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.

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).

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).

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.

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.