Fig. 5
Ibuprofen partially reverses POLY(I:C)-mediated consequences on iMG and cell proliferation. a Experimental timeline. At WK18.5, preMG are added to dissRO. After 9 days, cultures received fresh medium for control (CTRL, grey), POLY(I:C) (magenta), or POLY(I:C) and S(+)-ibuprofen (POLY(I:C) + IBU, blue) for 24 h before analysis. b, c PGE2 is released into the supernatant of iMG-dissRO (b) and iMG-3DRO (c). Boxplot of PGE2 concentration [pg/ml] after CTRL, POLY(I:C), and POLY(I:C) + IBU. Each symbol: an independent differentiation (n = 5). b One-way ANOVA with post-hoc Tukey’s test. c Kruskal–Wallis test with post-hoc Dunn’s test. d Release of TNF into the supernatant of iMG-dissRO. Boxplot of pixel intensity normalized to reference for CTRL, POLY(I:C), and POLY(I:C) + IBU. Each symbol is an independent differentiation (n = 3). One-way ANOVA with post-hoc Tukey’s test. e iMG-dissRO live imaging for 20 min after 24 h stimulation. iMG surveillance index normalized to the mean surveillance of the cells in CTRL with a 95% confidence interval. Data from five independent differentiations. Kruskal–Wallis test with post-hoc Dunn’s test. f, g iMG surface area in iMG-dissRO. f iMG surface rendering for CTRL (left), POLY(I:C) (middle), and POLY(I:C) + IBU (right), color-coded based on surface area: blue = 1000 µm2 to red = 6000 µm2. Scale bar: 50 µm. g Boxplot of individual iMG surface areas. iMG from five independent differentiations. Kruskal–Wallis test with post-hoc Dunn’s test. h–m, Proliferation rate of retinal cells in iMG-dissRO (h–j, m) and iMG-3DRO (k, l). h and k, Example ROI of iMG-dissRO (h) and cryostat sections focusing on retinal cup iMG-3DRO (k) at WK20 counterstained with the nuclei-dye Hoechst (blue) and immunostained for KI67 (green) for CTRL (left) and POLY(I:C) + IBU (right). Scale bar: 50 µm. i, j and l, m, Boxplot percent of KI67+-cells relative to Hoechst+-cells for CTRL, POLY(I:C), and POLY(I:C) + IBU excluding KI67+/iMG. Each symbol is an independent differentiation (n = 5). Single ROI of three biological replicates. i One-way ANOVA with post-hoc Tukey’s test. j Fold change of median percent of KI67+-cells relative to Hoechst+-cells compared to CTRL. One sample t-test and Student’s t-test. l Kruskal–Wallis test. m Boxplot percent of KI67+-cells relative to iMG in iMG-dissRO for CTRL and only IBU exposure (light-blue) for 24 h. Students’s t-test. For detailed statistical analysis, see Supplementary Table 4. ***p < 0.001. **p < 0.01. *p < 0.05. nsp > 0.05, not significant. CTRL untreated control, dissRO dissociated retinal organoid cultures without iMG, IBU S(+)-ibuprofen, iMG microglia-like cells, iMG-dissRO microglia integrated into dissociated retinal organoid culture, iMG-3DRO microglia assembled 3D-retinal organoids, KI67 marker of proliferation KI-67, PGE2 prostaglandin E2, POLY(I:C) polyinosinic: polycytidylic acid, POLY(I:C) + IBU POLY(I:C) and S(+)-ibuprofen, preMG microglia precursor cells, ROI region of interest, TNF tumor necrosis factor alpha, WK week after the start of 3DRO differentiation