Restoring NR4A3 function in neutrophils alleviates sepsis by limiting NF-κB-dependent NETs formation and organ damage

Sepsis, a life-threatening condition, involves excessive neutrophil extracellular traps (NETs) contributing to organ damage. The role of orphan nuclear receptor NR4A3 in modulating neutrophil NETosis via NF-κB during sepsis is poorly understood. This study aimed to elucidate NR4A3's function and therapeutic potential.

Single-cell RNA sequencing analysis was performed using GSE175453 and GSE167363 from GEO database, involving PBMCs from septic patients and controls. In vitro, ATRA-differentiated HL-60 neutrophils with NR4A3 overexpression were LPS-stimulated. In vivo, a cecal ligation and puncture (CLP) sepsis mouse model received AAV-mediated NR4A3 overexpression. Key readouts included NR4A3 expression, NF-κB activation, NETs markers (NE, CitH3, PADI4, MPO-DNA), inflammatory cytokines (TNF-α, IL-1β, IL-6), ROS generation, and intestinal tissue pathology.

scRNA-seq analysis revealed significant NR4A3 downregulation in neutrophils from septic patients. In vitro, NR4A3 overexpression in neutrophils significantly attenuated LPS-induced NF-κB (p65) activation, NETs formation, inflammatory cytokine production, and ROS generation. In vivo, AAV-mediated NR4A3 overexpression in CLP-induced septic mice ameliorated intestinal inflammation, suppressed p65 phosphorylation and NETs-related markers in the intestine, and reduced systemic inflammatory cytokine levels.

Our findings demonstrate that NR4A3 exerts a protective role in sepsis by inhibiting the NF-κB signaling pathway in neutrophils, thereby suppressing excessive NETs formation and the associated inflammatory cascade. Thus, NR4A3 represents a promising therapeutic target for mitigating NET-driven pathology in sepsis.