Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure

BACKGROUND: Previous studies have shown that mesenteric lymph (ML) has a crucial role in driving the systemic inflammatory response after trauma/hemorrhagic shock (T/HS). The specific mediators in the ML that contribute to its biological activity remain unclear despite decades of study. Exosomes are extracellular vesicles that are shed into body fluids such as serum and urine that can mediate intercellular communication. We hypothesized that exosomes are present in the ML after trauma/shock and are responsible for the biological activity of ML. METHODS: Male rats underwent cannulation of the vessels and mesenteric lymph duct. T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mmHg), followed by resuscitation. The ML was collected during three distinct time periods (pre-shock, shock, and resuscitation phase) and subsequently separated into exosome and supernatant fractions. Exosomes were characterized by electron microscope, nanoparticle tracking analysis, and immunoblotting. The biological activity of exosomes and supernatant of ML were characterized using a monocyte NF-kappa B reporter assay and by measuring macrophage intracellular TNF-alpha production. RESULTS: Exosomes were identified in ML by size and expression of the exosome markers CD63 and HSP70. The number of exosomes present in the ML was 2-fold increased during shock and 4-fold decreased in resuscitation phase compared to pre-shock. However, biological activity of exosomes isolated during the resuscitation phase was markedly increased and caused an 8-fold increase in monocyte NF-kappa B activation compared to supernatant. Macrophage TNF-alpha production was also increased after exposure to exosomes harvested in the resuscitation phase. The ML supernatant fraction had no effect on TNF-alpha production during any phase. CONCLUSIONS: Our findings show that exosomes, and not the liquid fraction of ML, are the major component triggering inflammatory responses in monocytes and macrophages after experimental T/HS. Copyright (C) 2016 Wolters Kluwer Health, Inc. All rights reserved.)