Hypoxia-driven macrophage heterogeneity in sepsis: single-cell transcriptomic insights into immune reprogramming and therapeutic targeting

Sepsis is characterized by profound immune dysregulation and metabolic remodeling, yet how hypoxia shapes macrophage-state diversity remains incompletely defined. Here, we integrated single-cell transcriptomic profiles from early- and late-stage sepsis with healthy controls to construct a hypoxia-centered immune atlas. The single-cell landscape identified 103,851 cells across 23 clusters, while hypoxia scoring showed that macrophages displayed the strongest hypoxic activity, particularly in early sepsis. Macrophages were further stratified into low-, intermediate- and high-hypoxic states, revealing a continuous transcriptional gradient rather than discrete subsets. High-hypoxic macrophages exhibited increased CytoTRACE-defined plasticity, occupied intermediate-to-late pseudotime states, and showed enhanced inflammatory and metabolic pathway activation. Cell–cell communication analysis positioned these macrophages as bidirectional signaling hubs, with amplified MIF, CCL, ANNEXIN and IL1-related interactions. Integrated high dimensional Weighted Gene Co-expression Network Analysis (hdWGCNA) and machine-learning analyses identified hypoxia-associated hub genes, among which TIPARP emerged as a key regulator linked to macrophage hypoxic remodeling. TIPARP was preferentially enriched in high-hypoxic macrophages and experimentally validated as responsive to hypoxia and inflammatory stimulation, with knockdown can enhance HIF1A and VEGF induction. Drug-sensitivity prediction further suggested state-specific vulnerabilities of hypoxia-adapted macrophages. Together, these findings define hypoxia as a central determinant of macrophage heterogeneity in sepsis and nominate TIPARP as a candidate target for immunometabolic intervention.