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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: J Bone Miner Res. 2020 Sep 11;36(1):199–214. doi: 10.1002/jbmr.4165

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

Amitabh Das 1,2, Xiaobei Wang 1,2, Jessica Kang 1, Alyssa Coulter 2, Amol C Shetty 3, Mahesh Bachu 4,5, Stephen R Brooks 6, Stefania Dell’Orso 6, Brian L Foster 7, Xiaoxuan Fan 8, Keiko Ozato 4, Martha J Somerman 2, Vivek Thumbigere-Math 1,2,*
PMCID: PMC8168257  NIHMSID: NIHMS1707355  PMID: 32804442

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 mice, Ly6Chi and Ly6Cint 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 pre-established transcripts, as well as de novo gain in enhancer activity and promoter changes. In the absence of 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.

Keywords: IRF8, monocyte, macrophage, osteoclast, bone marrow, RANK, RANKL, Ly6C, transcription factor, osteoclastogenesis

INTRODUCTION

Osteoclasts (OCs) are multinucleated giant cells that resorb bone and play an important role in the development and remodeling of skeleton. OCs that control fetal skeletal development and tooth eruption originate in fetal ossification centers from embryonic erythroid-myeloid progenitors (EMP)(1). In adults, OC maintenance and function are facilitated by serial fusion of hematopoietic stem cell (HSC)-derived circulating monocytes with long-lived OC syncytia(1).

Monocytes develop in the bone marrow (BM) from MDP-cMOP axis (monocyte and dendritic cell progenitors-common monocyte progenitors), and are subsequently released into circulation(2–4). Under the influence of microenvironmental cues, monocytes get recruited to peripheral tissues where they differentiate into a variety of cells including OCs, macrophages, and dendritic cells (DCs)(5). In humans and mice, functionally two distinct subsets of monocytes exist(6,7). The “inflammatory or classical monocytes” represent Ly6Chi monocytes in mice and CD14hi CD16− monocytes in humans, which can differentiate into M1 macrophages or TipDCs to produce pro-inflammatory cytokines and exhibit antimicrobial activities(3,8–10). In contrast, “nonclassical or patrolling monocytes” represent Ly6C− monocytes in mice and CD14low CD16hi monocytes in humans(11,12), which survey the luminal surfaces of blood vessels for dead cells and can differentiate into M2 macrophages to orchestrate tissue repair. Recent transcriptomic studies have highlighted that heterogeneity exists in mice and human monocytes and have identified a third intermediate population (Ly6Cint and CD14hi CD16low, respectively)(13–15). However, the precise function of this intermediate population is unknown.

Development of Ly6Chi monocytes is tightly regulated by lineage determining transcription factors (TFs) such as Irf8(16), Spi1 (PU.1)(17,18), Ccr2(8), and Klf4(16,19). Whereas, the generation of Ly6C− monocytes is controlled by Nr4a1 (Nur77)(20,21) and C/EBPβ(15,22). The development of Ly6Cint monocytes remains unknown.

Currently, it is unclear if all monocyte subsets exhibit osteoclastogenic potential. To date, limited studies have explored this question and the results are conflicting(23–30). Furthermore, the role of lineage determining TFs in regulating OC differentiation on a genome-wide scale remains poorly understood. IRF8, apart from playing an important role in myelopoiesis(16,31–33), also functions as a negative regulator of OC by inhibiting NFATc1, a master regulator of osteoclastogenesis(34–36). In this study, we utilized a novel Irf8 conditional knockout (Irf8 cKO) mouse model to characterize the importance of IRF8 in OC progenitor development and epigenetic regulation of OC differentiation. We show that Irf8 cKO mice contain severely diminished Ly6Chi monocytes but retain their ability to generate Ly6C− monocytes, and all three monocyte subsets in Irf8 cKO mice exhibit increased potential for OC formation. In contrast, Ly6Chi and Ly6Cint monocytes are the main source of OC formation in wild-type (WT) mice. Ly6Chi and Ly6Cint monocytes were found to express higher levels of RANK and CCR2, indicating that these cells constitute a primed source of pre-OCs. RNA-seq data overlapped with ChIP-seq data showed that the differentiation of Ly6Chi and Ly6Cint monocytes into OCs ensured significant transcriptomic changes specific to each subset, which was accompanied by de novo gain in enhancer activity and promoter changes. In Irf8 cKO mice, the transcriptomic and epigenetic changes are amplified and changes occurring during the progenitor stage may be critical for initiating early lineage commitment and priming monocyte subsets to differentiate into robust OCs. Collectively, our study provides critical insights into the complex network of TFs and active cis-regulatory elements that drive OC differentiation process in WT and Irf8 cKO mice.

MATERIALS AND METHODS

Detailed materials and methods are provided in the supplemental materials and methods section.

RESULTS

Generation of Irf8 cKO Mice and Characterization of HSCs:

Previously, IRF8’s role in osteoclastogenesis has been studied using Irf8 global knock-out (Irf8 gKO) mice(34,35,37). However, the severely altered population and properties of HSCs in Irf8 gKO mice (32,38), along with the development of chronic myelogenous leukemia and splenomegaly, has limited the genome-wide analysis of IRF8 function in OC precursors derived from a monocyte/macrophage lineage. Hence to overcome this limitation, we generated Irf8 cKO mice (Irf8fl/fl; Csf1rcre) by crossing Irf8fl/fl mice(39) with Csf1rcre mice(40) (Figure S1A–H). Deletion of IRF8 was confirmed by the absence of IRF8 transcript and protein expression in BM monocytes (BMMs) of Irf8 cKO mice (Figure S1E–F). The development of HSCs in Irf8 cKO mice was examined by flow cytometric analysis by comparing BM, blood, and spleen cells for expression of cell lineage markers. We observed that Irf8 cKO mice accumulate MDPs and cMoPs and have decreased monocyte and increased neutrophil population (Figures 1A–B and S2A–B). IRF8 is known to enforce monocyte development by opposing neutrophil lineage(41,42). In the absence of IRF8, myeloid progenitors accumulate at the MDP and cMoP stages and fail to generate their downstream population, instead aberrantly giving rise to neutrophils(41,43). In contrast to Irf8 gKO mice, we found that cMoPs and monocytes in Irf8 cKO mice were less severely affected, however the differences were not statistically significant (Figures 1A and S2A, BM cMoPs 0.3 vs 0.4 × 104; BM monocytes 0.21 vs 0.13 × 104; blood monocytes 0.74 vs 0.44 × 103; spleen monocytes 0.34 vs 0.32 × 104 (mean values)).

Figure 1. Characterization of HSCs in Irf8 cKO Mice:

Figure 1.

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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.

Additionally, IRF8 promotes the differentiation of common lymphoid progenitors (CLPs) to B cells(44,45), drives effector differentiation of CD8 T cells(46), and regulates the development of type 1 conventional DCs (cDC1s) and plasmacytoid DCs (pDCs)(33,42,47–49). Consequently, Irf8 gKO mice have reduced B/T-cells, completely lack cDC1s and pDCs, and are immunodeficient. In contrast to Irf8 gKO mice, we found that the development of B- and T-cells, cDCs, and pDCs were less severely affected in Irf8 cKO mice BM, blood, and spleen (Figures 1C–D and S2C–F).

Irf8 cKO Mice Exhibit Increased Osteoclastogenesis and Provide Novel Osteoclast Transcriptomic Results:

IRF8 deficiency in OC precursors but not osteoblasts is known to promote osteoclastogenesis(34,35). Hence, to study the effects of conditional deletion of Irf8 in OC precursors, we analyzed Irf8 cKO mice for abnormal bone and OC phenotypes. We noted that Irf8 cKO mice display severe osteoporosis accompanied by dramatic decreases in total amount of bone (39%), trabecular number (35%), and trabecular BMD (35%) when compared to WT mice (Figure 2A). Histological analysis showed a substantial increase in OC numbers and resorption activity in Irf8 cKO mice (Figure 2B). Increased OC-mediated bone resorption was supported by ~2.5-fold increase in serum C-terminal telopeptides type I collagen (CTX) levels in Irf8 cKO mice (Figure 2C).

Figure 2. Irf8 cKO Mice Exhibit Increased Osteoclastogenesis:

Figure 2.

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(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.

To measure OC formation in vitro, we cultured BMMs from WT and Irf8 cKO mice with M-CSF and RANKL for 6 days. Significantly increased number of OCs formed in Irf8 cKO cultures with cell size ~2-fold larger than WT cells, which was appended by a 3-fold increase in resorption activity (Figure 2D). Correspondingly, the mRNA and protein expression of NFATc1 and its downstream OC-related genes were strongly upregulated in Irf8 cKO OCs when compared to WT OCs (Figure 2E–F). Taken together, these results establish the loss of IRF8 regulatory function in Irf8 cKO mice, and Irf8 cKO mice present OC and skeletal phenotypes similar to Irf8 gKO mice.

Next, we aimed to understand if less altered HSCs or conditional deletion of Irf8 in monocyte/macrophage lineage provide a better model for studying genomewide effects of IRF8 on OC transcriptome. We performed RNA-seq on Irf8 WT, cKO, and gKO BMMs, preosteoclasts (PreOCs), and OCs (Figure S3A). 4726 RANKL-responsive genes were found to be commonly regulated among all groups (Figure S3B) and they clustered into four major categories with different enriched functions (Figure S3C). Transcripts enriched in Irf8 cKO mice were associated with hematopoietic cell differentiation, interferon signaling, chemokine receptor binding, and inflammatory response, suggesting that these functions are operational in Irf8 cKO mice when compared to Irf8 gKO mice (Figure S3D). We noted that a greater number of genes were significantly upregulated in Irf8 cKO OCs when compared to WT and Irf8 gKO OCs (Figure S3E–G). Several novel RANKL-responsive genes were identified in Irf8 cKO OCs that were diminished in Irf8 gKO OCs (61% vs 17%, Figure S3F), which included genes such as Ctsg, Eps8, Prr11, Mpo, Myb, Kif23, and Ccnb2. GSEA analyses comparing cell-specific transcript signatures obtained from Ingenuity Pathway Analysis (IPA) further illustrated that Irf8 cKO mice provide more precise/reliable OC-specific outcomes when compared to Irf8 gKO mice (Figure S3H). Altogether, these findings indicate that Irf8 cKO is an improved model over Irf8 gKO to study OCs, and it provides novel OC transcriptomic results that are deficient in Irf8 gKO mice.

All Monocyte Subsets in Irf8 cKO Mice Exhibit Increased Potential for Osteoclast Formation:

While the total number of monocytes were diminished in Irf8 cKO mice (Figures 1A and S2A), the animals exhibited increased osteoclastogenesis (Figures 2 and S3). This raises the question whether all monocyte subsets are equally diminished in Irf8 cKO mice or if particular monocyte subset development is relatively maintained, and if those subsets have increased potency for OC formation.

Flow cytometric analyses identified that Ly6Chi monocytes were significantly diminished in blood, spleen, and BM of Irf8 cKO mice when compared with WT mice (Figure 3A). Ly6Cint monocytes were also significantly reduced in blood and BM of Irf8 cKO mice, but not in spleen. The counts of Ly6C− monocytes were reduced in BM of Irf8 cKO mice, however their numbers in blood and spleen were not significantly different than WT 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:

Figure 3.

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(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.

Normally in WT mice, ~85% of BM monocytes are Ly6Chi, 6% are Ly6Cint, and 9% are Ly6C− cells(16,21,50). In blood, ~35% of monocytes are Ly6Chi, 20% are Ly6Cint, and 45% are Ly6C− cells. In comparison, we observed that in Irf8 cKO mice the ratios of Ly6C− and Ly6Cint monocytes to Ly6Chi monocytes were significantly higher (Figure 3A). In Irf8 cKO mice, increased number of OCs can be detected as early as 15 days of age (data not shown). We found that the ratios of Ly6C− and Ly6Cint monocytes to Ly6Chi monocytes were significantly higher at 15 days of age in Irf8 cKO BM and blood and remained elevated at every time point measured (15 days, 5 wks, 8.5 wks, 12 wks, data not shown). Altogether, these results indicate that Irf8 cKO mice retain their ability to generate Ly6C− monocytes, but at a reduced efficiency, and most of the circulating monocytes in Irf8 cKO mice are Ly6C− cells.

Next, we investigated the osteoclastogenic potential of Ly6Chi, Ly6Cint, and Ly6C− monocytes in WT and Irf8 cKO mice. Ly6Chi, Ly6Cint, and Ly6C− BM monocytes were isolated by FACS and cultured with M-CSF and RANKL. The sorted monocyte subsets exhibited varying phenotypic plasticity and differentiated into mature OCs much sooner than unsorted cells (4 days vs 6 days). In WT mice, Ly6Chi and Ly6Cint but not Ly6C− monocytes exhibited increased potential for OC formation and resorption activity (Figure 3B–C). In stark contrast, all monocyte subsets in Irf8 cKO mice robustly differentiated into mature OCs and the differences against respective WT subsets were-Ly6Chi = 1-fold, Ly6Cint = 2-fold, and Ly6C− = 6-fold (Figure 3B). Accordingly, Ly6Chi (1-fold), Ly6Cint (2-fold), and Ly6C− (9-fold) OCs from Irf8 cKO mice demonstrated significantly increased resorption activity when compared to respective WT subsets (Figure 3C). Altogether, these findings imply that IRF8 deficiency not only affects the development of selective monocyte subsets, but also promotes the osteoclastogenic potential of all three monocyte subsets.

Molecular Mechanisms Regulating the Osteoclastogenesis Potential of Monocyte Subsets in WT and Irf8 cKO mice:

To understand the plausible mechanisms shaping the varying osteoclastogenic potential of Ly6Chi, Ly6Cint, and Ly6C− monocytes in WT and Irf8 cKO mice, we examined the expression of cell surface receptors involved in OC signaling. We assessed the expression of RANK, CCR2, and CX3CR1 in BMMs by flow cytometry. In both WT and Irf8 cKO mice, we noted that Ly6Chi monocytes expressed high levels of RANK, Ly6Cint monocytes expressed moderate levels of RANK, and Ly6C− monocytes expressed low levels of RANK (Figure 4A). The expression of CCR2 followed a pattern similar to RANK in both WT and Irf8 cKO subsets. The expression of CX3CR1 was inverse to RANK and CCR2. These results indicate that the expression level of RANK, CCR2, and CX3CR1 may regulate the activation of OC signaling pathways and OC formation potential of distinct monocyte subsets. Indeed, in WT mice, Ly6Chi monocytes that express high levels of RANK and CCR2 differentiated into robust OCs, Ly6Cint monocytes that express moderate levels of RANK and CCR2 formed moderate OCs, and Ly6C− monocytes that express low levels of RANK and CCR2 formed minimal OCs (Figure 3B–C). These findings were corroborated by mRNA and protein expression of OC-related genes that correlated with the osteoclastogenic potential of Ly6Chi, Ly6Cint, and Ly6C− subsets (Figure 4B–C). Furthermore, in Ly6Chi cells, IRF8 is completely inhibited following RANKL stimulation, which leads to induction of NFATc1 and CTSK and subsequent OC formation. However, in Ly6C− cells, IRF8 does not get completely inhibited following RANKL stimulation, which prevents induction of NFATc1 and CTSK and subsequent OC formation in Ly6C− group.

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

Figure 4.

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(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.

While the expression of RANK, CCR2, and CX3CR1 in Irf8 cKO subsets were similar to WT mice, comparatively all monocyte subsets in Irf8 cKO mice formed increased OCs and the mRNA and protein expression of OC-related genes were strongly upregulated in Irf8 cKO OCs when compared to WT OCs. In depth analysis indicated that in Irf8 cKO mice, there is increased nuclear translocation of NFATc1 within 24 hours of RANKL stimulation (Figure 4D). The IAD domain of IRF8 is known to physically interact with the TAD-A domain of NFATc1 and inhibit NFATc1 nuclear translocation(34,51). In Irf8 cKO mice, the early release of NFATc1 from inhibition by IRF8 may enable higher auto-amplification of NFATc1, thus resulting in strong induction of NFATc1 expression and enhanced activation of downstream target genes that promote OC formation. Collectively, these results suggest that the osteoclastogenic potential of monocyte subsets may be regulated by the expression level of OC signaling receptors, and further influenced by IRF8-mediated NFATc1 nuclear translocation.

Transcriptional Program Governing the Osteoclastogenic Potential of Ly6Chi, Ly6Cint, and Ly6C− Monocytes Subsets in WT and Irf8 cKO mice:

To identify global transcriptional program governing the osteoclastogenic potential of Ly6Chi, Ly6Cint, and Ly6C− monocytes, and how it may be further influenced by IRF8 deficiency, we performed RNA-seq on sorted Ly6Chi, Ly6Cint, and Ly6C− cells from WT and Irf8 cKO mice. Cells were analyzed prior to (BMMs) and 4 days after RANKL stimulation (OCs) (Figure S4A).

During the course of OC differentiation, 4833 RANKL-responsive genes (FDR <0.01, >2-fold difference in any pairwise comparison among a total of 38,942 genes) were commonly regulated between WT and Irf8 cKO subsets (Figure S4B). Hierarchical clustering classified the genes into four major categories with different enriched functions (Figure S4B–C). Cluster I consisted of genes primarily upregulated in WT BMMs and were associated with type 1 IFN signaling, cytokine signaling, and innate immune response. Cluster II comprised of genes co-expressed by WT and Irf8 cKO BMMs, and were correlated with cellular response to cytokine stimulus, extracellular matrix organization, and neutrophil mediated immunity. Cluster III was defined by genes predominantly upregulated in Irf8 cKO BMMs and were associated with DNA replication and cell cycle pathway. Cluster IV was characterized by genes upregulated in both WT and Irf8 cKO OCs and comprised of genes involved in OC regulation such as Nfatc1, Ctsk, Oscar, Dcstamp, Acp5, Dnmt3a, and Calcr, etc. Cluster IV gene expression was prominent in Ly6Chi and Ly6Cint OCs in both genotypes, moderate in Irf8 cKO Ly6C− OCs, and diminished in WT Ly6C− OCs.

Amenable to its name as inflammatory monocytes, Ly6Chi BMMs exhibited distinct inflammatory signature when compared to Ly6C− BMMs, but also displayed augmented genes important for OC differentiation (Figure S4D). Similar findings were noted in Ly6Cint BMMs. These results indicate that the developmentally established transcriptome of Ly6Chi and Ly6Cint monocytes is likely a feature of committed OC precursors, thus predisposing them to respond intensely to RANKL stimulation (Figures S5A–B). This notion was further apparent when RANKL-stimulated Ly6Chi, Ly6Cint, and Ly6C− OCs were compared against their respective BMMs (Figure S5C). In WT mice, Ly6Chi OCs were markedly enriched for genes involved in OC differentiation and oxidative phosphorylation, Ly6Cint OCs were enriched for genes involved in DNA replication and RNA transport, and Ly6C− OCs were enriched for genes involved in chemokine signaling and cell adhesion (Figure S5D). In contrast, all three subset OCs in Irf8 cKO mice were overrepresented by genes involved in OC signaling, such as MAPK and PI3K-Akt signaling in Ly6Chi, TCA cycle and pyruvate metabolism in Ly6Cint, and PI3K-Akt-mTOR and EGFR1 signaling in Ly6C− (Figure S5D).

Subsequent direct comparison of Irf8 cKO vs WT subsets identified a greater number of genes important for OC differentiation were significantly regulated in all three subsets of Irf8 cKO BMMs and OCs (Figure 5A–B). Notably, in Irf8 cKO Ly6Chi, Ly6Cint, and Ly6C− BMMs, the basal expression of many established positive regulators of osteoclastogenesis were strongly enriched and negative regulators were diminished (Figure 5A). These results imply that genetic ablation of Irf8 initiates the OC differentiation program and lineage commitment of all three monocyte subsets at a very early stage. Consistent with BMMs, Irf8 cKO subset OCs further strongly expressed several positive regulators (e.g., Nfatc1, Acp5, Ctsk, Calcr, Mmp9, Dcstamp) of OC differentiation when compared to WT OCs (Figure 5A, C). Many of the differentially expressed genes (DEGs) contained IRF8 binding sites, suggesting that they may be direct transcriptional targets of IRF8.

Figure 5. Transcriptional Profiling of Ly6Chi, Ly6Cint, and Ly6C− BMMs and OCs in Irf8 cKO Mice:

Figure 5.

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(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.

Further, to investigate the effect of Irf8 deficiency on ontogeny and activity of OCs and OC precursors, we performed GSEA analysis, which showed strong enrichment of OC-specific signatures in Irf8 cKO Ly6Chi, Ly6Cint, and Ly6C− BMMs and OCs when compared to WT cells (Figure 5D). Collectively, these results corroborate the findings noted in Figure 4 and demonstrate that WT Ly6Chi and Ly6Cint monocytes developmentally contain OC-specific transcripts, which are augmented upon RANKL stimulation. Additionally, IRF8 deficiency initiates the OC differentiation program and lineage commitment at a very early stage. In Irf8 cKO mice, OC-specific transcripts in all three monocyte subsets are primed to robustly respond to RANKL stimulation and various OC signaling pathways are activated in each subset.

Induction of Active cis-Regulatory Elements Critical for Osteoclast Differentiation:

Currently, the epigenetic mechanisms governing the OC differentiation process remains poorly understood. To investigate the combinatorial activity of TFs, cis-regulatory elements, and the enhancer landscape that establish and maintain OC transcriptional identity, we profiled histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) and PU.1 and IRF8 binding by ChIP-seq. RNA-seq data from sorted Ly6Chi, Ly6Cint, and Ly6C− cells were overlaid with ChIP-seq data to identify epigenetic changes specific to each subset and genotype. Active promoters were identified according to proximity to transcription start sites (TSS) (<2 kb) and H3K4me3, while active enhancers were defined by their distance from TSS (>2 kb) and enrichment of both H3K4me1 and H3K27ac. Transcriptional repressors were identified by their distance from TSS (<2 kb) and enrichment of H3K27me3.

RNA-seq data indicated that majority (88%–90%) of the RANKL-responsive genes in WT monocytes had IRF8 occupancy (Figure S6A). Analysis of H3K4me3 marks for these IRF8 dependent genes (Figure S6B) identified several promoters that were either established (13%–24%) or lost (30%–37%) during the course of OC differentiation in WT Ly6Chi, Ly6Cint, and Ly6C− OCs (Figure S6C). Among them, 2–5% were active promoters that were established de novo (Figure S6D) and included OC-specific genes such as Ly6Chi - Ccr1, Myo1e, Angpt2, Prr11, Mt1; Ly6Cint - Ccr1, Angpt2, Prr11, Cenpf, Mt1; and Ly6C− - Mmp9, Mmp14, Myo1b, Mefv, Creg2. Subsequently, when H3K4me3 signal was analyzed for DEGs between Irf8 cKO vs WT mice (Figure 6A), we observed that Irf8 cKO BMMs (10%–38%) and OCs (17%–21%) had gained increased promoters compared to their respective WT cells (Figure 6B). Of the established promoters, 4–5% were active promoters specific to Irf8 cKO BMMs (Figure 6C) and included OC-related genes such as Ly6Chi - Elane, Trem3, Serpinb1a, Ly6c2, Myb, Rasgrp3; Ly6Cint - Elane, Phgdh, Rasgrp3, Dmd, Trem3, Myb; and Ly6C− - Rasgrp3, Siglec-E, Clec12a. Likewise, 6–10% were active promoters specific to Irf8 cKO OCs (Figure 6C) and included genes such as Ly6Chi - Dkk2, Cd4, Ffar4, Ltb, Cd69; Ly6Cint - Dkk2, Mpo, Elane, Camp, Mmp8, Siglec-E; and Ly6C− - Myl9, Itga11, Dlx3, Il34, Mmp8.

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

Figure 6.

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(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.

Analysis of H3K4me1 marks for IRF8-dependent DEGs identified de novo enhancer acquisitions in WT subset OCs (19%–31%) (Figure S6C). These gained enhancers were located in the vicinity of genes such as Nfatc1, Calcr, Ctsk, Oscar, Dcstamp, Dnmt3a, Vegfc, and Relb, which positively regulate OCs (Figures 7A and S7A). During BMM to OC differentiation, 23%–31% of the genes lost enhancer marks (Figure S6C) and they included mainly negative regulators of OCs such as Bcl6, Bcl2, Mafb, Irf4, Irf7, Cx3cr1, Nr4a1, and Klf6 (Figure S7B). Subsequently, when H3K4me1 signal was analyzed for DEGs between Irf8 cKO vs WT mice, we noted that Irf8 cKO BMMs and OCs had either acquired (BMMs=6%–18%, OCs=14%–17%) or lost (BMMs=14%–32%, OCs=32%–42%) more enhancers when compared to WT cells (Figure 6B). The gained enhancers in BMMs were located in the vicinity of OC-related genes such as Spp1, Atp6v0d2, Prr11, Mpo, Angpt2, Ccl3, Mmp12, and Msr1. The gained enhancers in OCs were located in the vicinity of genes such as Csf1, Calcr, Trem3, Ccr2, Il1r2, Atp6v0d2, Nfkbia, Cxcr2, Notch1, and Rasgrp3. To validate these results, we analyzed H3K27ac signal in WT and Irf8 cKO subsets. H3K27ac signal in enhancers followed a pattern similar to H3K4me1 (Figures S6C and 6B), corroborating that the establishment of enhancers in the vicinity of OC-specific genes is essential for enrichment of OC transcription (Figures 7A and S7A). Prominent changes were evident when H3K4me1 and H3K27ac marks were overlapped to identify active enhancers. Active enhancers were increasingly noted at BMM stage in Irf8 cKO Ly6Chi and Ly6Cint cells, and in contrast noted at OC stage in Irf8 cKO Ly6C− monocytes (Figure 6C). These active enhancer findings match with transcriptomic results (Figure 5A), indicating that in the absence of IRF8, enhancer establishment occurring during the progenitor stage may be critical for priming monocytes to differentiate into robust OCs, which is supported by the increased OC phenotype noted in Ly6Chi and Ly6Cint cells when compared to Ly6C− monocytes.

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

Figure 7.

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(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.

While H3K27ac is associated with active transcription, deposition of H3K27me3 by the polycomb repressive complex (PRC)2 is associated with chromatin-based gene silencing(52). To examine if IRF8 regulates OC-specific genes by methylation of H3K27 around the TSS, we assessed H3K27me3 marks. A global increase in H3K27me3 intensity was observed among the downregulated genes in both WT and Irf8 cKO subsets (Figure S6C–D and Figure 6B–C). Among the positive regulators of OCs, H3K27me3 intensity was significantly decreased at the OC stage when compared to their respective BMMs stage (Figures 7A and S7A). The concordant loss of H3K27me3 was associated with gain in promoters and enhancers and enrichment of transcription. In contrast, among the negative regulators of OCs, H3K27me3 intensity was significantly increased at the OC stage, which was associated with transcriptional repression (Figure S7B). All of these differences were stark in Irf8 cKO BMMs and OCs when compared to WT cells.

To identify potential TFs responsible for transcriptome and epigenome regulation during OC differentiation, we performed motif analysis within the identified active promoter and active enhancer regions. We identified both previously known and novel candidate regulators in WT and Irf8 cKO Ly6Chi, Ly6Cint, or Ly6C− BMMs and OCs (Figures S6E, 6D and 7B). Most of these TFs have known functions in OC differentiation, and are potential targets of IRF8 and PU.1. Previously, it has been reported that during osteoclastogenesis, PU.1 switches its transcription partner from IRF8 to NFATc1 and alters the chromatin binding regions, which is associated with changes in epigenetic profiles and gene expression(37). Therefore, we investigated the relationship between IRF8 and PU.1 binding sites in BMMs and OCs. Among the IRF8-dependent DEGs in WT subsets, 90% of them contained PU.1 binding sites (Figure S6C), highlighting the significant overlap between IRF8 and PU.1 binding sites in BMMs. The majority of PU.1 binding regions were shared between BMMs and OCs. Among the DEGs between Irf8 cKO vs WT mice, only a small fraction displayed enhanced PU.1 binding sites in Irf8 cKO subsets (Figure 7C). PU.1 peaks in OCs were enriched for histone marks in both WT and Irf8 cKO subsets. Figures 7A and S7 are representative of ChIP-seq data showing the co-localization of PU.1 with IRF8 and promoter, enhancer, and repressor marks in BMMs and OCs.

Subsequently, using RNA-seq data we compared predicted motif enrichment with TF expression in Ly6Chi, Ly6Cint, and Ly6C− subsets (Figure 7D). PU.1 and NFAT family of TFs were prominently expressed in OCs, confirming the requirement of these TFs for OC differentiation. In contrast, IRF8 and KLF family of TFs, which are known negative regulators of OC differentiation were decreased. Altogether, these data suggest that the Ly6Chi and Ly6Cint monocytes are transcriptionally and epigenetically programmed to differentiate in mature OCs when compared to Ly6C− anti-inflammatory monocytes. A combinatorial activity of TFs and cis-regulatory elements in the vicinity of several OC-specific genes are responsible for establishing and maintaining OC identity in these subsets. In Irf8 cKO mice, several promoters and enhancers established during the progenitor stage may be critical for initiating early lineage commitment and priming monocyte subsets to differentiate into robust OCs. In Irf8 cKO mice, while amplified transcriptomic and epigenetic changes are noted in well-known OC-specific genes, the stark differences against WT OCs are more visible in the lesser known OC-specific genes.

DISCUSSION

OCs are the only known bone resorbing cells in the body. Defective OC function leads to osteopetrosis, whereas, excessive OC activity leads to bone loss and osteoporosis. Understanding the molecular mechanisms that control OC development and function is vital for developing effective intervention and therapeutic strategies for OC-related disorders.

Previous attempts to gain an in-depth understanding of IRF8’s role in osteoclastogenesis have been limited by the extremely altered HSCs in Irf8 gKO mice(32,38). The Irf8 cKO mice generated in this study provide an improved mouse model as they exhibit less severely altered HSCs, but present OC and skeletal phenotypes similar to Irf8 gKO mice. Furthermore, the Irf8 cKO mice provide more precise and novel OC transcriptomic results when compared to Irf8 gKO mice. These results are in stark contrast to a previous study, which failed to notice in vivo skeletal or OC phenotypes in Irf8fl/fl; Lyz2cre mice, but observed increased TRAP+ OCs in vitro(53). It is unclear if the Irf8fl/fl; Lyz2cre mice in their study harbored extremely altered HSCs. These differences compared to our study could be related to different “macrophage targeting” Cre lines used and the efficacy and specificity in cell deletion(54). While our findings illustrate effective deletion of Irf8 in the monocyte/macrophage lineage of Irf8 cKO mice, the possibility that minor non-specific targeting of other immune cells could have occurred cannot be ruled out.

During myelopoiesis, IRF8 is sharply expressed at the MDP stage, and IRF8 interacts with C/EBPα to restrain MDPs and cMoPs from differentiating into neutrophils(41). IRF8 in turn binds to KLF4 to govern the differentiation of MDPs and cMoPs into monocytes(16). Consistent with the loss of IRF8 function, we noted that Irf8 cKO mice accumulate myeloid progenitors at cMoP stage and fail to effectively generate downstream monocytes, instead aberrantly giving rise to neutrophils. In-depth analysis indicated that Ly6Chi monocytes were severely diminished in Irf8 cKO mice, whereas, the generation of Ly6C− monocytes was relatively maintained. Developmentally, Ly6C− monocytes are considered to be derived from Ly6Chi monocytes(55,56). However, under certain circumstances Ly6C− monocytes can arise directly from MDPs or cMoPs independently of Ly6Chi monocytes(2,57). Perhaps, this explains the existence of Ly6C− monocytes in Irf8 cKO mice despite the absence of Ly6Chi monocytes. The existence of Ly6C− monocytes could be further explained by the fact that IRF8 is more essential for the development of Ly6C+ cells than Ly6C− monocytes(16).

As monocytes contribute to OC formation, it is expected that all monocyte subsets would exhibit osteoclastogenic potential. However, to date, only few studies have explored this subject and the results are conflicting. The osteoclastogenic potential of Ly6Cint monocytes has never been studied. Some groups have shown that in BM, CD11b−/low Ly6Chi progenitors (i.e., cMoPs) rather than CD11b+ Ly6Chi monocytes exhibit increased potential for OC formation(23,24). This is supported by the fact that CD11b and β2-integrin signaling negatively regulates OC differentiation by transiently repressing RANK expression and Nfatc1 transcription(26). In contrast, other studies have shown that CD11b+ Ly6Chi monocytes in BM and blood are far more efficient than Ly6C− monocytes in differentiating into mature OCs(25,27,28). In humans, it is firmly established that CD14hi CD16– classical monocytes but not nonclassical monocytes are the main source of OC formation(58,59). In agreement with the latter group of findings, we explicitly demonstrate that CD11b+ Ly6Chi and Ly6Cint monocytes in WT mice are transcriptionally and epigenetically programmed to differentiate in mature OCs when compared to Ly6C− monocytes. It is unclear if Ly6Chi, Ly6Cint, and Ly6C− monocytes exhibit varying osteoclastogenic potential in vitro vs in vivo, and in physiologic vs pathologic conditions. In inflammatory arthritis in mice, depending upon the experimental models used, previous studies have reported that either Ly6C− (29,30) or Ly6Chi (25,27) monocytes specifically migrate to the inflamed joints and contribute to bone erosion. Identifying specific subsets driving tissue destruction in a disease state is extremely critical for developing targeted anti-OC therapy.

Mechanistically, our findings suggest that the osteoclastogenic potential of monocyte subsets is regulated by OC signaling receptors such as RANK, CCR2 and CX3CR1. The binding of RANK receptor to its ligand RANKL triggers distinct signaling cascades and induces TFs such as NFATc1, c-Fos, and NF-κB to promote OC formation(60). Activation of CCR2 in OC precursors leads to increased RANK expression and predisposes cells to differentiate into mature OCs(61). CX3CR1 negatively correlate with Ly6C expression(50) and favors the maintenance of osteoclastic precursors, but not differentiated osteoclasts(62,63). It has been reported that increased expression of RANK is likely a feature of committed OC precursors, and human monocytes expressing high levels of RANK are predisposed to produce two-fold increased OCs when compared to cells expressing moderate or low levels of RANK(64). Consistent with these findings, we noted that Ly6Chi monocytes that expressed higher levels of RANK and CCR2 formed increased OCs when compared to Ly6Cint and Ly6C– monocytes in WT mice. Further in support of CCR2 influencing the osteoclastogenic potential of Ly6C+ monocytes, in vivo delivery of anti-CCR2 antibody (MC21) has been shown to selectively deplete Ly6Chi monocytes and inhibit OC formation and bone destruction(27). CX3CR1− OCs are known to exhibit higher inflammatory bone resorption(65), which is in agreement with reduced CX3CR1 noted in Ly6Chi subset. Collectively, our mechanistic studies provide critical insight into why Ly6Chi and Ly6Cint, but not Ly6C− monocytes exhibit osteoclastogenesis potential, and how it may be further augmented by increased NFATc1 nuclear translocation in the absence of IRF8.

Normally, TFs bind to promoters near TSS and distal enhancers to regulate chromatin signature and gene expression(66,67). Studying the states of active promoters and active enhancers is critical for understanding how gene expression patterns are established by TFs during cell differentiation. To date, the genomewide mapping of active cis-regulatory elements involved in OC differentiation has been very limited(37,68–70). In the present study, by overlapping RNA-seq data with ChIP-seq data, we identified cis-regulatory regions that are active during OC differentiation and are specific to each subset and genotype. During the course of BMMs to OC differentiation, several promoters and enhancers were either established or lost in WT and Irf8 cKO cells. The distribution and enrichment of promoters and enhancers were highly specific to the differentiation stage and varied between WT and Irf8 cKO BMMs and OCs. The de novo promoter and enhancer acquisitions in the vicinity of OC-specific genes integrated with H3K27me3-mediated transcriptional repression is extremely critical for establishing and maintaining OC transcriptional identity. We explicitly demonstrate that in a steady state, Ly6Chi and Ly6Cint monocytes are the OC forming cells and their osteoclastogenic potential is dictated by activation of pre-established transcripts, as well as de novo gain in enhancer activity and promoter changes. IRF8 deficiency further augments these transcriptomic and epigenetic changes in Irf8 cKO Ly6Chi and Ly6Cint subsets at an early developmental stage, priming them to display higher osteoclastogenic potential when compared to their WT counterparts. Ly6C− cells do not display osteoclastogenic potential in WT mice, however, in the absence of IRF8, active enhancers and transcriptional enrichment are established later on during the course of OC differentiation that shape their OC formation potential in Irf8 cKO mice. In Irf8 cKO mice, the gain in enhancers and promoters were noted in well-known OC-specific genes, however, the differences against WT cells were more visible in the lesser known OC-specific genes. These lesser known OC-specific genes are the unique features of Irf8 cKO when compared to Irf8 gKO mice.

DNA motif analysis identified several known and novel transcriptional regulators in WT and Irf8 cKO BMMs and OCs. Most of these TFs have known functions in OC differentiation, and are potential targets of IRF8 and PU.1. While the majority of PU.1 binding regions were shared between BMMs and OCs, it has been reported that PU.1 heterodimerizes with IRF8 to regulate BMM development(71), and switches its partner to MITF and NFATc1 to control OC differentiation(37,68). Similarly, we noted that among the DEGs in Ly6Chi, Ly6Cint, and Ly6C− BMMs, there was a significant overlap between IRF8 and PU.1 binding sites. It is anticipated that these identified PU.1 binding regions will be overlapped with NFATc1 binding sites in OCs. Despite repeated attempts, we were unable to obtain successful NFATc1 ChIP-seq results due to the lack of commercially available ChIP-grade NFATc1 antibody. Nevertheless, our results are in agreement with previous observations by Izawa et al, which showed that RANKL induced downregulation of Irf8 and upregulation of Nfatc1 is associated with significant histone modifications(37). Comparatively, the study by Izawa et al. had few shortcomings as ChIP-seq experiments did not include Irf8 deficient mice (except for H3K27ac) or appropriate input controls. Our study overcomes these shortcomings and provides significant new information about the important epigenetic and transcriptomic changes occurring at early developmental stages that prime the Irf8 cKO progenitors to differentiate into robust OCs.

In summary, to the best of our knowledge, this is the first study to investigate the osteoclastogenic potential of monocyte subsets in a novel Irf8 cKO mice and characterize the epigenetic mechanisms governing OC differentiation process in distinct monocyte subsets. Our study is a shift from the conventional approach of studying monocytes as a whole population and provides evidence of OC heterogeneity. Our study demonstrates the need for OC research to focus on individual monocyte/OC subsets in order to develop targeted therapy for OC-related disorders. Future studies should identify specific subsets driving tissue destruction in a disease state and how inflammatory cytokines may affect the osteoclastogenic potential of distinct monocyte subsets in the context of IRF8 deficiency. Also, it is important to determine whether estrogen deficiency, aging, or stress affect the osteoclastogenic potential of monocyte subsets and how it could be further impacted by IRF8 deficiency. While recent evidence indicate that aging and menopause can reprogram osteoclast precursors to exhibit aggressive bone resorption, the underlying transcriptomic and epigenetic mechanisms remains to be thoroughly investigated. This study provides critical insights into the important role of IRF8 in osteoclastogenesis and paves the way for future studies to address critical knowledge gap in OC research. Effective inhibition of IRF8 by molecules that target chromatin regulators may offer promising therapeutic approaches.

Supplementary Material

Supplementary Material

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
7-AAD BioLegend Cat# 420403
Anti-CD115 APC (eBio) (clone AFS98) eBioscience Cat# 17115282
Anti-CD117 PE-Cy7 (clone 2B8) BioLegend Cat# 105813
Anti-CD11b Brilliant Violet 421(clone M1/70) BioLegend Cat# 101236
Anti-CD11b APC-Cy7 (clone M1/70) BioLegend Cat# 101226
Anti-CD11c AF532 (clone N418) eBioscience Cat# 58011480
Anti-CD135 (Flt-3) PE-Cy5 (clone A2F10) BioLegend Cat# 135312
Anti-CD19 PE (clone 6D5) BioLegend Cat# 115507
Anti-CD19 Alexa Fluor 488 (clone CD5) BioLegend Cat# 115521
Anti-CD3 PE (clone 17A2) BioLegend Cat# 100205
Anti-CD3 APC (clone 17A2) BioLegend Cat# 100235
Anti-CD45 PE (clone I3/2.3) BioLegend Cat# 147712
Anti-CD45 Alexa Fluor 700 (clone 30-F11) eBioscience Cat# 56045180
Anti-CD45R (B220) PE (clone RA3–6B2) BioLegend Cat# 103208
Anti-CD45R (B220) Alexa Fluor 488 (clone RA3–6B2) eBioscience Cat# 53045280
Anti-IRF8 PerCP-eFluor 710 (clone V3GYWCH) eBioscience Cat# 46985280
Anti-Ly-6C eFluor 450 (clone HK1.4) eBioscience Cat# 48593282
Anti-Ly-6G PE-Cy7 (clone 1A8) BioLegend Cat# 127618
Anti-Ly-6G PE (clone 1A8) BD Biosciences Cat# 561104
Anti-MHC II (I-Ab) Alexa Fluor 488 (clone AF6 120.1) BioLegend Cat# 116410
Anti-PDCA-1 APC (clone ebio927) eBioscience Cat# 17317280
Anti-SiglecH PE-Cy7 (clone ebio440c) eBioscience Cat# 25033380
Anti-TER-119 PE (clone TER-119) BioLegend Cat# 116208
Anti-TER-119 PE-Dazzle 594 (clone TER-119) BioLegend Cat# 116243
Anti-biotin Microbeads Anti-TER-119 PE-Dazzle 594 Miltenyi Cat# 130100629
Anti-DC-STAMP antibody Millipore Cat# MABF39-I
Anti-NFAT2 antibody Abcam Cat# ab25916
Anti-Cathepsin K antibody Abcam Cat# ab19027
Anti-IRF8 antibody ThermoFischer Cat# 39–8800
Anti-beta Actin antibody Abcam Cat# ab8226
Anti-Rabbit IgG H&L antibody Abcam Cat# ab6721
Anti-Mouse IgG H&L antibody Abcam Cat# ab6728
Anti-H3K27ac antibody Abcam Cat# ab4729
Anti-Histone H3 (mono methyl K4) antibody Abcam Cat# ab8895
Anti-Histone H3 (tri methyl K4) antibody Abcam Cat# ab8580
Anti-Histone H3 (tri methyl K27) antibody Millipore Cat# 07–449
Anti-PU.1 antibody Laboratory of Michael Ostrowski (Carey et al., 2018)
Chemicals, Peptides, and Recombinant Proteins
Dynabeads Oligo(dT)25 ThermoFischer Cat# 61105
Dynabeads Protein G ThermoFischer Cat# 10004D
cOmplete, EDTA-free Protease Inhibitor Cocktail Sigma-Aldrich Cat# 11873580001
Proteinase K Solution ThermoFischer Cat# AM2546
High-Capacity cDNA Reverse Transcription Kit ThermoFischer Cat# 4374967
SYBR Green PCR Master Mix ThermoFischer Cat# 4309155
TRIzol Reagent ThermoFischer Cat# 15596026
ACK Lysing Buffer Quality Biological Cat# 118-156-101
Halt Protease Inhibitor Cocktail, EDTA-Free ThermoFischer Cat# 78425
Recombinant Murine M-CSF R&D systems Cat# 416-ML/CF
Recombinant Murine RANKL R&D systems Cat# 462-TEC/CF
Critical Commercial Assays
NEBNext Poly(A) mRNA Magnetic Isolation kit NEB Cat# E7490
NEBNext Ultra II Directional RNA Library Prep kit NEB Cat# E7760
NEBNext Multiplex oligos for Illumina NEB Cat# E7335
NEBNext Ultra II DNA library preparation kit NEB Cat# E7645
QIAquick PCR Purification Kit Qiagen Cat# 28104
RNeasy Mini Kit Qiagen Cat# 74104
Monocyte Isolation Kit (BM), mouse Miltenyi Cat# 130-100-629
RatLaps (CTX-I) EIA immunodiagnostic systems Cat# AC-06F1
High Sensitivity DNA ScreenTape Analysis (D1000) Agilent Cat# 5067–5587
Deposited Data
RNA-seq and ChIP-seq GEO GSE151483
Experimental models: Organisms/Strains
Mouse: Irf8fl/fl Laboratory of Keiko Ozato and Herbert Morse (Feng et al., 2011)
Mouse: Csf1rcre Jackson Laboratory Jax Stock# 021024; (Deng et al., 2010)
Mouse: Irf8 gKO and Irf8 gWT Laboratory of Keiko Ozato and Herbert Morse (Tamura and Ozato, 2002; Thumbigere-Math et al., 2019)
Mouse: Irf8 cWT (Irf8fl; Csf1rcre/-) Generated in this study N/A
Mouse: Irf8 cKO (Irf8fl/fl; Csf1rcre/+) Generated in this study N/A
Oligonucleotides
Primers for qPCR, see Table S1 This paper N/A
Software and Algorithms
GraphPad Prism 8 (version 8.0.1) GraphPad Software, Inc., Carlifornia https://www.graphpad.com
ImageJ ImageJ https://imagej.nih.gov/ij/
AnalyzePro 1.0 AnalyzeDirect https://analyzedirect.com
FCS Express 6 De Novo Software https://denovosoftware.com
Bowtie2 aligner version 2.4.0 Langmead and Salzberg, 2012 http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
HOMER Heinz et al., 2010 http://homer.ucsd.edu/homer/introduction/install.html
GSEA Broad Institute https://www.gsea-msigdb.org/gsea/index.jsp
R package: DESeq2 Anders and Huber, 2010 https://bioconductor.org/packages/release/bioc/html/DESeq2.html
Ingenuity Pathway Analysis N/A https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis/
Other
Cytek Aurora Cytek Biosciences N/A
BD FACS Aria II cell sorter BD Biosciences N/A
Zeiss Axio Observer 3 Microscope Carl Zeiss N/A
RNA-sequencing Genome Technology Unit, NIAMS/NIH N/A
ChIP-sequencing Genome Technology Unit, NIAMS/NIH N/A
Open in a new tab

ACKNOWLEDGEMENTS:

We thank Gustavo Gutierrez-Cruz and Faiza Naz of NIAMS for assistance with next-generation sequencing. We thank Kan Jiang of NIAMS for assistance with ChIP-seq data quality assessment. We thank Anup Mahurkar from Univ. of Maryland for critical input into ChIP-seq analysis. We thank Michael C. Ostrowski and Sudarshana M. Sharma from Medical University of South Carolina for providing us PU.1 antibody. We thank Bryan Hahn and Karen Underwood from Univ. of Maryland for assistance with cell sorting. We thank Satish Yesupatham from Univ. of Maryland School of Dentistry for assistance with FACS.

FUNDING SOURCES:

R00DE028439 and R03DE029258 to V.T.M.; start-up funds from Univ. of Maryland School of Dentistry to V.T.M.; University of Maryland Baltimore Institute of Clinical & Translational Research (ICTR) grant to V.T.M; R01DE027639 to B.L.F., and intramural funding to M.J.S. from NIAMS/NIH, and to K.O. from NICHD/NIH.

Footnotes

Conflict of interest: The authors have declared that no conflict of interest exists.

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