Migratory Activation of Primary Cortical Microglia upon Infection with Toxoplasma gondii

Abstract

Disseminated toxoplasmosis in the central nervous system (CNS) is often accompanied by a lethal outcome. Studies with murine models of infection have focused on the role of systemic immunity in control of toxoplasmic encephalitis, while knowledge remains limited on the contributions of resident cells with immune functions in the CNS. In this study, the role of glial cells was addressed in the setting of recrudescent Toxoplasma infection in mice. Activated astrocytes and microglia were observed in the close vicinity of foci with replicating parasites in situ in the brain parenchyma. Toxoplasma gondii tachyzoites were allowed to infect primary microglia and astrocytes in vitro. Microglia were permissive to parasite replication, and infected microglia readily transmigrated across transwell membranes and cell monolayers. Thus, infected microglia, but not astrocytes, exhibited a hypermotility phenotype reminiscent of that recently described for infected dendritic cells. In contrast to gamma interferon-activated microglia, Toxoplasma-infected microglia did not upregulate major histocompatibility complex (MHC) class II molecules and the costimulatory molecule CD86. Yet Toxoplasma-infected microglia and astrocytes exhibited increased sensitivity to T cell-mediated killing, leading to rapid parasite transfer to effector T cells in vitro. We hypothesize that glial cells and T cells, besides their role in triggering antiparasite immunity, may also act as “Trojan horses,” paradoxically facilitating dissemination of Toxoplasma within the CNS. To our knowledge, this constitutes the first report of migratory activation of a resident CNS cell by an intracellular parasite.

INTRODUCTION

Toxoplasma gondii is an obligate intracellular parasite that infects warm-blooded vertebrates. Up to one-third of the global human population is chronically infected (17). Initial infection occurs orally, and the tachyzoite form of the parasite disseminates widely in the organism. Differentiation of tachyzoites into cyst-stage parasites (bradyzoites), e.g., in the central nervous system (CNS), results in chronic lifelong asymptomatic infection. In individuals with acquired immune deficiencies, e.g., AIDS, or in individuals with prolonged immunosuppressive treatments, e.g., recipients of organ transplants, reactivation of the infection can lead to lethal toxoplasmic encephalitis (25). Conversion from bradyzoite cyst-stage parasites to fast-replicating tachyzoites is considered a prerequisite for reactivation and disseminated infection (30).

Toxoplasma tachyzoites actively invade and replicate within virtually any nucleated cell in the host (16) and efficiently establish infection in restricted organs, e.g., the CNS (1). Recent reports suggest that tachyzoites subvert the migratory properties of leukocytes, e.g., dendritic cells (DC), in peripheral tissues (21) and use shuttle leukocytes (“Trojan horses”) for systemic dissemination and to rapidly reach the CNS (5, 21, 22). Fast-replicating tachyzoites can infect and form slowly replicating bradyzoites in murine microglia, astrocytes, and neurons in vitro (23, 31).

Gamma interferon (IFN-γ) plays a crucial role in resistance to acute and chronic infection, and effector cells of hematopoietic and nonhematopoietic origin are required for host resistance (37). Microglia are considered resident brain tissue macrophage precursors which rapidly respond to tissue injury and inflammation (19, 27). They are likely an important source of IFN-γ and interact with CD4⁺ and CD8⁺ lymphocytes, thus contributing to acquired immunity in the CNS (33). Astrocytes also play important roles in resistance to T. gondii during chronic infection (reviewed in reference 36). Thus, multiple functions have been attributed to glial cells during Toxoplasma infection in murine models, including cytokine production and phagocytosis (3, 32, 33, 36). Microglial nodules in the CNS have also been described during Toxoplasma infection of humans (26). Yet the mechanisms for parasite dissemination locally in the CNS remain unknown, and little is known about the role of brain resident cells in the immunopathogenesis of Toxoplasma infection. The presence of glial cells in foci of Toxoplasma reactivation in mice led us to investigate putative roles for these cells in the local dissemination of parasites in the brain parenchyma. We provide evidence here that Toxoplasma-infected microglia exhibit minimal upregulation of immune signaling molecules and a dramatic migratory phenotype in vitro.

MATERIALS AND METHODS

Parasites and cell lines.

Tachyzoites from the T. gondii line CTGluc (type III) (8) and the green fluorescent protein (GFP)-expressing line PTG-GFPluc (type II) (13) were maintained by serial 2-day passaging in human foreskin fibroblast (HFF) monolayers. HFFs were propagated in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS), gentamicin (20 μg/ml; Gibco), glutamine (2 mM; Gibco), and HEPES (0.01 M; Gibco), referred to as complete medium (CM).

Experimental animals and immunosuppression.

Six- to 8-week-old male BALB/c mice, IFN-γ receptor-negative (IFN-γR^−/−) mice on a C57BL/6 background (15), and OT-I Rag1^−/− mice on a C57BL/6 background (14) and 1- to 3-day-old C57BL/6 pups were obtained from the Comparative Medicine Facility, Karolinska Institutet. Animals were kept under pathogen-free conditions. The Regional Animal Research Ethical Board approved all protocols involving animals. A reactivation model with immunosuppressed adult mice infected with the type III T. gondii strain CTGluc was described previously (8). Briefly, male BALB/c wild-type (wt) mice were infected intraperitoneally (i.p.) with 5 × 10⁴ freshly egressed tachyzoites (CTGluc). Mice were treated with dexamethasone 21-phosphate disodium salt (DXM; Sigma-Aldrich) in drinking water ad libitum. IFN-γR^−/− mice were infected i.p. with 0.5 × 10³ CTGluc parasites and treated with sulfadiazine sodium salt (Sigma-Aldrich) ad libitum as previously described (8).

In vitro-generated primary glial cells and macrophages.

Astrocytes were generated as follows. One- to 3-day-old C57BL/6 pups were euthanized, and their brains were dissected and cortices removed. Cortices were further washed in ice-cold Ca²⁺- and Mg²⁺-free Hanks' buffered salt solution (HBSS; Gibco), minced, and resuspended in ice-cold HBSS. After being washed, tissues were incubated for 15 min in HBSS containing 0.1% trypsin and resuspended in astrocyte medium containing DMEM F-12 (Gibco), 10% FBS, 1% G5 supplement (Gibco), and gentamicin (20 μg/ml; Gibco). Medium was changed every 2 to 3 days. Microglia were harvested as described previously (10). Briefly, confluent astrocyte monolayers were subcultivated in microglia medium containing DMEM F-12 (Gibco), 10% FBS, glutamine (2 mM; Gibco), and gentamicin (20 μg/ml; Gibco). Microglial cells were harvested from confluent astrocyte monolayers every 3 to 5 days by tapping the side of the culture flasks, removing loosely adherent microglia from astrocyte monolayers. Bone marrow-derived macrophages were generated as previously described (18). Briefly, cells from the bone marrow of C57BL/6 mice were grown on non-tissue culture-treated plates in CM containing 10% supernatant from the macrophage colony-stimulating factor (M-CSF)-secreting cell line L929. On day 7, loosely adherent cells were removed and discarded. Adherent cells were collected using a cell lifter and used for experimental setups.

Tissue preparation and immunofluorescence.

Brains were dissected, frozen in liquid nitrogen on O.C.T. compound (Tissue-Tek; Sakura), and prepared as previously described (8). Astrocytes were detected using a primary rat anti-glial fibrillary acidic protein antibody (anti-GFAP) (1:500; Zymed Laboratories) and secondary Alexa Fluor 594-conjugated chicken anti-rat IgG (1:400; Molecular Probes). Sections were costained with primary rabbit polyclonal anti-T. gondii tachyzoite antibodies (R14; diluted 1:500) (a gift from E. Linder, Statens Bakteriologiska Laboratorium, Solna, Sweden) and secondary Alexa Fluor 488-conjugated chicken anti-rabbit IgG (1:500; Molecular Probes) or tetramethyl rhodamine isocyanate (TRITC)–anti-rabbit IgG (1:500; Dako). Cryo-sections stained for microglia were fixed in 4% formalin for 40 min at room temperature (RT), washed in phosphate-buffered saline (PBS), and incubated for 1 h in 1% bovine serum albumin (BSA) and 0.3% Triton X-100. Sections were stained overnight at 4°C with primary rabbit anti-mouse calcium-binding adaptor molecule 1 (Iba1) (1:500; Wako Pure Chemical Industries) and secondary Alexa Fluor 594-conjugated chicken anti-rabbit IgG (1:300; Molecular Probes). Sections were costained with primary human polyclonal anti-T. gondii antibodies (WHO standard) (1:500; Statens Serum Institute, Copenhagen, Denmark) and secondary Alexa Fluor 488-conjugated goat anti-human IgG (1:300; Molecular Probes). Sections were mounted in fluorescence medium including DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories). CD8⁺ T cells were detected using anti-CD8a (Ly2; clone 53-6.7) (BD Bioscience Pharmingen). Alexa Fluor 594-conjugated chicken anti-rat IgG (Molecular Probes, Eugene, OR) was used as the secondary antibody at a dilution of 1:400. Sections were costained with 1:500-diluted rabbit polyclonal anti-T. gondii antibodies, using Alexa Fluor 488-conjugated chicken anti-rabbit IgG (Molecular Probes) as the secondary antibody.

For semiquantitative analysis of GFAP and Iba1 immunofluorescence, a four-point ranking scale was applied as described previously (9). Briefly, the ranking was based on a combination of cellular morphology and the overall density in the viewing field. The specific criteria for each rank were as follows: 0, normal morphology, <10% reactive cells; 1, mostly resting cells, about 30% reactive cells; 2, about 60% reactive, some resting cells present; and 3, 100% reactive cells, densely packed. Brain tissue sections from three infected and three uninfected BALB/c mice (8) were assessed. For each mouse, 15 fields of view (magnification, ×400) were chosen randomly in the frontal and parietal cortices to avoid localized bias.

Parasite replication assays.

Macrophages and microglia were incubated with freshly egressed T. gondii tachyzoites (PTG-GFPluc) at a multiplicity of infection (MOI) of 2 for 8, 16, and 24 h at 37°C and 5% CO₂. Samples were fixed in 4% paraformaldehyde for 15 min, washed in PBS, and then blocked in 3% BSA and 0.3% Triton X-100 for 30 min. Cells were stained with primary human polyclonal anti-T. gondii and secondary Alexa Fluor 488–anti-human IgG and mounted as indicated previously. Vacuole counts were determined by direct examination of 100 vacuoles in randomly selected fields of view by epifluorescence microscopy (Leica DMRB).

Transmigration assays.

Astrocytes, microglia, or macrophages were incubated with freshly egressed T. gondii tachyzoites (PTG-GFPluc) at the indicated MOI or with soluble extracts and reagents in CM for 6 h at 37°C and 5% CO₂. Cells were gently transferred to transwell filters (8-μm pore size; BD Bioscience) at a density of 0.5 × 10⁵ to 6 × 10⁵ cells/insert and then incubated for 18 h at 37°C and 5% CO₂. Reagents were added at the following final concentrations: cytochalasin D (CytD; Sigma-Aldrich), 0.5 μg/ml; lipopolysaccharide (LPS; Sigma-Aldrich), 100 ng/ml; pertussis toxin (PTX; Sigma-Aldrich), 2 μg/ml; cholera toxin (CT; List Biological Laboratories), 2 μg/ml; and IFN-γ (Preprotech), 1,000 U/μl (21). Excretory secretory antigen (ESA; 10 μg/ml) was prepared as previously described (21). T. gondii culture supernatant was collected from T. gondii-infected HFF monolayers after centrifugation of HFFs (10 μg/ml). Freshly egressed tachyzoites (PTG-GFPluc) were heat inactivated at 56°C for 30 min. For migration over astrocyte monolayers, astrocytes were plated onto transwell filters and cultured for at least 7 days (transcellular electrical resistance [TCER] of >192 Ω cm⁻², determined using an ohmmeter [Millipore]). Migrated cells were counted at a magnification of ×100 to ×200 by inverted fluorescence microscopy (Nikon Narishige microscope) or analyzed by flow cytometry (FACSCalibur; BD) as previously described (21). The frequency of transmigration was defined as the ratio of the number of transmigrated cells to the total number of cells added.

Cytotoxicity assays.

Splenocytes from OT-I Rag1^−/− mice were stimulated ex vivo with 1 μM SIINFEKL peptide for 5 days, after which the T cells were collected. Target cells were incubated for 1 h in the presence of Na₂O_4⁵¹Cr (Amersham, Oxford, United Kingdom) and 1 μM SIINFEKL peptide and then washed thoroughly in PBS. After 4 h of effector-target cell coincubation, cell culture supernatants were taken from these wells and analyzed on a gamma radiation counter (Wallac Oy, Turku, Finland). Specific lysis was calculated according to the following formula: % specific lysis = (experimental release − spontaneous release)/(maximum release − spontaneous release) × 100.

Tachyzoite (GFP⁺)-infected microglia and astrocytes loaded with SIINFEKL peptide or no peptide were mixed with stimulated CD8⁺ T cells from OT-I RAG1^−/− mice at a 10:1 ratio. After 2 h, the cells were stained with anti-CD8β antibody, fixed, and examined by flow cytometry.

Flow cytometry.

Staining was performed according to each manufacturer's instructions, using anti-CD8β, anti-CD11b, anti-CD54, anti-CD86, anti-I-A/I-E (major histocompatibility complex [MHC] class II), anti-H-2Kb (MHC class I), IgG1 (BD Bioscience, eBioscience, San Diego, CA), anti-CD200R, IgG2a (Serotec), streptavidin (Biolegend, San Diego, CA), and propidium iodide (PI; Sigma-Aldrich). All flow cytometry data were generated using a FACSCalibur flow cytometer (BD Bioscience).

Time-lapse microscopy.

Primary cortical microglial cells were seeded in a 96-well imaging plate (BD Biosciences) at a density of 20,000 cells/well. Cells in determined wells were incubated with freshly egressed GFP-expressing T. gondii tachyzoites (PTG-GFPluc) at an MOI of 2 for 18 h. Six fields of view in the wells containing uninfected cells and 6 fields of view in the wells containing infected cells were imaged for 1 h with a ×20 objective and a time lapse of 25 s, using an ImageXpress Micro wide-field high-content screening system (Molecular Devices). The bright-field and fluorescence image stacks were then assembled, processed, and formatted into AVI movies (10 fps) using ImageJ (http://rsbweb.nih.gov/ij/).

Statistical analyses.

Statistical analyses were performed using GraphPad Prism (version 4.0; GraphPad Software Inc.) and Minitab, version 15 (Minitab Inc., PA).

RESULTS

Glial cell activation in foci of Toxoplasma recrudescence in mouse cortex.

In order to investigate the nature of resident CNS cells infiltrating foci of parasite reactivation, brain tissue sections from BALB/c mice with recrudescent chronic infection were subjected to immunohistochemical analysis (8). Parasitic foci (Fig. 1A) exhibited an important component of leukocytic infiltration (CD45⁺ CD4⁺ CD8⁺ F4/80⁺) (8; data not shown). Importantly, significant increases in expression of the astrocyte marker GFAP and the microglia marker Iba1 were observed in the brain parenchyma of infected mice (Fig. 1E). Furthermore, parasites were often found in the close vicinity of cells expressing the microglia cell marker Iba1 (Fig. 1B) and of GFAP⁺ astrocytic populations (Fig. 1C). In addition, the localization of disperse single parasites in the parenchyma and in close association with the vasculature was indicative of dynamic spreading in the microenvironment (Fig. 1D). During reactivated infection (8), association of parasites with astrocytes and microglia was observed in both IFN-γR^−/− mice and wt mice (data not shown). None of the above findings were observed in brain tissue from wt mice with nonreactivated latent infection (data not shown). Taken together, these data indicate that reactivation of chronic CNS infection in the murine model involves a general activation of glial cells and that replicating tachyzoites can be observed in the vicinity of activated astrocytes and microglia.

Fig. 1.

Activated glial cells in association with foci of parasite reactivation. (A) Massive cell infiltration (DAPI) in brain parenchyma of a BALB/c mouse in association with a focus with replicating tachyzoites (red). Parasites were stained with polyclonal rabbit anti-T. gondii antibodies and TRITC–anti-rabbit IgG as indicated in Materials and Methods. Bar, 100 μm. (B) Microglia (Iba1⁺; red) in close proximity with tachyzoites (green). Microglia were stained with rabbit anti-mouse Iba1 and Alexa Fluor 594–anti-rabbit IgG. Tachyzoites were stained with human polyclonal anti-T. gondii antibodies and Alexa Fluor 488–anti-human IgG. Bar, 25 μm. (C) Activated astrocytes (GFAP⁺; red) in the vicinity of tachyzoites (green) in brain parenchyma. Astrocytes were stained using rat anti-mouse GFAP and Alexa Fluor 594–anti-rat IgG. Parasites were stained with polyclonal rabbit anti-T. gondii antibodies and Alexa Fluor 488. The rectangle indicates a focus with disperse tachyzoites (green) and activated astrocytes (red). The asterisk indicates a vacuole with intracellular replicating parasites (green) in the absence of GFAP⁺ astrocytes. Bar, 20 μm. (D) Activated astrocytes (GFAP⁺; red) and disseminating parasites (green). Staining was performed as described for panel C. White arrows indicate the lumens of blood vessels. Bar, 20 μm. (E) Mean ranking scores (with standard deviations [SD]) for immunoreactivity in the brain parenchyma for three uninfected animals and three infected animals, determined as indicated in Materials and Methods. *, P < 0.001 (Student's t test).

Primary microglia infected with T. gondii exhibit a migratory phenotype in vitro.

Recent findings indicate that Toxoplasma makes use of leukocytes as “Trojan horses” to disseminate in the organism (21, 22). To assess a putative role for glial cells in the local spreading of parasites, primary astrocytes and microglia derived from mouse brains were infected with tachyzoites (Fig. 2A and B), and migration of infected cells was assessed in a transwell system. In sharp contrast to astrocytes, microglia transmigrated in an infection dose-dependent fashion (Fig. 2C). In line with observations in DC and macrophages (20, 21), live intracellular tachyzoites were necessary for migratory activation of microglia, while other stimuli had little or no effect (Fig. 2D). Addition of cytochalasin D or pertussis toxin after infection by Toxoplasma abolished transmigration of DC, indicating a dependency on host cell actin polymerization and on Gi protein signaling (Fig. 2E). Time-lapse microscopy analysis confirmed that the hypermotility phenotype induced in infected microglia was characterized by random directional motility and by pronounced dynamic changes in the membrane morphology of the infected cells (see Videos S1 and S2 in the supplemental material). To test the potency of the migratory activation, infected microglia and infected bone marrow-derived macrophages were assessed for transmigration across confluent primary astrocyte monolayers. Infected microglia readily transmigrated across the monolayers, similar to macrophages (Fig. 3A). Next, replication of tachyzoites was analyzed in microglia and macrophages. There was no significant difference in replication rates of intracellular parasites between both cell types (Fig. 3B). We concluded that primary microglia are permissive to parasite replication and that live intracellular tachyzoites induce a potent transmigratory phenotype in microglia in vitro.

Fig. 2.

Microglia exhibit a migratory phenotype upon challenge with T. gondii. Primary murine astrocytes (A) and primary murine microglia (B) infected with freshly egressed GFP-expressing T. gondii tachyzoites (PTG-GFPluc) were assessed by inverted fluorescence microscopy. Bars, 15 μm and 10 μm, respectively. (C) Infected microglia exhibit enhanced migration at increasing MOIs. Astrocytes or microglia were preincubated for 6 h with PTG-GFPluc at the indicated MOIs. Transmigration of cells (migrated/added) in transwell filters was assessed as described in Materials and Methods. (D) Transmigration of microglia is induced by live intracellular tachyzoites. Primary murine microglial cells were preincubated for 6 h with PTG-GFPluc tachyzoites at an MOI of 3 (live T.g.), with heat-inactivated PTG-GFPluc tachyzoites (HI T.g.), with LPS (100 ng/ml), with Toxoplasma excretory secretory antigen (ESA; 10 μg/ml), with supernatant from infected fibroblast monolayers (HFF sup; 1:10 dilution), or with IFN-γ (1,000 U/μl). Cell migration (migrated/added) was assessed by optical counting of transmigrated cells across a transwell filter. Values represent means with SD for one representative experiment done in duplicate. (E) Treatment with cytochalasin D or pertussis toxin abolishes transmigration of infected microglia. Primary murine microglia were preincubated for 6 h with PTG-GFPluc tachyzoites at an MOI of 3 (T.g.). Cytochalasin D (CytD; 0.5 μg/ml), cholera toxin (CT; 2 μg/ml), or pertussis toxin (PTX; 2 μg/ml) was added after infection. Values represent means with SD for one representative experiment done in duplicate.

Fig. 3.

Microglia and macrophages transmigrate across astrocyte cell monolayers and are permissive to Toxoplasma infection. (A) Primary murine microglia and macrophages were preincubated for 6 h with freshly egressed T. gondii (T.g.) tachyzoites (PTG-GFPluc; MOI of 3), with LPS (100 ng/ml), or with CM. The cell suspensions were placed in transwells seeded with fully confluent astrocyte monolayers (TCER of >192 Ω cm⁻²) as indicated in Materials and Methods. Cell migration (migrated/added) was assessed by optical counting of transmigrated cells. Values represent means with SD for two independent experiments done in duplicate. *, P ≤ 0.05 (Student's t test). (B) Replication of tachyzoites in microglia and macrophages, assessed by vacuole size counts at the indicated time points as described in Materials and Methods. Nonsignificant differences were observed between groups (P > 0.05; Mann-Whitney U test).

Activation marker profile of tachyzoite-infected microglia differs from that of IFN-γ-stimulated microglia.

To test the activation of microglia upon infection, we examined infected (GFP⁺) cells for defined markers over time. An absence of upregulation of MHC class II molecules and of the costimulatory molecule CD86 was observed compared to the case for IFN-γ-stimulated cells (Fig. 4A and B). In contrast, expression of CD54/ICAM-1 and MHC class I molecules was upregulated compared to IFN-γ-stimulated cells. The expression of integrin CD11b and the microglia activation marker CD200R was high overall, without significant differences. Upon stimulation with IFN-γ, infected microglia maintained a relatively lower expression level of activation markers than the upregulation observed in uninfected IFN-γ-stimulated microglia. This effect was most pronounced for MHC class II molecules and CD86 (Fig. 4C). We concluded that upon tachyzoite infection in vitro, microglia exhibit minimal to moderate activation and no differential expression of MHC class II and CD86 molecules.

Fig. 4.

Characterization of cell surface markers of T. gondii-infected microglia. (A) Murine primary microglia were preincubated with freshly egressed T. gondii (T.g.) tachyzoites (PTG-GFPluc), with IFN-γ (1,000 U/μl), or with CM for 6 or 18 h before being stained and analyzed by flow cytometry. For Toxoplasma-infected cells, a gate was set for GFP⁺ cells (infection rate, >80%). Histograms show data for an isotype control (thin gray line) and a stained sample (shaded curve). The data displayed are representative of two independent experiments. (B) Mean fluorescence intensity (MFI) analysis of cell surface markers CD54, CD86, MHC class I, and MHC class II after 6 h and 18 h (x axis). For each marker and time point, the y axis indicates the mean intensity variation (%) for Toxoplasma-infected microglia (T.g.) or IFN-γ-stimulated microglia (IFNg) compared to microglia in CM. (C) Reduction of expression of cell surface markers in microglia incubated with freshly egressed tachyzoites (PTG-GFPluc; MOI of 2) and IFN-γ (1,000 U/μl) for 18 h relative to the expression in microglial cells exposed to IFN-γ. Mean percentages and standard errors of the means (SEM) for two independent experiments are shown.

Infected glial cells are targeted for cytotoxic attack by T cells.

Previously, we showed that DC infected with T. gondii exhibited increased sensitivity to T cells in vitro and in vivo (29). This sensitivity was due to perforin secretion and led to parasite egress from the DC and to subsequent infection of effector T cells. Interestingly, infiltration of CD8⁺ T cells was observed consistently in foci of reactivation in the brain parenchyma of infected mice (Fig. 5A and B). When CD8⁺ T cells from OT-I mice were added to infected microglia or astrocytes loaded with SIINFEKL peptide, an increase in the cytotoxicity of the infected cells was observed compared to that for uninfected cells (Fig. 5C and D). In line with our previous observation that targeting of infected cells by T cells or NK cells (28, 29) leads to parasite egress, effector T cells were readily infected by the egressing parasites after cytotoxic attack of the infected microglia or astrocytes (Fig. 5E). We concluded that infected microglia and astrocytes are more sensitive to T cell-mediated killing, which leads to parasite transfer between infected cells and effector T cells in vitro.

Fig. 5.

Toxoplasma-infected microglia and astrocytes exhibit increased sensitivity to T cell-mediated killing. (A) Immunofluorescence labeling of foci of recrudescence in mouse brain parenchyma showing the presence of infiltrating CD8⁺ T cells (red) and T. gondii (green). Staining was performed as indicated in Materials and Methods. (B) Uninfected brain stained as described for panel A. Bars, 23 μm. CD8⁺ T cell-mediated lysis was determined for SIINFEKL peptide-loaded microglia (C) and astrocytes (D) infected with Toxoplasma (+ Toxo) in comparison with that for uninfected cells. Data for one representative experiment of two are shown. (E) Parasite infection of CD8⁺ T cells after egress from infected microglia and astrocytes. Data for one representative experiment of two are shown.

DISCUSSION

Migration and focal activation of resident glial cells constitute an important response to CNS injury and infection (19, 27). Dissemination of parasites within biologically restricted organs, especially the CNS, is an important determinant of severe toxoplasmosis. Here we describe a novel migratory phenotype of primary microglia that is induced by intracellular parasites.

Following active invasion of microglia, intracellular tachyzoites rapidly induced migration of infected cells. In sharp contrast, primary astrocytes purified and cocultured with microglia prior to separation and infection were not responsive to induction of hypermotility, indicating that activation is cell type specific. Also indicative of the potency of this migratory induction, T. gondii-infected microglia readily migrated across confluent astrocytes, which constitute the brain parenchyma and part of the blood-brain barrier. Time-lapse microscopy demonstrated a random directional hypermotility in infected microglia, in the absence of chemotactic stimuli and accompanied by dynamic changes in membrane morphology. This is reminiscent of the recently described hypermotility of Toxoplasma-infected DC (21). The onset of hypermotility required active parasite invasion and parasite- or host cell-derived preparations; LPS or IFN-γ did not induce this type of migratory activation. This indicates that the activation is likely governed from the parasite's intracellular compartment. As recently described for DC (21), the phenomenon implicates Gi protein signaling. However, the precise mechanisms responsible for this intriguing migratory phenotype remain to be elucidated. There is mounting evidence that parasite-derived effector molecules are translocated to the infected host cell cytosol and exert potent effects on host cell functions (2, 35).

In foci of reactivation, a rich leukocytic infiltration with lymphocytic predominance was observed. Also, disperse parasites in the parenchyma were observed in the vicinity of microglia and activated astrocytes. IFN-γ is a central cytokine implicated in the activation of glial cells to control T. gondii infection, including inhibition of intracellular proliferation (4, 11). IFN-γ also induces the expression of MHC class I and II molecules in microglia and astrocytes that is required for antigen presentation. Studies indicate an activation of microglia during chronic Toxoplasma infection (12) and generally elevated MHC II expression levels in microglia during Toxoplasma encephalitis (6, 7). In contrast, a downregulation of MHC class II molecules in Toxoplasma-infected microglia after prolonged exposure to the parasite (44 h) was previously described (24). Here we show that downmodulation of MHC class II expression sets in very early after infection (<6 h), i.e., prior to significant parasite replication. Importantly, the finding that stimulation with IFN-γ did not fully restore the expression of MHC class II and CD86 molecules in infected cells is indicative of a potent downmodulatory effect. The downmodulation of MHC class II molecules could potentially affect the activation of naïve T helper cells as well as IFN-γ production by already activated Toxoplasma-specific CD4 T cells. Lower levels of CD86 could also affect the activation of naïve Toxoplasma-specific T cells. However, already activated CD8 T cells would still be able to recognize infected cells, since the downmodulation of MHC class I expression is less pronounced. By not markedly downmodulating MHC class I levels, CD8 T cells could still target infected cells, allowing egress from infected cells to antigen-specific CD8 T cells, as we have reported previously (28, 29). Further experimentation is needed to determine if Toxoplasma enhances its ability to avoid immuno-eradication by (i) maintaining low expression levels of MHC class II and CD86 molecules that would reduce the chances of abundant T cell activation and (ii) infecting T cells upon their lysis of infected glial cells in vivo.

A consequence of the observed cytotoxicity was a rapid transfer of parasites from infected glial cells to effector T cells in vitro. Given that determinant roles in Toxoplasma encephalitis have been attributed to T cells (34), the possibility of parasite transfer between glia and T cells is interesting in the context of reactivation of disease. It is tempting to speculate that infected hypermotile microglia might propagate the parasite locally and that transfer of parasites between different cell populations, e.g., cytotoxic T cells, could paradoxically contribute to local propagation and to systemic dissemination of tachyzoites. Thus, in a general encephalitic process, immunomodulatory effects may set in locally during the acute reactivation process as tachyzoites emerge in the parenchyma. The present findings are suggestive that infected hypermotile microglia may facilitate parasite transport in the brain parenchyma during reactivated infection. Whether this intriguing phenotype is also induced by the slowly replicating bradyzoite stage awaits further investigation.