The Identification and Experimental Validation of Monocyte/Macrophage-Related Inflammatory Hub Genes in Ventilator-Induced Lung Injury

Objective

This study aimed to identify monocyte/macrophage-related hub genes in ventilator-induced lung injury (VILI) by integrating transcriptomic analysis, immune infiltration analysis, weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) network analysis, and machine learning approaches, followed by experimental validation using a VILI animal model.

Methods

Seven mouse lung transcriptomic datasets related to VILI were obtained from the Gene Expression Omnibus (GEO) database and integrated after normalization and batch effect correction. Differential expression analysis was performed to identify VILI-associated genes. Immune infiltration analysis and WGCNA were applied to characterize changes in the pulmonary immune microenvironment and identify modules correlated with monocyte/macrophage infiltration. Key module genes were intersected with VILI-related differentially expressed genes (DEGs) and subsequently subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and PPI analyses. Based on monocyte/macrophage infiltration levels, samples were stratified, and candidate genes were screened using least absolute shrinkage and selection operator (LASSO) regression, support vector machine-recursive feature elimination (SVM-RFE), and the random forest algorithm. Receiver operating characteristic (ROC) curve analysis was then performed to evaluate the discriminatory performance of the selected genes. Finally, experimental validation was conducted using histological analysis, bronchoalveolar lavage fluid cytokine assays, quantitative real-time polymerase chain reaction (qPCR), and immunofluorescence staining.

Results

Lung tissues from VILI model mice showed marked remodeling of the immune microenvironment, characterized by increased proportions of monocytes/macrophages and neutrophils, along with reduced CD4 T cell- and innate lymphoid cell-related populations. The MEblack module was positively correlated with monocyte/macrophage infiltration (r = 0.64) and was selected as the key module. Intersecting MEblack genes with VILI-related differentially expressed genes identified 99 candidate genes mainly enriched in chemotaxis, inflammatory response regulation, leukocyte migration, and IL-17, TNF, and NF-κB signaling pathways. Stratification by monocyte/macrophage abundance further identified 82 common differentially expressed genes with similar enrichment patterns. Three machine-learning algorithms jointly prioritized Ccr1, Mmp9, and Il36g as core feature genes, all of which were upregulated in VILI. ROC analysis showed the strongest discriminatory performance for Ccr1 (area under the curve (AUC) = 0.987), followed by Il36g (AUC = 0.891) and Mmp9 (AUC = 0.883). Experimental validation confirmed inflammatory infiltration, structural lung injury, elevated IL-1β, IL-6, and TNF-α levels, increased expression of the three hub genes, and enhanced CCR1-positive signals in alveolar septal and perivascular regions.

Conclusions

VILI is associated with monocyte/macrophage infiltration and activation of inflammatory chemotaxis-related pathways. Ccr1, Mmp9, and Il36g may serve as monocyte/macrophage-related inflammatory feature genes, with Ccr1 showing particular promise as a candidate biomarker of pulmonary inflammatory activation in VILI.