Toxoplasma-Induced Hypermigration of Primary Cortical Microglia Implicates GABAergic Signaling

Toxoplasma gondii is a widespread obligate intracellular parasite that causes chronic infection and life-threatening acute infection in the central nervous system. Previous work identified Toxoplasma-infected microglia and astrocytes during reactivated infections in mice, indicating an implication of glial cells in acute toxoplasmic encephalitis. However, the mechanisms leading to the spread of Toxoplasma in the brain parenchyma remain unknown. Here, we report that, shortly after invasion by T. gondii tachyzoites, parasitized microglia, but not parasitized astrocytes, undergo rapid morphological changes and exhibit dramatically enhanced migration in 2-dimensional and 3-dimensional matrix confinements. Interestingly, primary microglia secreted the neurotransmitter γ-aminobutyric acid (GABA) in the supernatant as a consequence of T. gondii infection but not upon stimulation with LPS or heat-inactivated T. gondii. Further, microglia transcriptionally expressed components of the GABAergic machinery, including GABA-A receptor subunits, regulatory molecules and voltage-dependent calcium channels (VDCCs). Further, their transcriptional expression was modulated by challenge with T. gondii. Transcriptional analysis indicated that GABA was synthesized via both, the conventional pathway (glutamate decarboxylases GAD65 and GAD67) and a more recently characterized alternative pathway (aldehyde dehydrogenases ALDH2 and ALDH1a1). Pharmacological inhibitors targeting GABA synthesis, GABA-A receptors, GABA-A regulators and VDCC signaling inhibited Toxoplasma-induced hypermotility of microglia. Altogether, we show that primary microglia express a GABAergic machinery and that T. gondii induces hypermigration of microglia in a GABA-dependent fashion. We hypothesize that migratory activation of parasitized microglia by Toxoplasma may promote parasite dissemination in the brain parenchyma

Host-parasite interactions in the dissemination of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that infects virtually all warm-blooded organisms. Systemic dissemination of T. gondii in the organism can cause life-threatening infection that manifests as Toxoplasma encephalitis in immune-compromised patients. In addition, mounting evidence from epidemiological studies indicates a link between chronic Toxoplasma infection and mental disorders. To better understand the pathogenesis of toxoplasmosis, basic knowledge on the host-parasite interactions and the dissemination mechanisms are essential. Previous findings have established that, upon infection with T. gondii, dendritic cells (DCs) and microglia exhibit enhanced migration, which was termed the hypermigratory phenotype. As a result of this enhanced migration, DCs and microglia are used as vehicle cells for dissemination (‘Trojan horse’) which potentiates dissemination of T. gondii in mice. However, the precise mechanisms behind the hypermigratory phenotype remained unknown. In this thesis, we characterized host-parasite interactions upon infection with T. gondii and investigated the basic mechanisms behind the hypermigratory phenotype of T. gondii-infected DCs and microglia. In paper I, we observed that upon infection with T. gondii, DCs underwent rapid morphological changes such as loss of adhesiveness and podosomes, with integrin redistribution. These rapid morphological changes were linked to hypermotility and were induced by active invasion of T. gondii within minutes. T. gondii-infected DCs exhibited up-regulation of the C-C chemokine receptor CCR7 and chemotaxis towards the CCR7 chemotactic cue, CCL19. In paper II, we developed a 3-dimensional migration assay in a collagen matrix, which allowed us to characterize the hypermigratory phenotype in a more in vivo-like environment. The migration of T. gondii-infected DCs exhibited features consistent with integrin-independent amoeboid type of migration. T. gondii-induced hypermigration of DCs was further potentiated in the presence of CCL19 in a 3D migration assay. In paper III, we identified a parasite effector molecule, a Tg14-3-3 protein derived from parasite secretory organelles. Tg14-3-3 was sufficient to induce the hypermigratory phenotype. Transfection with Tg14-3-3-containing fractions or recombinant Tg14-3-3 protein induced the hypermigratory phenotype in primary DCs and in a microglial cell line. In addition, Tg14-3-3 localized in the parasitophorous vacuolar space and host 14-3-3 proteins were rapidly recruited around the parasitophorous vacuole. In paper IV, we found that mouse DCs dominantly express the L-type voltage-dependent calcium channel, Cav1.3. Cav1.3 was linked to the GABAergic signaling-induced hypermigratory phenotype. Pharmacological inhibition of Cav1.3 and knockdown of Cav1.3 abolished the hypermigratory phenotype in T. gondii infected DCs. Blockade of voltage-dependent calcium channels reduced the dissemination of T. gondii in a mouse model. In paper V, we showed that microglia, resident immune cells in the brain, also exhibited rapid morphological changes and hypermotility upon infection with T. gondii. However, an alternative GABA synthesis pathway was shown to be involved in the hypermigratory phenotype in microglia. In summary, this thesis describes novel host-parasite interactions, including host cell migratory responses and key molecular mechanisms that mediate the hypermigratory phenotype. The findings define a novel motility-related signaling axis in DCs. Thus, T. gondii employs GABAergic non-canonical pathways to hijack host cell migration and facilitate dissemination. We believe that these findings represent a significant step forward towards a better understanding of the pathogenesis of T. gondii infection.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p

Infection by <i>Toxoplasma gondii</i> Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix

<div><p><i>Toxoplasma gondii</i>, an obligate intracellular parasite of humans and other warm-blooded vertebrates, invades a variety of cell types in the organism, including immune cells. Notably, dendritic cells (DCs) infected by <i>T</i>. <i>gondii</i> acquire a hypermigratory phenotype that potentiates parasite dissemination by a ‘Trojan horse’ type of mechanism in mice. Previous studies have demonstrated that, shortly after parasite invasion, infected DCs exhibit hypermotility in 2-dimensional confinements <i>in vitro</i> and enhanced transmigration in transwell systems. However, interstitial migration <i>in vivo</i> involves interactions with the extracellular matrix in a 3-dimensional (3D) space. We have developed a collagen matrix-based assay in a 96-well plate format that allows quantitative locomotion analyses of infected DCs in a 3D confinement over time. We report that active invasion of DCs by <i>T</i>. <i>gondii</i> tachyzoites induces enhanced migration of infected DCs in the collagen matrix. Parasites of genotype II induced superior DC migratory distances than type I parasites. Moreover, <i>Toxoplasma</i>-induced hypermigration of DCs was further potentiated in the presence of the CCR7 chemotactic cue CCL19. Blocking antibodies to integrins (CD11a, CD11b, CD18, CD29, CD49b) insignificantly affected migration of infected DCs in the 3D matrix, contrasting with their inhibitory effects on adhesion in 2D assays. Morphological analyses of infected DCs in the matrix were consistent with the acquisition of an amoeboid-like migratory phenotype. Altogether, the present data show that the <i>Toxoplasma</i>-induced hypermigratory phenotype in a 3D matrix is consistent with integrin-independent amoeboid DC migration with maintained responsiveness to chemotactic and chemokinetic cues. The data support the hypothesis that induction of amoeboid hypermigration and chemotaxis/chemokinesis in infected DCs potentiates the dissemination of <i>T</i>. <i>gondii</i>.</p></div

Morphological characteristics and migration of <i>Toxoplasma</i>-infected DCs and non-infected DCs in the 3D matrix.

<p>(<b>A</b>) Representative micrographs in maximum intensity projection of unchallenged DCs in complete medium (CM, left), Challenged/By-stander DCs (middle) and Challenged/<i>T</i>. <i>gondii</i> DCs (right; PTG-GFP <b>type II</b>, MOI 3, green) in a 3D collagen matrix, stained with DAPI (blue), and Alexa Fluor 594-Phalloidin (red) to detect F-actin as indicated under Materials and Methods. In the middle micrograph, arrow indicates non-infected DC surrounded by an infected DC (green + red) and two extracellular <i>T</i>. <i>gondii</i> tachyzoites (green). Scale bars = 10 μm. (<b>B</b>) Graph shows, for each condition, the percentage of cells (mean ±SEM) that exhibit rounded phenotype, absence of membrane extensions and veils, respectively, related to the total cell population. The morphological criteria are specified under Materials and Methods. For each condition, a total of 50 cells/donor were analyzed from 3 different donors. (*: P < 0.05, **: P < 0.01, ns: P > 0.5, Paired <i>t</i>-test, Holm´s correction). (<b>C</b>) Compiled mean scores (± SD) based on morphological criteria as in (B). For each condition, a total of 50 cells/donor were analyzed from 3 different donors (*: P < 0.05, **: P < 0.01, Paired <i>t</i>-test, Holm´s correction). (<b>D</b>) Distribution of the total scores (% of total cell population) based on morphological criteria specified under Materials and Methods. For each condition, a total of 50 cells/donor from 3 donors were assessed. Significant differences were observed between tachyzoite-infected DCs and non-infected DCs (P < 0.0001; Fisher´s exact test) or by-stander DCs (P < 0.0001), while differences between non-infected DCs and by-stander DCs were non-significant (P > 0.05). (<b>E</b>) Representative 3D projection analysis of DCs challenged with <i>T</i>. <i>gondii</i> (PTG-GFP type II). The colored spheres indicate the position of cells in the defined 3D space. Infected cells and non-infected cells were defined and analyzed as indicated under Materials and Methods: co-localized signal/infected cell (actin: red; <i>T</i>. <i>gondii</i>: green) or absence of co-localization/by-stander cells (blue). The inset image represents a magnification of the white-dotted square. Data are representative from 4 independent experiments. (<b>F</b>) Mean migrated distances by unchallenged DCs (CM), and challenged non-infected DCs (By-stander) and infected DCs (<i>T</i>. <i>gondii</i>: PTG-GFP type II). Data represent compiled analysis of 500 randomly chosen cells per donor from 4 different donors. Bars indicate mean migrated distances (*: P < 0.05, ns: P > 0.05, Two-way ANOVA, Tukey´s HSD test). (<b>G</b>) Dot plots represent the distribution of migrated distances for individual DCs infected with <i>T</i>. <i>gondii</i> (type I: LDMluc; type II: PRU-RFP). For each condition, 100 single cells were randomly selected and analyzed from one representative donor. Bar indicates mean migrated distance. Asterisks indicate significant differences (**: P < 0.01; Paired <i>t</i>-test, Holm´s correction).</p

Migration in a 3D-matrix by DCs challenged with <i>T</i>. <i>gondii</i>.

<p>(<b>A</b> and <b>B</b>) Histograms represent the distribution of migrated distances for (<b>A</b>) unchallenged DCs in complete medium and (<b>B</b>) DCs pre-challenged with <i>T</i>. <i>gondii</i> (PRU-RFP, type II, MOI 3, 4h) as indicated under Materials and Methods. Arrows indicate, for each condition, the percentage (%) of cells in the matrix migrating < 80 μm or > 80 μm, respectively. 500 randomized cells from one representative donor are shown. The experiments were performed with DCs from 5 donors with similar results. Inset plots represent, for each condition, the 3D reconstruction assembly of z-stacks for the total cell population as indicated under Materials and Methods. (<b>C</b>) Mean migrated distances by unchallenged DCs and DCs challenged with <i>T</i>. <i>gondii</i> (PRU-RFP, type II), from 5 different donors. Data represent compiled analysis of 500 randomly chosen cells per donor. Bars indicate mean migrated distances. (***: P < 0.001; Paired t-test, Holm´s correction). (<b>D</b>) Dot plots represent the distribution of migrated distances of individual DCs challenged with <i>T</i>. <i>gondii</i> (type I: LDMluc; type II: PRU-RFP) related to unchallenged DCs. Bars indicate mean migrated distances. For each condition, 100 randomly chosen cells from one representative donor are shown. Significant differences were observed for challenged DCs (type I and II) versus unchallenged DCs (**: P < 0.01; Kruskal-Wallis test, Dunnett´s test). Performed with 3 donors with similar results. (<b>E)</b> Mean migrated distances of DCs challenged with <i>T</i>. <i>gondii</i> (type I: LDMluc; type II: PRU-RFP) as in (B) from 3 different donors. Data represent compiled analysis of 500 randomly chosen cells per donor. Bars indicate mean migrated distances. (*: P < 0.05; Paired t-test, Holm´s correction). (<b>F</b>) Mean migrated distances of DCs challenged with <i>T</i>. <i>gondii</i> (PRU-RFP, type II) as in (B) at indicated time points ± cytochalasin D (CytD). Data represent compiled analysis of 500 cells randomly chosen cells per donor from 3 different donors. (**: P < 0.01, ns: P > 0.05; Two-way ANOVA, Tukey´s HSD test).</p

Chemotaxis and hypermigration of <i>Toxoplasma</i>-infected DCs in a 3D matrix.

<p>(<b>A</b>) Schematic representation of the assay set-up with CCL19 added in the lower collagen matrix (dark blue). Cells were deposited onto the upper collagen matrix (light blue) as indicated under Materials and Methods. DCs were maintained in CM, pre-challenged with <i>T</i>. <i>gondii</i> (PRU-RFP, type II) or treated with LPS (final concentration 100 ng/ml) and deposited on top of the collagen layer in 96-well plates. After 24 h incubation, the localization of DAPI-labeled DCs in the gel was analyzed in 200 z-sections. (<b>B</b>) Plots indicate the assembly of z-stacks and colored structures indicate the localization of DCs in absence or presence of CCL19. (<b>C</b>) Dot plots represent the distribution of migrated distances for the different conditions. For each condition, 100 randomly chosen cells were analyzed from one representative donor. Performed with 5 donors with similar result. (<b>D</b>) Mean migrated distances of cells under same conditions as in B and C. Data represent compiled analysis of 500 randomly chosen cells per donor from 5 different donors. Bars indicate mean migrated distances. (*: P < 0.05, ns: P > 0.05; Paired <i>t</i>-test, Holm´s collection).</p

Adhesion and migration of <i>Toxoplasma</i>-infected DCs in the presence of integrin-blocking antibodies.

<p>(<b>A</b>) Bar graph shows average number of adhered cells per 100 mm² (± SD) for unchallenged DCs (CM) and <i>Toxoplasma</i>-challenged DCs (<i>T</i>. <i>gondii</i>, PRU-RFP <b>type II</b>, MOI 3, 4h) as indicated under Materials and Methods (*: P < 0.05; Paired <i>t-</i>test, Holm´s correction).(<b>B</b>) Bar graphs show the ratio of adhered cells treated with blocking antibodies compared to cells in CM from 3 donors. Unchallenged DCs (left/blue) and DCs challenged with <i>T</i>. <i>gondii</i> (right/red, PRU-RFP, MOI 3, 4h) were exposed to anti-human CD11a, anti-human CD11b, anti-human CD18, anti-human CD29, anti-human CD49b, as indicated under Materials and Methods, for 30 min and seeded on 1% BSA/serum-coated plates (CD11a, CD11b, CD18) or collagen-coated plates (CD29, CD49b). Mouse IgG1 κ Isotype (Isotype M, 10 μg/ml) or Rat IgG2b κ Isotype (Isotype R, 10μg/ml) were used as control antibodies (*: P < 0.05, **: P < 0.01, ***: P < 0.001,Two-way ANOVA, Dunnett´s test). (<b>C</b>) Mean migrated distances in 3D collagen matrix by cells exposed to blocking antibodies as in (B). Graphs show unchallenged DCs (left/blue) and DCs challenged with <i>T</i>. <i>gondii</i> (right/red, PRU-RFP, MOI 3, 4h). Data represent compiled analysis of 400 cells/donor (± SEM) from 4 donors. (ns: P > 0.05; Two-way ANOVA).</p

Revisiting the Plasmodium sporozoite inoculum and elucidating the efficiency with which malaria parasites progress through the mosquito

Abstract Malaria is initiated when infected anopheline mosquitoes inoculate sporozoites as they probe for blood. It is thought that all infected mosquitoes are equivalent in terms of their infectious potential, with parasite burden having no role in transmission success. In this study, using mosquitoes harboring the entire range of salivary gland sporozoite loads observed in the field, we demonstrate a strong and highly significant correlation between mosquito parasite burden and inoculum size. We then link the inoculum data to oocyst counts, the most commonly-used metric to assess mosquito infection in the field, and determine the efficiency with which oocyst sporozoites enter mosquito salivary glands. Taken together our data support the conclusion that mosquitoes with higher parasite burdens are more likely to initiate infection and contribute to onward transmission. Overall these data may account for some of the unexplained heterogeneity in transmission and enable more precise benchmarks for transmission-blocking interventions

Experimental set-up for DC migration in a 3D collagen matrix.

<p>(<b>A</b>) Schematic representation of the assay set-up with collagen matrix as indicated under Materials and Methods. DCs were maintained in complete medium (CM) and allowed to sediment on the top of the collagen matrix layer in 96-well plates. After 18 h incubation, the localization of DAPI-labeled DCs in the collagen gel was analyzed by confocal microscopy in 200 z-optical sections as indicated. (<b>B</b>) Plots represent, for each condition, the 3D reconstruction assembly of z-stacks as indicated under Materials and Methods. Colored structures indicate the localization of individual DCs (DAPI) at indicated time points ± cytochalasin D (CytD). (<b>C</b>) Dot plots represent the distribution of migrated distances for the different conditions. For each condition, 100 single cells were randomly selected and analyzed from one representative donor. Bar indicates mean migrated distance. Asterisks indicate significant differences (***: P < 0.001; non-significant (ns): P > 0.05 Kruskal-Wallis test, Dunnett´s test). (<b>D</b>) Mean migrated distances by DCs under same conditions as in B and C. Data represent compiled analysis of 500 randomly chosen cells per donor from 5 different donors. Bars indicate mean migrated distances (***: P < 0.001, ns: P > 0.05; Two-way ANOVA, Tukey´s HSD test).</p

Comparative intravital imaging of human and rodent malaria sporozoites reveals the skin is not a species‐specific barrier

Abstract Malaria infection starts with the injection of Plasmodium sporozoites into the host’s skin. Sporozoites are motile and move in the skin to find and enter blood vessels to be carried to the liver. Here, we present the first characterization of P. falciparum sporozoites in vivo, analyzing their motility in mouse skin and human skin xenografts and comparing their motility to two rodent malaria species. These data suggest that in contrast to the liver and blood stages, the skin is not a species‐specific barrier for Plasmodium. Indeed, P. falciparum sporozoites enter blood vessels in mouse skin at similar rates to the rodent malaria parasites. Furthermore, we demonstrate that antibodies targeting sporozoites significantly impact the motility of P. falciparum sporozoites in mouse skin. Though the sporozoite stage is a validated vaccine target, vaccine trials have been hampered by the lack of good animal models for human malaria parasites. Pre‐clinical screening of next‐generation vaccines would be significantly aided by the in vivo platform we describe here, expediting down‐selection of candidates prior to human vaccine trials