Integrated single-cell and bulk transcriptomic analyses reveal cDC1-centered ubiquitination dysregulation and identify UBE2F as a critical regulator in sepsis.

Abstract

BACKGROUND: Sepsis is a life-threatening syndrome with dysregulated immune responses and multiple organ dysfunction. However, precise diagnostic biomarkers and effective therapeutic targets for this syndrome are still lacking. Protein ubiquitination modulates inflammatory regulation and immune cell function, but the specific immune cell subsets that drive ubiquitination-associated immune dysregulation in human sepsis have not been clearly identified. METHODS: An integrated analysis was performed using 315,220 single cells from two single-cell RNA sequencing (scRNA-seq) datasets in conjunction with independent bulk transcriptomic cohorts. We quantified cell-type responsiveness using Augur, inferred intercellular communication via CellChat, and identified ubiquitination-related gene networks through weighted gene co-expression network analysis (WGCNA) and subsequent multi-algorithm feature selection. Functional validation was conducted with lipopolysaccharide (LPS)-stimulated murine dendritic cells (DCs) line - DC2.4and a cecal ligation and puncture (CLP) mouse model. RESULTS: conventional Dendritic cells (cDCs) were identified as the most transcriptionally perturbed immune population in sepsis, with subsequent subclustering revealing that the type-1 conventional dendritic cells (cDC1) subset specifically exhibited pronounced activation of ubiquitination signatures. Cell-cell communication analysis identified TNF signaling as a sepsis-specific pathway, in which cDC1 functions as a critical mediator predominantly via the TNF-TNFRSF1B axis. Four ubiquitination-related genes (CUL1, UBE2F, UBE2N and UBE3A) demonstrated reproducible diagnostic performance across three independent cohorts. Notably, UBE2F showed the strongest upregulation and functional relevance in sepsis models. Bothandexperiments showed that silencing UBE2F markedly suppressed dendritic cell activation, decreased proinflammatory cytokine production and organ injury, and ultimately improved survival in septic mice. CONCLUSIONS: Our results reveal cDC1 as a key immune cell subset involved in ubiquitination-mediated immune dysregulation in sepsis and suggests that UBE2F may serve as a potential diagnostic biomarker and therapeutic target.