Fig. 4: T. gondii-induced BBB inflammation promotes the sequestration of parasitized DCs.

A Experimental set ups. Infected CMTMR pre-labeled DCs (20 × 10⁶ DCs / 10 × 10⁶ cfu Tg, PRU-GFP) were inoculated via the ICA and brains collected 16 hpi. When indicated, mice were pre-treated with LPS ip or treated with heparin iv. B Confocal micrographs and corresponding 3D surfaces show T. gondii (PRU-GFP)-infected DCs (CMTMR⁺ GFP⁺, red/green) in the cortical vasculature (Evans blue, cyan) of LPS pre-treated animals. Scale bars: 10 µm. C Graph shows the absolute numbers (mean ± SEM) of infected DCs per cm² of cortical tissue in control (Tg), heparin and LPS pre-treated conditions. Data are from 20 cortical sections per condition from three independent experiments (n = 3 mice per condition). D, E Mean vascular luminal diameter (D) and distance to nearest vascular branching point (E), respectively, of infected DCs retrieved in the cortical microvasculature in LPS pre-treated mice. In box plot, center line indicates median. Box limits: 25^th and 75^th percentiles. Whiskers: maximum and minimum values. Data are from 70 infected DCs from 20 cortical sections from three independent experiments (n = 3 mice). F Experimental set up. Mice were inoculated ip with 2 × 10⁵ cfu of freshly egressed tachyzoites (ME49-RFP), (pre- ipTg) or control medium. After 48 h, infected CMTMR pre-labeled DCs (10 × 10⁶ DCs / 5 × 10⁶ cfu Tg, PRU-GFP) were inoculated via the ICA and brains collected 1 hpi. G Representative micrographs show T. gondii (PRU-GFP)-infected DCs (CMTMR⁺, GFP⁺) in cortical sections of control and pre-ip infected mice, respectively. Insets (a, b) show magnifications of white boxes and asterisks indicate infected DCs (CMTMR⁺ GFP⁺, red/green). Scale bars: 50 µm. H, I Graphs show the absolute numbers (mean ± SEM) of T. gondii (PRU-GFP)-infected DCs per cm² brain tissue at 1 hpi (H) and the relative expression (qPCR) of TgB1 gene in brain tissue (I), respectively. Data are from 30 cortical sections from three independent experiments (n = 3 mice). J Experimental set up. Freshly egressed RFP-expressing T. gondii (Tg) tachyzoites (ME49-RFP, 2 × 10⁵ cfu) or control medium were inoculated ip in mice, brains were extracted 24, 48 and 72 hpi and micro-vessels purified. K Relative mRNA expression (qPCR) of Icam1 (ICAM-1), Vcam1 (VCAM-1) and Elam1 (E-selectin) in brain micro-vessels at indicated time points. Data are expressed as mean ( ± SEM) from three independent experiments (n = 3 mice). L Representative micrographs show infected DCs (CMTMR⁺ GFP⁺) in the brain cortex of mice inoculated in the ICA with 10 × 10⁶ DCs / 5 × 10⁶ cfu Tg (PRU-GFP) plus α-ICAM-1 antibody or isotype. Mice were pre-infected ip with 2 × 10⁵ cfu type II ME49-RFP tachyzoites. Insets (a, b) show magnifications of the infected DCs (CMTMR⁺, GFP⁺) and asterisks show tachyzoites. Scale bars: 20 µm. M, N Graphs show the absolute numbers (mean ± SEM) of T. gondii (PRU-GFP)-infected DCs per cm² brain tissue at 1 hpi in mice following inoculation with DC/Tg plus α-ICAM-1 (M) or α-CD18 (N), respectively. Mice were pre-infected (pre- ip, ME49-RFP). Data are from 30 cortical sections per condition from three independent experiments (n = 3 mice). O, P Relative expression (qPCR) of T. gondii TgB1 gene in brain tissue of mice pre-ip (ME49-RFP) infected and inoculated in the ICA with infected CMTMR pre-labeled DCs (10 × 10⁶ DCs / 5 × 10⁶ cfu Tg, PRU-GFP) plus α-ICAM-1 (O) or α-CD18 (P), respectively. Data are expressed as mean ( ± SEM) from three independent experiments (n = 3 mice). C Kruskal–Wallis followed Dunn’s multiple comparison test, (H–N) 2-tailed Mann–Whitney U-test, (I–P) 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.