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

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

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

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

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

Endogenous RET is expressed in the embryonic neocortex.

<p>Immunohistochemistry of an E14.5 mouse forebrain coronal slice (<b>A</b>, Scale bars, 250 µm) and cortical plate (CP), intermediate zone (IZ) and ventricular zone (VZ) regions (<b>B</b>, Scale bars, 25 µm) for RET and TuJ1. Western blot (<b>C</b>), reverse transcription PCR (35 cycles) (<b>D</b>) and real-time PCR (<b>E</b>) analysis for RET in cortical tissue. Cerebellar tissue and NIH3T3 cells were used as controls. TATA-box binding protein (TBP) was the house keeping gene. (<b>F</b>) Representative single-cell Ca²⁺ recording of a RET^WT expressing primary cortical neuron loaded with Fura-2/AM and subsequently treated with GDNF (100 ng/ml).</p

GDNF induces cytosolic Ca²⁺ signaling through Tyr1015 of RET.

<p>(<b>A</b>) Schematic representation of GFP-tagged RET constructs. (<b>B</b>) Constructs expressed in HeLa cells. (<b>C–F</b>) Representative single-cell Ca²⁺ recordings of GFP positive HeLa cells loaded with Fura-2/AM and subsequently treated with GDNF (100 ng/ml). (<b>C</b>) Cells expressing the RET^WT construct responded to GDNF with either Ca²⁺ transient (top trace) or Ca²⁺ oscillations (bottom trace). Cells expressing RET¹⁰¹⁵ (<b>D</b>) or RET^1015/1062 (<b>F</b>) failed to trigger a Ca²⁺ response following GDNF exposure. Cells expressing RET¹⁰⁶² (<b>E</b>) responded to GDNF in a similar manner as cells expressing RET^WT.</p

GDNF-induced Ca²⁺ signaling phosphorylates ERK1/2 and CaMKII.

<p>(<b>A</b>–<b>D</b>) Western blot of HeLa cells transfected with RET^WT or RET¹⁰¹⁵ treated with GDNF (100 ng/ml). GDNF triggers time dependent phosphorylation of ERK1/2 (pERK1/2) in RET^WT cells that is suppressed by BAPTA (10 µM) (<b>A</b>). Less pERK1/2 is observed in cells transfected with RET¹⁰¹⁵ than RET^WT (<b>B</b>). GDNF-induced phosphorylation of CaMKII (pCaMKII) or pERK1/2 is suppressed when blocking PLC with U73122 (5 µM) (<b>C</b>) or knocking down PLCγ with siRNA (PLCγ-siRNA) (<b>D</b>). Treating RET^WT cells with the U73122 analogue U73343 (5 µM) had no effect on GDNF-activated pCaMKII or pERK1/2 (<b>C</b>). Increased Caspase-3 cleavage was not detected in cells treated with the inhibitors BAPTA or U73122 (<b>C</b>).</p

RET Tyr1015 mediates GDNF-stimulated migration <i>in vivo</i>.

<p>(<b>A</b>) Cartoon illustrating mouse embryo electroporation and GDNF-bead stimulated migration. (<b>B</b>–<b>D</b>) Migration of cortical progenitors in organotypic brain slices from embryos electroporated with RET^WT (<b>C</b>) or RET¹⁰¹⁵ (<b>D</b>) treated without beads (Control) or with beads (indicated with circles) soaked in PBS (Vehicle) or GDNF (500 ng/ml) placed in the cortical plate (CP). GFP positive RET^WT expressing progenitors (green) stimulated with GDNF beads (<b>B</b>, <b>C</b>) show significantly enhanced migration from the ventricular zone (VZ) towards the CP, as compared to Control, Vehicle, or inhibition of PLC with U73122 (5 µM). In RET¹⁰¹⁵ expressing progenitors GDNF beads failed to stimulate migration (<b>B</b>, <b>D</b>). Scale bars, 100 µm.</p