Decoding the ubiquitination-immunity axis in idiopathic pulmonary fibrosis: diagnostic insights and therapeutic implications

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with limited therapeutic options. Emerging evidence suggests that ubiquitination-related genes (URGs) may contribute to IPF pathogenesis, although their diagnostic and immunological significance remains poorly understood. Three bulk RNA sequencing (RNA-seq) datasets (GSE110147, GSE53845, and GSE32537) and one single-cell RNA-seq dataset (GSE122960) were obtained from the Gene Expression Omnibus database. GSE110147 and GSE53845 were merged as the training cohort to construct a URG-based diagnostic model using least absolute shrinkage and selection operator (LASSO) regression, while GSE32537 and GSE122960 served as external validation cohorts. Immune cell infiltration was assessed using the CIBERSORT algorithm. A bleomycin-induced mouse model of pulmonary fibrosis was used to validate hub URG expression. The pro-fibrotic role of ubiquitin D (UBD) was evaluated in vitro using Cell Counting Kit-8 (CCK-8), wound healing, and transwell assays in mouse primary lung fibroblasts (PLFs). Transwell-based neutrophil migration assays were used to assess the impact of UBD expression in PLFs on neutrophil infiltration. We developed a robust six-gene diagnostic signature with high predictive accuracy in both training and validation cohorts. Immune infiltration analysis revealed strong correlations between hub URGs and specific immune cell types. Among them, UBD showed the most significant upregulation in fibrotic lungs and was positively associated with neutrophil infiltration. Functional assays demonstrated that UBD silencing attenuated transforming growth factor β1 (TGFβ1)-induced fibroblast activation. Moreover, UBD knockdown significantly increased the expression levels of Cxcl2 and Cxcl3 in PLFs and promoted neutrophil migration. This study highlights the diagnostic and immunological relevance of URGs in IPF and identifies UBD as a key pro-fibrotic factor and neutrophil infiltration regulator, offering novel insights into IPF pathogenesis and potential therapeutic targets.