Death Receptor Ligation or Exposure to Perforin Trigger Rapid Egress of the Intracellular Parasite

This information is current as Emma K. Persson, Abela Mpobela Agnarson, Henrik of September 29, 2021. 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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 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. Downloaded from ellular cytotoxicity is an important immune defense Toxoplasma gondii is an obligate intracellular parasite of the phy- mechanism against intracellular pathogens. After target lum . T. gondii is one of the most wide spread human C recognition it can proceed via two main pathways, the parasites with an estimated 2 billion infected individuals. During the perforin/granzyme-dependent pathway or via expression of death acute phase of infection, parasites rapidly disseminate to establish a ligands, binding to death receptors on the target cells and leading life-long, often asymptomatic, chronic infection. Reactivated infec-

to apoptotic cell death. Many , including adenovirus, pox- tion in immunocompromised patients can result in severe disease, and http://www.jimmunol.org/ , herpesvirus, and papillomavirus, carry anti-apoptotic genes acute infection during pregnancy can cause damage in the developing presumably to prevent premature death of their host cells, be it an fetus (7). Within the host Toxoplasma is capable of infecting all types innate response to the infection or induced by immune cells upon of nucleated cells, including cells of the immune system (8). recognition (1). Recent reports indicate that other intracellular CD4ϩ and CD8ϩ T cells have been demonstrated to be important pathogens such as and certain are also able to in controlling T. gondii infection (reviewed in Ref. 9). It was later interfere with apoptosis induction and thereby protect their host shown that perforin-dependent cytotoxicity played a limited role in cells, and themselves, from cell death (1–3). resistance to T. gondii infection (10). Neither Fas nor TNF-receptors Apoptosis can be induced via the intrinsic or the extrinsic path- were subsequently demonstrated to be critical for control of T. gondii ways (4). The intrinsic pathway can be triggered by internal stress infection (11–13). A number of reports have since provided evidence by guest on September 29, 2021 signals in the cell resulting from DNA damage, damage to or- indicating that T. gondii can inhibit host cell apoptosis induced by ganelles, lack of essential growth factors, or infection. The extrin- CTL, death receptor ligation, or via the intrinsic pathway of apoptosis sic pathway is activated through death receptor ligation and is used (14–22). Based on these results it has been suggested that T. gondii- by T or NK cells to induce target cell killing in a specific manner. infected cells would be protected from apoptotic cell death and that Cytotoxic cells can also induce target cell death via the perforin- this could explain why neither cell-autonomous induction of apoptosis dependent granule exocytosis pathway (reviewed in Ref. 5). Also nor T cell-mediated assisted apoptosis would be effective defense nonlymphoid cells can, under certain circumstances such as in- mechanisms against Toxoplasma-infected cells (14–24). flammation, express death ligands and thereby induce apoptosis in Thus, in previous studies the absence of typical features of cells with which they interact (reviewed in Ref. 6). apoptosis in cells infected by Toxoplasma has been amply docu- mented 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 *Center for Infectious Medicine and †Center for Experimental Hematology, Depart- ment of Medicine, Karolinska Institutet, Karolinska University Hospital–Huddinge, not clear. In this study we have investigated this issue by studying Stockholm, Sweden; ‡Swedish Institute for Infectious Disease Control, Stockholm, the consequences of death receptor ligation and exposure to CTL Sweden; and §Department of Immunology, Juntendo University, School of Medicine, Tokyo, Japan in T. gondii-infected cells. We report that, early during the induc- tion of death receptor- or perforin-dependent cell death, parasites Received for publication August 22, 2007. Accepted for publication October 3, 2007. rapidly egress from their host cells resulting in necrotic cell death 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 of the host cells. Through this mechanism parasites avoid elimi- with 18 U.S.C. Section 1734 solely to indicate this fact. nation and are instead able to infect neighboring cells, including 1 This work supported by in part by grants from the Swedish Research Council (to the cytotoxic cells themselves. A.B.), the Swedish Cancer Society (to A.G.), and the Swedish Foundation for Stra- tegic Research. N.H. is the recipient of a postdoctoral fellowship from the Karolinska Institutet infection network. Materials and Methods 2 E.K.P. and A.M.A. contributed equally to this work. Mice 3 Address correspondence and reprint requests to Dr. Alf Grandien or Dr. Antonio Ϫ/Ϫ b Barragan, Center for Infectious Medicine, Department of Medicine, Karolinska In- C57BL/6, C57BL/6.perforin , C57BL/6.gld, and A.BY a H-2 congenic stitutet, Karolinska University Hospital–Huddinge, 141 86 Stockholm, Sweden. E- mice of the A/Sn background were maintained and bred at the animal mail addresses: [email protected] and [email protected] facility of Department of Microbiology, Tumor and Cell Biology, Karo- linska Institutet (Stockholm, Sweden). All procedures were performed with Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 relevant ethical permission according to local and national guidelines. www.jimmunol.org 8358 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) den- sity 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 Interna- tional). 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 ϫ 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 for6hatamultiplicity of infection of 1. The cells were washed thoroughly and had an infection rate of ϳ70%. 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. Downloaded from Cell lines and retroviral transduction 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 http://www.jimmunol.org/ 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 trans- fectant hTRAIL/2PK-3 (31). A20 cells were transduced with LXIN retro-

viruses (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). FIGURE 1. T. gondii infection does not protect host cells against death receptor-induced cell death. a, Flow cytometric profiles representing sur- Induction of apoptosis and redirected cytolysis vival of uninfected and T. gondii-infected A20 cells in the absence or by guest on September 29, 2021 presence of anti-Fas (10 h after addition of anti-Fas). b and c, Number of Agonistic anti-human or anti-mouse Fas mAbs Jo2 (BD Biosciences Pharmingen) and CH-11 (MBL International) were added at various con- live A20 (b) or Jurkat (c) cells in the absence or presence of anti-Fas (10 centrations to the cells as indicated. Alternatively, cells were coincubated h after addition of anti-Fas). Filled squares, T. gondii-infected cells; open with L5178Y, 2PK-3, mFasL/L5178Y, hFasL/L5178Y, or hTRAIL/2PK-3 circles, uninfected cells. The results represent the mean and 1 SD from cells at a 1:1 ratio. Intracellular calcium in A20 cells as a result of Fas three independent experiments (the frequency of parasite-infected cells at ligation was measured using preincubation of the cells with Fluo-4-AM the start of the culture in b and c was ϳ70%). (Calbiochem) at 1 ␮M for 30 min at 37°, after which the cells were ana- lyzed 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-Ala- stained with anti-CD45R/B220-allophycocyanin to detect A20 or 2PK-3 Asp-fluoromethyl ketone (z-VAD-FMK) at 10 ␮M (Enzyme System Prod- cells, anti-Thy1.2-PE to detect L5178Y cells, anti-human CD3-allophyco- ucts) added to cultures 20 min before Fas ligation or addition of T cells. T cyanin to detect Jurkat cells, or allophycocyanin- or PE-conjugated mAbs cell blasts were prepared from spleens of C57BL/6, C57BL/6 gld, and against CD4, CD8, or CD44 (reagents from BD Biosciences). Cell cycle C57BL/6.perforinϪ/Ϫ mice by culture for 2 days with 2 ␮g/ml Con A analysis was performed by 2 min of permeabilization with saponin (0.01%; (Amersham Biosciences) followed by 1 day of culture with 10 U/ml IL-2 w/v) and PI (50 ␮g/ml) followed by flow cytometry. (supernatant from transfected X63 cells) and were thereafter isolated over Lympholyte-M (Cedarlane Laboratories). CD4ϩ and CD8ϩ cells were ob- Real-time confocal microscopy tained through negative selection using magnetic beads (Miltenyi Biotech), Egress of intracellular tachyzoites upon Fas ligation was assessed with a and T cell blasts and T. gondii-infected A20 cells were cocultured at a ratio spinning disk confocal setup (Ultraview LCI-3 tandem scanning unit; of 1:1 in the presence of neutralizing anti-TRAIL (clone N2B2) mAb at 10 PerkinElmer) on an Axiovert 200 M (Carl Zeiss) connected to a charge- ␮ ␮ g/ml (32) in the presence or absence of 1 g/ml anti-CD3 mAb (clone coupled device camera (Orca ER; Hamamatsu). Cells were placed in 2C11). minichamber system (POCmini; LaCon) with heating stage. Image acqui- Detection of apoptotic cells and flow cytometry sition and analysis was performed with Openlab software (version 4.0.2; Improvision). Infected or noninfected cells were stained with annexin V-biotin (Roche Diagnostics) followed by staining with streptavidin-allophycocyanin (BD Results ␮ Biosciences Pharmingen) and propidium iodide (PI) (1 g/ml) and there- Fas ligation mediates cell death of Toxoplasma-infected host after analyzed on a FACSCalibur flow cytometer (BD Biosciences Immu- nocytometry Systems) or a CyAn ADP flow cytometer (DakoCytomation); cells the data were analyzed with CellQuest (BD Biosciences Immunocytometry To evaluate the ability of T. gondii to protect host cells against the Systems) or FlowJo software (Tree Star). In some experiments, cells were extrinsic pathway of apoptosis induction, the murine B cell lym- phoma cell line A20 and Jurkat, a human T lymphoma cell line, 4 Abbreviations used in this paper: m, murine (prefix); BMDC, bone marrow-derived were infected with the GFP-expressing virulent parasite line RH- dendritic cell; CrmA, cytokine response modifier A; FasL, Fas ligand; h, human (prefix); PI, propidium iodide; wt, wild type; z-VAD-FMK, benzyloxycarbonyl-Val- LDM and incubated with agonistic anti-Fas mAbs. Ten hours after Ala-Asp-fluoromethyl ketone. the addition of anti-Fas mAbs, cellular viability was evaluated by The Journal of Immunology 8359

FIGURE 2. Fas receptor ligation in T. gondii-infected A20 cells leads to necrotic-like cell death. a, An- nexin V/PI staining of uninfected (upper panel)orT. gondii-infected (lower panel) cell cultures 2 h after the addition of anti-Fas mAb. b, Ki- netics of annexin V/PI staining in un- infected (upper panels) and T. gon- dii-infected cultures (lower panels) in the presence (filled circles) or ab- sence (open squares) of anti-Fas. c, Analysis of GFP expression vs cell size (forward light scatter) in T. gon- dii-infected cultures in the absence or Downloaded from presence of anti-Fas mAb as a func- tion of time. Values in the graphs represent the percentage parasite in- fected cells (top), the percentage of noninfected cells (middle), and the percentage of free parasites (bottom). http://www.jimmunol.org/

examining the exposure of phosphatidyl serine on the cell surface by annexin V staining (Fig. 1a; showing A20 cells) and live cell by guest on September 29, 2021 counting (Fig. 1, b and c). Few live cells could be found in either uninfected (9%) or T. gondii-infected cultures (3%) after incuba- tion 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 re- ported 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 Vϩ and PIϪ, followed by the ap- pearance of annexin VϩPIϩ cells (Fig. 2, a and b, upper panels). However, in T. gondii-infected cultures the cells displayed a com- FIGURE 3. Fas receptor ligation in T. gondii-infected A20 cells leads to pletely different pattern that was characterized by a very rapid parasite egress. a, Ratio of free parasites vs live A20 cells, using cell Ϫ ϩ accumulation of atypical annexin V PIdim/ cells followed by the counting, as a function of time in the presence (filled circles) or absence appearance of annexin VϩPIϩ cells but low numbers of typical (open squares) of anti-Fas. b, Ratio of free vs intracellular parasites, using apoptotic (annexin VϩPIϪ) cells (Fig. 2, a and b, lower panels). flow cytometric analysis of A20 cells, as a function of time in the presence Thus, parasite-infected cells very rapidly acquired a necrotic-like (filled circles) or absence (open squares) of anti-Fas. c, Dose dependence phenotype following Fas ligation instead of the expected conven- 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 tional apoptotic phenotype. (open square). e, Ratio of free vs intracellular parasites, using flow cyto- The proportions of live infected and uninfected cells as well as metric analysis of murine dendritic cells, as a function of time in the pres- free parasites were also analyzed. In contrast to control cultures ence (filled circles) or absence (open squares) of soluble FasL (sFasL; (Fig. 2c, upper panels), the addition of anti-Fas mAb resulted in a diluted 1/4). f, Dose dependence of T. gondii egress in dendritic cells in the clear reduction in the proportion of live cells (Fig. 2c, lower pan- presence (filled circles) of soluble FasL or control supernatant (open els). The decrease in the proportions of infected live cells after Fas squares). 8360 CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII

FIGURE 4. Fas-mediated egress of T. gondii is de- pendent on caspase activity and free intracellular cal- cium. a, Intracellular calcium in A20 cells after the ad- dition 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-4- AM. b, Ratio free/intracellular parasites (toxo, Toxo- plasma gondii) after the addition of anti-Fas as a func- tion of time in the presence (filled circles) and absence (open squares) of BAPTA-AM. c, Ratio of free/intra- Downloaded from cellular parasites (toxo) as a function of time in the pres- ence (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. gon- dii-infected cell cultures in the presence (filled cir- cles) or absence (open squares) of BAPTA-AM. e, http://www.jimmunol.org/ Cell cycle analysis of T. gondii-infected GFPϩ or GFPϪ A20 cells in the presence or absence of z-VAD- FMK or BAPTA-AM 4 h after addition of anti-Fas. Per- centage of cells in subG0/G1is indicated. Below the his- tograms is shown the proportion of apoptotic (subG0/ G1) cells as a function of time. Filled squares indicate GFPϩ cells and open circles indicate GFPϪ cells. f,Ra- tio of free vs intracellular parasites, in A20 cells, as a function of time in the presence (open squares) or ab- sence (filled circles) of anti-Fas in calcium-free or cal- by guest on September 29, 2021 cium-containing culture medium.

ligation was paralleled by an increase in the proportion of free Rapid and synchronous egress of parasites from infected cells was parasites (Fig. 2c). observed as well as active parasite motility after the egress. To verify that the population defined by low forward light scat- Changes in host cell morphology were consistent with cell lysis ter and GFP positivity really represented free parasites, the num- (necrosis) after egress of the parasite (supplemental movies 1–3).5 bers of free parasites were counted using light microscopy. A good Although these data are in agreement with previous observa- correlation between data from counting and flow cytometry was tions concerning the lack of typical apoptotic features (in this obtained (Fig. 3, a and b). Fas-induced parasite egress was dose case lack of appearance of annexin Vϩ and PIϪ cells) in T. dependent (Fig. 3c) and was exhibited by both the highly virulent gondii-infected cells after death receptor ligation, we provide RH-LDM (type I) strain and the low virulence PTG/ME49 (type here an alternative explanation for this phenomenon. Our data II) strain (Fig. 3d). The B cell lymphoma cell line A20 may not indicate that rather than being the result of an active inhibition represent a cell type typically infected by T. gondii. Therefore, to of the apoptotic process by the parasite, the absence of apopto- approach a more physiological situation, primary BMDC were in- tic features is a secondary consequence of a rapid induction of fected with T. gondii and incubated with soluble recombinant necrosis because of early and violent parasite egress. As the FasL. In this setting, Fas ligation also resulted in rapid parasite appearance of free parasites and necrotic cells after Fas ligation egress (Fig. 3, e and f). To visualize the process, analysis using real-time confocal microscopy was performed and revealed a dra- matic egress of intracellular parasites as a result of Fas ligation. 5 The online version of this article contains supplemental material. The Journal of Immunology 8361

FIGURE 5. T. gondii rapidly infects cells expressing FasL or TRAIL upon coculture with infected target cells. a, Proportion of infected live (Thy1.2ϩGFPϩ) 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 (B220ϩ) 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 Downloaded from expressing FLIPL (filled triangles), Bcl-xL (filled circles), CrmA (open circles), or mock (open squares) as a function of time. e, Percentage of remaining ϩ 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,Asing, but open squares represent L5178Y cells and filled circles represent hFasL/L5178Y. i,Asing, but assaying B cell lymphoma cells 2PK-3 (open squares) and hTRAIL/2PK-3 (filled circles). MOI, Multiplicity http://www.jimmunol.org/ of infection. j, Proportion of infected live (Thy1.2ϩGFPϩ) hFasL/L5178Y (filled circles) and L5178Y (open squares) cells when cocultured with T. gondii-infected Jurkat cells. k, Proportion of infected live (B220ϩGFPϩ) hTRAIL/2PK-3 (filled circles) and 2PK-3 (open squares) cells when cocultured with T. gondii-infected Jurkat cells.

occurs considerably faster then the emergence of secondary ap- Fas-induced egress of T. gondii (Fig. 4, b and c). Furthermore, optotic cells (annexin VϩPIϩ) in uninfected cell cultures, two pretreatment of T. gondii-infected cell cultures with possibilities could be considered. The first possibility would be BAPTA-AM followed by Fas ligation resulted in predominantly that death receptor ligation would induce parasite egress, which apoptotic cell death, rather than necrotic cell death. (Fig. 4d). by guest on September 29, 2021 in turn would result in necrotic cell death of the host cell, i.e., Similar results were found using cell cycle analysis to quantify parasite egress would be an active process. The other possibility apoptosis (reflected by the number of sub-G0/G1 cells) in that would be that parasite egress would merely be a consequence of z-VAD-FMK completely blocked Fas-mediated apoptosis secondary apoptosis (annexin VϩPIϩ) that is known to occur in whereas incubation with BAPTA-AM resulted in increased vitro after primary apoptosis (annexin VϩPIϪ). This latter pos- numbers of apoptotic GFPϩ (T. gondii-infected) cells, consis- sibility is unlikely, as we failed to detect apoptotic cells before tent with the perseverance of parasites intracellularly (Fig. 4e). the appearance of necrotic cells and parasite egress. To provide To evaluate an eventual contribution of the influx of extracel- a more definitive answer to this question, we needed to address lular calcium during death receptor-induced parasite egress, ex- the mechanism behind this rapid parasite egress triggered by periments were performed using calcium-free medium in which death receptor ligation. parasites underwent Fas-induced egress with a comparable ef- ficiency as in normal calcium-containing culture medium (Fig. Death receptor-induced egress of T. gondii is dependent on 4f). Therefore, we concluded that the process of death receptor- caspase-mediated release of intracellular calcium induced T. gondii egress was an active process where the par- The natural egress of T. gondii from host cells can be mimicked asite responded to intracellular calcium mobilization as a con- experimentally using calcium ionophores (33) and can be in- sequence of caspase activation early in the apoptotic process. hibited using chelators of intracellular calcium such as Thus, parasite egress resulted in necrotic cell death of the BAPTA-AM (34). Apoptotic cell death is associated with, even host cell. if not dependent on, an increase in levels of intracellular cal- cium, possibly as a result of a breakdown of the endoplasmic reticulum membrane (35). Therefore, we tested whether this Death ligand-expressing cell lines rapidly become infected upon release of intracellular calcium in the early phase of host cell interaction with T. gondii-infected cells apoptosis could trigger egress of T. gondii. First, we verified Taken together, the data thus far suggest that death receptor liga- that Fas ligation in A20 cells resulted in a rapid increase of tion in T. gondii-infected cells, rather than leading to elimination intracellular calcium as measured using Fluo-4-AM, an effect of the parasite, could lead to the release of parasites in the micro- that was abolished in the presence of the pan-caspase inhibitor environment. It was therefore important to determine whether the z-VAD-FMK (Fig. 4a). To evaluate the involvement of free parasites were infectious after death receptor-induced egress. If intracellular calcium and its dependence on caspase activity this hypothesis was true, one would envisage that death ligand- during Fas-mediated parasite egress, infected A20 cells were expressing cells interacting with T. gondii-infected cells not only pretreated with z-VAD-FMK or BAPTA-AM. We found that would fail to eradicate the pathogen but potentially contribute to both z-VAD-FMK and BAPTA-AM completely blocked the local dissemination of the infection and even become infected 8362 CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII

FIGURE 6. Activated primary T cells rapidly get in- fected after interaction with T. gondii-infected target cells in Fas-dependent or perforin -dependent mecha- nisms. a, Representative flow cytometry profiles depict- ing infection of primary mitogen-activated wt or gld CD4ϩ T cells after incubation with T. gondii-infected A20 cells, for 1 or4hinthepresence or absence of anti-CD3, with or without preincubation with z-VAD- FMK. b, Proportion of infected live wt CD4ϩcells 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. c,Asinb but with CD4ϩ T cells from gld mice. d, Proportion of infected live wt CD8ϩcells 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,Asin Downloaded from d but with CD8ϩ T cells from gld mice. f, Proportion of infected live perforinϪ/Ϫ CD8ϩcells after coculture with T. gondii-infected A20-mock cells with anti-CD3 (filled circles) or without anti-CD3 (open squares). g, Propor- tion of infected live perforinϪ/Ϫ CD8ϩcells after cocul- ture with T. gondii-infected A20-FLIPL cells with anti-

CD3 (filled circles) or without anti-CD3 (open squares). http://www.jimmunol.org/ h,Asinf but with CD8ϩ T cells from gld mice. i,Asin g but with CD8ϩ T cells from gld mice. Live cells were gated using PI and eventual doublets were excluded from the analysis by gating on B220Ϫ cells and or gat- ing on single cells using a combination of forward light scatter and pulse width. by guest on September 29, 2021 themselves as well. To test this directly, we cocultured parasite- caspases as a necessary component in Fas-triggered parasite egress infected A20 cells with the murine T cell lymphoma L5178Y or and points to a negligible role for the intrinsic (Bcl-xL-inhibitable) the same cells transfected with murine Fas-ligand, mFasL/ pathway in this process. Furthermore, these results using the cel-

L5178Y, which are able to efficiently induce apoptosis in Fas- lular and viral apoptosis inhibitory proteins FLIPL and CrmA ar- expressing target cells (30). In contrast to the L5178Y cells, gue against eventual nonspecific effects of the pan-caspase inhib- mFasL/L5178Y cells coincubated with parasite-infected A20 cells itor z-VAD-FMK on T. gondii, used in Fig. 4. Next, we tested rapidly became infected with T. gondii (Fig. 5a). As expected, A20 whether our findings could be generalized to human cells and also cells were eliminated from the culture in the presence of mFasL/ to other death receptors than Fas. We found that cells transfected L5178Y cells but not when cocultured with L5178Y cells (Fig. with human Fas ligand, hFasL/L5178Y (Fig. 5j), or human 5b). To investigate the role of caspase activation in this process, TRAIL, hTRAIL/2PK-3 (Fig. 5k), rapidly became infected when A20 cells stably expressing human Bcl-xL (an inhibitor of the in- incubated with T. gondii-infected human Jurkat cells, in sharp con- trinsic pathway of apoptosis), human FLIPL (an inhibitor of death trast to mock-transfected cells. receptor mediated apoptosis), and CrmA (a poxvirus inhibitor of caspase-1 and caspase-8 activity) as well as mock-transduced con- trol cells were infected with T. gondii (29). The cells were then Primary T cells inducing death receptor- or perforin-dependent coincubated with mFasL/L5178Y cells and the number of infected cytotoxicity trigger T. gondii egress via different mechanisms effector cells was determined as a function of time. A20 cells ex- Next, we tested whether activated primary T cells inducing tar- pressing FLIPL or CrmA did not transfer the infection to the ef- fector cells as a result of Fas ligation, whereas A20 cells express- get cell death via the death receptor pathway or the perforin ing Bcl-x and mock-transduced A20 cells did (Fig. 5d). pathway also would lead to parasite egress and subsequent in- L ϩ Accordingly, a dramatic decrease of live A20-mock and A20- fection of the effector cells. Therefore, CD4 T cell blasts were prepared from wild-type (wt) and Fas ligand-deficient gld mice Bcl-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 and incubated with T. gondii-infected A20 cells in a redirected frequencies of infected mFasL/L5178Y cells differed between the cytotoxicity assay in the presence of neutralizing Abs against ϩ experiments shown in Fig. 5, a and d. This was because of differ- TRAIL. Wt CD4 T cells were rapidly infected in the presence ences in both the frequency of infected A20 cells and number of of anti-CD3, a process that could be blocked by z-VAD-FMK ϩ parasites per cell, which is illustrated in Fig. 5, c and f. The per- (Fig. 6, a and b). In contrast, gld CD4 T cells did not become missiveness of death ligand transfectants and mock-transfected infected after coculture with T. gondii-infected A20 cells (Fig. controls for T. gondii-infection was similar (Fig. 5, g–i). These 6, a and c). CD8ϩ T cell blasts also rapidly became infected results clearly implicate the activation of death receptor-associated after coculture with T. gondii-infected A20 cells in the presence The Journal of Immunology 8363

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 pro- files of T. gondii-infected CD8ϩ 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 CD8ϩ, CD8ϩCD44high (hi), and CD8ϩCD44low (lo) cells were gated and further analyzed for GFP expression as indicated. b, Per- centage of T. gondii-infected CD8ϩ, CD8ϩCD44high, and CD8ϩCD44low cells in primed and naive animals. c, Downloaded from Percentage of CD8ϩ, CD8ϩ CD44high, and CD8ϩCD44low cells recovered from primed and naive an- imals. d, Pearson’s test of covariance showed a significant correlation be- tween the proportion of T. gondii-in- http://www.jimmunol.org/ fected CD8ϩ T cells and the stimu- lation index (CD44high/CD44low) (p Ͻ 0.001, r ϭ 0.93; open circles, naive mice; filled circles, primed mice). by guest on September 29, 2021 of anti-CD3, but this could only partly be blocked by preincu- took advantage of the H-2b congenic mouse strain A.BY, which bation with z-VAD-FMK (Fig. 6d). When CD8ϩ T cell blasts mounts CTL responses to C57BL/6 mouse minor Ags presented on from gld mice were used they also rapidly got infected, but MHC class I (36). T. gondii-infected BMDC from C57BL/6 mice z-VAD-FMK had no detectable inhibitory capacity (Fig. 6e). were injected i.p. into A.BY mice, naive or previously primed with These results would indicate that T cell-mediated parasite irradiated splenocytes from C57BL/6 mice. Thus, an early primary egress could proceed both through a Fas-dependent and a response is compared with a secondary immune response. Peritoneal ϩ ϩ caspase-dependent pathway (used by CD4 and CD8 cells) exudate cells were obtained and the proportion of T. gondii-infected and a perforin-dependent, caspase-independent pathway used ϩ ϩ ϩ CD8 cells was determined by flow cytometry. Thirty-six hours after by CD8 cells. To directly test this, CD8 T cell blasts were injection of T. gondii-infected BMDC, transition from low to high prepared from perforin-deficient and FasL-deficient (gld) mice surface expression of CD44 indicated a vigorous activation of CD8ϩ and cocultured with T. gondii-infected A20-mock and A20- Ϯ ϩ T cells in primed mice compared with naive mice (49.5 7.0% vs FLIP cells in the presence or absence of anti-CD3. CD8 per- ϩ L 22.5 Ϯ 9.1%, p Ͻ 0.002). The percentage of CD8 CD44high T cells forinϪ/Ϫ cells rapidly became infected when cocultured with was increased 4.3 times in primed vs naive mice (Fig. 7, a–c). A parasite infected A20-mock cells (Fig. 6f) but not upon cocul- considerable fraction of CD8ϩ T cells in primed mice (12.0 Ϯ 3.0%) ture with infected A20-FLIPL cells (Fig. 6g), thus indicating ϩ became infected by the parasite as compared with in naive mice that in the absence of perforin expression CD8 cells only can (4.2 Ϯ 2.2%; Fig. 7, a–c). T. gondii-infected CD8ϩ cells displayed induce T. gondii egress via the death receptor pathway. As ex- high pected, FasL-deficient (but perforin-expressing) CD8ϩ cells almost exclusively an activated CD44 phenotype both in primed Ϯ high Ϯ were able to induce parasite egress and become infected, upon (22.8 4.5% infected cells among CD44 vs 1.8 0.4% in low Ϯ interaction with both T. gondii-infected A20-mock (Fig. 6h) and CD44 cells) and naive mice (13.2 4.5% infected among high low A20-FLIP cells (Fig. 6i). It could also be noted that the kinet- CD44 vs 2.0 Ϯ 1.2% in CD44 cells). The percentage of para- L ϩ ics of perforin-dependent T. gondii-infection were faster as site-infected, activated CD8 T cells in primed vs naive mice was compared with the Fas-dependent infection (compare Fig. 6, b increased 7.5 times. By comparing the proportions of T. gondii-in- ϩ and f vs e, h, and i). fected CD8 T cells with an index of activation (CD44high/CD44low), a strong positive correlation was found (Fig. 7d). ϩ Primed Ag specific CD8 T cells are preferentially infected by Thus, in this in vivo model, a high proportion of activated Ag- T. gondii upon challenge in vivo specific CTL became infected after interacting with T. gondii-in- Finally, we investigated whether T cells recognizing T. gondii-in- fected target cells. These results were in agreement with our data fected target cells in vivo would themselves become infected. We presented above using various in vitro systems. 8364 CYTOTOXIC CELLS INDUCE EGRESS OF INFECTIUOS T. GONDII

Discussion vivo, cause egress of infective parasites and can result in the pro- Induction of cell death is an important immune mechanism for ductive de novo infection of surrounding cells, including the eliminating cells that represent a threat to the integrity of the or- effector cells, after recognition of parasite-infected cells. It is thus ganism, e.g., cells infected by a pathogen. Consequently, intracel- possible that during Toxoplasma infection death receptor- and per- lular pathogens have evolved mechanisms to counteract elimina- forin-mediated parasite egress may contribute to parasite dissemina- tion secondary to the induction of cell death in host cells, often by tion both in peripheral tissues and systemically. As activated leuko- interfering with apoptosis induction and/or execution (1–3, 37). cytes have access to immunoprivileged sites, this process may also contribute to the establishment of infection in these locations. Cell-assisted death induced by cytotoxic lymphocytes in a spe- ϩ ϩ cific manner can proceed through two basic mechanisms: one Both CD4 and CD8 T cells have been shown to be important through the ligation of death receptors on target cells by death in the control of T. gondii infection (9). Our present results are ligands expressed on effector lymphocytes (CD4ϩ, CD8ϩ T cells, compatible with a scenario where T cell effector functions such as and NK cells) and the other dependent on the action of perforin cytokine production and B cell help, in combination with cytotox- and granzymes released by cytotoxic cells (CD8ϩ and NK cells) icity, would contribute to the control of T. gondii infection. Neither upon specific interactions with target cells. In this study we pro- perforin nor Fas ligands have been demonstrated to be critical vide evidence indicating that lymphocytes acting on T. gondii- during acute T. gondii infection (10–12). The absence of perforin, infected target cells via death ligand- or perforin/granzyme-depen- however, has been associated with increased numbers of brain dent cytotoxicity may not be effective in eradicating this cysts and earlier mortality during the chronic phase following in- fection with the type II strain, ME49 (10). Both IFN-␥ and B cells intracellular parasite. We show that T cells engaging any of these have shown to be necessary for the host to resist T. gondii infection Downloaded from pathways as a consequence of interactions with T. gondii-infected (13, 42, 43). Our results suggest that cellular cytotoxicity (death cells trigger the egress of infectious parasites with the ability to ligand mediated or perforin dependent) could be beneficial or infect surrounding cells, including the effector cells, although harmful for the host depending on the presence or absence of ad- through different mechanisms. Whereas death receptor-induced para- ditional immune mechanisms. In the absence of other immune site egress requires caspase activation in the infected target cell, per- mechanisms, cellular cytotoxicity would be inefficient in control- forin-dependent cytotoxicity induces parasite egress in a caspase-in-

ling Toxoplasma infection and could even promote parasite dis- http://www.jimmunol.org/ dependent manner, either as a consequence of the influx of semination. In the presence of additional effective immune mech- extracellular calcium, the efflux of potassium (38), or the caspase- anisms, however, cellular cytotoxicity could contribute to independent mobilization of intracellular calcium, possibly as a con- limitation of the infection. Considering the results referred to sequence of the action of granzymes or other components of cytotoxic above, it is reasonable to assume that the presence of neutralizing granules. As far as we are aware, this represents a previously unde- Abs would be one important factor determining the efficiency of scribed strategy for intracellular pathogens to cope with death recep- cellular cytotoxicity as a defense mechanism against T. gondii (10, tor-mediated apoptosis and cellular cytotoxicity. 13, 42, 43). At first, our results seemingly would be in contradiction with Increased FasL and TRAIL expression, both on hemopoietic and those of others concerning the ability of T. gondii to inhibit death nonhemopoietic cells, can be induced by stress or inflammation (6, by guest on September 29, 2021 receptor-mediated apoptosis (15, 20–22). Using different cell types 44). Inflammation also leads to increased numbers of activated leu- (primary dendritic cells and T and B cell lymphomas of human or kocytes in immunoprivileged sites including the CNS (45). It is there- murine origin) and two different parasite strains (RH and PTG), we fore conceivable that death receptor-induced or perforin-mediated provide substantial evidence that death receptor ligation in T. gon- egress could be involved during the reactivation of T. gondii in chron- dii-infected cells leads to caspase-dependent release of intracellu- ically infected individuals as a result of inflammatory reactions. Re- lar calcium, which in turn induces violent parasite egress with activation of T. gondii is associated with immunodeficiency and is an subsequent host cell necrotic cell death before the appearance of important problem in AIDS patients where it can be correlated to typical signs of apoptosis. A consequence of this when using var- decreased numbers of CD4ϩ T cells, assumed to be a consequence of ious readout methods for apoptosis (but not for cell death) could be defects in immune surveillance (46). An additional possibility would that intracellular components involved in finalizing the apoptotic be that the general inflammatory state, characteristic of HIV infected phenotype may become diluted out in the growth medium or that individuals and possibly of other immunodeficiencies, would result in intracellular conditions, including salt concentrations, pH, and energy increased expression of death ligands and therefore contribute to re- requirements, may not allow the apoptotic progression of the cell as a activation of T. gondii through death receptor-mediated parasite secondary effect of the preceding necrotic cell death. Alternatively, egress. Recent observations showing a positive correlation between sensitivity to Fas-induced cell death may vary in different cell types. viral load and TRAIL expression are compatible with such a scenario This, in conjunction with the absence of quantitative assessments of (47). the ability of T. gondii to protect against cell death in previous studies, Successful parasitic infection with host and parasite survival im- allow us to reconcile our new findings with previous data (15, 20–22), plicates a balance between parasite immune evasion strategies and although with different conclusions. host immune surveillance. The data presented herein concerning Early during T. gondii-infection, parasites rapidly spread in the the responses of T. gondii to death ligand- and perforin-depen- organism hematogenously and via lymphatics (25, 39). The infec- dent cytotoxicity adds a new dimension to this complex inter- tion results in the recruitment of intraepithelial lymphocytes to the play between parasite and host. Future investigations will de- gut (40) and inflammatory monocytic cells and neutrophils (41, 42) termine whether other intracellular pathogens, protozoa, to the peritoneal cavity, which become infected. Thus, it could be viruses, or bacteria would use similar mechanisms do deal with envisaged that this necrotic cell death induced by death receptor- death receptor-induced and perforin-dependent cell death as we or perforin-dependent parasite egress could contribute to inflam- here demonstrate for T. gondii. matory responses, leading both to tissue damage and the further spread of the parasite due to infection of invading leukocytes. Acknowledgments In this article we provide evidence that T cells interacting with We thank Drs. G. P. Nolan (Stanford) for the retroviral packaging line T. gondii-infected cells in an Ag-specific manner, in vitro and in Phoenix-Ampho, Vishva Dixit, (Tularik) for providing the CrmA gene, and The Journal of Immunology 8365

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