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Objectives and questions: Polytrauma is a leading cause of mortality worldwide and is associated with a profound systemic inflammatory response, in which polymorphonuclear neutrophils (PMNLs) are among the first immune cells to be recruited. The liver is a key metabolic and immunological organ in post-traumatic homeostasis. By releasing extracellular vesicles (EVs), PMNLs can interact with distant organs such as the liver and modulate inflammatory signaling pathways. This study aimed to investigate how PMNL-derived EVs, generated under different proinflammatory conditions, affect inflammatory and apoptotic signaling in a hepatocellular in vitro model.

Material and methods: In a prospective experimental ex vivo study, PMNLs were isolated from peripheral blood of healthy adult donors and stimulated for one hour with either interleukin-6 (IL-6-EVs) or a polytrauma cocktail (C3a, C5a, IL-1β, IL-6, IL-8 and TNF-α; PTC-EVs) mimicking the post-traumatic systemic inflammatory response; non-stimulated PMNLs treated with PBS (PBS-EVs) served as controls. EVs were isolated from cell culture supernatants, characterized by transmission electron microscopy and nanoparticle tracking analysis, and phenotyped using a MACSPlex EV panel and western blot. HepG2 liver cells were stimulated with 1 µg/mL EVs for 24 hours. Cell viability and apoptosis were assessed by flow cytometry (viability dye, Annexin V, CD95), and proinflammatory and proapoptotic gene expression (TNFα, IL6, BAX) was quantified. Experiments were performed with N=3 biological and n=3 technical replicates; group differences were analyzed using Kruskal-Wallis tests with Dunn post-hoc tests (p<0.05).

Results: IL-6-EVs significantly downregulated TNF-α expression versus controls, indicating an immunosuppressive profile. In contrast, PBS-EVs and PTC-EVs significantly increased IL-6 and BAX expression up to about 2-3-fold, consistent with a proinflammatory and proapoptotic shift. Overall cell viability and proportion of Annexin V-positive cells did not differ significantly between groups. However, within the Annexin V-positive population, PBS-EVs and PTC-EVs significantly increased Annexin V signal intensity, and CD95 surface expression was enhanced compared to the controls, indicating a susceptibility to Fas-mediated apoptosis.

Discussion and conclusions: EVs released from PMNLs under polytrauma-like conditions, as well as EVs from non-stimulated PMNLs, enhance proinflammatory and proapoptotic signaling in HepG2 cells and may contribute to liver injury as remote organ damage following trauma. In contrast, EVs derived from IL-6-stimulated PMNLs predominantly exerted immunosuppressive effects by attenuating TNF-α and appeared to partially counteract the proinflammatory actions of PBS-EVs and PTC-EVs. These findings highlight the pleiotropic nature of IL-6 signaling and suggest that a complex cytokine milieu, such as that represented by the polytrauma cocktail, is required to elicit the full spectrum of inflammatory responses in hepatic cells after trauma.