Microglia replacement by ER-Hoxb8 conditionally immortalized macrophages provides insight into Aicardi-Goutières Syndrome neuropathology

Abstract

Microglia, the brain's resident macrophages, can be reconstituted by surrogate cells - a process termed "microglia replacement." To expand the microglia replacement toolkit, we here introduce estrogen-regulated (ER) homeobox B8 (Hoxb8) conditionally immortalized macrophages, a cell model for generation of immune cells from murine bone marrow, as a versatile model for microglia replacement. We find that ER-Hoxb8 macrophages are highly comparable to primary bone marrow-derived (BMD) macrophages in vitro, and, when transplanted into a microglia-free brain, engraft the parenchyma and differentiate into microglia-like cells. Furthermore, ER-Hoxb8 progenitors are readily transducible by virus and easily stored as stable, genetically manipulated cell lines. As a demonstration of this system's power for studying the effects of disease mutations on microglia in vivo, we created stable, Adar1-mutated ER-Hoxb8 lines using CRISPR-Cas9 to study the intrinsic contribution of macrophages to Aicardi-Goutières Syndrome (AGS), an inherited interferonopathy that primarily affects the brain and immune system. We find that Adar1 knockout elicited interferon secretion and impaired macrophage production in vitro, while preventing brain macrophage engraftment in vivo - phenotypes that can be rescued with concurrent mutation of Ifih1 (MDA5) in vitro, but not in vivo. Lastly, we extended these findings by generating ER-Hoxb8 progenitors from mice harboring a patient-specific Adar1 mutation (D1113H). We demonstrated the ability of microglia-specific D1113H mutation to drive interferon production in vivo, suggesting microglia drive AGS neuropathology. In sum, we introduce the ER-Hoxb8 approach to model microglia replacement and use it to clarify macrophage contributions to AGS.

Keywords: Aicardi-Goutières Syndrome; ER-Hoxb8; macrophage; microglia; microglia replacement.

Figure 1:. 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

Figure 2:. 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 < 0.0001 Panel A created with BioRender.com/j85e198

Figure 3:. 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) as compared to unmanipulated in vivo microglia

Figure 4:. 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); statistics calculated at the 9 day time point (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 (L) or two-way (B, I, J, K) ANOVA with multiple comparisons; ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Figure 5:. 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; ns = not significant or p >= 0.05, ***p < 0.001, ****p < 0.0001

Figure 6:. Adar1 D1113H mutant ER-Hoxb8 macrophages persistently drive brain ISG expression.

(A) In vitro ER-Hoxb8 cell counts over time (p-values calculated via two-way ANOVA with multiple comparisons) (B) Multiplex bead array data for interferons, cytokines, and chemokines produced via ER-Hoxb8 macrophages (p-values calculated via independent two-sample t-test) (C) Sagittal sections of non-transplanted (tamoxifen (tam) sham control) Cx3cr1^CreERT; Csf1r^fl/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 Cx3cr1^CreERT; Csf1r^fl/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. All p-values characterized by: ns = not significant or p >= 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001