Unraveling the role of regulatory cell death in sepsis: an integrated analysis of bulk and single-cell sequencing data.

Background: Sepsis is a life-threatening condition characterized by systemic inflammation and dysfunction of multiple organs. Recently, regulatory cell death (RCD) has emerged as a distinct pathological feature and serve as a potential source of biomarkers or therapeutic targets in sepsis. Methods: Comprehensive transcriptomic datasets of sepsis were accessed from the Gene Expression Omnibus (GEO) database. Genes involved in 18 RCD pathways were compiled from databases and published literature. The limma package was utilized to identify differentially expressed genes (DEGs). The Gene Set Variation Analysis (GSVA), CIBERSORT, Weighted Gene Co-expression Network Analysis (WGCNA), and receiver operating characteristic (ROC) analyses were combined to identify key RCDs pathways. Core RCD-related DEGs (RRDs) were selected using Least Absolute Shrinkage and Selection Operator (LASSO), Support Vector Machine (SVM), and Random Forest (RF) machine learning methods. The expression patterns and diagnostic performance of the core RRDs were validated across multiple datasets and further confirmed through meta-analysis. Immune localization of RRDs was examined using single-cell transcriptomic data. Prognostic significance was evaluated using multivariate Cox analysis. Finally, the mRNA expression level was validated using quantitative real-time polymerase chain reaction (qRT-PCR). Results: Zinc Finger DHHC-Type Containing 3 (ZDHHC3), Chloride Intracellular Channel 1 (CLIC1), Glutathione S-Transferase Omega 1 (GSTO1), Biogenesis of Lysosomal Organelles Complex 1 Subunit 1 (BLOC1S1), and Toll-Like Receptor 5 (TLR5) were considered as core RRDs, with monocytes and neutrophils serving as the principal cell types responsible for their overexpression and likely contributing critically to their downstream biological effects. Among them, ZDHHC3 and TLR5 were identified as independent risk factors for sepsis. Their significantly elevated mRNA expression in septic mice was confirmed by qRT-PCR. Conclusion: Findings from this study underscored the crucial role of RCD pathways in the development of sepsis. Notably, ZDHHC3 and TLR5 were identified as novel and robust biomarkers for sepsis.