Fig. 6

The role of IL-33 is partly dependent on the polarization state of macrophages. A qPCR analysis of changes in the gene expression of the M2 macrophage marker CD206 and the M1 macrophage marker CD86 after IL-33 neutralizing antibody injection compared to the control groups (mean ± SD, n = 6). B Quantitative analysis of flow cytometry results of the proportion of differently polarized macrophages in skin single-cell suspensions after injection of IL-33-neutralizing antibody compared with controls (mean ± SD, n = 3). C Representative H&E staining, CD31 immunofluorescence staining, and Ki67 immunofluorescence images for the following conditions at 20 days post-irradiation: clearance of senescent cells, subcutaneous injection of recombinant IL-33 after clearance of senescent cells, and subcutaneous injection of recombinant IL-33 after clearance of macrophages with Clodronate; n = 3. Quantitative analyses were conducted by randomly selecting 12 high-power fields, and using ImageJ to compute the area with CD31 positive signals and the percentage of Ki67 positive cells. D Mouse bone marrow (BM) cells were isolated and cultured in the presence of M-CSF (20 ng/mL). After six days, macrophages were stimulated with LPS (100 ng/mL), IL-13 (10 ng/mL), IL-33 (20 ng/mL), or a combination of these agents for 48 h. Flow cytometry was used to analyze the surface markers CD206 for M2 macrophages and CD86 for M1 macrophages. Quantitative analyses of flow cytometry results for the proportions of M0, M1, and M2 macrophages are presented (mean ± SD, n = 3). E Primary fibroblasts or primary keratinocytes were stimulated with PBS, polarization-inducing medium containing IL-33 and IL-13 without macrophages, M0 macrophage-conditioned medium, or conditioned medium from M2 macrophages treated with IL-33 and IL-13. Flow cytometry was used to analyze the percentage of EdU⁺ proliferating cells, with results representing data from three repeated experiments (mean ± SD, n = 3). F Transwell assays revealed the macrophage-dependent ability of IL-33 to enhance cell migration. Representative images showing the migratory ability of primary keratinocytes (top) and primary fibroblasts (bottom) incubated under different conditions for 24 h. G Quantitative analysis of the number of migrating cells in the Transwell assay of keratinocytes and primary fibroblasts under different stimuli based on 12 randomly selected high magnification fields of view (mean ± SD). H Scratch assay demonstrated that IL-33-enhanced cell migration was dependent on macrophages. Representative images showing the migratory ability of primary keratinocytes (top) and primary fibroblasts (bottom) incubated under different conditions for 12 h. The red dashed lines indicated the edge of migrating cells. I Quantitative analysis of the wound closure ratio in the scratch assay of keratinocytes and primary fibroblasts under different stimuli based on 12 randomly selected high magnification fields of views (mean ± SD). When variance was met, a t-test was used for statistical analysis between two groups, while ANOVA was used for comparisons among three or more groups. If variance was not met, the Mann‒Whitney U test was used. *p < 0.05, **p < 0.01, ***p < 0.001. Bars represented 50 μm (F, H) and 100 μm (C). EdU stood for 5-ethynyl-2′-deoxyuridine, and BMDMs for bone marrow-derived macrophages