Background/Purpose: Behçet’s disease (BD) is a chronic, relapsing inflammatory disease characterized by complex immunopathogenesis and limited treatment options. Monocytes are known to play a significant role in BD, with studies indicating alterations in monocyte subsets and functions. However, the detailed transcriptional programs and subset-specific contributions of monocytes to BD pathogenesis remain to be fully characterized. In this study, we leveraged single-cell transcriptomics to uncover monocyte subset-specific immune alterations in BD.

Methods: We performed single-cell RNA sequencing using GEM-X technology (10X Genomics) on PBMCs from 50 individuals (38 BD patients, 12 matched healthy controls). Sample multiplexing was enabled by antibody-based cell hashing. Data preprocessing, normalization, and clustering were performed using Seurat v5. Differential expression, cell composition analyses, and pathway enrichment (GO and GSEA) were conducted to identify disease-associated profiles.

Results: After quality control filtering, we analyzed a total of 251,924 PBMCs, successfully characterizing all major immune cell populations. Focusing on monocyte subsets, we observed significant transcriptional alterations in BD patients. CD14⁺ monocytes exhibited a significant expansion compared to healthy controls as well as an upregulation of inflammatory and interferon-stimulated genes including IFI27, IFITM1/2, IFI6, and S100A9. Gene Ontology analysis showed enrichment of cytokine signaling and innate immune activation pathways. Gene Set Enrichment Analysis (GSEA) further highlighted hallmark pathway enrichment for inflammatory response and IL6/JAK/STAT3 signaling, consistent with known BD immunopathology. Similarly, CD16⁺ monocytes were expanded in BD but exhibited a distinct transcriptional program. Upregulated genes included cytotoxicity-associated effectors (PRF1, GIMAP4–8), chemotactic receptors (CCR5, CX3CR1, CMKLR1), inflammasome components (CASP1, PYCARD), and stress-response genes (HSPA1A, HSPA6). Increased expression of immune checkpoints (CD200R1, SLAMF6, BTLA) and ISGs (RSAD2, IFI44L, OAS1) suggested a pro-inflammatory, non-classical activation state in CD16⁺ monocytes. Functional enrichment pointed to immune effector functions, cell death, and migration pathways.

Conclusion: Our study provides a comprehensive single-cell map of PBMCs in Behçet’s disease, revealing profound transcriptional remodeling of monocyte subsets. While CD14⁺ monocytes display canonical inflammatory features, we identify an expansion of CD16⁺ monocytes with a distinct cytotoxic, chemotactic, and interferon-responsive profile. These findings highlight monocyte heterogeneity in BD and uncover candidate pathways that may guide future therapeutic strategies targeting innate immune dysregulation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/expansion-and-transcriptional-reprogramming-of-cd14%e2%81%ba-and-cd16%e2%81%ba-monocytes-in-behcets-disease/