Fig. 5.
In vivo deletion of Bin1 affects surface CD11c expression. A Experimental strategy for in vivo experiments involved three groups of mice: Bin1fl/fl (WT equivalent), Cx3cr1CreER (primary reference group), and Cx3cr1CreER-Bin1 cKO (experimental group). Mice were injected with tamoxifen for 5 consecutive days, then rested for four weeks to allow replenishment of Bin1 expression in peripheral monocytes. Mice then received saline or LPS for four consecutive days, and brains were harvested for flow cytometry / FACS, IHC, and cytokine assays, 24 h after the final injection. B Immunofluorescence staining in the piriform cortex demonstrates BIN1 expression in microglia (yellow arrows), oligodendrocytes (asterisks), and synapses (unlabelled) in mice with normal BIN1 expression (Cx3cr1CreER). Bin1 was deleted from the microglia of experimental mice (Cx3cr1CreER-Bin1 cKO), whilst oligodendrocytes and synaptic BIN1 were unaffected. C Mouse brain cells were labelled with APC-Cy7 α-CD11b, PE-Cy7 α-CD45, and BV421 α-CD11c. Single, mononuclear, live cells were gated, and microglia were sorted as CD11b+CD45INT population. D A representative flow cytometric image of each experimental group is depicted. E Flow cytometric analysis demonstrates that LPS administration in vivo caused an increase in the proportion of cells with high surface CD11c expression in all genotypes. The LPS effect was augmented by Cx3cr1 haploinsufficiency (Cx3cr1CreER); this additional increase was blunted by microglial Bin1 deletion (Cx3cr1CreER-Bin1 cKO). Two-way ANOVA found main effects for genotype (F2,17 = 32.98, p < 0.001) and LPS (F1,17 = 100.9, p < 0.001). There was a significant genotype*LPS interaction (F2,17 = 16.87, p < 0.001). F NanoString mRNA counts show that LPS increased Itgax transcript numbers (F1,16 = 27.014, p < 0.001). No differences between genotypes (F2,16 = 3.065, p = 0.075) and no genotype*LPS interactions (F2,16 = 1.052, p = 0.372) were found. Bin1 deletion did not attenuate Itgax transcript numbers. G NanoString analysis of mRNA from sorted microglia demonstrates that our cKO system resulted in approximately 50% decrease in microglial Bin1 expression (F2,17 = 13.14, p < 0.001), which was not affected by LPS (F2,17 = 0.712, p = 0.505), despite the main effect for LPS increasing Bin1 transcripts (F1,17 = 5.853, p = 0.027). Analysis of Cx3cr1 transcript numbers found a main effect for genotype (F2,17 = 43.802, p < 0.001), with post-hoc differences between Bin1fl/fl with Cx3cr1CreER (p < 0.001), Bin1fl/fl with Cx3cr1CreER-Bin1 cKO (p < 0.001), and Cx3cr1CreER with Cx3cr1CreER-Bin1 cKO (p = 0.043) demonstrating that the reduction in Cx3cr1 expression in the Cre line was partially attenuated by Bin1 deletion. No main effect for LPS treatment (F1,17 = 0.303, p = 0.589) and no genotype*LPS interaction (F2,17 = 0.515, p = 0.606) were found. All by two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001; by post-hoc t-test with Bonferroni correction for multiple comparisons. All data plotted as mean ± SEM