Fig 2: Analysis of microglial markers in SSLOW-infected P2Y12 KO and WT mice. a Immunofluorescence staining for P2Y12 (red) and IBA1 (green) showing lack of P2Y12 signal in non-infected and SSLOW-infected P2Y12 KO brains. Cortex is shown. b. Quantification of P2Y12 and IBA1 signal intensities in cortices of non-infected and SSLOW-infected WT and P2Y12 KO mice. Comparison by ordinary one-way ANOVA with Tukey’s multiple comparison test, n = 3–4 animals in each group, 3–6 fields of view per brain. c. TMEM119 (red) and IBA1 (green) co-immunostaining (left) and TMEM119 quantification (right) in the cortices of SSLOW-infected WT and P2Y12 KO mice. Comparison by unpaired Student’s t-test, n = 3 brains for each group, 3–7 fields of view per brain. In b and c, colors represent different brains, dots represent fields of view, average values for each brain are shown as circles. Means ± SD are marked by black lines. d–f RT-qPCR analysis of selected purinergic receptors enriched in microglia (d), neuroinflammatory markers (e), and markers of phagocytic activity (f). Comparison of non-infected WT, non-infected KO, SSLOW WT and SSLOW KO was performed using Brown-Forsythe ANOVA followed by Dunnett’s T3 multiple comparisons test, n = 6 brains per group; ns – not significant; non-infected WT vs. KO are not shown (not shown). Comparison of combined non-infected WT + KO vs. combined SSLOW WT + KO was performed using unpaired two-tailed t-test with Welch’s correction for C3, Ccl3, Ccl5, Il1a, Tmem119, Cd68, Trem2, Tlr2, P2rx4, P2rx7, P2ry13, P2ry6, and non-parametric two-tailed Mann-Whitney test for Cxcl10 and Mertk; n = 12 brains per combined group; **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars 50 μm

Fig 3: Microglial DGAT deletion upregulates cholesterol export transcriptional programs and increases the abundance of a Gpnmb-high, disease-associated myeloid subtype in male PS19 mice(A) Gating strategy used to isolate PU.1+ brain myeloid cells from frozen hippocampi of representative 9.5-month-old PCfD and CfD male mice treated with TAM or OIL. The groups comprise 3 pooled hippocampi, and each tissue was from an individual mouse.(B) UMAP plots of PU.1-sorted microglia with annotated clusters separated by experimental group.(C) Relative cluster frequencies across the groups.(D) Clec7a, Tmem119, and Gpnmb expression across PU.1 nuclei clusters.(E) Volcano plot of DEGs between PCfD OIL and PCfD TAM mice annotated with selected transcripts of interest.(F) Top and bottom 15 GSEA Reactome terms from analysis of DEGs between PCfD TAM vs. PCfD OIL groups ranked by normalized enrichment score (NES).

Fig 4: Hippocampal CD68 density is increased upon microglia-specific DGAT KO in male PS19 miceRepresentative images and percent area densities of (A) CD68, (B) Iba1, (C) Gfap, (D) Clec7a, and (E) Tmem119 in the hippocampus and entorhinal/piriform cortical area (EC/PC area) of 9.5-month-old male PCfD mice treated with OIL or TAM at 3 months of age (n = 16–20 per group). Scale bar: 500 μm. Mann-Whitney tests were used to determine statistical significance (CD68 hippocampus, p = 0.0222). Data are represented as the mean ± SEM. *p < 0.05.

Fig 5: MerTK-triggered phospholipase C signal is sufficient to induce microglial PU.1 and IRF8 expression.A Microglia were FACS-sorted from the optic nerves of 8-week-old C57BL/6 wild-type female mice (3 mice) and in vitro cultured in the presence of phosphatidylserine. The cells were visualized by the co-immunostaining of TMEM119 and IBA1, and representative images are shown. B, C 8-week-old Cx3cr1CreER/+; Mertkfl/fl and control Cx3cr1CreER/+; Mertk +/+ female mice were subjected to the 4-hydroxytamoxifen-induced Cre-recombinase activity. Microglia were FACS-sorted and in vitro cultured in the presence of phosphatidylserine. Microglial expression of IRF8 was assessed by co-immunostaining with CD11b. B Representative images are shown. C IRF8+ microglia were quantified. n = 3 mice per condition, mean ± SEM, two-sided Student’s t test. D–G Microglia were FACS-sorted from 8-week-old C57BL/6 wild-type female mice (3 mice per condition) and in vitro cultured in the presence of phosphatidylserine. The cells were then treated with the mTOR inhibitor rapamycin or the phospholipase C inhibitor U-73122. Microglial expression of IRF8 and phospho-SMAD2 (p-SMAD2) was assessed by co-immunostaining with CD11b. Representative images of IRF8 (D) or p-SMAD2 (F) are shown. Mean fluorescence intensities of IRF8 (E) and p-SMAD2 (G) in microglia were quantified. n = 50 cells, mean ± SEM, one-way ANOVA test. H–L 8-week-old Cx3cr1CreER/+; Rosa26-LSL-hM3D(Gq) and control Rosa26-LSL-hM3D(Gq) female mice were subjected to the 4-hydroxytamoxifen-induced Cre-recombinase activity and then treated with the chemogenetic ligand CNO. H Diagram of the experimental procedure. Note that the mice were not subjected to the model of optic nerve injury. Microglial expression of PU.1 (I) or IRF8 (J) in the optic nerves was assessed by co-immunostaining with CD11b, and representative images are shown. PU.1+ microglia (K) and IRF8+ microglia (L) were quantified. n = 5 mice for Rosa26-LSL-hM3D(Gq) and n = 6 mice for Cx3cr1CreER/+; Rosa26-LSL-hM3D(Gq), mean ± SEM, two-sided Student’s t test.