SAT-238 Effect of Testosterone on Immune Response in Monocytes After Lipopolysaccharide Stimulation

Abstract Disclosure: G. Ling: None. S. Dhindsa: None. M. Brennan: None. Background: The immune system is known to exhibit sexual dimorphism. Traditionally androgens are considered immunosuppressors and contribute to sex difference in vaccine response, susceptibility to autoimmune disease, and disease outcome. However, at the molecular level, how androgens affect cellular immunity, especially in humans, is not well understood. In this study, we evaluated the effect of testosterone on peripheral blood mononuclear cells (PBMC) after stimulation by lipopolysaccharide (LPS) using single cell RNA sequencing technology. Methods: We collected PBMC from 3 hypogonadal males and cultured them ex vivo in the presence or absence of 30 nM testosterone for 48 hours. We then treated them with or without 20 ng/ml LPS for one hour. We harvested the RNA from 12 samples (3 individuals, 4 conditions). Single cell transcriptomes were captured by 10x Genomic fixed RNA profiling. Seurat, an R toolkit along with additional bioinformatic tools to perform cell annotation and Gene Set Enrichment Analysis (GSEA), were used to analyze single cell sequencing data. Here we present our findings on monocytes. Results: After low-quality cells were excluded, a total of 152,092 cells were included in the final dataset for analysis. UMAP dimensionality reduction, cell clustering and annotation were applied to the normalized, integrated count data, allowing us to identify seven main cell types: B, CD4+ T, CD8+ T, monocytes, natural killer, basophils and dendritic cells. As we expected, LPS stimulation activated canonical pro-inflammatory pathways in monocytes with or without testosterone. Differential gene expression (DGE) using Seurat FindMarkers function showed that in LPS-stimulated monocytes, there are 66 genes differentially expressed with an adjusted p-value < 0.05, and 1,170 genes differentially expressed with a p-value < 0.05 following testosterone treatment. GSEA demonstrated testosterone treatment negatively affected interferon alpha and gamma pathways (NES -2.91, -2.74, FDR 0.000, 0.000), whereas there were no gene sets positively enriched at FDR < 25%. Closer examination of the enrichment plots of interferon alpha and gamma pathways showed that most of the genes contributing to downregulation of pathways are closer to the middle portion of the graphs, suggesting that while testosterone affects multiple genes, its effect on each individual gene is modest. Conclusion: using LPS-stimulated PBMC from hypogonadal males, we were able to demonstrate that 48-hour testosterone treatment downregulated interferon pathways through its inhibitory effects on multiple genes expression levels. Presentation: Saturday, July 12, 2025