Chronic gouty arthritis (GA) is characterized by sustained synovial inflammation driven by the deposition of monosodium urate crystals, which ultimately results in joint structural damage and functional impairment. The cellular heterogeneity, spatial architecture, and underlying metabolic regulatory mechanisms of this synovitis remain poorly delineated. Riboflavin (vitamin B₂) serves as a precursor for the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are integral to redox metabolism and inflammatory signaling. While dysregulation of riboflavin metabolism has been implicated in chronic inflammatory states, its precise role and molecular mechanisms within the synovial microenvironment of gouty arthritis are unknown. This study integrated single-cell and spatial transcriptomic profiling to deconvolute the cellular composition and transcriptional landscape of the knee synovium in chronic gouty arthritis, with a specific focus on elucidating the contribution and regulatory network of riboflavin metabolic dysfunction, thereby informing potential diagnostic and therapeutic strategies.

Synovial tissues were obtained from patients diagnosed with chronic gouty arthritis and from individuals with non-inflammatory joint pathologies . Single-cell RNA sequencing (scRNA-seq) was performed to comprehensively characterize cellular subpopulations, identify differentially expressed genes (DEGs), and assess the expression profiles of genes associated with riboflavin metabolism. Spatial transcriptomics (ST) was utilized to map the topographic distribution of inflammatory cell subsets and to investigate spatial correlations with riboflavin metabolism-related gene expression. An integrative analytical pipeline was applied to combine scRNA-seq and ST datasets, enabling the construction of a cellular interaction network and the spatial validation of key gene expression and cell localization patterns.

scRNA-seq analysis resolved multiple synovial cell subsets, including distinct macrophage and fibroblast populations. GA patients exhibited a significant expansion of SPP1⁺/MMP9⁺ macrophages, pro-inflammatory fibroblasts, and CD4⁺ regulatory T cells compared to controls. These cell subsets demonstrated heightened expression of canonical pro-inflammatory mediators, including IL1B and TNF. Critical genes involved in riboflavin metabolism (RFK and ENPP1) were dysregulated within these pro-inflammatory populations. Notably, RFK expression was markedly downregulated, indicative of impaired conversion of riboflavin to its active coenzyme forms (FMN/FAD). This metabolic perturbation was associated with compromised cellular antioxidant capacity, exacerbated oxidative stress, and subsequent activation of the NF-κB signaling pathway, thereby amplifying the release of inflammatory cytokines. ST revealed co-enrichment of pro-inflammatory cell clusters, inflammatory gene signatures, and riboflavin metabolism dysfunction-related transcripts within discrete synovial inflammatory infiltrates. Spatial co-localization was observed between SPP1⁺ macrophages and pro-inflammatory fibroblasts, with their respective gene expression profiles showing significant correlation, collectively establishing a "metabolic-inflammatory" micromilieu. Pathway enrichment analysis confirmed significant overrepresentation of genes involved in inflammatory response and riboflavin metabolism pathways among the spatial DEGs.

The synovial tissue in chronic gouty arthritis is characterized by distinct cellular heterogeneity and a pronounced dysregulation of riboflavin metabolism. Riboflavin metabolic dysfunction, exemplified by the downregulation of RFK, disrupts the biosynthesis of essential redox cofactors, attenuates antioxidant defenses, and potentiates NF-κB pathway activation. This metabolic aberration cooperates with expanded pro-inflammatory cell populations (SPP1⁺ macrophages and activated fibroblasts) to establish and maintain a pro-inflammatory synovial microenvironment, thereby driving the chronicity of the disease. The integrated application of single-cell and spatial transcriptomics has delineated the regulatory role and mechanistic basis of riboflavin metabolism in gouty synovitis, nominating it as a plausible target for therapeutic intervention and outlining a novel direction for the development of targeted treatment modalities.