Figure 8. Repopulating myeloid cells are transcriptionally distinct and mount a differential response to inflammatory stimulus compared to homeostatic microglia.

(A) Two-month-old WT control or 28 day recovery mice were given intraperitoneal injections of either PBS or LPS (0.33 mg/kg) and then collected at 6 or 24 hr post injection. Controls, which were mice that did not receive LPS, are referred to as 0 hr post LPS. Myeloid cells were extracted from whole brain hemispheres, isolated using FACS gating for CD11b⁺CD45^int and processed for RNA-seq. (B) Volcano plots displaying the fold change of genes (log2 scale) and their significance (y axis, -log10 scale) between control vs. 28 day recovery mice. (C) Gene ontology chord plot of DEGs between control and 28 day recovery myeloid cells. (D) Heatmap showing expression of genes enriched in DAM, HSC/BM-derived cells, canonical microglia, BAM, PAM, and WAM signatures in control and 28 day repopulating myeloid cells. (E–F) Representative immunofluorescence 20x images of IBA1⁺ (red) and AXL⁺ (green, I) or CLEC7A⁺ (green, J) cells shown in areas with high repopulating cell deposition in control, 7, 14, and 28 day recovery mice. (G) Principal component analysis plot of extracted control and 28 day recovery cells, across time (0 hr, 6 hr, 24 hr) and treatment (+/- LPS), depicting the separation of groups into six clusters. (H) Heatmap of selected time-series cluster analysis of control and 28 day recovery cells. Provided number indicates number of genes per cluster. (I) Time-series cluster analysis of control vs. 28 day recovery myeloid cell response (during WM repopulation) to LPS challenge following 14 day PLX3397 (600 ppm in chow; from H). Clusters showing distinct responses to LPS between control and 28 day WM repopulated cells, across time, were plotted as eigengene values, along with the top represented genes within each cluster. Data are represented as mean ± SEM (n=3–5). *p < 0.05, ** p < 0.01, *** p < 0.001. CP, caudoputamen; CC, corpus callosum; CTX, cortex; LV, lateral ventricle; PirCTX, piriform cortex. Gene expression data can be explored at http://rnaseq.mind.uci.edu/green/alt_repop_lps/gene_search.php.

Figure 8—figure supplement 1. WM repopulating myeloid cells are transcriptionally distinct from homeostatic microglia.

(A) FACS gating strategy for CD11b⁺CD45^int myeloid cells isolated from half brains. (B–C) Quantification of CD45 and CD11b intensities of extracted myeloid cells, respectively. (D–E) Quantification of forward- and side- scatter values of extracted myeloid cells, respectively. (F–G) Two-month-old WT mice treated with vehicle (blue) or PLX3397 (600 ppm in chow) for 7 or 14 days to stimulate GLOBAL (red) and WM (orange) repopulation, respectively. Microglia were isolated via FACS at 28 days recovery, RNA was extracted, and gene expression analysis was performed using RNA-seq. (F) Volcano plots displaying the fold change of genes (log2 scale) and their significance (y axis, -log10 scale) between control vs. GLOBAL repopulation microglia in 28 day recovery mice. (G) Heatmap of identified differentially-expressed genes (DEGs) between control vs. 28 day myeloid cells following 14 days of PLX3397 (600 ppm in chow; from Figure 2F), showing distinct expression patterns in control (blue), WM repopulated cells (orange), and GLOBAL repopulated cells (red). (H) Heatmap of full time-series cluster analysis of control and 28 day recovery cells. Provided number indicates number of genes per cluster. Data are represented as mean with individual data points (n=3).