Fig 2: CD11cHI Retinal Microglia Are More Resistant to Loss or Inhibition of CSF1R Signaling(A) Tamoxifen dosing regimen.(B) Confocal images of retinal wholemounts of control at P9 (tamoxifen at P4, P6, and P8) (left) and homozygous floxed at P8 (tamoxifen at P4 and P6) (right). Scale bars, 100 µm. Experiment repeated twice, n = 4 animals each.(C) PLX3397 (PLX) dosing regimen.(D) Confocal images of retinal wholemounts at P7 from vehicle- (left) and PLX-treated (right) animals. Scale bar, 100 µm.(E) Representative flow cytometry plot and gate for collecting GFP+CD45+ microglia in vehicle- (left) and PLX-treated (right) animals.(F) Percent microglia (CD45+GFP+) of total single cells by flow cytometry. (±SEM; n = 8 each). Two-tailed unpaired t test t(14) = 4.438, ***p = 0.0006.(G) Percent CD11cHIof total CD45+GFP+ microglia by flow cytometry. (±SEM; n = 8 each) Two-tailed unpaired t test t(14) = 4.662, ***p = 0.0004.(H) Log-transformed relative gene expression by qPCR (±SEM) in sorted microglia from PLX-treated retinas relative to vehicle controls. WT (n≥8 except n = 5 Cd68, Lamp1, Lyz2, P2ry12); PLX (n = 9 except n = 8 for Itgax, n = 5 Cd68, Lamp1, Lyz2, P2ry12). Two-tailed unpaired t test *p ≤ 0.05, **p ɛ≤ 0.01, ***p ≤ 0.001. #, Welch’s correction.(I) HCR of vehicle- and PLX-treated retinas. Scale bars, 50µm.(J) Ratio (±SEM) of PLX-treated over genotyped-matched control density (CD45+Cd11b+/singlets). WT (n = 17); Trem2 KO (n = 6); Bax KO (n = 5). One-way BrownForsythe ANOVA F(2,24.29) = 11.76, p = 0.0003 and Games-Howell’s multiple comparisons test. *p ≤ 0.05, ***p ≤ 0.001.(K) Percent (±SEM) of CD11cHImicroglia in total CD45+CD11b=population after PLX by flow cytometry. WT littermates (n = 5), Trem2 KO (n = 7); Bax KO (n = 5). One-way ANOVA F(2,14) = 24.55, p < 0.0001 and Tukey’s multiple comparisons test ***p = 0.0005, ****p < 0.0001.See also Figures S3 and S4.

Fig 3: Increased recruitment of MT4-MMP-null patrolling monocytes in incipient atherosclerotic lesions. a Representative orthogonal XZ view images of whole-mount-stained lesser curvature of the aortic arch from Ldlr–/– mice transplanted with MT4-MMP+/+ or MT4-MMP–/– BM cells and fed a HFD for 3 days. Samples were stained for CD115 (magenta) and Ly6C (green); elastin autofluorescence (green) and nuclei (Hoechst, blue). The bar graph (right) shows the quantification of the number of patrolling (CD115+Ly6C–) and classical monocytes (CD115+Ly6C+) in the aorta lumen; n = 6 mice per genotype in two independent experiments. b Representative confocal microscopy images of whole-mount-stained lesser curvature of the aortic arch from Ldlr–/– mice adoptively transfer with Cx3cr1Gfp/+ MT4-MMP+/+ or Cx3cr1Gfp/+ MT4-MMP–/– patrolling monocytes and fed a HFD for 3 days. Samples were stained for GFP (red) and CD31 (gray); elastin autofluorescence (green) and nuclei (Hoechst, blue). A z-stack of the confocal microscopy sections close to the lumen (with an inset of CD31 staining) is shown to the top and the orthogonal XZ view of the merged images to the bottom. Scale bar, 20 µm. The bar graph (right) shows the quantification of the number of transferred monocytes (GFP+) in the aorta lumen; n = 9 mice per genotype in two independent experiments. Data were tested by Student’s t-test. Results are expressed as mean ± SEM.*p < 0.05, **p < 0.01

Fig 4: Anti-CCR2 antibody reduces monocyte populations in the blood without affecting cognitive behavior. WT mice received 4 injections of αCCR2 antibody every 4 days, while control mice were untreated. Mice were euthanized 3 days after the 4th injection, and blood was collected. (A) Flow cytometry gating strategy for monocyte (top) and memory CD4+ T cell (bottom) populations in the blood of WT animals. (B) Flow cytometry analyses of Ly6G− CD115+ myeloid cells (two-tailed Student’s t-test: t(14) = 2.256, *p = 0.0406), (C) Ly6C+ myeloid populations (two-tailed Student’s t-test: Ly6Chi t(14) = 3.764, **p = 0.0021; Ly6Cmed t(14) = 2.442, *p = 0.0285), (D) CD4+ T cells, and (E) memory CD4+ T cell populations in control and αCCR2-injected groups, n = 8 mice per group. (F) Flow cytometry gating strategy and (G) analysis of Tregs in the blood of WT mice, n = 10 mice per group. Data are presented as mean ± s.e.m. *p < 0.05, **p < 0.01. (H-J) Male WT mice received 4–5 injections of αCCR2 antibody every 4 days, while control mice remained untreated. Behavioral testing was carried out during the 4 days following the last injection. (H) T-maze: Willingness to explore a novel environment is presented as percent of time spent by each mouse in a novel arm divided by the total time spent in all three arms (the novel arm and two familiar arms). (I) Spontaneous alternation in the Y maze: The spontaneous alternation behavior of the mice is presented as percent alternation: number of alternations divided by number of possible triads (see Methods). (J) Novel Object Recognition (NOR): The memory recognition is presented as percent time the mouse interacted with the novel object divided by the total time spent with both objects. n = 6 mice per group. Data are presented as mean ± s.e.m. One-way ANOVA was used for the analyses

Fig 5: Effects of the Npc1 loss-of-function mutation in hematopoietic cells on blood, splenic, and bone marrow myeloid cells in WTD-fed Ldlr−/− mice. Ldlr−/− mice were transplanted with Npc1wt or Npc1mut BM and fed a chow diet for 9 weeks, followed by WTD for 9 weeks. A: Total monocytes, monocyte subsets, and neutrophils in blood from mice fed WTD for 4 weeks, assessed by flow cytometry. BM (B and C) and spleen (D and E) were collected after 9 weeks of WTD feeding, and monocytes and neutrophils were assessed by flow cytometry. B and D: Representative flow cytometry plots of monocytes and neutrophils as percentage of total CD45+ leukocytes and Ly6Clo and Ly6Chi monocyte subsets as percentage of CD115+ monocytes in BM (B) and spleen (D). Neu denotes neutrophils. Total monocytes, monocyte subsets, and neutrophils in BM (C) and spleen (E). n = 15–16 mice per genotype. Data are shown as mean ± SEM. ∗∗∗P < 0.001 by t-test.