LOXL1, THY1, and TYMS define an annotation-derived hemoglobin-associated immunotranscriptomic signature in osteoarthritis cartilage.

Background: Osteoarthritis (OA) is increasingly recognized as a whole-joint disease involving cartilage degeneration, synovial inflammation, subchondral bone remodeling, and immune-associated inflammatory signaling. Non-erythroid hemoglobin (Hb)-associated transcriptional programs have recently been implicated in chondrocyte hypoxic adaptation, but their relationship with immune-associated transcriptomic remodeling in OA cartilage remains unclear. This study aimed to identify annotation-derived Hb-associated transcriptomic biomarkers in OA cartilage and to characterize their associations with inferred immune-related transcriptomic features. Methods: Human cartilage transcriptomic datasets were integrated from Gene Expression Omnibus (GEO) to construct a harmonized training cohort, with independent datasets used for external validation. After batch-effect correction, OA differentially expressed genes were intersected with a GeneCards-derived Hb-associated annotation gene set. Weighted gene co-expression network analysis, together with the least absolute shrinkage and selection operator and other complementary machine-learning approaches, was applied to identify hub Hb-associated OA genes. A multivariable logistic-regression nomogram was constructed and evaluated by receiver operating characteristic analysis across cohorts. Immune pathway activity and immune-cell-associated transcriptomic signatures were inferred using GSVA, CIBERSORT, ssGSEA, and correlation analyses. Experimental validation was performed using qRT-PCR in an interleukin-1β-stimulated inflammatory chondrocyte model. Results: Three Hb-associated hub genes-LOXL1, THY1, and TYMS-were identified through integrative transcriptomic analysis and consistently upregulated in OA cartilage across training and independent validation cohorts. Immune profiling suggested an immune-activated transcriptomic state in OA cartilage, characterized by increased macrophage-associated signals, reduced resting NK and CD4 memory T cells, elevated activated immune cell signatures, and enhanced antigen presentation-related and immune regulation-related functions. Expression of these Hb-associated hub genes showed strong positive correlations with macrophage- and activated T-cell-related signatures and antigen presentation-associated immune functions. A nomogram based on the three genes demonstrated high discriminative performance across cohorts, with an AUC of 0.974 in the training cohort. qRT-PCR in an IL-1β-stimulated C28/I2 chondrocyte model supported the inflammatory inducibility of LOXL1, THY1, and TYMS. Conclusion: This study identifies LOXL1, THY1, and TYMS as reproducible annotation-derived Hb-associated tissue-level transcriptomic biomarkers in OA cartilage. Their expression is associated with macrophage- and activated T-cell-related transcriptomic signatures, providing an immunotranscriptomic link between annotation-derived Hb-associated gene programs and immune remodeling in OA.