Fig 1.
DC hypermotility and transmigration are abrogated at low extracellular Ca2+ concentration.
(A) Representative motility plot analysis of DCs challenged with T. gondii in Ca2+-free medium, 1% FBS ± CaCl2 (1.8 mM). DCs were pre-incubated with freshly egressed T. gondii PRU-RFP tachyzoites (MOI 3, 4 h) in complete medium (CM) as described in Materials and Methods. Red and black track plots indicate infected DCs (RFP+) and non-infected DCs (RFP-), respectively. (B) Bar graph represents median velocity of DCs from 3 independent experiments (n = 60 cells per experiment) performed as in (A). Asterisks indicate significant differences (*: p < 0.001, Pairwise Wilcoxon rank-sum test, Holm correction). (C and D) Histograms show distributions of accumulated distances migrated by T. gondii-infected DCs in the presence (C) or absence (D) of extracellular Ca2+. Vertical red lines indicate, for each condition, the median distance migrated by cells. Significant differences in distances migrated were observed between the conditions (p < 0.001, Wilcoxon rank-sum test, Holm correction). Data are representative of 3 independent experiments. (E) Transmigration frequency of DCs incubated with freshly egressed T. gondii tachyzoites (MOI 3) for 6 h in CM. Transmigration assay was performed in Ca2+-free medium or in CM as described under Materials and Methods. Data represent means (± SD) of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.01, One-way ANOVA, Tukey’s HSD test). (F) Normalized velocity of DCs incubated with freshly egressed T. gondii PRU tachyzoites (MOI 3) for 3 h in CM and treated with NiCl2 for 1 h. Data represent median velocities (± SD) from 2 independent experiments (n = 60 cells per experiment) normalized against a non-infected control (horizontal line). Asterisks indicate significant differences vs. non-treated control (*: p < 0.05, **: p < 0.001, Steel’s Many-one Rank test, Holm correction).
Fig 2.
GABA elicits Ca2+ influx transients in DCs.
(A) Representative pseudocolor micrographs of live cell Ca2+ imaging of DCs loaded with 2 μM Fluo-8H/AM as described in Materials and Methods. Continuous line indicates perfusion time of GABA (10 mM) and dashed line indicates perfusion time of ATP (50 μM). For each micrograph color scale indicate fluorescence intensity at indicated time point (min:sec). (B) Relative fluorescence intensity of DCs as in (A). The lines indicate perfusions of GABA (10 mM) after 3 min and ATP (50 μM) after 6.5 min, respectively. Displayed data are 3 representative traces from one experiment. (C) Relative fluorescence intensity of DCs as in (A) and (B). Displayed data are 5 superposed representative traces from the same experiment. Experiments were performed 3 times with similar results (n = 15–20 cells per experiment).
Fig 3.
Exogenous GABA and Ca2+ receptor agonism reconstitutes DC hypermotility.
(A) Mass spectrometry analysis of DCs challenged with tachyzoites (PRU, MOI 3) for 16 h ± GABAergic inhibition (SC, 50 μM; SNAP, 50 μM). Cell supernatant was analyzed as indicated under Materials and Methods. Mod. R indicates modified Krebs-Ringer’s solution. Data are representative of 3 independent experiments. (B) Representative motility plot analyses of DCs incubated with tachyzoites (PTG, MOI 3) for 2 h with SC (50 μM), SNAP (50 μM), ± GABA (5 μM) or Bay K8644 (10 μM). (C) Compiled motility analysis of DCs under same conditions as in (B). Motility assays were performed as described under Materials and Methods. Data represent median velocities ± SD of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.01, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction).
Fig 4.
VDCC blockade abolishes GABA-mediated hypermigration of infected DCs that is not rescued by exogenous GABA or membrane depolarization.
(A) Motility analysis of DCs incubated with PRU tachyzoites for 3 h and treated for 1 h with nifedipine (10 μM) or PPADS (100 μM). Data represent median velocities ± SD of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.001, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction). (B) Transmigration frequency of DCs challenged with PRU tachyzoites for 5 h followed by treatment (1 h) with nifedipine (30 μM) or PPADS (100 μM). Data represent means ± SD of 3 independent experiments performed in duplicate. Asterisks indicate significant differences (*: p < 0.01, ns: p ≥ 0.05, One-way ANOVA, Tukey’s HSD test). (C) Representative motility plot analyses of DCs incubated with tachyzoites (PRU, MOI 3) for 2h with SNAP (50 μM), ± GABA (5 μM) or nifedipine (Nif, 10μM) ± GABA (5 μM). (D) Motility analysis of DCs incubated with PRU tachyzoites and treated for 2 h with SC (50 μM), SNAP (50 μM) or nifedipine (10 μM), ± GABA (5 μM). Data represent median velocities ± SD of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.001, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction). (E) Motility analysis of DCs incubated with PRU tachyzoites and treated as in (D) ± KCl (25 mM). Data represent median velocities ± SD of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.001, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction).
Fig 5.
Expression of VDCCs in murine DCs and link to hypermigration of Toxoplasma-infected DCs.
(A) RT-PCR using primers against CaV α1 subunits CaV1.1, 1.2, 1.3, 1.4, as detailed in Materials and Methods. Gel shows template negative (C), whole brain lysate (brain) and DC cDNA. Data are representative of 3 independent experiments. (B) qPCR using primers against α1 subunits of CaV1.1, 1.2, 1.3, 1.4, 2.1, 2.2, 2.3, 3.1 and 3.2 as detailed in Materials and Methods. Each graph depicts expression levels in DCs derived from one individual mouse (n = 6). ΔCt values were calculated with TBP as reference gene. (C) Compiled qPCR analysis as in (B). ΔCt values are given as means ± SEM of 6 independent experiments performed in duplicate. (D) Compiled qPCR analysis as in (C) for T. gondii-challenged DCs. ΔCt values are given as means ± SEM of 4 independent experiments performed in duplicate. (E) Western blot of lysates from primary astrocytes, unchallenged DCs (DCs), DCs challenged with T. gondii tachyzoites (DCs+ Toxo) and tachyzoite lysate (Toxo lysate) immunoblotted with CaV1.3 mAb as detailed in Materials and Methods. GAPDH was used as loading reference. (F) Immunocytochemistry of DCs incubated with GFP-expressing T. gondii tachyzoites (green), stained with CaV1.3 monoclonal antibody (red) and DAPI (blue) as described in Materials and Methods. Scale bars: 10 μm. (G) Motility analysis of DCs challenged with T. gondii tachyzoites (PTG, 3 h, MOI 3) followed by treatment (1 h) with selective CaV1.3 inhibitor CPCPT (1 μM) or benidipine (10 μM), respectively. Data represent median velocities ± SD of 3 independent experiments. Asterisks indicate significant differences (*: p < 0.001, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction). (H) Transmigration frequency of DCs challenged with T. gondii tachyzoites followed by treatment (1 h) with CPCPT (10 μM) or benidipine (40 μM), respectively. Data represent means ± SD of 3 independent experiments performed in duplicate. Asterisks indicate significant differences (*: p < 0.02, One-way ANOVA, Tukey’s HSD test).
Fig 6.
CaV1.3 gene silencing abolishes DC hypermotility.
(A) Live cell imaging of DCs transduced with EGFP-expressing lentiviral vectors (green) carrying shRNA targeting CaV1.3 (shCav1.3), CaV1.2 (shCav1.2) or a non-related target (Control shRNA, shLuc) as indicated under Materials and Methods. DCs were challenged with RFP-expressing tachyzoites (PRU, MOI 3, 4 h, red). Arrowheads indicate representative infected cells expressing EGFP-reporter (red + green) assessed in the assay. Scale bar: 50 μm. (B) Relative Cav1.3 expression in DCs transduced with shCav1.3, shCav1.2 and control shLuc related to mock-transduced DCs (polybrene-treated). Data represents mean ± SEM from 4 independent experiments. (*: p < 0.05, ns: p ≥ 0.05, Student´s t-test). (C) Expression of Cav1.3 protein after treatment with polybrene and transduction with shLuc, shCav1.2 or shCav1.3, analyzed by Western blotting as indicated under Materials and Methods. Data is representative of 3 independent experiments. (D) Motility plots of DCs transduced with lentiviral vectors carrying shRNA targeting CaV1.2, CaV1.3 or control shLuc and challenged with T. gondii tachyzoites (PRU, MOI 3) as indicated under Materials and Methods. “Polybrene” indicates mock-transduced DCs. Data is representative of 4 independent experiments. (E) Motility analysis of DCs transduced as in (D). Data represent mean velocities ± SD of 4 independent experiments. Asterisks indicate significant differences (*: p < 0.001, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction). (F) Motility analysis of DCs transduced with recombinant lentiviral vectors carrying shRNA targeting Cav1.3. DCs were challenged with T. gondii tachyzoites ± SC (50 μM, GABA-deprived) ± GABA (5 μM). CM indicates complete medium. Data represent median velocities ± SD of 2 independent experiments. Asterisks indicate significant differences (*: p < 0.01, ns: p ≥ 0.05, Pairwise Wilcoxon rank-sum test, Holm correction).
Fig 7.
Parasite loads in non-perfused and perfused mice upon VDCC inhibition in adoptively transferred Toxoplasma-infected DCs.
(A) C57BL/6 mice were challenged with 5x104 cfu of freshly egressed PTGluc tachyzoites (Free Toxo), 5x104 cfu of tachyzoite-challenged DCs (Toxo-DC Benidipine (-)) or 5x104 cfu tachyzoite-challenged DCs treated with benidipine (Toxo-DC Benidipine (+)). Photonic emissions were assessed by BLI on days 1–4 post-inoculation intraperitoneally. Color scales indicate photon emission (photons/s/cm2/sr) during 180 s exposures. Data are representative of 2 mice from each group (n = 4/group). (B) Ex vivo photonic emissions from spleen, MLN and brains of mice as in (A) on day 5 post-inoculation. Representative data from 2 mice from each group are shown. Scale as in (A). (C, D and E) Parasite loads in spleen, MLN and brain, respectively, on days 1–5 post inoculation quantified by plaquing assays as indicated under Materials and Methods. C57BL/6 mice were inoculated intraperitoneally with 105 cfu of tachyzoite (PTGluc)-challenged DCs ± benidipine pre-treatment, as indicated. Organ extraction was performed without blood perfusion or posterior to perfusion, as indicated. Open circles indicate individual mice. Box-plot and whiskers graphs represent the lower, upper quartiles and median (*: p < 0.05, ns: p ≥ 0.05, Student´s t-test, n = 4).
Fig 8.
Impact of VDCC inhibition on parasite loads and cell-associated parasite numbers in spleen, blood and peritoneum 24 h post-inoculation in mice.
(A) Parasite loads in spleen, blood and peritoneum, as indicated, 24 h post-inoculation of 5x106 T. gondii (PTGluc)-challenged DCs ± benidipine in C57BL/6 mice. For each tissue, dot plots show parasite numbers quantified by plaquing assays and bar graphs show parasite quantification by qPCR, as indicated under Material and Methods. Data represents mean ± SD from 3 independent experiments. (*: p < 0.05, ns: p ≥ 0.05, Student´s t-test, n = 6). (B) Flow cytometry analysis of spleen, blood and peritoneal fluid 24 h post-inoculation of 5x106 T. gondii (PTGluc, GFP-expressing)-challenged DCs ± benidipine, as indicated under Materials and Methods. Bivariate dot plots show, for each condition, GFP+ cells and CD11c+ cells, following gating on live CD3- CD19- GR1-NK1.1- CD11b+ cells. Gatings indicate percentage of GFP+ cells related to the total cell population. Bar graphs indicate, for each condition, the percentage of GFP+ cells from 3 independent experiments (*: p < 0.05, ns: p ≥ 0.05, Student´s t-test, n = 5–6).
Fig 9.
Schematic representation of the proposed mechanism for the initiation of the hypermigratory phenotype in Toxoplasma-infected DCs.
(1) Active invasion by T. gondii (Tg) of the host cell across its plasma membrane (PM) involves secretory processes. Inside the host cell, T. gondii resides in the parasitophorous vacuole (PV). (2) Parasite invasion sparks an increase in GABA synthesis by host-cell glutamate decarboxylase (GAD) that can be inhibited by semicarbazide (SC) [8]. Synthesized GABA is secreted through GABA transporters (GAT). Inhibition of GABA synthesis (SC) or GABA secretion (SNAP) abolishes DC hypermotility [8]. (3) GABA activates GABAA receptor (GABAAR) channels on the host cell surface by an autocrine loop, leading to chloride (Cl-) efflux. The GABAAR antagonist bicuculine inhibits hypermotility and exogenous GABA (exoGABA) reconstitutes hypermotility. Hypothetically, the Cl- gradient is maintained by cation-chloride co-transporters (CCC). (4) Efflux of Cl- leads to membrane depolarization (-). Induced membrane depolarization by potassium chloride (KCl) or GABA can also reconstitute hypermotility. (5) Voltage-dependent calcium channels (VDCCs; primarily Cav1.3) open in response to membrane depolarization, leading to Ca2+ influx. Blockade of membrane-bound calcium channels by nickel (Ni), inhibition of VDCCs by nifedipine or benidipine, specific inhibition of Cav1.3 (CPCPT) or ablation of CaV1.3 expression by shRNA (CaV1.3 shRNA) have inhibitory effects on hypermotility, while the VDCC agonist Bay K 8644 (Bay K) induces motility and reconstitutes hypermotility upon GABAergic inhibition. Upon Cav1.3 blockade, GABA cannot induce or reconstitute hypermotility. (6) Hypothetically, the Ca2+ transient generated may be propagated to intracellular Ca2+ channels and stores. The altered Ca2+ signaling pattern may activate the downstream migratory machinery and MAP kinase regulators (e.g. 14-3-3), leading to cytoskeletal rearrangements and shifting the cell into a hypermotile state.