Exosomes: multitask cargo carriers modulating innate immunity to viruses

Chronic infection with hepatitis B virus (HBV) is a major cause of cirrhosis and liver cancer worldwide. HBV is a smart DNA virus that replicates through an RNA intermediate. The viral genome translocates to the nucleus where it is converted into covalently closed circular DNA (cccDNA) serving as a template for viral transcription.¹ Acute HBV infection is usually self-limited in adults but HBV cccDNA may persist for life in the host's hepatocytes as a mini-chromosome often resulting in occult infection, which may reactivate in case of immune suppression. Although HBV eradication is theoretically possible, there are currently no drugs capable of achieving complete viral clearance when chronic infection is established. Viral control occurs through coordinated innate² and adaptive³ immune responses. Although the latter are thought to be instrumental in getting to grips with most viral infections including HBV, the former, that were traditionally viewed as having a critical role in the initial containment of viraemia in acute infections, are now gaining substantial credit as fine tuners of T-cell responses to regulate immunopathology. Moreover, the rigid partition between the two branches of immune responses is losing ground as cells traditionally assigned to innate immunity, such as natural killer (NK) cells, have been shown to bear classical adaptive features such as long-lived memory or fine specificity.⁴

NK cells represent a cornerstone in the defense against pathogens and are tightly regulated by several membrane receptors able to switch the NK function toward activation or inhibition after ligand recognition and interaction.⁵ Their ability to contribute to control HBV has been clearly demonstrated in humans;⁶ however, during chronic HBV infection, multiple studies have shown that NK cells are defective in the production of cytokines, such as interferon-γ (IFNγ) and tumor necrosis factor-α, impairing their non-cytolytic antiviral capacity.⁷ In addition, NK cells fulfill negative tasks in chronic HBV infection by inhibiting antiviral T-cell responses, via rapid, contact-dependent killing of HBV-specific T cells.⁸ Further, NK-cell depletion as well as TNF-related apoptosis-inducing ligand and the NK group 2D pathway blockade improves HBV-specific T-cell function, providing incremental evidence for a critical role of NK cells in HBV infection.⁹

Taking advantage from studies indicating that exosomes can transfer viral genetic material to cells,¹⁰ Yang et al. in this issue of Cellular and Molecular Immunology¹¹ provide novel and interesting data demonstrating that, similarly to other (mostly RNA) viruses, HBV nucleic acids and proteins can be transferred to liver cancer and NK-cell lines as well as in primary human NK cells, independent of HBV-specific receptors (Figure 1). They further show that NK-cell function is impaired following exosome-mediated transfer of HBV material, resulting in reduced expression of degranulation markers and impaired IFNγ production. Such functional defects were paralleled by phenotypic changes indicating selectively reduced activating receptor (NKp44) and increased inhibitory receptor (NKG2A) expression. Importantly, they also show that exosome-mediated transfer of HBV material predominantly suppressed retinoic acid-inducible gene 1 (RIG-I), which resulted in inhibition of NF-κB and MAP kinase pathways. These results are consistent with recent data showing intrahepatic HBV sensing by RIG-I,¹² shedding more light on a hitherto unrecognized mechanism of HBV-induced crippling of innate immunity. Recognition of HBV by intracellular pattern recognition receptors has been a rather controversial area. Early studies showed that although hepatitis C virus (HCV) ‘makes a lot of noise', for example, induces substantial amounts of type I IFNs in infected cells, HBV produces no clearly appreciable changes in innate immune response genes of HBV-infected chimpanzees in the first weeks of infection.¹³ For this reason, HBV has for long been considered a ‘stealth' virus, gaining access virtually unchecked to hepatocytes because of failure to induce and actively suppress intracellular innate responses.

Figure 1.

Proposed mechanisms of exosome-mediated propagation of HBV infection and modulation of NK-cell responses. HBV-infected hepatocytes release fully competent virions that can be recognized by HBV receptor such as the NTCP and transmit the infection to neighboring hepatocytes. Infection can also be conveyed by viral nucleic acid-loaded exosomes originated by HBV-infected hepatocytes in a receptor-independent manner. Exosomes regulate NK-cell responses by dampening intracellular innate sensing by RIG-I, which results in down-modulation of the NKp44 activating receptor and up-regulation of the NKG2A inhibitory receptor. NK-cell phenotypic changes are accompanied by reduced IFN-γ production and impaired cytolytic activity. Which of the many components of the exosome cargo is responsible for impaired innate immune responses is still unknown. HBV, hepatitis B virus; IFN-γ, interferon-γ NK, natural killer; NTCP, sodium-taurocholate co-transporting polypeptide; RIG-I, retinoic acid-inducible gene 1.

Transmission and regulation of viral infections by exosomes has been known for quite a while. Exosomes represent the smallest (40–100 nm) type of extracellular vesicles secreted by sundry cells and are derived from endosomes containing membrane-bound vesicles. The endosomal sorting complexes required for transport machinery has a pivotal role in the biogenesis of this process. Exosomes regulate cell–cell communication via horizontal transfer of their cargo, including proteins, messenger RNAs and microRNAs (miRNAs).¹⁴ Viral infections can be readily transmitted by exosomes loaded with viral nucleic acids with no need for virus–receptor interaction. Notably, replication competent HCV RNA can be successfully transferred by exosomes¹⁵ and antiviral responses may be profoundly influenced by exosome-mediated transfer. For instance, liver cells stimulated by IFNα-release exosomes that contain antiviral molecules and can attenuate HBV replication.¹⁶ Moreover, exosome-mediated communications have a broad effect on the functionality of cells in the vicinity of infected cells, including those that are refractory to productive viral infection, such as dendritic cells,¹⁷ and most likely, NK cells. However, which of the many diverse components of exosome cargoes originated from HBV-infected cells is responsible for down-modulation of HBV sensing and weakening of NK-cell responses is still unknown, but miRNAs derived from the viral genome are high in the list of candidates. Unfortunately, this was not addressed by Yang's study.

Can exosomes be exploited to carry therapeutic cargoes to infected cells? Targeted delivery is indeed another area where the potential of exosomes remains to be explored. To this end, one can envisage to turn exosomes into delivery vehicles of drug/small interfering/miRNA-based therapeutics to infected cells, paving the way to an exciting and nascent research topic in virology.