Ιmmunosuppressive factors in small extracellular vesicles as mediators of immune regulation after hepatitis C virus treatment with direct acting antivirals

Intercellular communication mediated by Extracellular Vesicles (EVs), including small EVs (sEVs) and exosomes, is systematically used by viruses, such as the Hepatitis C Virus (HCV), for establishing viral infection, spread and persistence through immune system deregulation. HCV causes severe liver disease. Direct-acting antivirals (DAAs) eradicate the virus, but host immunity fails to fully recover. To investigate the role of HCV-associated sEV cargo in the immune response of chronic Hepatitis C (CHC) patients with characterised liver fibrosis, pre- and post-DAA treatment, we isolated serum EVs and peripheral blood mononuclear cells (PBMCs) from these patients. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) determined EV size/concentration and shape, respectively. sEV cargo was analysed for the presence of immunosuppressive factors transforming growth factor-β (TGF-β) and programmed death-ligand 1 (PD-L1) by immunoblotting and enzyme-linked immunosorbent assays (ELISA). Our data showed that DAA treatment reduced the HCV-mediated increase in sEV-TGF-β from mild to advanced fibrosis. sEV-PD-L1 expression was up-regulated from mild fibrosis to advanced fibrosis and cirrhosis and remained elevated irrespectively of DAA administration. PBMC phenotyping, performed by flow cytometry, revealed unchanged levels of cytotoxic T-cells in all conditions examined. Furthermore, the regulatory T-cells (Tregs) were decreased significantly in mild fibrosis, but they were found expanded in advanced fibrosis and cirrhosis after DAA treatment, compared to infected controls. Multiple linear regression models revealed a strong reciprocal relationship between Tregs and sEV-PD-L1 levels. Overall, our data suggest an intricate interplay between Tregs and sEV-PD-L1, which could assist with the maintenance of an immune suppression state and chronic tissue remodelling in CHC patients. Post-treatment, this remaining immunosuppressive viral signature could deteriorate liver disease in patients with advanced fibrosis and cirrhosis, possibly promoting the development or progression of hepatocellular carcinoma (HCC) in susceptible individuals cured of HCV.