NETs drive CD4⁺ T cell dysfunction in sepsis via mitochondrial ROS and impaired calcium signalling

Abstract

Sepsis is a life-threatening condition with high mortality, in which sepsis-induced immunosuppression—characterized by CD4+ T cell dysfunction and apoptosis—worsens clinical outcomes. The mechanistic link between neutrophil extracellular traps (NETs) and CD4+ T cell fate in sepsis remains poorly elucidated. We integrated plasma analysis from 50 sepsis patients and 31 healthy controls, a murine cecal ligation and puncture (CLP) sepsis model, and in vitro assays with Jurkat cells and primary CD4+ T cells. We investigated the effects of NETs on CD4+ T cell calcium signaling and mitochondrial function, and verified the therapeutic efficacy of DNase I combined with the mitochondrial-targeted antioxidant mitoTEMPO. Circulating NETs were significantly elevated in sepsis patients, positively correlated with disease severity (SOFA score) and negatively correlated with peripheral CD4+ T cell counts. NETs disrupted intracellular calcium homeostasis by inhibiting store-operated calcium entry, suppressing calcineurin activity and NFAT nuclear translocation, thus impairing T cell activation and IL-2 production. Additionally, NETs induced mitochondrial dysfunction via excessive mitochondrial reactive oxygen species (mtROS) production and reduced mitochondrial calcium uptake, leading to loss of mitochondrial membrane potential, structural damage, and subsequent CD4+ T cell apoptosis. In CLP mice, monotherapy with either DNase I or mitoTEMPO partially attenuated CD4+ T cell injury, while the combination significantly improved 7-day survival compared with monotherapy or vehicle, reduced plasma organ injury markers, and alleviated splenic lymphocyte apoptosis. The NETs-mtROS axis represents an important pathway driving CD4+ T cell death in sepsis. Dual targeting of NETs and mitochondrial oxidative stress holds promising therapeutic potential for ameliorating sepsis-induced immunosuppression.