Fig. 3

The expression of HIF-1α in microglia in innate immune memory models. A, B RNA-seq analysis of the striatum between mice treated with 2xLPS or 4xLPS. TR, Training; TL, tolerance. A The volcano plot of differentially expressed genes (DEGs). B The gene ontology (GO) enrichment analysis of DEGs. C The summary heatmap of RT-qPCR results based on M1/M2-associated genes and inflammatory genes in mouse striatum. n = 4–5/group. D Protein levels of HIF-1α in BV2 cells treated with LPS for 12 h, detected by Western Blot. n = 7–10/group. Differences were analyzed by unpaired t-test. **p < 0.01. E A representative 3D reconstruction image of activated microglia showing the colocalization of IBA1 (green) and HIF-1α (Magenta). Scale bar, 5 µm. F Co-localization of HIF-1α, CD16/32 and IBA1 in striatal microglia. Representative images from confocal microscopy revealed the colocalization of HIF-1α (Magenta) and CD16/32(red) in IBA1⁺ (green) microglia. White arrow: HIF-1α⁻ CD16/32⁻ cell; yellow arrow: HIF-1α⁺ CD16/32⁺ cell; magenta arrow: HIF-1α⁺ CD16/32⁻ cell; red arrow: HIF-1α⁻ CD16/32⁺ cell. Scale bar, 20 µm. G Representative images of IBA1/HIF-1α/CD16/32 triple immunostaining in mouse striatum. Scale bar, 50 µm. Small plots in the upper left quadrant showed the zoomed details. Scale bar, 20 µm. The experimental schedule was described as in Fig. 2A. (Ctrl, Control; TR, Training; TL, Tolerance). H Quantification of the numbers of HIF-1α⁺, CD16/32⁺ and HIF-1α⁺CD16/32⁺ cells among all the IBA1-immunolabeled cells. n = 3–5/group. Differences were analyzed by one-way ANOVA followed by LSD multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, vs control groups. I The percentage of HIF-1α⁺, CD16/32⁺ and HIF-1α⁺ CD16/32⁺ cells within the total IBA1⁺ cells in mouse striatum. n = 3–5/group. Differences were analyzed by one-way ANOVA followed by LSD multiple comparison tests. **p < 0.01, ***p < 0.001, vs control groups. ^#p < 0.05. J The relative proportion of sub-populations of IBA1-immunolabeled cells in the acute innate immune memory mouse model. The difference of HIF-1α⁻CD16/32⁻ microglia between groups was marked in the figure. n = 3–5/group. Differences were analyzed by one-way ANOVA followed by LSD multiple comparison tests. ***p < 0.001 vs control groups. K The relative proportion of IBA1-immunolabeled HIF-1α⁺ and HIF-1α⁻ cells in acute innate immune memory model. The difference in HIF-1α⁻ microglia between groups was marked in the figure. n = 4–5/group. Differences were analyzed by one-way ANOVA followed by LSD multiple comparison tests. *p < 0.05, **p < 0.01 vs control groups. #p < 0.05. L Representative images of IBA1/HIF-1α/CD16/32 triple immunostaining in the striatum of mice treated with NS, 1xLPS, or 4xLPS for two or five weeks. Scale bar, 50 µm. Small plots in the upper left quadrant showed the zoomed details. Scale bar, 20 µm. M Quantification of the numbers of HIF-1α⁺, CD16/32⁺ and HIF-1α⁺CD16/32⁺ cells in all IBA1-immunolabeled cells. n = 3–4/group. Differences were analyzed by two-way ANOVA followed by LSD multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, vs control groups. ^#p < 0.05, ^##p < 0.01. N The percentages of HIF-1α⁺, CD16/32⁺ and HIF-1α⁺CD16/32⁺ in the total IBA1⁺ cells in mouse striatum. n = 3–4/group. Differences were analyzed by two-way ANOVA followed by LSD multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001, vs control groups. ^##p < 0.01. O The relative proportions of HIF-1α⁻ CD16/32⁻ (white), HIF-1α⁺ CD16/32⁺ (yellow), HIF-1α⁺ CD16/32⁻ (green), and HIF-1α⁻ CD16/32⁺ (blue) in all IBA1-immunolabeled cells in acute innate immune memory mouse models. The difference of HIF-1α⁻ CD16/32⁻ microglia in 2w and 5w groups, compared to Control groups, was marked by asterisks (*) inside the figure, and difference of HIF-1α⁻ CD16/32⁻ microglia between 2 and 5w groups was indicated by pound sign (#). n = 3–4/group. Differences were analyzed by one-way ANOVA followed by LSD multiple comparison tests. **p < 0.01, ***p < 0.001, vs control groups. #p < 0.05