Infection by Toxoplasma gondii Induces Amoeboid-Like Migration of Dendritic Cells in a Three-Dimensional Collagen Matrix
Fig 3
Morphological characteristics and migration of Toxoplasma-infected DCs and non-infected DCs in the 3D matrix.
(A) Representative micrographs in maximum intensity projection of unchallenged DCs in complete medium (CM, left), Challenged/By-stander DCs (middle) and Challenged/T. gondii DCs (right; PTG-GFP type II, MOI 3, green) in a 3D collagen matrix, stained with DAPI (blue), and Alexa Fluor 594-Phalloidin (red) to detect F-actin as indicated under Materials and Methods. In the middle micrograph, arrow indicates non-infected DC surrounded by an infected DC (green + red) and two extracellular T. gondii tachyzoites (green). Scale bars = 10 μm. (B) Graph shows, for each condition, the percentage of cells (mean ±SEM) that exhibit rounded phenotype, absence of membrane extensions and veils, respectively, related to the total cell population. The morphological criteria are specified under Materials and Methods. For each condition, a total of 50 cells/donor were analyzed from 3 different donors. (*: P < 0.05, **: P < 0.01, ns: P > 0.5, Paired t-test, Holm´s correction). (C) Compiled mean scores (± SD) based on morphological criteria as in (B). For each condition, a total of 50 cells/donor were analyzed from 3 different donors (*: P < 0.05, **: P < 0.01, Paired t-test, Holm´s correction). (D) Distribution of the total scores (% of total cell population) based on morphological criteria specified under Materials and Methods. For each condition, a total of 50 cells/donor from 3 donors were assessed. Significant differences were observed between tachyzoite-infected DCs and non-infected DCs (P < 0.0001; Fisher´s exact test) or by-stander DCs (P < 0.0001), while differences between non-infected DCs and by-stander DCs were non-significant (P > 0.05). (E) Representative 3D projection analysis of DCs challenged with T. gondii (PTG-GFP type II). The colored spheres indicate the position of cells in the defined 3D space. Infected cells and non-infected cells were defined and analyzed as indicated under Materials and Methods: co-localized signal/infected cell (actin: red; T. gondii: green) or absence of co-localization/by-stander cells (blue). The inset image represents a magnification of the white-dotted square. Data are representative from 4 independent experiments. (F) Mean migrated distances by unchallenged DCs (CM), and challenged non-infected DCs (By-stander) and infected DCs (T. gondii: PTG-GFP type II). Data represent compiled analysis of 500 randomly chosen cells per donor from 4 different donors. Bars indicate mean migrated distances (*: P < 0.05, ns: P > 0.05, Two-way ANOVA, Tukey´s HSD test). (G) Dot plots represent the distribution of migrated distances for individual DCs infected with T. gondii (type I: LDMluc; type II: PRU-RFP). For each condition, 100 single cells were randomly selected and analyzed from one representative donor. Bar indicates mean migrated distance. Asterisks indicate significant differences (**: P < 0.01; Paired t-test, Holm´s correction).