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. Author manuscript; available in PMC: 2015 Jun 9.
Published in final edited form as: Immunol Cell Biol. 2014 Dec 23;93(5):508–513. doi: 10.1038/icb.2014.106

Toxoplasma gondii-infected natural killer cells display a hypermotility phenotype in vivo

Norikiyo Ueno 1, Melissa B Lodoen 1, Graeme L Hickey 2, Ellen A Robey 3, Janine L Coombes 4,*
PMCID: PMC4446200  NIHMSID: NIHMS643357  PMID: 25533287

Abstract

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells, and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. While in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here, we use a physiologically relevant oral model of T. gondii infection, in conjunction with two-photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared to uninfected “bystander” NK cells, these infected NK cells showed faster, more directed, and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA-1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii-infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host.

Keywords: natural killer cell, NK cell, Toxoplasma gondii, two-photon, integrin, motility, LFA-1, CD11a, lymph node

Introduction

Toxoplasmosis is a common zoonosis caused by the obligate intracellular protozoan parasite, Toxoplasma gondii. Initial infection occurs orally, but the parasite rapidly traverses tissues and biological barriers, disseminating widely through the host.

T. gondii is capable of invading any nucleated cell, including cells of the immune system1. Immune cells are often highly motile and adept at traversing biological barriers, and it is thought that T. gondii makes use of these existing properties to reach distant tissues2-5. For example, dendritic cells, CD11b+ cells and T cells have been shown to promote parasite dissemination in vivo2,4,6. Furthermore, in vitro assays reveal that T. gondii actively manipulates the migratory patterns of the cells it invades. Infected myeloid cells become “hypermotile”, displaying rapid cytoskeletal rearrangement, impaired adhesion to extra-cellular matrix, and increased chemotaxis2,7-12. Alterations in monocyte rolling and trans-endothelial migration through endothelial barriers under shear stress have also recently been described13,14. These behavioural changes are often accompanied by changes in the expression, activation, or clustering of integrins 7,13-15. While these observations are suggestive of manipulations in cell behaviour that would allow T. gondii to travel through tissues and across barriers more easily, a “hypermotility” phenotype in invaded cells has not yet been directly observed in vivo. Given the important role played by the tissue environment in regulating immune cell motility, a tractable in vivo assay will be crucial to understanding how T. gondii manipulates immune cell motility to enhance its spread.

NK cells play a protective role in T. gondii infection, but are susceptible to direct invasion by the parasite16-23. We have recently shown that NK cells are recruited to foci of T. gondii infection in the subcapsular sinus of the lymph node, where their migration and localisation are regulated by α2β1-integrin mediated interactions with collagen17. Here we demonstrate that T. gondii invades NK cells and alters their migration in lymph nodes, providing direct evidence for a T. gondii-induced immune cell hypermotility phenotype in vivo.

Results

T. gondii-infected NK cells display a hypermotility phenotype in vivo

Direct infection of immune cells by T. gondii results in a hypermotility phenotype in in vitro assays 2,8,9,11-13. However, two-photon laser-scanning microscopy (TPLSM) analysis of T cells and neutrophils migrating in intact living tissues has shown that the motility of the parasitized cells does not differ significantly from their uninfected counterparts6,24,25. We recently showed that NK cells accumulate in foci of T. gondii infection beneath the lymph node capsule17. In these experiments, we consistently observed that a small proportion of these NK cells contained parasites. We therefore assessed the impact of direct invasion by T. gondii on NK cell behavior in intact, living tissues.

To detect and visualize NK cells, we used mice in which one copy of the Ncr1 gene had been replaced with a GFP reporter26. These mice were infected via the physiologically relevant oral route with tissue cysts of the type II Prugniaud strain engineered to express tdTomato, allowing us to monitor infection levels in NK cells by flow cytometry6. Five days after oral infection, 0.72±0.14% of NK cells in the draining mesenteric lymph nodes contained parasites (Figure 1a,b). This was greater than the proportion of T cells containing parasites (0.20±0.03%), or the proportion of infected cells in lymph node as a whole (0.21±0.03%, Figure 1a-b). Nevertheless, the relative abundance of T cells in the lymph node when compared to NK cells meant that they accounted for a high proportion of T. gondii infected cells (Figure 1b). NK cells containing intact parasites could be readily visualized in mesenteric lymph node sections (Figure 1c). In some cases, multiple parasites were observed within a single NK cell (Figure 1c).

Figure 1. T. gondii-infected NK cells display a hypermotility phenotype in vivo.

Figure 1

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(a) Flow cytometric analysis of mesenteric lymph node at day 5 following oral infection is shown. Plots show gating of live, single cells into T cell (CD3+) and NK cell (NKp46+CD3-) populations (top row). The percentage of cells in each population containing T. gondii is then determined by gating on parasite fluorescence (blue numbers, bottom row). (b) Graphs show the percentage of the indicated cell population that contains T. gondii (mean ± SEM of five mice), and the percentage of T. gondii infected cells that are T cells or NK cells. (c) Fluorescence microscopy of the mesenteric lymph node from an Ncr1GFP/+ mouse 6 days after oral infection is shown. NK cells are green, T. gondii is pink. (d) Individual time points and tracks from a TPLSM movie showing a T. gondii-infected NK cell migrating in the mesenteric lymph node 4 days after oral infection are shown. NK cells are green, T. gondii is red. An infected NK cell is highlighted with yellow arrows/red track, and uninfected NK cells with grey arrows/tracks. Corresponds to supplementary movie 1. (e-g) Graphs show the average speed (e) confinement index (f) and arrest coefficient (g) of individual NK cells. For each condition data are pooled from 5 imaging volumes obtained over the course of three independent experiments (n=3, days 4-5 post infection). **p <0.001.

We then used TPLSM to compare the motility of T. gondii-infected and uninfected “bystander” NK cells in mesenteric lymph nodes of orally infected mice (Figure 1d, Supplementary Movie 1). Using a standard linear regression model, T. gondii infected NK cells migrated 6.00 μm/min faster than non-infected cells after adjustment for differences between mice(95% Confidence Interval(CI): 4.10 - 7.90; P<0.001, Figure 1e). The linearity of the path taken by a cell can be described by the confinement index (maximum displacement/path length), where higher values indicate more linear migration. Using the linear regression model, the confinement index was 0.203 units greater in the T. gondii-infected cells (95% CI: 0.093 - 0.313; P<0.001, Figure 1f). The arrest coefficient is the percentage of time that a cell's speed falls below 5 μm/min, and is generally high when NK cells form stable contacts with target cells or immotile tissue structures. The arrest coefficient was smaller by an absolute value of 50.64 percentage points in the T. gondii-infected cells, indicating that stable contacts with immotile cells or structures in the lymph node were greatly reduced (95% CI: 31.63 - 69.66; P<0.001, Figure 1g). This faster, more directed, and more persistent migratory behavior allows NK cells to cover more ground, potentiating spread of the parasite.

Infected NK cells display impaired spreading and integrin clustering

NK cells use integrins to form low motility contacts with the extracellular matrix and target cells. For example, VLA-2 (CD49b:CD29, α2β1) mediates low motility contacts between NK cells and collagen fibers in the lymph node, while LFA-1 (CD11a:CD18, αLβ2) is involved in NK cell adhesion to, and killing of, target cells 17,27-31. The increased motility of T. gondii-infected NK cells could therefore be explained by parasite-driven alterations in integrin expression or activity.

Integrin activity is regulated by conformational changes to the receptor and by dynamic alterations in expression, trafficking, clustering, or distribution32. Our initial experiments demonstrated that oral infection did not alter NK cell surface expression of a panel of integrins tested, including the CD11a subunit of LFA-1 (Figure 2a, b, Supplementary Figure 1).

Figure 2. Infected NK cells display impaired integrin clustering and cell spreading.

Figure 2

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(a) Flow cytometric analysis of CD11a expression on NK cells in mesenteric lymph nodes at day 5 following oral infection is shown. Plots are derived from concatenated samples from 4 individual mice analysed. Infected NK cells are shown in red, and bystander NK cells in grey. (b) Graph shows the median fluorescence intensity of CD11a on the indicated cell populations (mean ± SEM of four mice). (c-d) Immunofluorescence analysis of CD11a distribution on the NK cell surface in response to ICAM-1 ligand. Uninfected and T. gondii-infected NK cells were settled onto immobilized mouse ICAM-1/Fc. After 15 to 30 minutes, samples were fixed and stained to detect surface CD11a by fluorescence microscopy. Z-sections from the cell base to the cell top were acquired at intervals of 0.5 μm. Representative fluorescent and differential interference contrast micrographs from three independent experiments are shown. CD11a is shown in red, the parasites in green, and the nuclei in blue. Corresponds to supplementary movie 2. (e-f) Differences in CD11a distribution and surface area between uninfected and infected cells were quantified as ratios of their respective values at the cell base to the cell center (nuninfected = 37, ninfected = 37 cells, from three indeoendent experiments). Red bars show the mean.

To assess whether T. gondii infection alters integrin clustering, we infected NK cells with T. gondii and seeded the NK cells onto ICAM-1 coated cover-glass13. CD11a (LFA-1) localization was determined by confocal imaging of the NK cells from the point of contact with the ICAM-1-coated surface, to the top of the cell, at 0.5 μm intervals (Figure 2c). In uninfected NK cells CD11a clustered in the contact zone between the NK cell and the ICAM-1-coated surface. However, in infected cells, CD11a was more evenly distributed over the entire surface of the cell (Figure 2c-e, Supplementary Movie 2). Furthermore, while uninfected cells showed evidence of spreading at the point of contact with the ligand, the infected cells were more rounded in morphology (Figure 2f).

Given the important role played by integrins in the formation of low motility contacts with target cells and the ECM, the observed reduction in cell spreading and redistribution of integrin in T. gondii-infected NK cells is consistent with the absence of low motility behavior we observe in these cells in vivo.

Discussion

Infection with T. gondii has significant socio-economic costs, both in terms of severe disease in the human population and economic losses in farming. Understanding how the parasite spreads through the host will be important in the design of novel vaccines and therapeutics aimed at minimising the burden of infection in the brain, or preventing trans-placental transmission to the developing foetus. Here, we used a physiologically relevant oral model of T. gondii infection to show that T. gondii-infected NK cells display a hypermotility phenotype in vivo. Our data provide (1) crucial support for the hypothesis that T. gondii manipulates immune cell motility to spread through its host and (2) a cellular and anatomical context to understand how the parasite achieves this in complex tissues.

Our data reveal that impaired cell spreading and CD11a/LFA-1 clustering in T. gondii-infected NK cells is a possible mechanism for their altered motility in tissues. Intermediate levels of integrin-mediated adhesion are usually optimal for cell migration, whereas too much or too little adhesion can negatively impact cell motility33. Thus, the reduction in cell spreading and LFA-1 clustering observed in T. gondii-infected NK cells is consistent with the changes in motility observed in vivo, and implies that NK cells are constantly using LFA-1 to contact other cells or structures in tissues. Similarly, LFA-1 has been implicated in the intranodal migration of T cells, while in vitro studies have shown that LFA-1 triggers asymmetrical NK cell spreading and migration 34-36. Interestingly, enhanced transmigration was not observed in parasitised human NK cells migrating in vitro, suggesting that the anatomical context in which migration takes place is an important contributing factor to the hypermotility phenotype in NK cells8. Infection of other immune cell populations by T. gondii has also been associated with changes in the expression, activation, or clustering of integrins 7,13-15. For example, T. gondii-infected macrophages display a reduction in adhesiveness to extracellular matrix components, which is accompanied by reduced surface expression of multiple integrins, including LFA-17. Furthermore, T. gondii-infected human monocytes, which rolled at higher speeds and for longer distances over endothelial cells, displayed impaired LFA-1 clustering and cell spreading13.

Our results raise the possibility that hypermotile NK cells play an important role in facilitating the spread of T. gondii through the host. This idea is supported by an earlier study demonstrating that NK cells become infected following lytic contacts with infected dendritic cells, but are not susceptible to lysis by other NK cells23. Although it has been shown that adoptive transfer of T. gondii-infected immune cells results in higher infection loads when compared to inoculation with free parasites, an important question is whether hypermotility of endogenous NK cells, or other immune cells, contribute to the spread of infection in a natural setting2,4. This type of experiment is complicated by the protective roles that immune cells also play in infection. A better understanding of how T. gondii alters immune cell motility is therefore necessary to design experiments to address this question. Following initial infection in the small intestine, T. gondii spreads through the intestine, from the intestine to the lymph nodes and blood, and from the blood to the brain, to muscle, and across the placenta to the developing fetus. Different immune cell populations display distinct migratory pathways, and we favour the idea that the parasite utilises different immune cell populations at different stages in this process. For example, neutrophils have been implicated in the luminal spread of parasites through the small intestine, preventing T cell egress from lymph nodes reduces spread of the parasite to the spleen, and CD11b+ cells are implicated in the delivery of parasites to the brain4,6,24. While the exact role of NK cells remains to be determined, the role of uterine NK cells in trans-placental transmission of infection is of particular interest in this respect. The ability to directly visualize hypermotility in infected immune cells in an in vivo infection model will provide an important platform for these studies.

Methods

Mice

CBA/J mice were purchased from The Jackson Laboratory. Ncr1GFP/+ mice were a gift from Dr. O. Mandelboim (The Hebrew University of Jerusalem)26. Mice were housed under specific pathogen-free conditions at the AALAC-approved animal facility in the University of California, Berkeley. Animal experiments were approved by the Animal Care and Use Committee of the University of California, Berkeley.

T. gondii Infections

Type II Prugniaud parasites engineered to express tdTomato and ovalbumin were used for oral infections6. Brain homogenates were prepared from CBA/J mice infected i.p. with 400 tachyzoites 3-6 weeks previously. Cysts were counted after staining with Dolichos Biflorus Agglutinin (Vector Laboratories) and 50 cysts were administered by gavage to Ncr1GFP/+ mice. For infections in vitro, type II Prugniaud parasites engineered to express GFP were used. Murine NK cells were enriched from the spleen of wild-type C57BL/6 mice by negative magnetic selection (Stemcell Technologies). Tachyzoites were added to purified NK cells at a multiplicity of infection of 2, and the mixture was incubated for three hours at 37 °C.

Two-photon Imaging

Two-photon imaging was performed on mesenteric lymph nodes from Ncr1GFP/+ mice4–5 days after oral infection. Lymph nodes were explanted and perfused in warmed oxygenated media, as previously described37. Images were acquired using a custom-built microscope with a Spectra-Physics MaiTai laser (tuned to 920 nm), and a 20×/0.95 Nikon objective. Emission light was separated with 495nm, 510nm and/or 560nm dichroics, and collected with photomultiplier tube detectors. To minimize spectral overlap a bandpass filter, HQ 450/80 M, was used. In some cases, imaging data were subjected to post-acquisition processing to limit spectral cross-talk or background signal. Nonspecific background signal was subtracted using a digital mask generated on an unrelated channel using Imaris software, and/or a Gaussian filter was applied. Any adjustments made to brightness or contrast were linear and applied to the whole image. The x, y, z coordinates of individual cells over time were obtained with Imaris Bitplane software. Motility parameters were calculated with MATLAB. There is a partial overlap between the raw datasets (image files) used to compile Figure 1e-g in this paper, and Figure 2e of reference 17.

Fluorescence Microscopy

Six days after oral infection with tdTomato-expressing parasites, mesenteric lymph nodes from Ncr1GFP/+ mice were prepared for fluorescence microscopy as previously described16. Images were acquired using a Nikon Eclipse TE2000-E. Immunofluorescence microscopy of surface integrins was performed as previously described13. Briefly, uninfected and T. gondii-infected NK cells were settled onto ICAM-1-coated cover-glass. Cells were then fixed with paraformaldehyde and stained with a monoclonal antibody against mouse CD11a (M17/4, Biolegend) and Alexafluor 594-conjugated anti-rat secondary antibody (Life Technologies). Cover-glasses were mounted onto slides with Vectashield with DAPI (Vector Labs) and imaged using the 60× objective lens of a Nikon Eclipse Ti fluorescent microscope. Micrographs were analyzed using ImageJ software, and the fluorescence intensities and cell surface areas were plotted using GraphPad Prism software.

Flow cytometry

Single cell suspensions were prepared from mesenteric lymph nodes of Ncr1GFP/+ mice 5 days after oral infection with tdTomato-expressing parasites. Cells were stained with a fixable Aqua Live/Dead dye (Molecular Probes), then with antibodies to mouse CD3ε (145-2C11, Ebioscience) and CD11a (M17/4, Ebioscience). Data was acquired using a BD LSR II and analyzed with FlowJo software.

Statistics

Unless otherwise noted, values are expressed as mean ± SEM. For Figure 1b, levels of significance were calculated by one-way ANOVA with Tukey's post-hoc tests. For Figure 2e, the student's two tailed t-test with Welch's correction was used (both GraphPad Prism). For analysis of cell motility data, linear regression models were fit to each observed motility parameter, with adjustment for mouse and a binary indicator of whether the cell was infected with T. gondii or not. The adjustment for mouse accounts for heterogeneity between mice, although this is not of interest. The estimated model coefficient for infection is interpreted as the difference in motility parameter between an infected cell and non-infected cell. The normality of fitted model residuals were visually inspected using quantile-quantile plots and a suitable transformation applied to the motility data where appropriate. For confinement index, inspection of the residuals suggested a log-transformation was appropriate to satisfy normality assumptions. After adjustment for variation in individual mice, the difference in log-confinement index between T. gondii infected and non-infected cells was 0.607 (95% CI: 0.286 - 0.929; P<0.001). As the arrest coefficient is a percentage, the use of linear regression methods is a limitation. A Mann-Whitney U-test comparing the arrest coefficient between infected and non-infected cells (after pooling data from all mice) also confirmed a statistically significant difference (P<0.001). The Mann-Whitney U-test also yielded a significant difference for speed (P=0.002) and confinement (P<0.001) Regression analyses were done using the R statistical computing language version 3.0.2 (R Foundation for Statistical Computing, Vienna, Austria, 2013). Differences were considered significant at p < 0.05. and are indicated with an asterisk (*p<0.05, **p<0.001, ***p<0.0001). “ns” is not significant.

Supplementary Material

1

Supplementary Figure 1. (a) Flow cytometric analysis of integrin expression on NK cells in mesenteric lymph nodes at day 5 following oral infection is shown. Plots are derived from concatenated samples from 3-4 individual mice analysed. Infected NK cells are shown in red, and bystander NK cells in grey. (b) Graphs shows the median fluorescence intensity of integrin expression on the indicated cell populations (mean ± SEM of 3-4 mice).

2

Supplementary Movie 1. TPLSM of mesenteric lymph node 4 days after oral infection with T. gondii. NK cells are shown in green, and T. gondii in red. The tracks of uninfected NK cells are shown in white, and of a parasitized NK cell in red. Corresponds to Figure 1d.

Download video file (2.3MB, mov)
3

Supplementary Movie 2. 3-D reconstruction of surface CD11a on a T. gondii-infected NK cell (right) and an adjacent uninfected bystander NK cell (left) adhered to immobilized ICAM-1. CD11a is shown in red, the parasites in green, and the nuclei in blue. Corresponds to Figure 2c-f.

Download video file (2.4MB, mov)

Acknowledgments

We thank the following colleagues for their valuable contributions to this study: Paul Herzmark for imaging expertise and Shiao Chan for technical assistance. This work was funded by a Wellcome Trust ISSF to the University of Liverpool (097826/Z/11/A); Sir Henry Wellcome Postdoctoral Fellowship WT085494 (J.L.C.); Royal Society Research Grant RG130129 (J.L.C); American Heart Association 10SDG3140025 (M.B.L.); American Heart Association Postdoctoral Fellowship 13POST14580034 (N.U.); National Institutes of Health, USA R01AI065537 (E.A.R).

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Associated Data

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Supplementary Materials

1

Supplementary Figure 1. (a) Flow cytometric analysis of integrin expression on NK cells in mesenteric lymph nodes at day 5 following oral infection is shown. Plots are derived from concatenated samples from 3-4 individual mice analysed. Infected NK cells are shown in red, and bystander NK cells in grey. (b) Graphs shows the median fluorescence intensity of integrin expression on the indicated cell populations (mean ± SEM of 3-4 mice).

NIHMS643357-supplement-1.pdf (597.2KB, pdf)
2

Supplementary Movie 1. TPLSM of mesenteric lymph node 4 days after oral infection with T. gondii. NK cells are shown in green, and T. gondii in red. The tracks of uninfected NK cells are shown in white, and of a parasitized NK cell in red. Corresponds to Figure 1d.

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3

Supplementary Movie 2. 3-D reconstruction of surface CD11a on a T. gondii-infected NK cell (right) and an adjacent uninfected bystander NK cell (left) adhered to immobilized ICAM-1. CD11a is shown in red, the parasites in green, and the nuclei in blue. Corresponds to Figure 2c-f.

Download video file (2.4MB, mov)

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