Fig. 1: Sequestration of infected PBMCs and DCs in the brain microvasculature.
A Experimental set up. Freshly egressed GFP-expressing T. gondii (Tg) tachyzoites (PRU-GFP, 2 × 105 cfu) were inoculated ip in mice and organs were collected 3 or 6 days post-inoculation (dpi). B Representative flow cytometry plots of GFP vs forward scatter (FSC) show percentage of tachyzoite-associated PBMCs (GFP+) in unchallenged and infected mice 3 and 6 dpi (n = 3 mice). C Representative micrographs of brain cortex at 3 or 6 dpi. Mice were injected iv with α-CD45-Alexa 647 prior to organ extraction. Arrows indicate leukocyte (CD45+, red)-associated tachyzoites (GFP+, green). Dotted lines delimit cortical capillaries. Asterisk indicates tachyzoite (GFP+) non-associated to a leukocyte (CD45-). Lower panel insets show magnification of the leukocyte- associated tachyzoites. Scale bars: 10 µm. D Cartoon shows microsurgical procedure, as detailed in Methods, with injection of cell suspensions (DC/Tg) into the internal carotid artery (ICA), followed by brain extraction and tissue sectioning (50 µm thickness). Common carotid artery (CCA) and external carotid artery (ECA) are indicated. Created in BioRender. Pairoto, M. (2025) https://BioRender.com/e26i498. E Experimental set up. CFSE or CMTMR pre-labeled DCs were challenged in vitro with T. gondii (RH-GFP, MOI 1; ME49-RFP, MOI 2; CFSE-prelabelled CTG, MOI 2) to obtain a DC infection frequency of ∼50%. Infected DCs (~20 × 106 DCs / ~ 10 × 106 cfu Tg) were slowly (5 min) inoculated into the brain circulation via the ICA as detailed in Methods. Brains were extracted 16 h post-inoculation (hpi). F Confocal micrographs, with corresponding 3D surface analyses, show the localization of Tg type I RH (GFP, green)-infected DCs (CMTMR, red), Tg type II ME49 (RFP, red)-infected DCs (CFSE, green) and Tg type III CTG (CFSE, green)-infected DCs (CMTMR, red) in relation to the vascular marker Evans blue (cyan). Arrows indicate infected DCs magnified in the insets, respectively. Scale bars: 10 µm. G 3D surface analysis as in (F) of microvessel (cyan) with infected DC (red) with T. gondii vacuoles (green). Microvessel lumen diameter (5,04 µm) and vascular branching point are indicated. H, I Luminal diameter (H) and distance to nearest vascular branching point (I) of cortical microvessels containing T. gondii type I, II, and III-infected DCs, respectively. In box plots, center line indicates median. Box limits: 25th and 75th percentiles. Whiskers: maximum and minimum values. Data are from 86 (TgI), 61 (TgII), 47 (TgIII) -infected DCs per condition from three independent experiments (TgI n = 4 mice; TgII, TgIII n = 3 mice). J. Bar graph shows mean ( ± SEM) numbers of T. gondii type I, II or III-infected DCs related to cortical area, from 30 (TgI), 28 (TgII) and 27 (TgIII) cortical sections per condition from 3 independent experiments (n = 3 mice per condition). K Bar graph shows the relative attachment frequency of T. gondii type I- or type II-infected DCs to polarized brain endothelial cells (bEnd.3) in vitro. Data are expressed as mean ( ± SEM) from three independent experiments (n = 3 biological replicates). Statistical analyses: (H–J) Kruskal–Wallis test followed Dunn’s post-hoc test (K) 2-tailed unpaired Student’s t-test, numeric p-values are indicated, ns: non-significant, p > 0,05. Source data are provided as a Source Data file.
