Fig 2: Hallmarks of microglial activation in DS microglia. (A) UMAP of microglia from all processed samples, colored by microglial subcluster. (B) Heatmap displaying key differentially expressed genes used to define microglial subclusters. (C) Fraction of total microglia from each sample that clustered in each of the four major microglia subclusters. (D) Violin plots of gene expression for hallmark microglial activation genes from DS vs. Ctrl and DS-young vs. Ctrl-young cohorts; adjusted P value using Bonferroni correction on Wilcoxon rank sum test. (E) Western blot for CX3CR1 and quantification relative to GAPDH. Asterisk denotes statistical significance in unpaired t test (P = 0.011). (F) Violin plots of gene expression for C1q complement genes, ADGRG1, and RUNX1. (G) Volcano plots for total DEGs in microglia (gray), DEGs from HSA21 that are triplicated in the Dp16 mouse model but not Tc1 or Ts1Rhr (green), and all other HSA21 microglial DEGs (purple) (48–50).

Fig 3: In vitro validation of PtenY68H microglia pathology. a Representative immunofluorescence staining of Pten+/+ and PtenY68H/+ primary microglia with Pten (red), Iba1 (red), C1q (green), Trem2 (green), Cx3cr1 (green), and DAPI (blue). N = 7. b-e Quantification of IF staining in panel a for Pten (P = 0.0006; b), Iba1 (P = 0.038; c), C1q (P = 0.004; d), Trem2 (P = NS; e), and Cx3cr1 (P = NS; f). g Representative image of phagocytosis assay comparing Pten+/+ and PtenY68H/+ microglia. h Quantification of panel g for proportion of active phagocytic microglia over total microglia. P = 0.01 i Quantification of panel g of average bead counts per phagocytic microglia. P = 0.005. P value key: *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: Effect of PDTC on the expression of CX3CR1 mRNA in TNFa-induced BV-2 microglial cells. PDTC-treatment group had lower CX3CR1 mRNA expression than other groups did. A) Bands of RT-PCR of CX3CR1 mRNA expression. B) Statistical analysis of relative density of RT-PCR bands is shown between different groups. Results are expressed as mean± SEM (n=4). #p<0.05 vs control cells; *p<0.05 vs PDTC and TNFa treated cells.

Fig 5: The expression of Plscr1 is inhibited by IL-13 and Type 2 inflammation.(A) WT mice were subjected to HDM administration, lung Plscr1 mRNA expression was assessed by quantitative real-time PCR. Values are mean± SEM with 7–9 mice in each group. Data was assessed with unpaired Student’s t-test. **P = 0.01. (B) Lungs from WT mice with or without HDM challenges were sectioned, SPC, CX3CR1, or Gata-3 was labeled with red fluorescence (Alexa Fluor 594) and Plscr1 was labeled with green fluorescence (Alexa Fluor 488). Nuclei are stained with DAPI (blue). Images are representative of 3 mice. (C) Whole lung lysates from WT (IL-13 Tg (–)) and IL-13 Tg (+) mice were isolated and Plscr1 protein level was then evaluated by Western immunoblot analysis as noted. (D) BAL inflammatory cells were isolated from WT mice. Cells were treated with 30 ng/mL IL-13, and Plscr1 protein level was evaluated using Western immunoblot analysis as noted.