(A) Representative micrographs of mBMDCs infected by T. gondii tachyzoites (PRU-RFP, arrowheads) and by-stander non-infected BMDCs, stained for F-actin (Alexa Fluor 488 Phalloidin) and nuclei (DAPI). Scale bars, 20 μm, inset image 10 μm. (B) Representative motility plots of unchallenged mBMDCs (green) and T. gondii- or N. caninum-infected mBMDCs (red and orange, respectively). X- and y-axes show distances in μm. (C) Box-and-whisker dot plots show, for each condition in (B), median velocities (μm/min) of cells shown in motility plots (n = 3–6 independent experiments). (D) Motility plots of hMonocytes, hMoDCs and hMDCs, respectively, unchallenged (blue) or infected with T. gondii (PRU-RFP, red). (E) Quantifications of velocities (μm/min) in (D) are shown as box-and-whisker dot plots (n = 3–6 independent experiments). (F) Normalized mean velocities of unchallenged and T. gondii-infected hMDCs from human donors (n = 5 independent donors, 50–60 cells per donor). (G) Relative mRNA expression (2-ΔCt) of GABA synthesis enzymes (GAD65 and GAD67) and catabolic enzyme (GABA-T) in unchallenged mBMDCs, hMoDCs, hMDCs and hMonocytes, respectively, determined by real-time qPCR. (H) Corresponding heat maps for the indicated cell types show (%) transcriptional changes upon challenge with T. gondii relative to unchallenged cells at indicated time-points, as detailed in Materials and methods. (X) indicates no amplification (n = 3–6 independent experiments). (I) GABA (ng/ml) secreted in supernatants of hMoDCs challenged with T. gondii (ME49/PTG) at indicated time-points (n = 3 human donors). (J) GABA (ng/ml) secreted in supernatants of mBMDCs, hMonocytes and hMoDCs, respectively, challenged with different strains of T. gondii (PRU and RH) or N. caninum (Nc-1 and Nc-Liverpool) was quantified by ELISA (n = 6–8 independent experiments). Bar graphs show mean + SEM. Statistical significance was tested by Mann-Whitney test for (C, E, F), ordinary one-way ANOVA with Dunnett’s multiple comparison test for (J) and paired t-test for (I), *p<0.05, **p<0.01, ***p<0.001, ns p≥0.05.