Fig. 5: T. gondii effectors TgWIP and GRA15 modulate the sequestration of parasitized DCs. | Nature Communications

Fig. 5: T. gondii effectors TgWIP and GRA15 modulate the sequestration of parasitized DCs.

From: ICAM-1/CD18-mediated sequestration of parasitized phagocytes in cortical capillaries promotes neuronal colonization by Toxoplasma gondii

Fig. 5

A. Experimental set up. CMTMR pre-labeled DCs were challenged with GFP-expressing wild type (WT), ΔGRA15 or ΔTgWIP tachyzoites, followed by inoculation into the brain circulation via ICA (20 × 106 DCs / 10 × 106 cfu Tg). Brains were collected 16 hpi. B, C Graphs show the absolute numbers (mean ± SEM) of infected DCs (CMTMR+, GFP+) per cm2 cortical tissue for PRU-WT versus PRUΔGRA15 (B) and RH-WT versus RHΔTgWIP (C), respectively. Data are from 30 cortical sections per condition from three independent experiments (n = 3 mice). D, E Relative expression (qPCR) of T. gondii TgB1 gene in brain tissue of mice inoculated with DC/Tg PRU-WT versus PRU ΔGRA15 (D) and RH-WT versus RH ΔTgWIP, respectively. Data are expressed as mean ( ± SEM) 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). After 48 h, infected CMTMR pre-labeled DCs (10 × 106 DCs / 5 × 106 cfu Tg, WT, ΔGRA15 or ΔTgWIP) were inoculated via the ICA and brains collected 1 hpi. G, H Graphs show the absolute numbers (mean ± SEM) of infected DCs (CMTMR+, GFP+) per cm2 cortical tissue for PRU-WT versus PRUΔGRA15 (G) and RH-WT versus RHΔTgWIP (H), respectively. Data are from 30 cortical sections per condition from three independent experiments (n = 3 mice). I, J Relative expression (qPCR) of T. gondii TgB1 gene in brain tissue of pre- ip infected mice inoculated with DC/Tg PRU-WT versus PRUΔGRA15 (I) and RH-WT versus RHΔTgWIP (J). Data are expressed as mean ( ± SEM) from three independent experiments (n = 3 mice). K Experimental set up. Infected CMTMR pre-labeled DCs (2 × 104 DCs / 1 × 104 cfu Tg, WT or ΔGRA15) were inoculated via the ICA and brains collected 7 dpi. L. Relative cerebral parasite loads in brain and spleen, respectively, upon challenge with PRU-WT and PRUΔGRA15, determined by plaquing assays. Data show number (mean ± SEM) of plaques per gram of tissue from four independent experiments (n = 4 mice). M Experimental set ups. Infected CMTMR pre-labeled DCs (2 × 104DCs / 1 × 104 cfu Tg, PRU-WT) were inoculated via the ICA and brains collected 7 dpi. When indicated, mice were pre-treated with LPS ip or treated with heparin iv. N Relative cerebral parasite loads in brain and spleen, respectively, of mice challenged with PRU-WT and pre/treated with LPS or heparin, determined by plaquing assays. Data show number (mean ± SEM) of plaques per gram of tissue from three independent experiments (n = 4 mice). O Experimental set up. Mice were inoculated ip with 10 × 106 cfu of freshly egressed non-replicative tachyzoites (RH-CPS-mCherry), (pre-ip Tg CPS). After 48 h, infected DCs (10 × 104DCs / 5 × 104 cfu Tg, PRU-WT) plus α-ICAM-1 or isotype control were inoculated via the ICA. Brains were collected 7 dpi. P Cerebral parasite loads in brain and spleen, respectively, of mice challenged with DC/Tg PRU-WT plus α-ICAM-1 (or isotype), determined by plaquing assays. Mice were pre-infected (pre- ip, RH-CPS mCherry). Data show number (mean ± SEM) of plaques per gram of tissue from five independent experiments. For each experiment, mice were challenged and treated pairwise (α-ICAM-1 or isotype control) and datapoints are color-coded accordingly (n = 5 mice). B–H 2-tailed Mann–Whitney U-test, (D–P) 2-tailed unpaired Student’s t-test, (N) One-way ANOVA followed by Bonferroni’s multiple comparison test, numeric p-values are indicated, ns: non-significant, p > 0,05. Source data are provided as a Source Data file.

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