Comparison of ER-Hoxb8 to BMD macrophages in vitro.

(A) Schematic for creation of bone marrow-derived (BMD) and ER-Hoxb8 cells (B) Brightfield images of BMD and ER-Hoxb8 macrophages plated in the presence of 30ng/mL mouse CSF1 and differentiated for seven days (scale bar = 100um) (C) Dot plot representing CD45/CD11B levels (pre-gated on live, singlet, leukocyte) by flow cytometry (D) Heatmap showing Log2 CPM of canonical macrophage (top) and non-macrophage (bottom) immune cell genes (E) Whole transcriptome comparison between BMD and ER-Hoxb8 macrophages, depicting best fit line and coefficient of determination (one dot = one gene) (F) Volcano plots comparing all genes or those with CPM > 1 (Log2FC >= 2, FDR < 0.05); blue = upregulated in ER-Hoxb8 macrophages, red = upregulated in BMD macrophages

Panel A created with BioRender.com/w42j197.

Engraftment potential of ER-Hoxb8 compared to BMD macrophages after intracranial transplantation in Csf1r−/− hosts.

(A) Schematic for in vivo Csf1r−/− transplant experiments (B) Rendered tile stitches of Csf1r−/− brains after intracranial injection of GFP+ bone marrow (left) or ER-Hoxb8 (right) progenitor cells (C) Percent of total brain area tiled by donor cells; n = five to seven biological replicates per group; each dot = one biological replicate (average area across three matched sagittal sections) (D) Immunostaining of cortical brain region 12-16 days post-intracranial injection (red = IBA1, green = endogenous GFP, blue = DAPI; scale bar = 100um; inset scale bar = 5um) (E) Cortical density calculations (cells per mm2) between groups; n = five to seven biological replicates per group; each dot = one biological replicate (average density across three regions of interest across three matched sagittal sections). All p-values calculated via one-way ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Panel A created with BioRender.com/j85e198.

ER-Hoxb8 macrophages become microglia-like cells (MLCs) after engraftment in the Csf1r−/− brain.

(A) Histogram of TMEM119 surface staining by flow cytometry (pre-gated on live, singlet, leukocyte, CD45+/CD11B+) for brain-engrafted cells 14 days post-intracerebral transplantation; Mg = WT Microglia, BM = BMD MLCs, Hox = ER-Hoxb8 MLCs (B) PCA plot comparing in vitro macrophages from Figure 1 with in vivo macrophages; Mg = WT Microglia, BM = BMD MLCs, Hox = ER-Hoxb8 MLCs (C) Whole transcriptome comparison between WT microglia, BMD, and ER-Hoxb8 macrophages in vivo, depicting best fit line and the coefficient of determination (D) Comparison of microglia signature genes (Cronk, et al. JEM (2018)) depicting best fit line and coefficient of determination (E) In vitro and in vivo Log2 CPM gene expression of ten canonical microglia/myeloid genes for bone marrow (left) and ER-Hoxb8s (right)

Adar1 mutation prevents macrophage-lineage cell expansion and causes interferon induction, rescued by JAKi or Ifih1 mutation.

(A) Schematic of ADAR1 locus, depicting exons, alternative start sites for p150 and p110 isoforms, and sgRNA targets (B) ER-Hoxb8 cell counts over differentiation time course (C) Immunostaining of in vitro, eight-day differentiated macrophages comparing control (NTC) and Adar1 guide-transduced macrophages (red = CD11B, blue = DAPI; scale bar = 100um) (D) PCA plot of progenitors and macrophages in vitro (E) Volcano plots showing differentially expressed genes between Adar1 KO and NTC progenitors and macrophages (CPM > 1, Log2FC >= 2, FDR < 0.05) (F) Heatmap showing the Log2FC (Adar1 KO values over NTC values) for relevant interferon-stimulated genes (G) Heatmap showing Log2 CPM of canonical macrophage (top) and non-macrophage (bottom) immune cell genes (H) Heatmap showing Log2FC (Adar1 KO over NTC expression) for interferon-stimulated genes in macrophages treated with baricitinib (I) ER-Hoxb8 cell counts over differentiation time course, comparing the effect of baricitinib on Adar KO and NTC lines (dosages = 0uM, 0.00064uM, 0.16uM, 0.4uM, and 10uM) (J) Interferon, cytokine and chemokine production after treatment with baricitinib via cytokine bead array (K) ER-Hoxb8 cell counts over differentiation time course, comparing NTC, Adar1 KO, and Adar/Ifih1 double KO (dKO) lines - dotted NTC and Adar1 KO lines are equivalent to those shown in panel (B) (L) Interferon, cytokine and chemokine production via cytokine bead array, comparing NTC, Adar KO, and Adar/Ifih1 dKO lines. All p-values calculated via one-way ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Adar1 mutation prevents ER-Hoxb8 engraftment in the Csf1r−/− mouse, partially rescued by Ifih1 deletion.

(A) Representative rendering of donor cell engraftment (scale bar = 1000um) with inset microscopy of GFP+ donor cell engraftment (green = endogenous GFP, blue = DAPI; scale bar = 100um) for control cells (TLR4 KO and NTC) harvested 7-15 days post-injection (dpi), (B) Adar1 KO (sgRNA #1) cells (harvest details in (E)), (C) Adar1 KO (sgRNA #2) cells harvested 9-12dpi, and (D) Adar1/Ifih1 double KO (dKO) cells harvested 10-15dpi (rendered dots in two right brains enlarged 5x for visualization) (E) Percent of total brain area tiled with cells between groups (numbers denote “n” per group); Adar1 KO (#1) cells include pooled data (brains injected with 300k cells/hemisphere, harvested at 10-15 days post-injection (dpi; n = 3); brains injected with 300k cells/hemisphere, harvested at 4-8dpi (n=8); brains injected with 50k cells/hemisphere, harvested at 13dpi (n = 4); and brains injected with 100k cells/hemisphere pre-treated with 0.5uM Baricitinib, mice treated daily with 1mg/kg Baricitinib, harvested at 5dpi (n = 1)); asterisk indicates samples where engraftment is present but does not meet criteria for tiled brain area, as exemplified in (D); p-values calculated via one-way ANOVA with multiple comparisons

Adar1 D1113H mutant ER-Hoxb8 macrophages persistently drive brain ISG expression.

(A) In vitro ER-Hoxb8 cell counts over time (p-values calculated via one-way ANOVA with multiple comparisons) (B) Multiplex bead array data for interferons, cytokines, and chemokines produced via ER-Hoxb8 macrophages (p-values calculated via one-way ANOVA with multiple comparisons) (C) Sagittal sections of non-transplanted (tamoxifen (tam) sham control) Cx3cr1CreERT; Csf1rfl/fl brains (left) and Adar1 D1113H mutant brains (right) at age P15 and P28-31; nuclei (blue, DAPI), Isg15 (white via RNA in situ hybridization (ISH)); scale bar = 1000um; red arrow depicts location of corresponding closeup images below, showing IBA1 (red, protein stain), Cre (green, ISH), Isg15 (purple, ISH), and nuclei (teal, DAPI); scale bar = 20um; see Supplemental Figure 7A for further corresponding closeup images (D) Sagittal sections of Cx3cr1CreERT; Csf1rfl/fl brains intracranially transplanted with WT ER-Hoxb8s (left) and Adar1 D1113H ER-Hoxb8s (right) at 13 and 27 days post-injection (dpi); nuclei (blue, DAPI), Isg15 (white, ISH); scale bar = 1000um; red arrow depicts location of corresponding closeup images below, showing IBA1 (red, protein stain), Cre (green, ISH), Isg15 (purple, ISH), and nuclei (teal, DAPI); scale bar = 20um; see Supplemental Figure 7A for further corresponding closeup images

Extended comparison of ER-Hoxb8 to BMD macrophages in vitro, relating to Figure 1.

(A) Gating strategy for Figure 1C (B) Flow cytometry histograms and median fluorescence intensity (MFI) of CD11B (left) and CD45 (right), relating to Figure 1C (C) Heatmap showing Log2 CPM gene expression levels of macrophage genes across groups; progenitor and macrophage groups are as represented in Figure 1A, BM Mono = monocytes isolated from BM, 4div ER-Hoxb8 = ER-Hoxb8s collected after four days of in vitro differentiation (D) PCA plot combining bulk RNA sequencing data amongst all groups (E) Log2 CPM values of top 10 differentially expressed genes by Log2FC (CPM > 1, Log2FC >= 2, FDR < 0.05)

Extended comparison of ER-Hoxb8 to BMD macrophages after intracranial transplantation in Csf1r−/− hosts, relating to Figure 3.

(A) Histogram of TMEM119 surface staining by flow cytometry (pre-gated on live, singlet, leukocyte, CD45+/CD11b+) for seven-day differentiated in vitro BMD and ER-Hoxb8 macrophages (B) Gating strategy for in vivo TMEM119 histogram shown in Figure 3A (pre-gated on live, singlet, leukocyte, GFP) of WT microglia and intracranially transplanted (ICT) cells (BM or ER-Hoxb8s) (C) Unsupervised hierarchical cluster dendrogram, related to Figure 3B (distance method = euclidean; cluster method = complete) (D) Comparison of “batch” by PCA plots for harvest days (left), sorter used (middle), and host mouse sex (right) (E) Quantification of GFAP percent area covered; n = 3 biological replicates per group; each dot = one biological replicate (average area across three matched sagittal sections); p-values calculated via one-way ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

CRISPR-Cas9 editing of ER-Hoxb8 progenitors, relating to Figures 4 and 5.

(A) Schematic for Tlr4 knockout using Cas9+/− ER-Hoxb8s and sgRNA viral transduction (B) Post-editing trace decomposition charts (via TIDE Analysis) for Tlr4 KO macrophages (C) Histogram of TLR4 surface staining by flow cytometry (pre-gated on live, singlet, leukocyte) for eight day-differentiated in vitro BMD and ER-Hoxb8 macrophages (D) TLR4 median fluorescence intensity (MFI) normalized to FMO levels using flow cytometry data shown in C (E) TNFa production, comparing control, LPS, and R848 treated samples (eight-day differentiation, 9.5h LPS or R848, 100ng/mL, n = four replicates per condition); p-values calculated via one-way ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Panel A created with BioRender.com/q68t656.

Evidence for gene knockout (KO), Adar1 sgRNA #2 results, and extended bead array data, relating to Figure 4.

(A) Heatmap of Log2 CPM expression of seven causal AGS genes in BMD and ER-Hoxb8 macrophages (B) Post-editing trace decomposition charts (via TIDE Analysis) for Adar1 KO cells targeted with two distinct guides (C) ER-Hoxb8 cell counts over time (D) Immunostaining of in vitro, eight-day differentiated macrophages comparing control (NTC) and Adar1 sgRNA #2-transduced macrophages (red = CD11B, blue = DAPI; scale bar = 100um) (E) Heatmap showing the Log2FC (Adar1 KO values over NTC values) for relevant interferon-stimulated genes for both progenitors and macrophages at baseline (F) Validation of in vitro ER-Hoxb8 cell counts with vehicle or 10uM baricitinib treatment using Adar1 sgRNA #2 (G) Interferon, cytokine and chemokine production for ER-Hoxb8 macrophages grown with or without baricitinib, via cytokine bead array (H) Trace decomposition charts (via TIDE Analysis) for Adar1/Ifih1 double KO (dKO) cell lines (I) Interferon, cytokine and chemokine production for ER-Hoxb8 macrophages with or without subsequent Ifih1 KO. All p-values calculated via one-way ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Adar1 mutation prevents long-term ER-Hoxb8 engraftment in the Cx3cr1CreERT; Csf1rfl/fl mouse, relating to Figure 5.

(A) Schematic for in vivo Cx3cr1CreERT; Csf1rfl/fl transplant experiments (B) Representative rendering of donor cell engraftment (scale bar = 1000um) with inset microscopy of GFP+ donor cell engraftment (green = endogenous GFP, blue = DAPI; scale bar = 100um) for control cells (TLR4 KO, NTC) harvested 15 days post-injection, (C) Adar1 KO cells harvested seven days post-injection, (D) Adar1/Ifih1 double KO (dKO) cells harvested 13-15 days post-injection, and (E) Adar1 KO cells harvested three days post-injection

Panel A created with BioRender.com/n75g136.

Evidence for Cre expression, relating to Figure 6.

(A) Dual immunostaining/RNA in situ hybridization (ISH) showing IBA1 (red, protein stain), Cre (green, ISH), and nuclei (blue, DAPI); scale bar = 100um (B) magnified image of a repopulated microglia from the non-transplanted (tam sham control) Cx3cr1CreERT; Csf1rfl/fl brain shown in (A); scale bar = 10um

Extended Isg15 RNA in situ hybridization (ISH), relating to Figure 6.

(A) Corresponding dual immunostaining/RNA ISH depicted by red arrows in Figures 6C/D, showing IBA1 (red, protein stain), Isg15 (purple, ISH), and nuclei (teal, DAPI); scale bar = 100um; white arrow depicts location of corresponding close up image; scale bar = 20um (B) Representative rendering of donor cell engraftment in the Csf1r−/− brain (scale bar = 1000um) with inset microscopy of IBA1+ donor cell engraftment (red = IBA1, blue = DAPI; scale bar = 100um) for FVB Osb-GFP WT ER-Hoxb8s and Adar1 D1113H mutant ER-Hoxb8s harvested 13-14 days post-injection (C) Dual immunostaining/RNA ISH for samples shown in (B); tile scale bar = 1000um; inset = Isg15 (pink, ISH), nuclei (blue, DAPI); scale bar = 100um