Download Toxoplasma gondii Intracellular Parasite Perforin Trigger Rapid

Death Receptor Ligation or Exposure to
Perforin Trigger Rapid Egress of the
Intracellular Parasite Toxoplasma gondii
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of June 18, 2017.
Emma K. Persson, Abela Mpobela Agnarson, Henrik
Lambert, Niclas Hitziger, Hideo Yagita, Benedict J.
Chambers, Antonio Barragan and Alf Grandien
J Immunol 2007; 179:8357-8365; ;
doi: 10.4049/jimmunol.179.12.8357
http://www.jimmunol.org/content/179/12/8357
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The Journal of Immunology is published twice each month by
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Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Supplementary
Material
The Journal of Immunology
Death Receptor Ligation or Exposure to Perforin Trigger
Rapid Egress of the Intracellular Parasite Toxoplasma gondii1
Emma K. Persson,2*‡ Abela Mpobela Agnarson,2*‡ Henrik Lambert,*‡ Niclas Hitziger,*‡
Hideo Yagita,§ Benedict J. Chambers,* Antonio Barragan,3*‡ and Alf Grandien3*†
The obligate intracellular parasite Toxoplasma gondii chronically infects up to one-third of the global population, can result in
severe disease in immunocompromised individuals, and can be teratogenic. In this study, we demonstrate that death receptor
ligation in T. gondii-infected cells leads to rapid egress of infectious parasites and lytic necrosis of the host cell, an active process
mediated through the release of intracellular calcium as a consequence of caspase activation early in the apoptotic cascade. Upon
acting on infected cells via death receptor- or perforin-dependent pathways, T cells induce rapid egress of infectious parasites able
to infect surrounding cells, including the Ag-specific effector cells. The Journal of Immunology, 2007, 179: 8357– 8365.
*Center for Infectious Medicine and †Center for Experimental Hematology, Department of Medicine, Karolinska Institutet, Karolinska University Hospital–Huddinge,
Stockholm, Sweden; ‡Swedish Institute for Infectious Disease Control, Stockholm,
Sweden; and §Department of Immunology, Juntendo University, School of Medicine,
Tokyo, Japan
Received for publication August 22, 2007. Accepted for publication October 3, 2007.
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 supported by in part by grants from the Swedish Research Council (to
A.B.), the Swedish Cancer Society (to A.G.), and the Swedish Foundation for Strategic Research. N.H. is the recipient of a postdoctoral fellowship from the Karolinska
Institutet infection network.
2
E.K.P. and A.M.A. contributed equally to this work.
3
Address correspondence and reprint requests to Dr. Alf Grandien or Dr. Antonio
Barragan, Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital–Huddinge, 141 86 Stockholm, Sweden. Email addresses: [email protected] and [email protected]
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. T. gondii is one of the most wide spread human
parasites with an estimated 2 billion infected individuals. During the
acute phase of infection, parasites rapidly disseminate to establish a
life-long, often asymptomatic, chronic infection. Reactivated infection in immunocompromised patients can result in severe disease, and
acute infection during pregnancy can cause damage in the developing
fetus (7). Within the host Toxoplasma is capable of infecting all types
of nucleated cells, including cells of the immune system (8).
CD4⫹ and CD8⫹ T cells have been demonstrated to be important
in controlling T. gondii infection (reviewed in Ref. 9). It was later
shown that perforin-dependent cytotoxicity played a limited role in
resistance to T. gondii infection (10). Neither Fas nor TNF-receptors
were subsequently demonstrated to be critical for control of T. gondii
infection (11–13). A number of reports have since provided evidence
indicating that T. gondii can inhibit host cell apoptosis induced by
CTL, death receptor ligation, or via the intrinsic pathway of apoptosis
(14 –22). Based on these results it has been suggested that T. gondiiinfected cells would be protected from apoptotic cell death and that
this could explain why neither cell-autonomous induction of apoptosis
nor T cell-mediated assisted apoptosis would be effective defense
mechanisms against Toxoplasma-infected cells (14 –24).
Thus, in previous studies the absence of typical features of
apoptosis in cells infected by Toxoplasma has been amply documented in vitro by using defined cell lines. However, whether the
lack of characteristic attributes of apoptosis in T. gondii-infected
cells correlates with the inhibition of cell death of infected cells is
not clear. In this study we have investigated this issue by studying
the consequences of death receptor ligation and exposure to CTL
in T. gondii-infected cells. We report that, early during the induction of death receptor- or perforin-dependent cell death, parasites
rapidly egress from their host cells resulting in necrotic cell death
of the host cells. Through this mechanism parasites avoid elimination and are instead able to infect neighboring cells, including
the cytotoxic cells themselves.
Materials and Methods
Mice
C57BL/6, C57BL/6.perforin⫺/⫺, C57BL/6.gld, and A.BY a H-2b congenic
mice of the A/Sn background were maintained and bred at the animal
facility of Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet (Stockholm, Sweden). All procedures were performed with
relevant ethical permission according to local and national guidelines.
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C
ellular cytotoxicity is an important immune defense
mechanism against intracellular pathogens. After target
recognition it can proceed via two main pathways, the
perforin/granzyme-dependent pathway or via expression of death
ligands, binding to death receptors on the target cells and leading
to apoptotic cell death. Many viruses, including adenovirus, poxvirus, herpesvirus, and papillomavirus, carry anti-apoptotic genes
presumably to prevent premature death of their host cells, be it an
innate response to the infection or induced by immune cells upon
recognition (1). Recent reports indicate that other intracellular
pathogens such as protozoa and certain bacteria are also able to
interfere with apoptosis induction and thereby protect their host
cells, and themselves, from cell death (1–3).
Apoptosis can be induced via the intrinsic or the extrinsic pathways (4). The intrinsic pathway can be triggered by internal stress
signals in the cell resulting from DNA damage, damage to organelles, lack of essential growth factors, or infection. The extrinsic pathway is activated through death receptor ligation and is used
by T or NK cells to induce target cell killing in a specific manner.
Cytotoxic cells can also induce target cell death via the perforindependent granule exocytosis pathway (reviewed in Ref. 5). Also
nonlymphoid cells can, under certain circumstances such as inflammation, express death ligands and thereby induce apoptosis in
cells with which they interact (reviewed in Ref. 6).
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CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII
Parasites and infection
Toxoplasma lines RH-LDM (25) (which was used in all experiments if not
indicated otherwise) and PTG/ME49 (26) were maintained by serial 2-day
passage in human foreskin fibroblast monolayers. A20 and Jurkat cell lines
were infected at a multiplicity of infection of 4 for 4 h. After infection, live
cells (density: 1.0409 –1.0643 mg/ml) were separated from free parasites
(density 1.0877–1.0994 mg/ml) by Percoll (Amersham Biosciences) density centrifugation.
Dendritic cells and immunizations
Bone marrow-derived dendritic cells (BMDC) were generated as described
previously (27). Bone marrow cells were cultured in DMEM (Invitrogen
Life Technologies) containing 10 ng/ml rGM-CSF (BioSource International). After 6 days of culture, the cells were harvested and used for
infection. For immunizations to minor histocompatibility Ags, A.BY mice
were immunized twice over 14 days with 25 ⫻ 106 irradiated splenocytes
from either A.BY (denoted “naive”) or C57BL/6 mice (denoted “primed”).
Ten days after the final immunization, BMDC from C57BL/6 mice were
infected with Toxoplasma for 6 h at a multiplicity of infection of 1. The
cells were washed thoroughly and had an infection rate of ⬃70%. The
dendritic cells were injected i.p. and 36 h later the peritoneal cavity was
rinsed and the infection of T cells was examined by flow cytometry.
A20 is a Fas-sensitive murine B cell lymphoma (28) and was maintained
in RPMI 1640 complete medium as described (29). In some experiments,
DMEM medium without calcium (Invitrogen Life Technologies) was used.
In these cases, the FCS had been extensively dialyzed against PBS. Jurkat
is a Fas-sensitive human T cell lymphoma and was maintained in RPMI
1640 complete medium. The murine T cell lymphoma L5178Y and the
murine (m)4 and human (h) Fas ligand (FasL) transfectants mFasL/L5178Y
and hFasL/L5178Y were maintained in complete RPMI 1640 medium (30)
as were the murine B cell lymphoma 2PK-3 and the human TRAIL transfectant hTRAIL/2PK-3 (31). A20 cells were transduced with LXIN retroviruses (BD Biosciences Pharmingen) expressing human Bcl-xL, human
FLIPL, or cytokine response modifier A (CrmA) produced by transient
transfection of Phoenix-Ampho packaging cells as described (29).
Induction of apoptosis and redirected cytolysis
Agonistic anti-human or anti-mouse Fas mAbs Jo2 (BD Biosciences
Pharmingen) and CH-11 (MBL International) were added at various concentrations to the cells as indicated. Alternatively, cells were coincubated
with L5178Y, 2PK-3, mFasL/L5178Y, hFasL/L5178Y, or hTRAIL/2PK-3
cells at a 1:1 ratio. Intracellular calcium in A20 cells as a result of Fas
ligation was measured using preincubation of the cells with Fluo-4-AM
(Calbiochem) at 1 ␮M for 30 min at 37°, after which the cells were analyzed using flow cytometry. Where indicated, intracellular calcium was
chelated using 50 ␮M BAPTA-AM (Calbiochem) added 20 min before Fas
ligation. Caspase activity was blocked using benzyloxycarbonyl-Val-AlaAsp-fluoromethyl ketone (z-VAD-FMK) at 10 ␮M (Enzyme System Products) added to cultures 20 min before Fas ligation or addition of T cells. T
cell blasts were prepared from spleens of C57BL/6, C57BL/6 gld, and
C57BL/6.perforin⫺/⫺ mice by culture for 2 days with 2 ␮g/ml Con A
(Amersham Biosciences) followed by 1 day of culture with 10 U/ml IL-2
(supernatant from transfected X63 cells) and were thereafter isolated over
Lympholyte-M (Cedarlane Laboratories). CD4⫹ and CD8⫹ cells were obtained through negative selection using magnetic beads (Miltenyi Biotech),
and T cell blasts and T. gondii-infected A20 cells were cocultured at a ratio
of 1:1 in the presence of neutralizing anti-TRAIL (clone N2B2) mAb at 10
␮g/ml (32) in the presence or absence of 1 ␮g/ml anti-CD3 mAb (clone
2C11).
Detection of apoptotic cells and flow cytometry
Infected or noninfected cells were stained with annexin V-biotin (Roche
Diagnostics) followed by staining with streptavidin-allophycocyanin (BD
Biosciences Pharmingen) and propidium iodide (PI) (1 ␮g/ml) and thereafter analyzed on a FACSCalibur flow cytometer (BD Biosciences Immunocytometry Systems) or a CyAn ADP flow cytometer (DakoCytomation);
the data were analyzed with CellQuest (BD Biosciences Immunocytometry
Systems) or FlowJo software (Tree Star). In some experiments, cells were
4
Abbreviations used in this paper: m, murine (prefix); BMDC, bone marrow-derived
dendritic cell; CrmA, cytokine response modifier A; FasL, Fas ligand; h, human
(prefix); PI, propidium iodide; wt, wild type; z-VAD-FMK, benzyloxycarbonyl-ValAla-Asp-fluoromethyl ketone.
FIGURE 1. T. gondii infection does not protect host cells against death
receptor-induced cell death. a, Flow cytometric profiles representing survival of uninfected and T. gondii-infected A20 cells in the absence or
presence of anti-Fas (10 h after addition of anti-Fas). b and c, Number of
live A20 (b) or Jurkat (c) cells in the absence or presence of anti-Fas (10
h after addition of anti-Fas). Filled squares, T. gondii-infected cells; open
circles, uninfected cells. The results represent the mean and 1 SD from
three independent experiments (the frequency of parasite-infected cells at
the start of the culture in b and c was ⬃70%).
stained with anti-CD45R/B220-allophycocyanin to detect A20 or 2PK-3
cells, anti-Thy1.2-PE to detect L5178Y cells, anti-human CD3-allophycocyanin to detect Jurkat cells, or allophycocyanin- or PE-conjugated mAbs
against CD4, CD8, or CD44 (reagents from BD Biosciences). Cell cycle
analysis was performed by 2 min of permeabilization with saponin (0.01%;
w/v) and PI (50 ␮g/ml) followed by flow cytometry.
Real-time confocal microscopy
Egress of intracellular tachyzoites upon Fas ligation was assessed with a
spinning disk confocal setup (Ultraview LCI-3 tandem scanning unit;
PerkinElmer) on an Axiovert 200 M (Carl Zeiss) connected to a chargecoupled device camera (Orca ER; Hamamatsu). Cells were placed in
minichamber system (POCmini; LaCon) with heating stage. Image acquisition and analysis was performed with Openlab software (version 4.0.2;
Improvision).
Results
Fas ligation mediates cell death of Toxoplasma-infected host
cells
To evaluate the ability of T. gondii to protect host cells against the
extrinsic pathway of apoptosis induction, the murine B cell lymphoma cell line A20 and Jurkat, a human T lymphoma cell line,
were infected with the GFP-expressing virulent parasite line RHLDM and incubated with agonistic anti-Fas mAbs. Ten hours after
the addition of anti-Fas mAbs, cellular viability was evaluated by
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Cell lines and retroviral transduction
The Journal of Immunology
8359
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FIGURE 2. Fas receptor ligation
in T. gondii-infected A20 cells leads
to necrotic-like cell death. a, Annexin V/PI staining of uninfected
(upper panel) or T. gondii-infected
(lower panel) cell cultures 2 h after
the addition of anti-Fas mAb. b, Kinetics of annexin V/PI staining in uninfected (upper panels) and T. gondii-infected cultures (lower panels)
in the presence (filled circles) or absence (open squares) of anti-Fas. c,
Analysis of GFP expression vs cell
size (forward light scatter) in T. gondii-infected cultures in the absence or
presence of anti-Fas mAb as a function of time. Values in the graphs
represent the percentage parasite infected cells (top), the percentage of
noninfected cells (middle), and the
percentage of free parasites (bottom).
examining the exposure of phosphatidyl serine on the cell surface
by annexin V staining (Fig. 1a; showing A20 cells) and live cell
counting (Fig. 1, b and c). Few live cells could be found in either
uninfected (9%) or T. gondii-infected cultures (3%) after incubation with anti-Fas mAbs for 10 h. Infection with the PTG/ME49
strain yielded similar results (data not shown). Thus, under these
experimental conditions Fas ligation consistently led to cell death
of infected cells.
Death receptor ligation leads to rapid parasite egress and host
cell necrosis
To analyze the possible dichotomy between the previously reported lack of apoptotic features and the absence of inhibition of
cell death reported here, we determined the kinetics of Fas-induced
cell death in uninfected and T. gondii-infected A20 cells using a
combination of annexin V and PI staining. Uninfected A20 cells
treated with anti-Fas mAb displayed typical features of apoptotic
cells, rapidly becoming annexin V⫹ and PI⫺, followed by the appearance of annexin V⫹PI⫹ cells (Fig. 2, a and b, upper panels).
However, in T. gondii-infected cultures the cells displayed a completely different pattern that was characterized by a very rapid
accumulation of atypical annexin V⫺PIdim/⫹ cells followed by the
appearance of annexin V⫹PI⫹ cells but low numbers of typical
apoptotic (annexin V⫹PI⫺) cells (Fig. 2, a and b, lower panels).
Thus, parasite-infected cells very rapidly acquired a necrotic-like
phenotype following Fas ligation instead of the expected conventional apoptotic phenotype.
The proportions of live infected and uninfected cells as well as
free parasites were also analyzed. In contrast to control cultures
(Fig. 2c, upper panels), the addition of anti-Fas mAb resulted in a
clear reduction in the proportion of live cells (Fig. 2c, lower panels). The decrease in the proportions of infected live cells after Fas
FIGURE 3. Fas receptor ligation in T. gondii-infected A20 cells leads to
parasite egress. a, Ratio of free parasites vs live A20 cells, using cell
counting, as a function of time in the presence (filled circles) or absence
(open squares) of anti-Fas. b, Ratio of free vs intracellular parasites, using
flow cytometric analysis of A20 cells, as a function of time in the presence
(filled circles) or absence (open squares) of anti-Fas. c, Dose dependence
of T. gondii egress after Fas ligation in A20 cells. d, Fas-mediated egress
of PTG/ME49 T. gondii in A20 cells (filled circles) and without anti-Fas
(open square). e, Ratio of free vs intracellular parasites, using flow cytometric analysis of murine dendritic cells, as a function of time in the presence (filled circles) or absence (open squares) of soluble FasL (sFasL;
diluted 1/4). f, Dose dependence of T. gondii egress in dendritic cells in the
presence (filled circles) of soluble FasL or control supernatant (open
squares).
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CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII
ligation was paralleled by an increase in the proportion of free
parasites (Fig. 2c).
To verify that the population defined by low forward light scatter and GFP positivity really represented free parasites, the numbers of free parasites were counted using light microscopy. A good
correlation between data from counting and flow cytometry was
obtained (Fig. 3, a and b). Fas-induced parasite egress was dose
dependent (Fig. 3c) and was exhibited by both the highly virulent
RH-LDM (type I) strain and the low virulence PTG/ME49 (type
II) strain (Fig. 3d). The B cell lymphoma cell line A20 may not
represent a cell type typically infected by T. gondii. Therefore, to
approach a more physiological situation, primary BMDC were infected with T. gondii and incubated with soluble recombinant
FasL. In this setting, Fas ligation also resulted in rapid parasite
egress (Fig. 3, e and f). To visualize the process, analysis using
real-time confocal microscopy was performed and revealed a dramatic egress of intracellular parasites as a result of Fas ligation.
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FIGURE 4. Fas-mediated egress of T. gondii is dependent on caspase activity and free intracellular calcium. a, Intracellular calcium in A20 cells after the addition of anti-Fas as a function of time with (filled
circles) and without z-VAD-FMK (open squares) as
measured by flow cytometry after staining with Fluo-4AM. b, Ratio free/intracellular parasites (toxo, Toxoplasma gondii) after the addition of anti-Fas as a function of time in the presence (filled circles) and absence
(open squares) of BAPTA-AM. c, Ratio of free/intracellular parasites (toxo) as a function of time in the presence (filled circles) or absence (open squares) of
z-VAD-FMK after the addition of anti-Fas. d, Kinetics
of annexin V/PI staining after Fas ligation in T. gondii-infected cell cultures in the presence (filled circles) or absence (open squares) of BAPTA-AM. e,
Cell cycle analysis of T. gondii-infected GFP⫹ or
GFP⫺ A20 cells in the presence or absence of z-VADFMK or BAPTA-AM 4 h after addition of anti-Fas. Percentage of cells in subG0/G1is indicated. Below the histograms is shown the proportion of apoptotic (subG0/
G1) cells as a function of time. Filled squares indicate
GFP⫹ cells and open circles indicate GFP⫺ cells. f, Ratio of free vs intracellular parasites, in A20 cells, as a
function of time in the presence (open squares) or absence (filled circles) of anti-Fas in calcium-free or calcium-containing culture medium.
Rapid and synchronous egress of parasites from infected cells was
observed as well as active parasite motility after the egress.
Changes in host cell morphology were consistent with cell lysis
(necrosis) after egress of the parasite (supplemental movies 1–3).5
Although these data are in agreement with previous observations concerning the lack of typical apoptotic features (in this
case lack of appearance of annexin V⫹ and PI⫺ cells) in T.
gondii-infected cells after death receptor ligation, we provide
here an alternative explanation for this phenomenon. Our data
indicate that rather than being the result of an active inhibition
of the apoptotic process by the parasite, the absence of apoptotic features is a secondary consequence of a rapid induction of
necrosis because of early and violent parasite egress. As the
appearance of free parasites and necrotic cells after Fas ligation
5
The online version of this article contains supplemental material.
The Journal of Immunology
8361
occurs considerably faster then the emergence of secondary apoptotic cells (annexin V⫹PI⫹) in uninfected cell cultures, two
possibilities could be considered. The first possibility would be
that death receptor ligation would induce parasite egress, which
in turn would result in necrotic cell death of the host cell, i.e.,
parasite egress would be an active process. The other possibility
would be that parasite egress would merely be a consequence of
secondary apoptosis (annexin V⫹PI⫹) that is known to occur in
vitro after primary apoptosis (annexin V⫹PI⫺). This latter possibility is unlikely, as we failed to detect apoptotic cells before
the appearance of necrotic cells and parasite egress. To provide
a more definitive answer to this question, we needed to address
the mechanism behind this rapid parasite egress triggered by
death receptor ligation.
Death receptor-induced egress of T. gondii is dependent on
caspase-mediated release of intracellular calcium
The natural egress of T. gondii from host cells can be mimicked
experimentally using calcium ionophores (33) and can be inhibited using chelators of intracellular calcium such as
BAPTA-AM (34). Apoptotic cell death is associated with, even
if not dependent on, an increase in levels of intracellular calcium, possibly as a result of a breakdown of the endoplasmic
reticulum membrane (35). Therefore, we tested whether this
release of intracellular calcium in the early phase of host cell
apoptosis could trigger egress of T. gondii. First, we verified
that Fas ligation in A20 cells resulted in a rapid increase of
intracellular calcium as measured using Fluo-4-AM, an effect
that was abolished in the presence of the pan-caspase inhibitor
z-VAD-FMK (Fig. 4a). To evaluate the involvement of free
intracellular calcium and its dependence on caspase activity
during Fas-mediated parasite egress, infected A20 cells were
pretreated with z-VAD-FMK or BAPTA-AM. We found that
both z-VAD-FMK and BAPTA-AM completely blocked the
Fas-induced egress of T. gondii (Fig. 4, b and c). Furthermore,
pretreatment of T. gondii-infected cell cultures with
BAPTA-AM followed by Fas ligation resulted in predominantly
apoptotic cell death, rather than necrotic cell death. (Fig. 4d).
Similar results were found using cell cycle analysis to quantify
apoptosis (reflected by the number of sub-G0/G1 cells) in that
z-VAD-FMK completely blocked Fas-mediated apoptosis
whereas incubation with BAPTA-AM resulted in increased
numbers of apoptotic GFP⫹ (T. gondii-infected) cells, consistent with the perseverance of parasites intracellularly (Fig. 4e).
To evaluate an eventual contribution of the influx of extracellular calcium during death receptor-induced parasite egress, experiments were performed using calcium-free medium in which
parasites underwent Fas-induced egress with a comparable efficiency as in normal calcium-containing culture medium (Fig.
4f). Therefore, we concluded that the process of death receptorinduced T. gondii egress was an active process where the parasite responded to intracellular calcium mobilization as a consequence of caspase activation early in the apoptotic process.
Thus, parasite egress resulted in necrotic cell death of the
host cell.
Death ligand-expressing cell lines rapidly become infected upon
interaction with T. gondii-infected cells
Taken together, the data thus far suggest that death receptor ligation in T. gondii-infected cells, rather than leading to elimination
of the parasite, could lead to the release of parasites in the microenvironment. It was therefore important to determine whether the
parasites were infectious after death receptor-induced egress. If
this hypothesis was true, one would envisage that death ligandexpressing cells interacting with T. gondii-infected cells not only
would fail to eradicate the pathogen but potentially contribute to
local dissemination of the infection and even become infected
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FIGURE 5. T. gondii rapidly infects cells expressing FasL or TRAIL upon coculture with infected target cells. a, Proportion of infected live
(Thy1.2⫹GFP⫹) mFasL/L5178Y (filled circles) or L5178Y (open squares) when cocultured with T. gondii-infected A20 cells at an E:T ratio of 1:1. b,
Percentage of remaining live A20 cells (B220⫹) when cocultured with mFasL/L5178Y (filled circles) or L5178Y (open squares) as a function of time. c,
Depiction of the degree of infection of the A20 cells used in a and b (thick line). The intensity of GFP expression in free parasites (thin line) serves as
a reference. The percentage of parasite-infected cells is indicated. d, Infection of mFasL/L5178Y cells when cocultured with T. gondii-infected A20 cells
expressing FLIPL (filled triangles), Bcl-xL (filled circles), CrmA (open circles), or mock (open squares) as a function of time. e, Percentage of remaining
live A20 cells (B220⫹) expressing FLIPL (filled triangles), Bcl-xL (filled circles), CrmA (open circles), or mock (open squares) when cocultured with
mFasL/L5178Y cells. f, Depiction of the degree of infection of A20-mock cells used in d and e (thick line). The intensity of GFP expression in free parasites
(thin line) serves as a reference. The percentage of parasite-infected cells is indicated. The degree of infection was similar for A20-mock, A20-FLIPL,
A20-Bcl-xL, and A20-CrmA. g, T cell lymphoma cells L5178Y (open squares) and mFasL/L5178Y (filled circles) were exposed to increasing numbers of
T. gondii tachyzoites (RH-LDM) and infection was assayed 18 h later using flow cytometry. h, As in g, but open squares represent L5178Y cells and filled
circles represent hFasL/L5178Y. i, As in g, but assaying B cell lymphoma cells 2PK-3 (open squares) and hTRAIL/2PK-3 (filled circles). MOI, Multiplicity
of infection. j, Proportion of infected live (Thy1.2⫹GFP⫹) hFasL/L5178Y (filled circles) and L5178Y (open squares) cells when cocultured with T.
gondii-infected Jurkat cells. k, Proportion of infected live (B220⫹GFP⫹) hTRAIL/2PK-3 (filled circles) and 2PK-3 (open squares) cells when cocultured
with T. gondii-infected Jurkat cells.
8362
CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII
themselves as well. To test this directly, we cocultured parasiteinfected A20 cells with the murine T cell lymphoma L5178Y or
the same cells transfected with murine Fas-ligand, mFasL/
L5178Y, which are able to efficiently induce apoptosis in Fasexpressing target cells (30). In contrast to the L5178Y cells,
mFasL/L5178Y cells coincubated with parasite-infected A20 cells
rapidly became infected with T. gondii (Fig. 5a). As expected, A20
cells were eliminated from the culture in the presence of mFasL/
L5178Y cells but not when cocultured with L5178Y cells (Fig.
5b). To investigate the role of caspase activation in this process,
A20 cells stably expressing human Bcl-xL (an inhibitor of the intrinsic pathway of apoptosis), human FLIPL (an inhibitor of death
receptor mediated apoptosis), and CrmA (a poxvirus inhibitor of
caspase-1 and caspase-8 activity) as well as mock-transduced control cells were infected with T. gondii (29). The cells were then
coincubated with mFasL/L5178Y cells and the number of infected
effector cells was determined as a function of time. A20 cells expressing FLIPL or CrmA did not transfer the infection to the effector cells as a result of Fas ligation, whereas A20 cells expressing Bcl-xL and mock-transduced A20 cells did (Fig. 5d).
Accordingly, a dramatic decrease of live A20-mock and A20Bcl-xL cells, but not of A20-FLIPL or A20-CrmA cells, could be
seen in these cultures (Fig. 5e). Also, it could be noted that the
frequencies of infected mFasL/L5178Y cells differed between the
experiments shown in Fig. 5, a and d. This was because of differences in both the frequency of infected A20 cells and number of
parasites per cell, which is illustrated in Fig. 5, c and f. The permissiveness of death ligand transfectants and mock-transfected
controls for T. gondii-infection was similar (Fig. 5, g–i). These
results clearly implicate the activation of death receptor-associated
caspases as a necessary component in Fas-triggered parasite egress
and points to a negligible role for the intrinsic (Bcl-xL-inhibitable)
pathway in this process. Furthermore, these results using the cellular and viral apoptosis inhibitory proteins FLIPL and CrmA argue against eventual nonspecific effects of the pan-caspase inhibitor z-VAD-FMK on T. gondii, used in Fig. 4. Next, we tested
whether our findings could be generalized to human cells and also
to other death receptors than Fas. We found that cells transfected
with human Fas ligand, hFasL/L5178Y (Fig. 5j), or human
TRAIL, hTRAIL/2PK-3 (Fig. 5k), rapidly became infected when
incubated with T. gondii-infected human Jurkat cells, in sharp contrast to mock-transfected cells.
Primary T cells inducing death receptor- or perforin-dependent
cytotoxicity trigger T. gondii egress via different mechanisms
Next, we tested whether activated primary T cells inducing target cell death via the death receptor pathway or the perforin
pathway also would lead to parasite egress and subsequent infection of the effector cells. Therefore, CD4⫹ T cell blasts were
prepared from wild-type (wt) and Fas ligand-deficient gld mice
and incubated with T. gondii-infected A20 cells in a redirected
cytotoxicity assay in the presence of neutralizing Abs against
TRAIL. Wt CD4⫹ T cells were rapidly infected in the presence
of anti-CD3, a process that could be blocked by z-VAD-FMK
(Fig. 6, a and b). In contrast, gld CD4⫹ T cells did not become
infected after coculture with T. gondii-infected A20 cells (Fig.
6, a and c). CD8⫹ T cell blasts also rapidly became infected
after coculture with T. gondii-infected A20 cells in the presence
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FIGURE 6. Activated primary T cells rapidly get infected after interaction with T. gondii-infected target
cells in Fas-dependent or perforin -dependent mechanisms. a, Representative flow cytometry profiles depicting infection of primary mitogen-activated wt or gld
CD4⫹ T cells after incubation with T. gondii-infected
A20 cells, for 1 or 4 h in the presence or absence of
anti-CD3, with or without preincubation with z-VADFMK. b, Proportion of infected live wt CD4⫹cells after
coculture with T. gondii-infected A20 cells with antiCD3 in the absence (filled circles) or presence of zVAD-FMK (filled triangles) or without anti-CD3 (open
squares) as a function of time. c, As in b but with CD4⫹
T cells from gld mice. d, Proportion of infected live wt
CD8⫹cells after coculture with T. gondii-infected A20
cells with anti-CD3 in the absence (filled circles) or
presence of z-VAD-FMK (filled triangles) or without
anti-CD3 (open squares) as a function of time. e, As in
d but with CD8⫹ T cells from gld mice. f, Proportion of
infected live perforin⫺/⫺ CD8⫹cells after coculture with
T. gondii-infected A20-mock cells with anti-CD3 (filled
circles) or without anti-CD3 (open squares). g, Proportion of infected live perforin⫺/⫺ CD8⫹cells after coculture with T. gondii-infected A20-FLIPL cells with antiCD3 (filled circles) or without anti-CD3 (open squares).
h, As in f but with CD8⫹ T cells from gld mice. i, As in
g but with CD8⫹ T cells from gld mice. Live cells were
gated using PI and eventual doublets were excluded
from the analysis by gating on B220⫺ cells and or gating on single cells using a combination of forward light
scatter and pulse width.
The Journal of Immunology
8363
of anti-CD3, but this could only partly be blocked by preincubation with z-VAD-FMK (Fig. 6d). When CD8⫹ T cell blasts
from gld mice were used they also rapidly got infected, but
z-VAD-FMK had no detectable inhibitory capacity (Fig. 6e).
These results would indicate that T cell-mediated parasite
egress could proceed both through a Fas-dependent and a
caspase-dependent pathway (used by CD4⫹ and CD8⫹ cells)
and a perforin-dependent, caspase-independent pathway used
by CD8⫹ cells. To directly test this, CD8⫹ T cell blasts were
prepared from perforin-deficient and FasL-deficient (gld) mice
and cocultured with T. gondii-infected A20-mock and A20FLIPL cells in the presence or absence of anti-CD3. CD8⫹ perforin⫺/⫺ cells rapidly became infected when cocultured with
parasite infected A20-mock cells (Fig. 6f) but not upon coculture with infected A20-FLIPL cells (Fig. 6g), thus indicating
that in the absence of perforin expression CD8⫹ cells only can
induce T. gondii egress via the death receptor pathway. As expected, FasL-deficient (but perforin-expressing) CD8⫹ cells
were able to induce parasite egress and become infected, upon
interaction with both T. gondii-infected A20-mock (Fig. 6h) and
A20-FLIPL cells (Fig. 6i). It could also be noted that the kinetics of perforin-dependent T. gondii-infection were faster as
compared with the Fas-dependent infection (compare Fig. 6, b
and f vs e, h, and i).
Primed Ag specific CD8⫹ T cells are preferentially infected by
T. gondii upon challenge in vivo
Finally, we investigated whether T cells recognizing T. gondii-infected target cells in vivo would themselves become infected. We
took advantage of the H-2b congenic mouse strain A.BY, which
mounts CTL responses to C57BL/6 mouse minor Ags presented on
MHC class I (36). T. gondii-infected BMDC from C57BL/6 mice
were injected i.p. into A.BY mice, naive or previously primed with
irradiated splenocytes from C57BL/6 mice. Thus, an early primary
response is compared with a secondary immune response. Peritoneal
exudate cells were obtained and the proportion of T. gondii-infected
CD8⫹ cells was determined by flow cytometry. Thirty-six hours after
injection of T. gondii-infected BMDC, transition from low to high
surface expression of CD44 indicated a vigorous activation of CD8⫹
T cells in primed mice compared with naive mice (49.5 ⫾ 7.0% vs
22.5 ⫾ 9.1%, p ⬍ 0.002). The percentage of CD8⫹CD44high T cells
was increased 4.3 times in primed vs naive mice (Fig. 7, a– c). A
considerable fraction of CD8⫹ T cells in primed mice (12.0 ⫾ 3.0%)
became infected by the parasite as compared with in naive mice
(4.2 ⫾ 2.2%; Fig. 7, a– c). T. gondii-infected CD8⫹ cells displayed
almost exclusively an activated CD44high phenotype both in primed
(22.8 ⫾ 4.5% infected cells among CD44high vs 1.8 ⫾ 0.4% in
CD44low cells) and naive mice (13.2 ⫾ 4.5% infected among
CD44high vs 2.0 ⫾ 1.2% in CD44low cells). The percentage of parasite-infected, activated CD8⫹ T cells in primed vs naive mice was
increased 7.5 times. By comparing the proportions of T. gondii-infected CD8⫹ T cells with an index of activation (CD44high/CD44low),
a strong positive correlation was found (Fig. 7d).
Thus, in this in vivo model, a high proportion of activated Agspecific CTL became infected after interacting with T. gondii-infected target cells. These results were in agreement with our data
presented above using various in vitro systems.
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FIGURE 7. T cells primed against
A.BY minor histocompatibility Ags
rapidly get infected after challenge
with T. gondii-infected dendritic
cells from A.BY mice, in vivo. a,
Representative flow cytometry profiles of T. gondii-infected CD8⫹ cells
36 h after injection i.p. of 2 million
infected dendritic cells from A.BY
mice into primed or naive C57BL/6
mice. Total CD8⫹, CD8⫹CD44high
(hi), and CD8⫹CD44low (lo) cells
were gated and further analyzed for
GFP expression as indicated. b, Percentage of T. gondii-infected CD8⫹,
CD8⫹CD44high, and CD8⫹CD44low
cells in primed and naive animals. c,
Percentage of CD8⫹, CD8⫹
CD44high, and CD8⫹CD44low cells
recovered from primed and naive animals. d, Pearson’s test of covariance
showed a significant correlation between the proportion of T. gondii-infected CD8⫹ T cells and the stimulation index (CD44high/CD44low)
(p ⬍ 0.001, r ⫽ 0.93; open circles,
naive mice; filled circles, primed
mice).
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CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII
Discussion
Acknowledgments
We thank Drs. G. P. Nolan (Stanford) for the retroviral packaging line
Phoenix-Ampho, Vishva Dixit, (Tularik) for providing the CrmA gene, and
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Induction of cell death is an important immune mechanism for
eliminating cells that represent a threat to the integrity of the organism, e.g., cells infected by a pathogen. Consequently, intracellular pathogens have evolved mechanisms to counteract elimination secondary to the induction of cell death in host cells, often by
interfering with apoptosis induction and/or execution (1–3, 37).
Cell-assisted death induced by cytotoxic lymphocytes in a specific manner can proceed through two basic mechanisms: one
through the ligation of death receptors on target cells by death
ligands expressed on effector lymphocytes (CD4⫹, CD8⫹ T cells,
and NK cells) and the other dependent on the action of perforin
and granzymes released by cytotoxic cells (CD8⫹ and NK cells)
upon specific interactions with target cells. In this study we provide evidence indicating that lymphocytes acting on T. gondiiinfected target cells via death ligand- or perforin/granzyme-dependent cytotoxicity may not be effective in eradicating this
intracellular parasite. We show that T cells engaging any of these
pathways as a consequence of interactions with T. gondii-infected
cells trigger the egress of infectious parasites with the ability to
infect surrounding cells, including the effector cells, although
through different mechanisms. Whereas death receptor-induced parasite egress requires caspase activation in the infected target cell, perforin-dependent cytotoxicity induces parasite egress in a caspase-independent manner, either as a consequence of the influx of
extracellular calcium, the efflux of potassium (38), or the caspaseindependent mobilization of intracellular calcium, possibly as a consequence of the action of granzymes or other components of cytotoxic
granules. As far as we are aware, this represents a previously undescribed strategy for intracellular pathogens to cope with death receptor-mediated apoptosis and cellular cytotoxicity.
At first, our results seemingly would be in contradiction with
those of others concerning the ability of T. gondii to inhibit death
receptor-mediated apoptosis (15, 20 –22). Using different cell types
(primary dendritic cells and T and B cell lymphomas of human or
murine origin) and two different parasite strains (RH and PTG), we
provide substantial evidence that death receptor ligation in T. gondii-infected cells leads to caspase-dependent release of intracellular calcium, which in turn induces violent parasite egress with
subsequent host cell necrotic cell death before the appearance of
typical signs of apoptosis. A consequence of this when using various readout methods for apoptosis (but not for cell death) could be
that intracellular components involved in finalizing the apoptotic
phenotype may become diluted out in the growth medium or that
intracellular conditions, including salt concentrations, pH, and energy
requirements, may not allow the apoptotic progression of the cell as a
secondary effect of the preceding necrotic cell death. Alternatively,
sensitivity to Fas-induced cell death may vary in different cell types.
This, in conjunction with the absence of quantitative assessments of
the ability of T. gondii to protect against cell death in previous studies,
allow us to reconcile our new findings with previous data (15, 20 –22),
although with different conclusions.
Early during T. gondii-infection, parasites rapidly spread in the
organism hematogenously and via lymphatics (25, 39). The infection results in the recruitment of intraepithelial lymphocytes to the
gut (40) and inflammatory monocytic cells and neutrophils (41, 42)
to the peritoneal cavity, which become infected. Thus, it could be
envisaged that this necrotic cell death induced by death receptoror perforin-dependent parasite egress could contribute to inflammatory responses, leading both to tissue damage and the further
spread of the parasite due to infection of invading leukocytes.
In this article we provide evidence that T cells interacting with
T. gondii-infected cells in an Ag-specific manner, in vitro and in
vivo, cause egress of infective parasites and can result in the productive de novo infection of surrounding cells, including the
effector cells, after recognition of parasite-infected cells. It is thus
possible that during Toxoplasma infection death receptor- and perforin-mediated parasite egress may contribute to parasite dissemination both in peripheral tissues and systemically. As activated leukocytes have access to immunoprivileged sites, this process may also
contribute to the establishment of infection in these locations.
Both CD4⫹ and CD8⫹ T cells have been shown to be important
in the control of T. gondii infection (9). Our present results are
compatible with a scenario where T cell effector functions such as
cytokine production and B cell help, in combination with cytotoxicity, would contribute to the control of T. gondii infection. Neither
perforin nor Fas ligands have been demonstrated to be critical
during acute T. gondii infection (10 –12). The absence of perforin,
however, has been associated with increased numbers of brain
cysts and earlier mortality during the chronic phase following infection with the type II strain, ME49 (10). Both IFN-␥ and B cells
have shown to be necessary for the host to resist T. gondii infection
(13, 42, 43). Our results suggest that cellular cytotoxicity (death
ligand mediated or perforin dependent) could be beneficial or
harmful for the host depending on the presence or absence of additional immune mechanisms. In the absence of other immune
mechanisms, cellular cytotoxicity would be inefficient in controlling Toxoplasma infection and could even promote parasite dissemination. In the presence of additional effective immune mechanisms, however, cellular cytotoxicity could contribute to
limitation of the infection. Considering the results referred to
above, it is reasonable to assume that the presence of neutralizing
Abs would be one important factor determining the efficiency of
cellular cytotoxicity as a defense mechanism against T. gondii (10,
13, 42, 43).
Increased FasL and TRAIL expression, both on hemopoietic and
nonhemopoietic cells, can be induced by stress or inflammation (6,
44). Inflammation also leads to increased numbers of activated leukocytes in immunoprivileged sites including the CNS (45). It is therefore conceivable that death receptor-induced or perforin-mediated
egress could be involved during the reactivation of T. gondii in chronically infected individuals as a result of inflammatory reactions. Reactivation of T. gondii is associated with immunodeficiency and is an
important problem in AIDS patients where it can be correlated to
decreased numbers of CD4⫹ T cells, assumed to be a consequence of
defects in immune surveillance (46). An additional possibility would
be that the general inflammatory state, characteristic of HIV infected
individuals and possibly of other immunodeficiencies, would result in
increased expression of death ligands and therefore contribute to reactivation of T. gondii through death receptor-mediated parasite
egress. Recent observations showing a positive correlation between
viral load and TRAIL expression are compatible with such a scenario
(47).
Successful parasitic infection with host and parasite survival implicates a balance between parasite immune evasion strategies and
host immune surveillance. The data presented herein concerning
the responses of T. gondii to death ligand- and perforin-dependent cytotoxicity adds a new dimension to this complex interplay between parasite and host. Future investigations will determine whether other intracellular pathogens, protozoa,
viruses, or bacteria would use similar mechanisms do deal with
death receptor-induced and perforin-dependent cell death as we
here demonstrate for T. gondii.
The Journal of Immunology
Hans-Gustaf Ljunggren, Manuchehr Abedi-Valugerdi, Jelena Levitskaya,
Kari Högstrand, and Göran Möller for discussions.
Disclosures
The authors have no financial conflict of interest.
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