Background/Purpose: Rheumatoid arthritis (RA) is characterized by chronic inflammatory erosions and female predominant disease. Androgen, the dominant sex hormone in males, is protective against bone loss. We have previously shown that androgen limits erosions in a TNF-mediated model of RA and potentially targets osteoclast precursor (OCP). Here, we investigated androgen’s effect on osteoclast differentiation and hypothesized that androgen negatively regulates osteoclastogenesis in precursor cells.

Methods: Bone marrow derived cells were harvested from a 3-month-old male C57BL/6J mouse and plated in media containing either 10-9M of 5ɑ-dihydrotestosterone (DHT) or vehicle control and M-CSF. After 48 hours, RANK-L, and human TNFɑ (hTNFɑ) were added to media and cells were incubated for 24 hours. Adherent cells were captured with the 10X Genomics Chromium system to undergo single-cell RNA-sequencing (scRNA-seq). Cell typing was determined by the mouse cell atlas and “FindAllMarkers” command in Seurat. Gene ontology (GO) analysis was performed by determining the differentially expressed genes (DEGs) between vehicle and DHT-treated samples in each cluster and submitting them to the DAVID Bioinformatics database. Pseudotime trajectory analysis was performed with the Monocle3 R package. Transcription factor (TF) analysis was performed by generating gene modules between clusters using Monocle3 and the pseudotime dataset. DEGs from this analysis were inputted into the iRegulon package in Cytoscape to determine TFs. DEGs between samples were illustrated in a volcano plot. Using the same cell culture protocol, RT-qPCR was performed on DHT and vehicle-treated samples to compare gene expression.

Results: In the scRNA-seq dataset, 8 clusters were defined as myeloid-lineage cells (Fig. 1A-B). The GO terms that were downregulated with DHT treatment and conserved throughout the clusters included terms associated with translation (Fig. 2A). In comparison, terms upregulated with DHT treatment included innate immune response and response to interferon (Fig. 2B). Pseudotime trajectory analysis of the vehicle and DHT-treated samples showed a difference in differentiation trajectory, where differentiation is decreased in proliferating OCPs with DHT treatment (Fig. 3A-B). TF analysis between our clusters of interest exhibit greater expression of the TF Irf7 and other interferon-related genes with DHT (Fig 3C-D). The top DEG in the DHT-treated sample for most clusters was Isg15 (Fig. 3E-F). Relative Isg15 expression was also significantly increased in DHT-treated OCPs (Fig. 3G).

Conclusion: Androgen treatment in an inflammatory in vitro system of osteoclastogenesis caused a change in the expression of interferon genes and the trajectory of osteoclast differentiation. DHT treatment upregulated interferon signaling, particularly Isg15 expression, which has been found to negatively regulate osteoclastogenesis (1). This increase in expression may potentially limit differentiation and therefore erosive disease. Further studies will involve examining how androgen targets interferon-related genes and their role in osteoclastogenesis.1) MacLauchlan et al. Proc Natl Acad Sci U S A 120(15) 2023.

Figure 1. Myeloid Lineage Cells in an Inflammatory Osteoclast Culture. Bone marrow derived cells from a male C57BL/6J mouse were plated and treated with either DHT or vehicle control in osteoclast culture media and collected for single-cell RNA-sequencing. Myeloid lineage populations were subset out from the dataset and defined as 8 unique cluster as illustrated in the uniform manifold approximation and projections (UMAPs) for vehicle-treated (A) and DHT-treated samples (B).

Figure 2. Androgen Treatment Alters the Biological Processes in Myeloid Lineage Cells. Differentially expressed genes (DEGs) between the vehicle and DHT-treated samples in each cluster were used to determine the gene ontology (GO) terms downregulated (A) and upregulated with DHT treatment (B). GO terms that are downregulated with DHT treatment include translation-related terms throughout majority of the clusters and terms that are upregulated with DHT treatment include immune defense response and interferon-related terms throughout majority of the cluster.

Figure 3. Interferon-Related Gene Expression is Increased with Androgen Treatment. Pseudotime trajectory analysis was performed on vehicle-treated (A) and DHT-treated (B) myeloid lineage cells. Cluster 6 (monocyte progenitors) was the root trajectory. Trajectory analysis of the vehicle-treated sample displays the progression of differentiation through monocyte and macrophage populations into differentiating OCPs and osteoclasts. In comparison, the DHT-treated subset illustrates differentiation from monocytes to M2-like differentiating OCPs and osteoclasts, with proliferating OCP clusters determined to be less differentiated. Transcription factor gene expression was analyzed between cluster 3 (M2-like macrophages) to 0 (proliferating OCPs) in the DHT-treated sample (C), and the transcription factor Irf7 was found to be decreased in cluster 0, along with Stat1 and Isg15 (D). In comparison, vehicle-treated cells did not express Irf7 or Isg15 and had lower expression of Stat1 overall. Differentially expressed genes (DEGs) of the DHT-treated sample were exhibited in a volcano plot (E), with Isg15 as the most upregulated DEG. A violin plot illustrates that Isg15 is differentially expressed with DHT treatment in all clusters (F). RT-qPCR of DHT and vehicle-treated cell culture confirmed increased relative expression of Isg15 in DHT-treated cells (G). Plots and analysis were performed with the Cytoscape software and Monocle3, Seurat and EnhancedVolcano R packages. Bars show mean ± standard deviation. Comparisons in RT-qPCR analysis were made with an unpaired t-test. ** = p < 0.01.

« Back to ACR Convergence 2025

ACR Meeting Abstracts - https://acrabstracts.org/abstract/androgen-upregulates-interferon-signaling-in-osteoclast-differentiation-during-inflammatory-state/