Integrative Bulk and Single-Cell Transcriptomic Analyses Identify NFKBIE as a Candidate Gene Associated with IS-II Neutrophil Differentiation in Lung Ischemia-Reperfusion Injury.

Objective: People undergoing lung transplantation may develop lung ischemia-reperfusion injury (LIRI), in which neutrophils contribute to the inflammatory response. However, the molecular features associated with distinct neutrophil states remain incompletely understood. This study aimed to identify candidate molecular features associated with the immunosuppressive-II (IS-II) neutrophil state, with particular attention to NFKBIE, an NF-κB inhibitor. Methods: We integrated bulk transcriptomic and single-cell RNA-sequencing data. Candidate genes were identified using co-expression analysis and machine-learning methods. We further examined cell-cell communication and the association of NFKBIE with neutrophil states using pseudotime analysis and computational gene perturbation. We validated NFKBIE expression in a mouse model of LIRI using RT-qPCR and Western blotting and assessed lung injury by histological examination. Results: A total of 553 differentially expressed genes and a key disease-associated module containing 4,456 genes were identified, with predominant enrichment in inflammation- and immunity-related pathways (eg, TNF and NF-κB). Five overlapping candidate transcriptomic markers, CXCL3, CYLD, NFKBIE, SLC30A1, and KBTBD8, were identified. The five-gene model showed strong discrimination in the discovery cohort (AUC = 0.986) and also showed strong discrimination in the separate GSE127003 validation dataset (AUC = 0.999, 95% CI 0.996-1.000). Single-cell analysis showed that neutrophils were the largest annotated cell population in both CIT and post-reperfusion samples, whereas the IS-II neutrophil subtype was markedly enriched after reperfusion; NFKBIE was most highly expressed in IS-II neutrophils. Pseudotime trajectory analysis further indicated that elevated NFKBIE expression was associated with a later differentiation state toward the IS-II phenotype, while computational NFKBIE perturbation predicted alterations in several perturbation-associated genes. In vivo validation confirmed that both NFKBIE mRNA and protein levels were upregulated in lung tissue after reperfusion. Conclusion: The five-gene transcriptomic model showed reproducible discriminatory performance in a separate validation dataset. NFKBIE was consistently associated with the IS-II neutrophil state across multiple computational analyses, while in vivo experiments confirmed its increased expression after reperfusion. These findings nominate NFKBIE as a candidate gene associated with IS-II neutrophil differentiation and provide a hypothesis-generating basis for further functional investigation.