Monocyte Subsets With High Osteoclastogenic Potential and Their Epigenetic Regulation Orchestrated by IRF8

Abstract

Osteoclasts (OCs) are bone-resorbing cells formed by the serial fusion of monocytes. In mice and humans, three distinct subsets of monocytes exist; however, it is unclear if all of them exhibit osteoclastogenic potential. Here we show that in wild-type (WT) mice, Ly6C^hi and Ly6C^int monocytes are the primary source of OC formation when compared to Ly6C^- monocytes. Their osteoclastogenic potential is dictated by increased expression of signaling receptors and activation of preestablished transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of interferon regulatory factor 8 (IRF8), a transcription factor important for myelopoiesis and osteoclastogenesis, all three monocyte subsets are programmed to display higher osteoclastogenic potential. Enhanced NFATc1 nuclear translocation and amplified transcriptomic and epigenetic changes initiated at early developmental stages direct the increased osteoclastogenesis in Irf8-deficient mice. Collectively, our study provides novel insights into the transcription factors and active cis-regulatory elements that regulate OC differentiation. © 2020 American Society for Bone and Mineral Research (ASBMR).

Keywords: BONE MARROW; ChIP-seq; IRF8; LY6C; MACROPHAGE; MONOCYTE; OSTEOCLAST; RANK; RANKL; RNA-seq.

Figure 1.. Characterization of HSCs in Irf8 cKO Mice:

Flow cytometric analysis of major immune cells in BM, blood, and spleen. Pseudocolor plots show cell population in percentages and bar graphs show absolute counts. Data are representative of at least four independent experiments. The data are presented as the mean ± STD (n = 4–5 mice per genotype). One-way ANOVA and post-hoc Tukey’s test was used for comparisons among groups. See also Figure S2 for extensive flow cytometric analysis of HSCs. Cells were gated as described in Supplemental Methods.

Figure 2.. Irf8 cKO Mice Exhibit Increased Osteoclastogenesis:

(A) Micro-CT analysis of femurs (8.5-week-old mice). Top, longitudinal view; middle, axial view of the cortical bone in midshaft; bottom, axial view of the trabecular bone in metaphysis. Scale bar, 0.5 mm. Bar graphs show bone morphometric analysis of femurs. BV/TV, bone volume per tissue volume; Tb.N, trabecular number; Tb. BMD, trabecular bone mineral density. n = 5–6 mice per genotype. (B) Histological analysis of proximal femurs from 8.5-week-old mice (TRAP-stained; red arrows indicate OCs). Scale bar, 1000 μm. n = 5 mice per genotype. (C) Serum CTX-1 levels were measured in 8.5 to 9.5-week-old mice. n = 6–7 mice per genotype. (D) Top panel, TRAP-stained cells show OC formation. Bottom panel shows pit formation ability of OCs. Scale bar, 1000 μm. Bar graphs show quantified number of average cell size of TRAP⁺ cells and percentage area of resorption in each group. (E) RT-qPCR analysis of OC-specific genes in BMMs, Pre-OCs, and OCs. (F) Immunoblot analysis of OC-specific proteins. Day0=BMMs, Day3=PreOCs, and Day6=OCs. (D-F) Data are representative of three independent experiments performed in triplicates. The data are presented as the mean ± STD. One-way ANOVA and post-hoc Tukey’s test was used for comparisons among groups. See also Figure S3 for novel osteoclast transcriptomic results in Irf8 cKO mice.

Figure 3.. Irf8 cKO Mice Retain Development of Circulating Ly6C⁻ Monocytes and All Monocyte Subsets in Irf8 cKO Mice Exhibit Increased Potential for Osteoclast Formation:

(A) Flow cytometric analysis of monocyte subsets in blood, spleen, and BM of WT and Irf8 cKO mice. Pseudocolor plots show cell population in percentages and bar graphs show absolute counts. Data are representative of at least four independent experiments (n = 4–5 mice per genotype). (B) TRAP-stained cells show OC formation potential of monocyte subsets. Data are mean of three independent experiments performed in triplicates. Scale bar, 1000 μm. (C) Pit formation activity of OCs. Data are representative of three independent experiments performed in triplicates. Scale bar, 1000 μm. The data are presented as the mean ± STD. A Student’s t test was used for comparations between two groups, and One-way ANOVA with post-hoc Tukey’s test was used for comparisons among more than two groups.

Figure 4.. Osteoclastogenic Potential of Monocyte Subsets is Regulated by Cell Surface Receptors and Increased Nuclear Translocation of NFATc1 in Irf8 cKO Mice:

(A) Histograms display the expression of cell surface markers in monocyte subsets (without RANKL). Bar graphs show median ± IQR frequencies of at least three independent experiments. (B) mRNA expression of OC-specific genes in distinct subsets. (C) Immunoblot analysis of OC-specific proteins in distinct subsets. (D) Immunoblot analysis of cytoplasmic and nuclear NFATc1 expression at 0 hours and 24 hours after RANKL stimulation. The data are presented as the mean ± STD. One-way ANOVA and post-hoc Tukey’s test was used for comparisons among groups.

Figure 5.. Transcriptional Profiling of Ly6C^hi, Ly6C^int, and Ly6C⁻ BMMs and OCs in Irf8 cKO Mice:

(A) Volcano plot of transcriptomic changes between Irf8 cKO vs WT subsets (one-way ANOVA, >2-fold change, FDR<0.01). red=up, green=down, back=no significant change. (B) Heatmap showing GO term enrichment for genes in each cluster. (C) Gene expression changes for randomly selected osteoclast-specific markers (n=51). Shaded heat map on the right indicates the presence of one or more IRF8 binding sites. (D) GSEA analysis of osteoclast-specific signatures in Irf8 cKO vs WT subsets. See also Figures S4–S5 for detailed transcriptomic analysis.

Figure 6.. Histone Modifications Regulated by IRF8 Deficiency During Osteoclastogenesis:

(A) Heatmap illustrating H3K4me3, H3K4me1, H3K27ac, H3K27me3, and PU.1 binding signal in DEGs between Irf8 cKO vs WT BMMs and OCs. (B) Scatter plots show the correlation between histone modifications and genes either upregulated or downregulated in Irf8 cKO vs WT BMMs and OCs. Gain in histone marks indicated by + and loss of histone marks indicated by –. Data are represented as log₂. (C) Heatmap depicting number of active promoters, active enhancers, and repressors acquired in Irf8 cKO vs WT BMMs and OCs. Numbers in parenthesis indicate percentages. (D) Homer motif analysis of active promoter and active enhancer regions enriched in Irf8 cKO vs WT BMMs. See also Figure S6 for additional histone modification data.

Figure 7.. The Epigenetic Landscape of WT and Irf8 cKO BMMs and OCs:

(A) UCSC Genome Browser tracks showing normalized tag-density profiles at key OC-specific genes. Note the enrichment of H3K4me1 and H3K27ac marks at the Nfatc1 and Ocstamp loci in Irf8 cKO OCs when compared to WT OCs. (B) Homer motif analysis of active promoter and active enhancer regions enriched in Irf8 cKO vs WT OCs. (C) Scatter plots show the correlation between PU.1 binding and genes either upregulated or downregulated in Irf8 cKO vs WT BMMs and OCs. Data are represented as log₂. (D) RNA-seq expression of TFs predicted to bind motifs identified in Figures 6D, 7B and S6E. See also Figures S7 for examples of IGV tracks.