Single-cell multi-OMICs and functional immune profiling reveal increased innateness and interferon-driven pathways in Behçet's disease skin and blood

Behçet's Disease (BD) is a chronic, multi-system inflammatory disorder with both autoimmune and autoinflammatory features, arising in genetically predisposed individuals exposed to environmental triggers. While prior studies have largely focused on immune dysfunctions in peripheral blood, including increased Th17, Th1, and cytotoxic CD8+ T cells. Innate immune cells, including natural killer (NK) cells and γδ T cells have also been implicated in BD pathophysiology. Despite these advances, a significant gap remains in understanding the immune landscape of BD, particularly in affected skin tissues, one of the cardinal sites of disease pathogenesis. To date, no comprehensive study has integrated single-cell, molecular, and functional analyses to characterize immune dysregulation in both the blood and skin of BD patients. To address this gap, we performed single-cell RNA sequencing (scRNAseq), TCR sequencing, flow cytometry, and functional analyses on BD skin tissue and PBMCs, comparing them with healthy controls. Our scRNAseq and flow cytometry analyses revealed significant enrichment of plasmacytoid dendritic cells (pDCs), mucosal-associated invariant T (MAIT) cells, NK cells, ILC3s, and memory B cells in BD skin lesions. These populations were associated with robust type I immune responses and heightened expression of cytotoxic molecules, including granzyme B, granzyme K, and perforin. Effector memory CD4+ and CD8+ T cells exhibited a cytotoxic and inflammatory profile, characterized by increased production of IFN-γ, GZMK, and CCL5, while pathway analysis indicated significant activation of the JAK-STAT signaling pathway. Additionally, myeloid cells demonstrated upregulation of inflammasome-related genes linked to enhanced toll-like receptor signaling, as revealed by scRNAseq. Functional studies showed increased IFN-γ and TNF-α expression in Th, Tc, and MAIT cells in BD skin, contrasting with their decreased expression in blood Th cells. Overall, our findings reveal that innate immune cells and innate T lymphocytes, in conjunction with an activated Th1/Tc1 pathway, play a central role in BD pathogenesis. This study highlights the intricate immune dysregulation underlying BD, offering insights into potential therapeutic targets for this complex disease.