T cell-related diagnostic model and the underlying mechanism related to PRF1-mediated glycolysis in sepsis: evidences from single-cell, bulk transcriptomics, and experiment validation.

AIM: Sepsis is a serious systemic inflammatory response. We aimed to construct a T cell-related diagnostic model for sepsis and uncover the underlying mechanisms. METHODS: Through downloading the single-cell RNA-sequencing (scRNA-seq) and RNA-seq data from online source, a series of bioinformatics methods including principal component analysis (PCA), differential expression analysis, and least absolute shrinkage and selection operator (LASSO) was used for selection of the T cell-related signatures. Then a diagnostic model was constructed and receiver operator characteristic (ROC) curve was used for evaluation of the diagnostic ability. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for the function and pathways enrichment analysis. Metabolic flux analysis was performed to investigate the underlying metabolic mechanism. Finally, cecal ligation and puncture (CLP)-induced mouse sepsis model and LPS-induced RAW264.7 macrophage cell sepsis model were utilized to investigate the PRF1-mediated glycolysis mechanism in sepsis. RESULTS: scRNA-seq emphasized the important roles of T cell especially CD8(+) T cell in sepsis. T cell-related differential genes were mainly enriched in T cell behavior and infection-related functions. A 15-gene diagnostic model related to T cell was constructed and validated to be effective in predicting the sepsis status. The most overexpressed gene PRF1 in T cell was related to glycolysis-related metabolic process including G3P → 3PD, 3PD → pyruvate, G3P → PRPP, and G6P → G3P. PRF1, inflammatory factors (TNF-α and IL-1β), lactate level, and glycolysis-related markers (PFKFB3, PKM2, and GLUT1) were increased and the ratio of CD4(+)/CD8(+) T cells was decreased in vivo and in vitro sepsis models. But PRF1 knockdown significantly decreased the inflammatory factors (TNF-α and IL-1β), lactate levels, and glycolysis-related markers (PFKFB3, PKM2, and GLUT1) in sepsis. CONCLUSION: A 15-gene T cell-related diagnostic model for sepsis is constructed and PRF1 is confirmed to be an effective indicator and therapeutic target for sepsis, mainly functions in glycolysis.