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[Preprint]. 2023 Jun 13:2023.06.12.544691. [Version 1] doi: 10.1101/2023.06.12.544691

Figure 6: Enhanced pro-inflammatory signaling is coordinated with phagocytosis in P2ry6+/+ microglia.

Figure 6:

(A) Outline of transcriptomics experiment using FACS-based microglia isolation 3 days after KA-SE from whole brain. (B) Top differentially expressed genes (DEGs) in microglia between WT-KO genotypes (log2FC>1.0 and BH-adjusted p<0.1). (C) Key Gene Ontology (GO) terms based upon the DEG set in (B). Details are provided in the Star Methods section. (D) Protein-level evaluation of hippocampal and cortical microglia using high-parameter flow cytometry 5 days after KA-SE. (E) Mode-normalized, representative histograms and intensity distributions for microglial P2Y12 and Cx3Cr1 expression. Two-Way ANOVA with Sidak’s post-hoc test (box plots display the mean and interquartile range with whiskers denoting min to max). (F) UMAPs of TNFα, Pro IL-1β, and CD68 expression, or NeuN/CD68 co-detection in hippocampal microglia (boxes highlight a distinct population between genotypes). (G) Frequency of marker expression by microglia in cortex and hippocampus. One-Way ANOVA with Tukey’s post-hoc test (normalized to mean naïve frequency per cohort/batch; dot: one mouse). (H) Pseudocolor plots displaying the NeuN gate, based upon an FMO, and application of that gate onto hippocampal microglia. (I) Psuedocolor plot with gates to isolate microglial populations which are CD68Low, CD68High, and CD68High SSCHigh. (J) Evaluation of key signal intensity differences between P2ry6+/+ hippocampal microglia which are CD68Low, CD68High, and CD68High SSCHigh. One-Way ANOVA with Tukey’s post-hoc test (dot: one mouse, where dot shape denotes cohort; ****p<0.0001). (K) Representative immunofluorescent imaging of DAPI, IBA1, and IL-1β expression in WT CA1 one week after KA-SE. Scale bar, 20µm. (L) Correlations between Pro IL-1β intensity and NeuN intensity inside of microglia. Simple linear regression (dot: one mouse). (M) Comparison of different CD68 population frequency between genotypes in the hippocampus. Two-Way ANOVA with Sidak’s post-hoc test (dot: one mouse, where dot shape denotes cohort). Data were aggregated from N=6 naïve mice (4 WT, 2 KO) N=8 D5 P2ry6+/+ mice and N=7 D5 P2ry6−/− mice across two independent cohorts.