Extracellular vesicles derived from patients with liver cirrhosis and acute-on-chronic liver failure show distinct phenotype leading to stronger immunomodulatory and cytotoxic function

Liver cirrhosis (LC) and acute-on-chronic liver failure (ACLF) are characterized by systemic inflammation and dysfunctional immune responses. In the present study, we aimed to explore the phenotype and the role of extracellular vesicles (EVs) on hallmarks of disease development and progression. Patients with compensated or decompensated LC or ACLF were recruited from a prospective, monocentric cohort study at the University Hospital Essen. Precipitation method was used to isolate EVs from EDTA plasma and ascites fluid. EVs were characterized regarding particle size and concentration by Nanoparticle Tracking Analysis (NTA), protein cargo by flow cytometry, western blot, and mass spectrometry as well as RNA content by smallRNA sequencing. The role of EVs was assessed by in vitro stimulation of immune cells such as neutrophils, monocytes, NK, B and T cells and liver cells such as primary human hepatocytes (PHHs) and hepatic stellate cells (HSCs). Particle concentration in the blood of patients was increased in LC but decreased in ACLF patients, whereas the average size increased with disease progression. EDTA EVs of LC and ACLF patients showed lower expression of exosome-specific markers CD9, CD63, CD81 and higher amounts of liver cell-specific markers (e. g. ASGPR1, CD163) compared to healthy donor EVs. Immune cell marker and smallRNAs were mainly decreased on and in blood-derived EVs with disease progression. In functional assays, EDTA EVs of LC and – to a stronger extend – of ACLF patients led to changes in the composition of T cell populations, e. g. by downregulation of CD197 expression on T cells enabling tissue infiltration. The EVs even induced mitochondrial dysfunction in T cells and reduced the cells’ viability. Furthermore, LC and ACLF EVs led to decreased viability of primary human hepatocytes. Besides, plasma-derived EVs of LC and ACLF patients increased activation of B cells and neutrophils. Concluding, EVs from patients with LC and ACLF were less differentiated compared to those of healthy controls. It seems that EVs of LC patients were more unspecific released from cells compared to healthy donors shown by decreased exosome-specific marker expression. LC- and ACLF-derived EVs induced hallmarks in LC or ACLF development and progression such as immune cell dysfunction, mitochondrial dysfunction,