NETosis in the Kidney, Liver, and Lung of Mice With Cecal Ligation and Puncture-Induced Sepsis and the Ameliorating Effects of Adipose-Derived Stem Cell Exosomes.

Abstract

Sepsis-induced organ failure involves dysregulated neutrophil responses, including neutrophil extracellular trap (NET) formation, via NETosis. Exosomes generated from adipose-derived stem cells (ADSCs) have shown potential in sepsis treatment. This study characterized organ-specific NETosis in the kidney, liver, and lungs using a murine cecal ligation and puncture (CLP) model and assessed the modulatory effects of ADSC exosomes. Male C57BL/6 mice were grouped as follows: control, CLP, and CLP with ADSC exosome therapy (n = 6 per group). Plasma cell-free DNA (cfDNA) and tissue citrullinated histone H3 (CitH3) levels were measured at 16 h postoperatively. Confocal immunofluorescence was performed by co-staining for CitH3 and Ly6G. Organ-derived Ly6G-enriched neutrophils were subjected to quantitative reverse transcription polymerase chain reaction for seven NETosis-related genes: CYBB, Padi4, H2ac20, H2bc21, Nlrc4, Nlrp3, and Ripk3. CLP significantly elevated plasma cfDNA and lung CitH3 levels, whereas ADSC exosome treatment reduced both. Kidney and liver CitH3 levels remained unchanged across the groups. Confocal imaging confirmed active NETosis predominantly in the lung, with minimal signals in the kidneys and liver. In kidney and liver neutrophils, only Nlrp3 and Ripk3 were significantly upregulated by CLP. In the lungs, all seven genes were significantly elevated; ADSC exosomes significantly reduced Padi4, H2ac20, Nlrp3, and Ripk3. CLP-induced NETosis was more pronounced in the lungs at the protein and transcriptional levels, where ADSC exosomes broadly suppressed NETotic pathways. NETosis reduction by ADSC exosomes was not observed in renal and hepatic neutrophils, indicating their possible protective role in sepsis within the pulmonary system.