosciences) and 10 nM TOPRO-3 (Invitrogen).

NK cell cytotoxicity

We determined the specific lytic activity of NK cells by means of a 4 h 51Cr-labeled YAC-1 murine lymphoma target release assay. YAC-1 target cells were labeled by incubation with 51Cr (ICN Pharmaceuticals) for 1 h at 37°C in an incubator with an atmosphere containing 5% CO2. Labeled cells were washed and added to wells containing effector cells. The plates were incubated at 37°C for 4 h in an incubator containing 5% CO2 in air. Supernatants were collected to measure the radioactivity released. Cytotoxicity was expressed as the percentage of cells specifically lysed, according to the formula: (cpm experimental release – cpm spontaneous release)/(cpm maximum release – cpm spontaneous release) x 100. Spontaneous release from labeled cells in control medium was always below 10%. Maximum release was obtained by the lysis of target cells with 10% Triton X-100 (Sigma-Aldrich). Total splenic, hepatic, or purified NK cells were used as effector cells. Experiments were performed in triplicate at various E:T ratios. The optimal E:T cell ratio was determined for a range of E:T ratios from 10:1 to 0.1:1. The experimental results obtained during infection are presented with the same E:T ratio in a given organ.

Hepatic NK cell purification

For functional assays, cell suspensions were prepared from a pool of livers removed from sporozoite-infected C57BL/6N mice on day 10. Liver cells were incubated with anti-FcR mAb (2.4G2; BD Pharmingen), and subsequently stained with PE-conjugated anti-CD8{alpha} (53-6.7) and anti-CD19 (1D3) mAbs (BD Pharmingen). Stained cells were washed in PBS supplemented with 0.5% FCS and 2.5 mM EDTA and incubated with anti-PE microbeads (Miltenyi Biotec). CD8{alpha}- and CD19-positive cell populations were depleted using the Depletes AutoMACS program (Miltenyi Biotec). Before staining for cell sorting, we checked that PE-positive cells had been depleted by flow cytometry. Cells were then incubated with FITC-conjugated anti-CD5 mAb (BD Pharmingen) and PE-conjugated anti-NK1.1 mAb.

NK1.1+ CD5– cells were sorted by flow cytometry, using a MoFlo cell sorter (BD Biosciences). Cell purity was between 94 and 99%. Finally, cells were counted (viability, as estimated by eosin incorporation, was >99%), washed and resuspended in complete William’s medium E before addition to primary infected hepatocyte cultures.

Primary cultures of hepatocytes and P. yoelii infection in vitro

Hepatocytes were prepared from C57BL/6N mice as previously described (34). A hepatic lobe was removed and perfused with HEPES buffer and a collagenase solution (1.2 FALGPA U/ml, collagenase type IV; Sigma-Aldrich). Cells were washed once in complete William’s medium E (Invitrogen Life Technologies) supplemented with 10% FCS, 1 mM glutamine, 100 U/ml penicillin-streptomycin, and then overlaid on 60% Percoll (Pharmacia Biotech). Hepatocytes were recovered by centrifugation for 3 min at 800 x g at room temperature and washed once in complete Williams’ medium E. Cell viability was assessed by eosin staining (>90%), and purity was estimated by studying cell morphology (>95%). Hepatocytes were then dispensed into Labteck plastic culture wells (Nunc), coated with collagen to optimize cell adhesion and viability (rat tail collagen; Sigma-Aldrich), at a concentration of 8 x 104 cells per well. After 24 h of incubation, the medium was removed, and we added 1.5 x 105 sporozoites in complete Williams’ medium E, enriched with antibiotics (Williams’ medium E supplemented with 10% FCS, 1 mM glutamine, 200 U/ml penicillin and streptomycin, 1/1000 gentamicin) to each well. Plates were incubated for a further three hours and the medium was replaced by fresh complete Williams’ medium E.

In vitro assay of parasite liver stage

Sorted hepatic NK1.1+ CD5– cells from infected animals were added 3 h after the infection of hepatocyte cultures with sporozoites, in various ratios, to the culture wells, to assess inhibition of the intrahepatic development of the parasite in vitro. The cultures were incubated for 45 h and were then fixed in methanol. We counted the number of schizonts after immunofluorescence staining with an immune serum-specific for P. yoelii liver-stage parasites.

Statistical analysis

Statistical analyses were performed with StatView software (SAS Institute), using Mann-Whitney U test. Kaplan-Meier tests were used for the statistical analysis of survival curves. In both tests, p values <0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Dynamic and phenotypic characterization of NK cells during P. yoelii infection

The primary infection of B6 mice with 4000 sporozoites of P. yoelii 265By induces a nonlethal infection that resolves within 3 weeks and is characterized by a peak of parasitemia around day 15. We analyzed the dynamics of the NK cells response by flow cytometry, for various anatomical sites, during infection. In the spleen, the number of cells in the total lymphoid population, defined by a gate based on forward light scatter (FSC) and side light scatter increased gradually with time after the initiation of infection (from 108 ± 14 x 106 cells at day 0 to 193 ± 24 x 106 cells 10 days postinfection (p.i.)). Despite this increase, the number of NK cells fell below that in control animals after 5 days of infection (Fig. 1A). By contrast, in the liver, the total lymphoid population and the absolute number of NK cells increased after day 3 of infection. By day 10, the number of hepatic NK cells was 8 to 10 times the initial number in control mice (Fig. 1B). Thus, splenic and hepatic NK cells displayed opposite dynamics during infection. We observed no major change in the number of NK cells in the bone marrow and the peritoneal cavity after infection (data not shown). As the NK cell response was particularly strongly affected in the spleen and the liver, we subsequently focused our studies on these two organs.


Figure 1
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  		FIGURE 1. NK cell numbers in spleen and liver. We injected 8- to 12-wk-old B6 mice i.v. with 4000 sporozoites and counted lymphoid cells in the spleen (A) and liver (B) at the times indicated after infection. We surface-stained cells for the CD3 and NK1.1 markers. The data presented correspond to the means + SD of the number of NK cells determined as the percentage of NK1.1+ CD3– in a gate of lymphoid cells, for 3 mice for each time point. Representative data of 1 of 5 independent experiments with similar results are shown.

 
We then investigated the activation status of splenic and hepatic NK cells. NK cell activation can be assessed by measuring the increase in cell size during viral infection (35). We therefore determined the proportion of blasting NK1.1+ CD3– cells by flow cytometry, by looking at the distribution of FSC intensity (Fig. 2A). In the spleen, NK cells underwent blastogenesis after 4 days p.i. (Fig. 2B). On day 14 p.i., half the splenic NK cells were blastoid. By contrast, in the liver, only a small proportion of NK cells were undergoing blastogenesis on day 7 p.i. (Fig. 2C). We also examined surface expression of the early activation marker CD69, which is up-regulated on T, B, and NK cells shortly after stimulation (36, 37, 38). In uninfected mice, a significant percentage of NK cells were nonetheless found to express CD69 (22.6 ± 4.8% in the spleen and 47.3 ± 5.7% in the liver). The proportion of NK cells that were CD69+ cells increased with time of infection and peaked on day 6 p.i. in both organs (Fig. 3). Concerning another marker of activation, CD25 (IL-2R{alpha}), we did not detect any CD25 expression on NK cells from naive or infected mice (data not shown). Our data show that, during P. yoelii infection, blastogenesis is linked to activation for splenic NK cells, whereas hepatic NK cells seem to proliferate with a slight increase in size (Fig. 2).


Figure 2
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  		FIGURE 2. Blastogenesis of NK cells during P. yoelii infection. Flow cytometry analysis of splenic and hepatic lymphoid cells from naive and infected B6 mice using fluorescent anti-NK1.1 and anti-CD3 Abs. The NK1.1+ CD3– gated cells were analyzed on the basis of FSC distribution (A). Filled histogram represents the FSC distribution of splenic NK cells from a naive mouse and solid lines indicate the FSC distribution of the splenic NK cells from an infected mouse (A). The proportion of blasting NK cells (percentage in M1) was determined during the course of infection in the spleen (B) and liver (C). The data shown are the means ± SD of three mice for each time point and representative of at least 3 independent experiments. *, Significant difference from the corresponding control mice d0 (p < 0.05).

 

Figure 3
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  		FIGURE 3. Expression of CD69 on NK cells in the spleen and liver during the course of infection. Mice were infected with sporozoites, and at various times after infection, cells were isolated from the spleen and liver and incubated with fluorescent mAbs directed against NK1.1, CD3, and CD69. We analyzed CD69 expression on NK1.1+ CD3– gated cells. Data are means of the percentage ± SD for each group of 3 mice per point tested. The dat a are representative of at least three independent experiments. For statistical significance determinations, we compared the value obtained in naive animals (day 0) with those for the infected animals: *, p < 0.05.

 
NK cells are a heterogeneous population expressing various receptors, including the Ly49 receptors, which can be used to identify subpopulations. We investigated whether there was a particular NK cell subpopulation specifically affected during infection. We used flow cytometry to analyze surface expression of the Ly49A, Ly49C/I, and Ly49G2 receptors (which transduce inhibitory signals) and the Ly49D receptor (which transduces activating signals) on splenic and hepatic NK cells from naive (day 0) and infected mice. As expected, we identified several subpopulations of Ly49+ NK cells in naive mice (Fig. 4). Ly49A+ NK cells were under-represented with respect to other Ly49+ receptors among splenic and hepatic NK cells from naive C57BL/6 mice. Overall, the proportion of a given Ly49+ population on NK cells was similar for both organs in naive mice (day 0 on Fig. 5, A and B). Some NK cells seem to express more than one Ly49 receptor, as the sum of the percentages of Ly49+ cells among NK cells exceeded 100 (39). In the spleens of infected mice, both the percentage (Fig. 5A) and the absolute number (Fig. 5C) of cells expressing Ly49 markers gradually decreased in the total NK cell population with time of infection. Indeed, the percentage of Ly49C/I-, Ly49D-, and Ly49G2-positive NK cells dropped from around 40% in naive animals to around 10% 10 days after parasite inoculation. In contrast, the percentages of the various Ly49+ NK subsets in the liver remained fairly stable during P. yoelii infection (Fig. 5B), and the absolute number of cells in each Ly49+ NK subpopulation increased (Fig. 5D). Thus, the various subpopulations of NK cells, defined on the basis of the expression of a given Ly49, evolved similar during infection within a given organ, but the patterns of change were opposite in the spleen and liver.


Figure 4
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  		FIGURE 4. FACS analysis of Ly49 expression on NK cells in the spleen of naive B6 mice. Spleen cells from B6 mice were stained with anti-CD3, anti-NK1.1 and anti-Ly49 Abs. Expression of Ly49 receptors was analyzed by gating on NK1.1+ CD3– cells. The percentages of Ly49-positive cells in the NK population are given on each histogram. The data are representative of at least 10 independent experiments.

 

Figure 5
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  		FIGURE 5. Dynamics of the Ly49 repertoire of NK cells in the spleen and liver of B6 mice after P. yoelii infection. Splenocytes and hepatic lymphocytes were isolated from B6 mice at the indicated time points after P. yoelii infection and stained with anti-CD3, anti-NK1.1 and anti-Ly49 Abs. NK cells were defined by a gate on NK1.1+ CD3- cells. The data shown are the means of the percentages ± SD of the various Ly49 subsets in the spleen (A) and liver (B), and the absolute numbers of the various Ly49 subsets in the spleen (C) and liver (D). Data are means ± SD from 1 representative experiment of 3 independent experiments.

 
We next investigated whether the disappearance of NK cells in the spleen was due to apoptosis by analyzing the labeling of annexin V (Fig. 6A). We did not observe any difference in the percentage of apoptotic NK cells (annexin V+ TOPRO-3–) after 2 h of culture at 4° or 37°C, between splenic cells from naive as well as infected mice. However, after 2 h of culture at 37°C in presence of staurosporine, an inductor of apoptosis, we observed a significant increase in the percentage of apoptotic NK cells among cells isolated from day 7 infected mice (Fig. 6A). To determine whether the apoptosis was associated to Fas-mediated mechanism, we analyzed the surface expression of CD95 by flow cytometry on splenic NK cells after infection. The proportion of CD95+ NK cells in the spleen started to increase from day 5 p.i. and reached 80 ± 2% on day 14 p.i. (Fig. 6B). Double staining with anti-CD69 and anti-CD95 Abs showed that all CD95+ NK cells were CD69+ (data not shown). In contrast, hepatic NK cells did not express CD95 (Fig. 6B). Thus, the NK cells elicited in the spleen and the liver during P. yoelii infection are activated but have different phenotypes and behaviors.


Figure 6
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  		FIGURE 6. Analysis of apoptosis and CD95 expression on NK cells during P. yoelii infection. A, Spleen cells were isolated from naive (day 0) and day 7 infected B6 mice, and incubated for 2 h at 4° or 37°C, in the presence or absence of staurosporine. Then, cells were surface stained with anti-CD3, anti-NK1.1, and anti-annexin V mAbs, and subsequently resuspended in a solution containing TOPRO-3, before FACS analysis. Histograms represent the percentages of apoptotic cells (annexin V+ TOPRO-3– cells) within the NK cell population (as defined by a gate on CD3– NK1.1+ cells). B. Hepatic and splenic cells were isolated from B6 mice at the indicated time points following infection, and stained with anti-CD3, anti-NK1.1 and anti-CD95 mAbs. The percentages of CD95+ cells was determined among the NK cells which were defined by a gate on CD3– NK1.1+. %Data are means ± SD of the percentages for each group of 3 mice per time point and are representative of, at least, 3 independent experiments.

 
Pattern of cytokines produced by NK cells during P. yoelii infection

We investigated whether the phenotypic differences between splenic and hepatic NK cells during P. yoelii infection were linked to different functions. We investigated NK cell function during P. yoelii infection, by flow cytometry, assessing the modification of the pattern of production of TNF-{alpha}, IFN-{gamma}, and IL-4 after incubation 1 h with brefeldin A and intracellular staining of splenic and hepatic NK cells at various time points after sporozoite injection. During the course of infection, the number of splenic NK cells producing IFN-{gamma} and/or TNF-{alpha} increased significantly at day 10 p.i. (Fig. 7A). Hepatic NK cells produced IFN-{gamma} from day 7 p.i. (Fig. 7B), whereas the number of NK cells secreting TNF-{alpha} was lower but significantly higher than day 0 and 10 p.i. As expected, no IL-4-producing NK cells were detected in the spleen or liver of control or infected mice (Fig. 7).


Figure 7
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  		FIGURE 7. Expression of cytokines by splenic and hepatic NK cells during P. yoelii infection. Hepatic and splenic cells were isolated from B6 mice at the indicated time points following infection. After 1 h of culture in the presence of brefeldin A, cells were surface-stained with anti-CD3 and anti-NK1.1 mAbs. Then, they were fixed, permeabilized, and intracellularly stained with anti-IFN-{gamma}, anti-TNF-{alpha}, and anti-IL-4 mAbs, before FACS analysis. The data presented are means ± SD of the numbers of splenic (A) and hepatic (B) NK cells positively stained for the different cytokines. Each time point is representative of 3 mice. The data are representative of three independent experiments. *, p < 0.05 between day 0 and infected mice.

 
Cytotoxic activities of NK cells isolated from P. yoelii-infected mice

We further characterized the function of NK cells during P. yoelii infection by investigating whether the activation of these cells was linked to their cytotoxic activity. We measured in vitro the cytotoxic activity of P. yoelii-activated NK cells on various cell targets: YAC-1 cells, P. yoelii-infected hepatocytes and P. yoelii-infected erythrocytes.

We analyzed whether NK cells from the liver and spleen of naive and infected B6 mice at various time points after sporozoite injection lysed YAC-1 in a dose-dependent manner. We determined effector NK cell number by FACS analysis (NK1.1+ CD3–) in splenic or hepatic cell suspensions. We used various E:T ratios, starting at 12:1 and then following a series of 1 in 3 dilutions (data not shown). In naive mice, splenic and hepatic NK cells at E:T ratios of 4:1 and 1:1 respectively, displayed similar levels of basal cytotoxic activity (Fig. 8; day 0). The cytotoxic activity of splenic NK cells from infected mice did not vary significantly during infection, whereas that of hepatic NK cells increased from day 3 p.i. to day 7 p.i. (67.6 ± 12.7%) and decreased thereafter. Similar results were obtained if sorted hepatic NK cells were used as effector cells.


Figure 8
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  		FIGURE 8. Dynamics of cytotoxicity in spleen and liver. Cytotoxicity of splenic and hepatic NK cells from C57BL/6 mice infected with sporozoites on day 0. Single-cell suspensions were prepared at various times after infection. 51Cr-labeled YAC-1 target cells were incubated with effector cells at various E:T ratios, ranging from 10:1 to 0.1:1, in a 4 h 51Cr release assay, and the percentage of specific lysis was determined. The data presented in A are the mean percentages for cytotoxicity in the spleen with an E:T ratio of 4:1 during infection. The data presented in B are the mean percentages for cytotoxicity in the liver with an E:T ratio of 1:1. Data are means ± SD of the percentage of specific lysis obtained for a group of 3 mice tested per day after infection for a given E:T ratio. The data are representative of 3 independent experiments. *, Significant difference from the corresponding control mice d0 (p < 0.05).

 
Then we investigated the capacity of sorted NK cells (NK1.1+ CD5–) from the livers of day 10 P. yoelii sporozoite-infected mice to inhibit parasite development in primary cultures of C57BL/6 hepatocytes (Fig. 9). We observed a significant decrease in schizont numbers in culture wells to which NK cells were added at different E:T cell ratios (Fig. 9A). This decrease was proportional to the number of effector cells added and was accompanied by a decrease in parasite size. Diameter of the intrahepatic schizonts in experimental wells (n = 66) was significantly smaller (p = 0.0029) (Fig. 9B) than that in control wells (n = 54) (Fig. 9C).


Figure 9
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  		FIGURE 9. Inhibition of the intrahepatic development of P. yoelii by liver NK cells in vitro. NK cells from day 10 infected mice were layered at different ratios onto syngeneic hepatocyte cultures 3 h after sporozoite inoculation. Cultures were stopped 45 h later. A, Histograms represent the percentage of inhibition of the mean percentage of reduction in parasite numbers from duplicate wells compared with control wells. The difference between control and experimental wells is statistically significant (*, p < 0.0001). B and C, Pictures of 48-h intrahepatic schizonts from in vitro infected cultures of B6 hepatocytes, stained with an immune serum specific for P. yoelii liver stage revealed with goat anti-mouse IgG-FITC, using the same magnification (x400). Representative schizonts found in control wells (n = 54) (C) and experimental wells (n = 66) (B), where hepatic NK cells were added. The diameter of the intrahepatic schizonts in experimental wells was significantly smaller (p = 0.0029) (B) than that in control wells (C).

 
NK cells have been shown to interact with P. falciparum-infected human erythrocytes (40, 41). This led us to investigate whether NK cells from naive or infected mice were also cytotoxic to normal or parasitized erythrocytes. Neither splenic, nor hepatic NK cells from naive or infected mice lysed these cells in vitro (data not shown). NK cells can also be activated via their Fc receptors (CD16). We therefore investigated whether the Abs contained in sera from P. yoelii-infected mice taken on day 10 of infection mediated the ADCC of NK cells against infected erythrocytes in vitro. Splenic and hepatic NK cells from naive or infected mice displayed no cytotoxic activity on day 10 p.i., with or without day 10 sera from infected mice (data not shown).

Comparison of the survival of B6, RAG2–/– and RAG–/– {gamma} c–/– mice following infection with P. yoelii

Our ex vivo and in vitro results showed that NK cells are activated during P. yoelii infection, produced Th1-type cytokines, displayed cytotoxicity against YAC-1 cells and inhibited parasite liver stages in vitro. To investigate whether these activated NK cells influence the outcome of the infection in vivo, we compared the survival and the parasitemia of C57BL/6 wild-type mice, RAG2–/– mice (deficient in T, NKT, and B cells) and RAG2–/– {gamma} c–/– mice (devoid of T, NKT, B and NK cells) following infection with P. yoelii sporozoites or pRBCs.

RAG2–/– and RAG2–/– {gamma} c–/– (infected with 4000 P. yoelii sporozoites or 106 pRBCs) died from hyperparasitemia whereas wild-type C57BL/6 mice survived (Fig. 10, A and B) and their parasitemia resolved (Fig. 10, E and F). Following sporozoite infection, RAG2–/– {gamma} c–/– mice died earlier than RAG2–/– mice (Fig. 10A). On the contrary, following infection with P. yoelii pRBC, RAG2–/– and RAG2–/– {gamma} c–/– mice behaved similarly and died similar lengths of time after infection (Fig. 10B). Thus, NK cells may play a role in controlling the early stage of parasite development, and the hepatic phase in particular, in infections initiated with 4000 sporozoites. We then investigated whether the number of NK cells present in the RAG2–/– mice was sufficient to control the infection if fewer sporozoites were used to initiate infection. We infected RAG2–/– and RAG2–/– {gamma} c–/– mice with 400 or 40 sporozoites (Fig. 10, C and D). Even with as few as 40 sporozoites, RAG2–/– mice did not efficiently control development of the parasite liver stage; they developed parasitemia and died later (Fig. 10D). However, the difference in survival between the two mouse strains following infection with 400 and 40 sporozoites was significant (p < 0.003).


Figure 10
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  		FIGURE 10. Comparison of survival curves for B6, RAG2–/– and RAG2–/– {gamma} c–/– mice infected with P. yoelii. C57BL/6 mice were infected by i.v. injection with 4000 (A and F) 400 (C), or 40 (D) sporozoites, or by i.p. injection with 106 P. yoelii-pRBC (B and E), and survival and parasitemia were monitored. The data presented were obtained by analysis of 8 mice per group in A, B, and C and 5 mice per group in D. The differences between survival curves were analyzed by log-rank (Mantel-Cox) tests. A statistically significant difference in survival between RAG2–/– and RAG2–/– {gamma} c–/– mice was observed when 400 or 40 sporozoites were used for infection (p < 0.003). The difference observed in A and D, using 4000 sporozoites or pRBC, was not statistically significant (p ≤ 0.3). Representative levels of parasitemia (means + SD) of the same groups of mice infected with pRBC (E) or 4000 sporozoites (F).

 
These data clearly show that NK cells are involved in effector mechanisms leading to host resistance to early developmental stages of P. yoelii. Nevertheless, NK cells alone do not totally control the infection.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
It has been suggested that NK cells are involved in the development of protective innate immunity through their production of IFN-{gamma} (15, 42, 43, 44, 45). However, most studies on the role of NK cells in the immune response to malaria in mice have been conducted with irradiated sporozoites or blood-stage parasites. We therefore investigated whether infections initiated with fully competent sporozoites also activated the effector functions of NK cells. Phenotypical and functional studies showed that the NK cell response induced in C57BL/6 mice following infection with P. yoelii sporozoites differs in the spleen and liver. In both these organs, NK cells are activated during infection, as shown by the increase in expression of the CD69 surface marker (36, 37, 38). Like previous studies (46), we did not detect CD25 molecules on the surface of naive or activated NK cells, in the liver or spleen during P. yoelii infection.

Splenic NK cells increased in size during their activation (35), but their total number decreased. Such a contraction of the NK cell compartment has also been observed in viral infection (47) and a decrease in the number of NK cells has been described in P. falciparum-infected patients with acute malaria (48). We thought that the decrease in size of the NK cell population in the spleen might be due to migration to other anatomical sites, or to death by apoptosis. We looked for NK cells in the bone marrow, peritoneal cavity, blood and liver of infected mice, and observed an increase in the number of NK cells only in the liver. However, this increase did not counterbalance the decrease observed in the spleen. So, it seems that activated splenic NK cells undergo death by apoptosis rather than they migrate to other organs during infection. Splenic NK cells displayed up-regulation of the expression of the CD95 membrane receptor following activation. Splenic NK cells from day 7 infected mice are more sensitive to apoptosis induced by staurosporine than naive splenic NK cells, as measured by FITC-annexin V staining. By contrast, in the liver, the number of activated NK cells increased during the course of infection. The activation of these cells was also accompanied by an increase in size, but the proportion of large NK cells was smaller than that in the spleen. These results suggest that the NK cell activation process in P. yoelii-infected mice differs in the spleen and liver.

NK cells are a heterogeneous population defined in mice on the basis of their repertoire of expressed Ly49 molecules transducing inhibitory or activating signals that modulate the threshold of NK cell activation (49). We studied the various NK cell subpopulations during P. yoelii infection by looking at the pattern of expression of members of the Ly49 family. The percentage of Ly49-expressing NK cells in the spleen decreased during infection (Fig. 5A). This decrease may be due to the expansion of a new population of NK cells expressing Ly49 receptors not detected with the anti-Ly49 Abs used. We do not observed newly emerging NK cells expressing CD94/NKG2 (50, 51). It is also possible that NK cells ceased to express the Ly49 receptors we studied. It has been recently shown that the NK Ly49 repertoire undergoes major alterations in response to certain cytokines (52).

Each subpopulation of NK cells in the liver, defined on the basis of its Ly49 repertoire, displayed changes similar to those for the total NK population because we observed no expansion of a given Ly49+ subpopulation, as has been observed in murine cytomegalovirus (MCMV) infection (53). In this viral infection model, an NK cell population expressing the Ly49H activating receptor was expanded, and this expansion was correlated with resistance to the infection involving interaction with a ligand encoded by the virus (54, 55). These studies provided the first evidence that NK receptors can directly recognize a molecule encoded by a pathogen. In our model of Plasmodium infection, parasite-infected cells, or the parasite itself, may express an unknown ligand for a Ly49 receptor. This ligand could be an MHC class I-like molecule encoded by Plasmodium, as recently described in MCMV infection (54, 55). However, a BLAST search of the P. yoelii genome (TIGR database) revealed no significant similarity with murine class I gene sequences.

Previous studies with various Plasmodium species have highlighted the contribution of IFN-{gamma} produced by NK cells to control the infection (8, 15, 25, 56). However, these studies were conducted with blood-stage parasite infections. We found that infection with sporozoites led to the production of IFN-{gamma} and TNF-{alpha} by hepatic and splenic NK cells.

These data obtained show a modification of NK cells potentialities to produce cytokines following primary plasmodial infection. They also indicate that Plasmodium pre-erythrocytic stages, directly or indirectly, stimulate cytokines production by NK cells during the first few days of infection, as seen in viral diseases (57). The IFN-{gamma} production is involved in controlling parasite development in our model of nonlethal primary P. yoelii infection, as has been shown for blood-stage infection (15, 42, 43, 44, 45) or in exoerythrocytic stage of P. berghei infection (58).

We observed an increase of cytotoxic activity to YAC-1 cells in vitro of hepatic NK cells from infected mice whereas the cytotoxic activity of splenic NK cells stay at the level of naive mice. As YAC-1 cytotoxicity was maximal with NK cells from the liver of P. yoelii sporozoite-infected mice on day 7, we investigated the potential targets of these cells in vivo. We tested the cytotoxicity in vitro of hepatic NK cells on P. yoelii 265By blood stages with or without immune sera from mice previously infected with the same parasite strain. Hepatic NK cells had no cytotoxic effects on normal or pRBCs, even in the presence of sera from infected mice (data not shown), on the contrary to what has been described for P. falciparum blood stages in vitro (40). These results are consistent with recent published observations that some subpopulations of human NK cells form stable conjugates with P. falciparum-infected RBCs without inducing a cytotoxic response (41). Alternatively, as pRBCs circulate and are captured in the spleen, this organ might be expected to display efficient cytotoxic effector mechanisms mediated by splenic NK cells against the parasite. However, this was found not to be the case. These results obtained in vitro are strongly supported by our in vivo observations that RAG2–/– (deficient in T, NKT and B cells) and RAG2–/– {gamma} c–/– (deficient in T, NKT, B and NK cells) mice behave similarly in response to infection initiated with P. yoelii blood stages. By contrast, hepatic NK cells isolated from the liver of P. yoelii sporozoite-infected mice on day 10 efficiently inhibited growth of the parasite in primary hepatocyte cultures, as demonstrated by smaller number and size of hepatic schizonts in experimental wells than in control wells. Effector mechanisms leading to the inhibition of hepatic schizont development mediated by IFN-{gamma} play a role in host defenses against Plasmodium liver stages (59). The proportion of hepatic NK cells producing IFN-{gamma} increased during the course of P. yoelii infection. However, we cannot exclude the possibility of a direct NK cell-mediated cytotoxic mechanism. These in vitro results corroborate our in vivo results demonstrating that RAG2–/– {gamma} c–/– mice, which lack NK cells, are significantly more sensitive to infection with P. yoelii sporozoites than are RAG2–/– mice. However, NK cells are not sufficient to control subsequent development of the parasite in the liver and blood, as the mice died from hyperparasitemia between days 20 and 40. If infection was initiated with fewer sporozoites (400 and 40), RAG2–/– {gamma} c–/– mice controlled the infection better than following infection with 4000 sporozoites. Although the innate immune response mediated by NK cells in Plasmodium-infected mice did not lead to the total eradication of the parasite by the host, it did seem to control parasite load at the beginning of the infection by targeting the pre-erythrocytic phase, but not the erythrocytic stage.

Much remains unknown concerning the mechanisms underlying NK cell activation during Plasmodium infection, including the nature of the signals activating these cells in particular. In P. falciparum infection, optimal activation of human NK cells has been shown to require the integration of two or more signals, transduced through different receptors. One signal is cytokine-mediated and the other is dependent upon contact with a target cell (26, 41). However, despite efforts to dissect the various signal transduction pathways (60, 61, 62), no receptor has yet been identified as essential for the triggering of cytotoxicity or cytokine secretion by NK cells. Thus, further investigations in this infection model are required to define the signals, receptors and cells involved in induction of the NK cell response.

The compartmentalization of the NK response, together with the phenotypic and functional differences observed in the spleen and liver of infected mice may be influenced by environment. An organ-specific NK cell response has already been observed during infections with lymphocytic choriomeningitis virus (63) and MCMV (64). In viral infection models, splenic NK cells were mainly cytotoxic, whereas hepatic NK cells produced cytokines. In the P. yoelii infection model, the opposite situation appeared to occur, with only hepatic NK cells displaying cytotoxic activity. The observed differences between viral infections and malaria may also reflect specific features of the host immune response and parasite interaction.

This study is the first to investigate the behavior and role of the NK cell response in the outcome of a primary Plasmodium infection initiated by the injection of live sporozoites. These results shed light on the organ specificity of the NK cell response during malaria. They also demonstrate, for the first time, the efficiency in vitro and in vivo of effector mechanisms mediated by NK cells targeting the parasite liver stage but not the erythrocytic stage.


   Acknowledgments
 
We thank Olivier Gorgette for help with statistical analyses. We also thank Paul Brey, Isabelle Thiery, and Catherine Bourgouin for the production of Anopheles stephensi mosquitoes (Institut Pasteur, Paris).


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the "Programme de Recherche Fondamentale en Microbiologie, Maladies Infectieuses et Parasitaires (PRFMMIP) AO 2000" of the French Ministry of Research. J.P.D. is supported by Institut National de la Santé et de la Recherche Médicale and the Ligue Nationale contre le Cancer. Back

2 Address correspondence and reprint requests to Dr. Jacques Roland, Département d’Immunologie, Unité d’Immunophysiopathologie Infectieuse, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France. E-mail address: jroland{at}pasteur.fr Back

3 Abbreviations used in this paper: pRBC, parasitized RBC; MCMV, murine cytomegalovirus; p.i., post infection; FSC, forward light scatter. Back

Received for publication May 23, 2005. Accepted for publication April 14, 2006.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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