Mesenchymal stem cell transplantation alleviated TBI-induced lung injury by inhibiting PAD4-dependent NET formation.

introductionTraumatic brain injury (TBI) affects millions of people worldwide and often results in significant extracranial complications, particularly acute respiratory distress syndrome (ARDS). The mechanisms underlying TBI-induced lung damage remain poorly understood, and current treatment options are limited.

objectivesThis study aimed to investigate the therapeutic potential and mechanisms of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) transplantation for alleviating TBI-induced lung injury and improving neurological function. Specifically, we sought to determine the role of neutrophil extracellular traps (NETs) in TBI-induced lung injury and whether hUC-MSCs improve acute lung injury (ALI) by inhibiting NET formation.

methodsTBI-associated ARDS in patients was diagnosed based on chest computed tomography (CT) imaging and relevant physiological and biochemical parameters. Bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) samples from TBI patients were collected to evaluate neutrophil activation and its correlation with the severity of pulmonary injury. A TBI mouse model was established using the Controlled Cortical Impact (CCI) method. 12 h post-injury, hUC-MSCs were administered via intravenous injection. Neurological function was assessed using the modified Neurological Severity Score (mNSS) and balance beam test. Lung and brain tissue injury were evaluated by histological staining, oxygen saturation monitoring, and micro-CT. Neutrophil infiltration and NET formation were detected in PB, BALF, and lung tissue by flow cytometry, immunofluorescence, and Western blotting. To further elucidate the direct regulatory effects of hUC-MSCs on neutrophils in vitro, neutrophils isolated from the PB of TBI patients were co-cultured with hUC-MSCs. The formation of NETs and reactive oxygen species (ROS) was subsequently quantified.

resultsWe initially assessed neutrophil activation and NET formation in PB and BALF from TBI patients. The results revealed that neutrophils in PB were activated, with even more pronounced activation observed in BALF. Simultaneously, NET formation in PB was significantly elevated. A strong positive correlation was identified between the extent of neutrophil infiltration in BALF and the severity of pulmonary injury. In the CCI-induced TBI mouse model, hUC-MSC transplantation notably improved neurological function and alleviated pathological brain damage. Additionally, hUC-MSC administration increased SpO2, reduced lung injury scores, and partially restored the ultrastructural integrity of type II alveolar epithelial cells. Mechanistic studies demonstrated that hUC-MSC transplantation effectively suppressed neutrophil infiltration, NET formation, and the expression of peptidyl arginine deiminase 4 (PAD4), a crucial enzyme involved in NETosis. Remarkably, hUC-MSCs showed superior efficacy in mitigating TBI-induced ALI compared to pharmacological approaches targeting PAD4 inhibition or DNase-mediated NET degradation. Moreover, in vitro co-culture experiments confirmed that hUC-MSCs directly inhibited both NET production and ROS generation by peripheral neutrophils isolated from TBI patient.

conclusionOur findings demonstrate that hUC-MSCs significantly alleviate TBI-induced lung injury by inhibiting neutrophil infiltration and NET formation, offering potential therapeutic benefits for treating TBI-associated lung complications. These results highlight the clinical potential of hUC-MSCs in addressing both neurological and pulmonary damage in TBI patients.