Fig. 3: Initial ferroptosis impairs the maturation of dendritic cells.

a MCA205 cells with depleted levels of GPX4 and doomed for ferroptosis were co-incubated with dendritic cells for 16 h. To address the role of each ferroptosis stage on the dendritic cell maturation, iGPX4KD cells at different stages of cell death were used. In all conditions iGPX4KD cells reached terminal stage during the co-culture. b The analysis of the maturation of dendritic cells incubated with ferroptotic cells. The levels of CD86, CD40, PD-L1 and percentage of dendritic cell population expressing high levels of MHCII was assessed by flow cytometry measurements. Data from n = 3 independent samples for CD40 and MHCII measurement and n = 4 independent samples for CD86 and PD-L1. Data are presented as floating bar plots with box bounds representing the range and center showing the median of obtained measurements. One-way ANOVA, with Dunnett’s post-hoc test analyzing comparison to ‘0 h’ sample. c The analysis of cytokine production from the dendritic cells incubated with ferroptotic cells. Data from n = 4 independent biological replicates and presented as floating bars with bar limits showing the range and the center describing the median. One-way ANOVA, with Dunnett’s post-hoc comparing results from the co-cultures to the untreated bone marrow-derived dendritic cells (BMDC). d The analysis of phosphatidylserine exposure on the surface of the ferroptotic cells at different stages of cell death. Representative contour plot of flow cytometry analysis using Annexin V and 7-AAD. Bar graphs show the mean ± SEM of n = 3 independent experiments. e The level of phagocytosis of untreated and undergoing the initial or terminal ferroptosis. CypHer-labeled MCA205 cells were incubated with CFSE-labeled bone marrow-derived dendritic cells (BMDC) for 2 h. Afterwards the phagocytosis was determined by the detection of CypHer fluorescence in CFSE-stained BMDC. Cytochalasin D and Fer1 were used as inhibitors of phagocytosis and lipid peroxidation respectively. Data from n = 3 independent biological samples and is presented as floating bars with bounds representing the range and the center showing the median of % of phagocytic BMDC. Two-way ANOVA with Dunnett’s post-hoc test shows the comparison to the untreated condition with the same inhibitor. Contour plots represent the stages of cell death (upper panel) and the gating strategy for phagocytosis detection. f The analysis of the dendritic cells phagocytosis of the terminal ferroptotic cells with and without PS blockage by Annexin V. Data presented as median and range and come from n = 3 biological replicates, two-sided t-test, ns—not significant. g The analysis of dendritic cells phagocytosis of terminal ferroptotic cells with blocked calreticulin. Data presented as floating bars with bounds representing the range and the center showing the median of n = 3 biological replicates, two-sided t-test, ns—not significant. h The microscopy analysis of lipid droplets accumulation using BODIPY 493/503 nm probe. Bone marrow-derived dendritic cells (BMDC) were incubated with iGPX4KD cells at different stages of cell death O/N. Afterwards, cells were fixed and visualized on confocal microscope. Box plot shows the analysis of the relative volume of detected lipid droplets in the BMDC for each condition and is presented as a box showing median (center line), 25th and 75th percentile (box bounds) and range of the observed results (whiskers) from n = 3 independent experiments, where each dot represents one cell in the analyzed images. One-way ANOVA with Dunnett’s post-hoc test analyzing the comparison to the untreated BMDC. i The flow cytometry analysis of BODIPY 493/503 nm accumulation in the BMDC after the incubation with untreated or ferroptotic iGPX4KD cells. Data presented as floating bar plots with bounds representing the range and the center showing the median of values generated from n = 4 independent experiments. One-way ANOVA with Dunnett’s post-hoc test analyzing the comparison of ferroptosis conditions to ‘0 h’ condition.