Fig. 3: Eosinophils inhibit osteoclast differentiation and function in vitro.
a, b Quantification, viability (DAPI gated on BMMs) (a), and representative images (b) of TRAP-positive polynucleated (≥3 nuclei or ≥5 nuclei) WT osteoclasts co-cultured with different ratios of eosinophils (Eos) compared with unstimulated control (n = 5). Scale bar, 100 μm. c, d Quantification, viability (DAPI gated on BMMs) (c), and representative images (d) of TRAP-positive polynucleated (≥3 nuclei or ≥5 nuclei) WT osteoclasts co-cultured with different dilutions of eosinophil supernatant (Eos spn) compared with unstimulated control (n = 5). Scale bar, 100 μm. e–h Bulk RNA-seq. analysis of WT osteoclasts stimulated with eosinophils (2:1 Eos/BMMs ratio; OCsEo) or eosinophil supernatant (1:2 dilution; OCsEoS) compared with unstimulated control (OCsCtr) (n = 4). Principal component analysis (PCA) visualizing the patterns of the individual samples of the 3 different groups (e). Volcano plots showing differentially expressed genes (DEG) between the groups OCsEo vs. OCsCtr, OCsEoS vs. OCsCtr and OCsEoS vs. OCsEo (f). Statistically significantly upregulated genes are orange and downregulated genes are purple. Heat map showing osteoclast-associated DEGs between the aforementioned groups (g). Heat map showing phagocytosis- and eosinophil-associated DEGs between the aforementioned groups (h). i, j Quantification (i) and representative images (j) of the demineralization activity of WT osteoclasts cultured on hydroxyapatite-coated plates following the stimulation with eosinophils (1:1 Eos/BMMs ratio) or eosinophil supernatant (1:2 dilution) compared with unstimulated control (n = 5). Scale bar, 100 μm. Data are shown as mean ± SEM. Symbols represent individual mice. P values were determined by one-way ANOVA Dunnett’s test (3a, 3c, 3i) for multiple comparisons. Asterisks mark statistically significant difference (**P < 0.01, ***P < 0.001, ****P < 0.0001). Differential expression analysis between 2 groups was performed with log2 fold change cut off 0.5. The resulting P values were adjusted using the Benjamini–Hochberg approach for controlling the false discovery rate (FDR). Genes with an adjusted P value (Padj) less than 0.05 were assigned as differentially expressed (2f–h). Source data are provided as a Source data file.
