CD63hi macrophages define a conserved terminal inflammatory state driving lung immunopathology in severe COVID-19

Abstract

Background Severe COVID-19 is characterized by profound lung immune dysregulation, yet the pathogenic states and regulatory mechanisms of monocyte-derived macrophages (MDMs) remain poorly defined due to substantial phenotypic heterogeneity across studies. Methodology We integrated multiple single-cell RNA sequencing datasets from COVID-19 lung tissues and bronchoalveolar lavage fluid (BALF) samples to systematically characterize macrophage heterogeneity and differentiation dynamics across anatomical compartments. Trajectory inference, transcription factor activity analysis, and m6A-associated regulatory profiling were performed to delineate molecular programs underlying macrophage state transitions. Cross-dataset validation analyses were conducted to assess the conservation and clinical relevance of identified macrophage states. Functional roles of candidate regulators were further investigated through ELAVL1 silencing in monocyte-derived macrophages in vitro, while the pathogenic potential of CD63hi macrophages was evaluated using adoptive transfer experiments in an acute lung injury mouse model. Results We define a conserved CD63hi terminal inflammatory macrophage state markedly expanded in severe COVID-19 lungs. CD63hi macrophages exhibited enhanced inflammatory cytokine expression together with elevated glycolysis- and hypoxia-associated programs, representing a terminal inflammatory differentiation state along the monocyte-to-MDM trajectory. This population was reproducibly detected across independent datasets and preferentially enriched in lung tissues, with strong associations with disease severity and adverse clinical outcomes. Transcriptional analysis identified JUND activity as progressively increased during macrophage differentiation toward the CD63hi state. In parallel, ELAVL1 was associated with inflammatory and metabolic reprogramming programs. ELAVL1 silencing suppressed pro-inflammatory cytokine expression, attenuated glycolytic and hypoxia-related pathways, and shifted macrophages toward an immune-regulatory phenotype accompanied by reduced CD63 expression. Functionally, adoptive transfer of CD63hi macrophages exacerbated lung inflammation in vivo, resulting in increased cytokine production and neutrophil infiltration. Conclusions In conclusion, our study defines a conserved CD63hi macrophage inflammatory state in severe COVID-19 and reveals its association with metabolic reprogramming, lung immunopathology, and disease severity. ELAVL1 emerges as an important RNA regulatory factor associated with inflammatory macrophage remodeling, highlighting the potential of targeting macrophage regulatory networks as a strategy for modulating pathological inflammation in severe viral infection.