Functional and transcriptional responses of chicken macrophages to hypoxia

The emergence of wooden breast (WB) myopathy, marked by palpable hardness and excessive fibrosis in the affected pectoralis major muscle of broilers, has greatly affected poultry industry. Hypoxia-triggered dysfunction of macrophages play a crucial role in inflammation-driven tissue fibrosis. Nevertheless, how hypoxia-induced changes in chicken macrophages contribute to WB development remain poorly understood. In this study, we established an in vitro hypoxia model using chicken macrophages HD11 cells and characterized their functional and transcriptional alterations. The results demonstrated that hypoxia enhanced macrophage proliferation while suppressing their migration. RNA-seq analysis revealed 418, 1,462, and 2,670 differentially expressed genes (DEGs) at 12 h, 24 h, and 48 h post-hypoxia exposure, respectively. The protein-protein interaction (PPI) network indicates that DEG-enriched modules were primarily involved in immune response, cytoskeletal regulation, and metabolism. Key hub genes, including MMP2, MMP9, CCL4, CCL5, and CXCL12, were identified across different time points. Functional enrichment analyses revealed that prolonged hypoxia first induced acute stress in HD11 cells, which then progressively transitioned toward an immune-activation phenotype. This transition was evidenced by enrichment of DEGs in lysosomal, cytokine and immune-regulatory pathways, concomitant with up-regulated glycolysis and down-regulated antioxidant pathways. Furthermore, sustained hypoxia augmented the extracellular matrix (ECM)-remodeling capacity of HD11 cells, as reflected by elevated expression of ECM-associated genes and pathways, accompanied by functional reprogramming of cytoskeletal and membrane proteins. This research elucidates functional and transcriptional changes of chicken macrophages in response to hypoxia, demonstrating that hypoxia enhances the immune response and ECM-remodeling capacity of macrophages, which can provide theoretical insights for targeting interventions to control WB myopathy via macrophage mechanisms.