Table 7_Comprehensive analysis of the role of diverse programmed cell death patterns in sepsis.xlsx

BackgroundSepsis, a life-threatening condition with persistently high mortality, involves dysregulated immune responses and programmed cell death (PCD). However, the specific roles and interactions of diverse PCD pathways in sepsis pathogenesis remain incompletely understood. This study aimed to systematically characterize PCD patterns and their clinical relevance in sepsis.
MethodsWe integrated three bulk transcriptomic datasets (81 controls, 165 sepsis patients) and one single-cell RNA sequencing (scRNA-seq) dataset (4 controls, 4 early sepsis patients, 52,315 cells) from public databases. Gene set variation analysis (GSVA) quantified activity of 13 PCD pathways. Immune infiltration was assessed via single-sample gene set enrichment analysis (ssGSEA). A cell death-associated signature (CDS) risk score was developed using least absolute shrinkage and selection operator (LASSO) regression. scRNA-seq analysis identified cell-type-specific PCD activation and intercellular communication using Seurat, AUCell, and CellPhoneDB. Additionally, an independent RNA-seq cohort generated from our own sequencing of sepsis patients and healthy controls was used for external validation.
ResultsTranscriptomic analysis identified 5,591 differentially expressed genes enriched in immune and cell death pathways. Four PCD pathways-ferroptosis, disulfidptosis, NETosis, and entotic cell death-were significantly upregulated in sepsis and strongly correlated with immune cell infiltration, such as activated dendritic cells and neutrophils. The CDS risk score, based on 18 core PCD genes, showed excellent diagnostic accuracy across both public microarray datasets (AUC = 0.961 and 0.844) and our independent high-throughput RNA-seq dataset (AUC = 0.975). scRNA-seq revealed monocytes as dominant effectors, exhibiting heightened activation of ferroptosis, entotic death, and netotic pathways alongside metabolic reprogramming, including enhanced glutathione metabolism and oxidative phosphorylation (OXPHOS). Furthermore, monocyte-centric intercellular communication was dysregulated in sepsis, featuring upregulated MIF-CXCR4, ANXA1-FPR2, and HLA-KIR signaling axes.
ConclusionsBy integrating public microarray and single-cell transcriptomic data with independent high-throughput sequencing validation, this study analysis identifies ferroptosis, disulfidptosis, netotic death, and entotic death as key dysregulated PCD pathways in sepsis, with monocytes serving as central hubs integrating PCD, metabolic reprogramming, and immune communication. The CDS risk score provides a robust diagnostic and stratification tool. Targeting monocyte-driven PCD-metabolism-communication networks offers promising avenues for precision immunotherapy in sepsis.

背景：脓毒症（Sepsis）是一种致死性疾病，病死率持续居高不下，其核心病理机制涉及免疫应答失调与程序性细胞死亡（programmed cell death, PCD）。然而，多样化的程序性细胞死亡通路在脓毒症发病机制中的具体作用及相互调控关系仍未完全阐明。本研究旨在系统解析脓毒症中程序性细胞死亡的模式及其临床相关性。

方法：本研究从公共数据库中整合了3个批量转录组（bulk transcriptomic）数据集（81例健康对照、165例脓毒症患者）以及1个单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据集（4例健康对照、4例早期脓毒症患者，共52315个细胞）。采用基因集变异分析（gene set variation analysis, GSVA）量化13种程序性细胞死亡通路的活性；通过单样本基因集富集分析（single-sample gene set enrichment analysis, ssGSEA）评估免疫浸润状态。利用最小绝对收缩和选择算子（least absolute shrinkage and selection operator, LASSO）回归构建细胞死亡相关特征（cell death-associated signature, CDS）风险评分。借助Seurat、AUCell及CellPhoneDB工具对scRNA-seq数据进行分析，明确细胞类型特异性的程序性细胞死亡激活特征与细胞间通讯网络。此外，本研究通过自主测序获取的脓毒症患者与健康对照的独立RNA-seq队列作为外部验证集。

结果：转录组分析共筛选出5591个差异表达基因，这些基因显著富集于免疫应答与细胞死亡通路。4种程序性细胞死亡通路——铁死亡（ferroptosis）、二硫化物应激死亡（disulfidptosis）、中性粒细胞胞外陷阱形成（NETosis）及胞吞性细胞死亡（entotic cell death）——在脓毒症患者中显著上调，且与活化树突状细胞、中性粒细胞等免疫细胞的浸润程度呈强相关性。基于18个核心程序性细胞死亡基因构建的CDS风险评分，在公共微阵列数据集（曲线下面积AUC分别为0.961与0.844）及本研究自主的高通量RNA-seq数据集（AUC=0.975）中均表现出优异的诊断效能。scRNA-seq分析显示，单核细胞是脓毒症中主要的效应细胞群，其铁死亡、胞吞性细胞死亡及中性粒细胞胞外陷阱形成通路的激活水平显著升高，同时伴随代谢重编程，包括谷胱甘肽代谢与氧化磷酸化（oxidative phosphorylation, OXPHOS）增强。此外，脓毒症中以单核细胞为核心的细胞间通讯网络失调，具体表现为MIF-CXCR4、ANXA1-FPR2及HLA-KIR信号轴的表达上调。

结论：本研究通过整合公共微阵列、单细胞转录组数据，并结合自主高通量测序的独立队列进行验证，明确了铁死亡、二硫化物应激死亡、中性粒细胞胞外陷阱形成及胞吞性细胞死亡为脓毒症中关键的失调程序性细胞死亡通路，且单核细胞是整合程序性细胞死亡、代谢重编程与免疫通讯的核心枢纽。CDS风险评分可作为可靠的脓毒症诊断与分层工具。靶向单核细胞驱动的程序性细胞死亡-代谢-通讯网络，有望为脓毒症的精准免疫治疗提供新的思路与途径。