Exosomes target HBV-host interactions to remodel the hepatic immune microenvironment

Chronic hepatitis B poses a significant global burden, modulating immune cells, leading to chronic inflammation and long-term damage. Due to its hepatotropism, the hepatitis B virus (HBV) cannot infect other cells. The mechanisms underlying the intercellular communication among different liver cells in HBV-infected individuals and the immune microenvironment imbalance remain elusive. Exosomes, as important intercellular communication and cargo transportation tools between HBV-infected hepatocytes and immune cells, have been shown to assist in HBV cargo transportation and regulate the immune microenvironment. However, the role of exosomes in hepatitis B has only gradually received attention in recent years. Minimal literature has systematically elaborated on the role of exosomes in reshaping the immune microenvironment of the liver. This review unfolds sequentially based on the biological processes of exosomes: exosomes’ biogenesis, release, transport, uptake by recipient cells, and their impact on recipient cells. We delineate how HBV influences the biogenesis of exosomes, utilizing exosomal covert transmission, and reshapes the hepatic immune microenvironment. And based on the characteristics and functions of exosomes, potential applications of exosomes in hepatitis B are summarized and predicted. Graphical Abstract


Introduction
The widespread use of HBV vaccines in many countries, especially due to improved vaccination rates among newborns and maternal-infant blocking [1,2], has paved the way for the potential future elimination of HBV.Despite current antiviral treatments, complete eradication of the virus remains unachievable [3].Approximately 250 million people worldwide remain infected with HBV, with an estimated 17 million fatalities from chronic hepatitis B projected by 2030 [4].Current research indicates that HBV modulates immune cells to cause chronic inflammatory damage, ultimately leading to liver fibrosis and cancer through repeated injury and repair processes [5,6].However, as a hepatotropic virus, HBV primarily relies on liver cells to replicate and synthesize components of the virus.The mechanisms by which HBV regulates across cell types and leads to immune cell dysfunction and disruption of the immune microenvironment remain unclear.
Exosomes, 30-150 nm extracellular vesicle.The cargo and quantity of exosomes can fluctuate in response to the physiological and pathological conditions of the internal environment.Therefore, exosomes can serve as diagnostic biomarkers, reflecting the status of donor cells [7].Moreover, they can influence the function and phenotype of recipient cells and serve as targets for therapeutic interventions [8][9][10].With the advancements in exosome research, it has been discovered that exosomes contribute to the transportation and immune modulation of HBV.Key events in the study of exosomes in HBV include the following milestones: Li et al. first identified the therapeutic potential of exosomes in HBV in 2013, demonstrating that exosomes could transport interferon to HBV-infected hepatocytes, exerting antiviral effects [11]; Yang et al. in 2017 first demonstrated that exosomes could carry and transfer HBV proteins and nucleic acids [12]; In 2023, it was first discovered that exosomes contain intact HBV viral particles and facilitate HBV transmission [13]; cccDNA, as the critical factor in HBV's sustained infection and the current inability of antiviral drugs to cure it, was addressed by Zeng et al. in 2024, who found that exosomes could serve as carriers for gene editing to eliminate cccDNA [14].
Considering the dual nature of exosomes, we comprehensively analyzed the pathogenic mechanisms and therapeutic applications of exosomes in the progression of hepatitis B. This review is based on the biological processes of exosomes: their biogenesis (Fig. 1), release, transportation, uptake by recipient cells, and impact on recipient cells.We delineate how HBV influences the secretion and transportation of exosomes (Fig. 2).These exosomes act on immune cells and reshape the hepatic immune microenvironment.(Fig. 3).Based on the characteristics and functions of exosomes, we summarize and predict the potential applications of exosomes in hepatitis B (Fig. 4).

HBV utilizes the release and transport of exosomes
The process of exosome biogenesis is complex, but how does HBV intelligently engage in and utilize the release and transport of exosomes?This section explores how HBV engages and utilizes exosome biogenesis.Furthermore, it explores the covert transportation of HBV-modified exosomes and their selective binding to recipient cells.

HBV regulates exosome biogenesis to promote the viral release Biogenesis of exosomes
Exosome biogenesis begins with forming early endosomes through the inward budding of the parent cell membrane, subsequently fusing to create early sorting endosomes (ESEs) [15].ESEs undergo a complex sorting, release, and assembly process, eventually maturing into Fig. 1 Exosome biogenesis, the structure and contents of exosomes.The cell membrane invaginates to encapsulate extracellular material, forming early endosomes, which then fuse to form early sorting endosomes (ESEs).ESEs undergo a complex sorting, releasing, and assembling process, ultimately maturing into late sorting endosomes (LSEs).The membrane invagination of LSEs gives rise to intraluminal vesicles (ILVs), leading to the formation of multivesicular bodies (MVBs).MVBs secrete ILVs into the extracellular environment through exocytosis.Regarding the structure and contents of exosomes: they have a diameter of approximately 30-150 nm, encapsulating various cargoes such as proteins, non-coding RNAs, DNA, and more.The outer membrane of exosomes is composed of phospholipid bilayers and surface proteins, including specific marker proteins such as CD63 late-sorting endosomes (LSEs).The membrane invagination of LSEs gives rise to intraluminal vesicles (ILVs), which subsequently lead to the formation of multivesicular bodies (MVBs) [16,17].By undergoing exocytosis, MVBs secrete ILVs into the extracellular environment (Fig. 1).Exosome biogenesis consists of membrane fusion processes and the sorting and regulation of cargo under the control of cellular endosomal sorting complex required for transport (ESCRT)-dependent or ESCRTindependent pathways [18].Moreover, similarities exist between the release pathways of viruses and exosomes [19].Research has extensively documented that viruses participate in exosome sorting and utilize exosomes for release and trafficking in viral infectious diseases [20][21][22].

HBV is involved in the biogenesis of exosomes
HBV and exosomes exhibit a shared mechanism for budding and releasing from cells.For instance, Hepatitis B virus surface antigen (HBsAg) accumulates in the endoplasmic reticulum (ER), potentially being released as subviral particles (SVPs) [23], through the ER Golgi secretion pathway.The released SVPs can enter endosomes and autophagosomes (AP) [24].Moreover, HBV particles and other viral components contribute to the formation of the ESCRT complex and utilize it for secretion from infected hepatocytes [25].HBV infection can lead to significant and specific alterations in the protein content of exosomes [26].Although the underlying mechanisms of HBV entry into exosomes remain unknown, the current evidence suggests that HBV participates in exosome biogenesis.

HBV affects and exploits the biogenesis of exosomes
As depicted in Fig. 2, HBV influences and exploits the biogenesis of exosomes to enhance infectivity, maintain secretory secrecy of HBV, and facilitate replication.HBV genetic material, antigens, and even HBV viral particles can be loaded into exosomes during the process of cargo sorting (Table 1).HBV-induced endoplasmic reticulum stress may increase the sorting of SVPs or HBsAg into exosomes and stimulate exosome production [23].Furthermore, Hepatitis B X (HBx) mRNA and protein can be sorted into exosomes, and notably, HBx, particularly HBx mutant X15, provokes specific alterations in neutral sphingomyelinase 2 (nSMnase2), thereby augmenting enzyme activity and subsequently enhancing exosome biogenesis [27], which facilitates the horizontal transfer of viral genes and production to support viral transmission.Conversely, the inhibition of exosome generation hinders the release of exosome-encapsulated HBV [13].Deleting exosome membrane protein, CD63 accumulates large hepatitis B virus surface antigen (LHB) and substantially decreases infectivity [28].Thus, exosomes can function as viral vectors.Exosomes with HBV modifications facilitate the release of HBV and its associated components and exhibit an increased quantity of released exosomes, contributing to the infectivity of HBV.
Additionally, HBV can exploit the biogenesis of exosomes to achieve the covert release of viral components not readily detected by HBsAg, thereby increasing the risk of cancer.For example, the rtS78T HBV polymerase mutation, which generates a premature stop codon at sC69 (rtS78T/sC69* mutation), significantly intensifies viral replication and reduces susceptibility to Entecavir (ETV) and Tenofovir Disoproxil Fumarate (TDF), with truncated HBs protein and impaired HBsAg detection, but maintains exosome-mediated viral secretion [29], consequently covertly elevating the risk of carcinogenesis.In addition, HBV can also utilize exosome production to promote the exocytosis of apolipoprotein B mRNA editing catalytic polypeptide-like 3G (APOBEC3G) [30].The human APOBEC3 protein is a potent retroviral inhibitor recognized as a restriction factor for HBV [31].In contrast, HBV can mediate its activation by reducing intracellular levels of APOBEC3G, and these diminished APOBEC3G levels are not dependent on proteasomal or lysosomal degradation.On the contrary, they facilitate sorting APOBEC3G into exosomes and promote exocytosis.

Exosomes transport HBV components and enter receptor cells
As discussed in the preceding section, HBV infection influences the biogenesis of exosomes and loads viral components into exosomes.Next, we explore how exosomes carrying viral components achieve stable Fig. 4 Potential applications of exosomes in hepatitis B. markers of diagnosis, progression and prognosis of hepatitis B and its related liver diseases, immunomodulators of chronic hepatitis B, therapeutic targets, treatment of hepatitis B-related liver cancer resistance to chemotherapeutic agents, prediction of hepatitis B drug responsiveness, HBV gene editing vectors, HBV vaccine transport in the extracellular fluid, how they enter recipient cells, and whether the internalization of exosomes by recipient cells is specific (Fig. 2).

The structure and characteristics of exosomes protect HBV components from degradation
Exosomal membranes have a stable phospholipid bilayer featuring higher levels of sphingolipids, unsaturated lipids, and cholesterol than cell membranes [32,33].These lipids are unevenly distributed between the inner and outer layers [34], contributing to the stability of exosomes.Although still debated, pH has been proposed to play an important role in exosome membrane stability [35].Stable membranes render exosomes rigid and resistant to degradation, making them effective carriers of proteins and nucleic acids [36].
Furthermore, in addition to their stable membrane structure, how do these exosomes carrying aberrant components evade recognition and clearance by immune cells?This question may be attributed to the regulation of exosomes on the immune system.Some tumor-derived exosomes downregulate surface major histocompatibility complex I (MHC-I) levels, leading to CD8 + T cell dysfunction [37].Similarly, certain tumor-derived exosomes carry increased programmed death ligand 1 (PD-L1) on their surface, which is an inhibitory checkpoint molecule hindering the function of cytotoxic T cells and macrophages [38].Furthermore, exosomes from hepatitis B-infected liver cells can induce the upregulation of PD-L1 in monocytes [39].While the exact mechanisms remain unclear, the induction of immune tolerance is likely to facilitate the smoother transport of hepatitis B viral components or tumor-derived materials.

Mechanisms by which exosomes enter recipient cells
Current research suggests that different recipient cells may employ various mechanisms for exosome entry, primarily through endocytosis and membrane fusion [40].However, some studies propose that receptor (direct) interaction is also a mode of action with recipient cells.This can occur through the direct binding of transmembrane ligands on the surface of exosomes with surface receptors on recipient cells, leading to downstream signaling cascades to activate target cells [41].For instance, exosomes released by dendritic cells express surface tumor necrosis factor (TNF), Fas ligand (FasL), and TNFrelated apoptosis-inducing ligand (TRAIL), which can interact with TNF receptors on tumor cells, triggering caspase activation and apoptosis [42].Through receptor interactions, signaling pathways can be directly activated to influence the function of target cells, or they can initiate subsequent internalization processes.For example, different liver cells exhibit varying efficiencies in absorbing exosomes bearing surface hepatitis B large surface antigen (LHBS).Liver cells possessing the NTCP receptor can demonstrate more efficient internalization and transport of exosomes through an LHBs/NTCP-dependent mechanism [13].
The mechanism of endocytosis is rapid and energyconsuming.Exosomes can be identified inside cells after approximately 15 min of co-culture [43].Lowering the temperature significantly reduces the ability of endocytosis, indicating that intake is an energy-requiring process [44].Endocytosis can be further classified into five types: Clathrin-mediated endocytosis (CME), caveolin-mediated endocytosis, macropinocytosis, phagocytosis, and lipid-raft-mediated endocytosis [45].
Caveolin-mediated endocytosis, also known as caveolin-dependent endocytosis (CDE), does not rely on clathrin.Caveolae are subdomains of the plasma membrane glycolipid rafts, enriched with cholesterol, sphingolipids, and caveolin proteins.Caveolae can expand and envelop to form vesicles, enabling the internalization of exosomes.Inhibition of caveolin-1 can significantly impair B cell uptake of extracellular vesicles [49], while upregulation of caveolin-1 expression in neurons can enhance the uptake of extracellular vesicles [50].
Macropinocytosis involves the formation of membrane ruffles, extending from the cell surface to surround the extracellular fluid area.Subsequently, through fusion with itself or with the plasma membrane, exosomes within this extracellular fluid area are completely internalized [51].This mechanism can intake large volumes of fluid, with the maximum diameter of extracellular vesicles taken up exceeding 200 nm [45].Since this process relies on rac1, its small-molecule inhibitor NSC23766 can suppress the uptake of extracellular vesicles by microglia [52].Exosomes derived from macrophages bind to the T cell immunoglobulin and mucin receptor 1 (TIM-1) and enter HBV-infected liver cells through CME and macropinocytosis, effectively transferring interferonalpha (IFN-α) induced anti-HBV activity [53].However, this TIM-1-mediated uptake mechanism via CME and macropinocytosis is not applicable to cell types such as HepG2 and LX-2 [53].Mechanisms of internalization from different types of receptor cells still require further exploration.
Phagocytosis is initiated by the binding of particles to surface proteins (such as scavenger receptors) or through specific receptor interactions, triggering actin polymerization to form vesicles that enclose exosomes.Phagocytosis includes professional phagocytic cells (polymorphonuclear leukocytes, monocytes, and macrophages) and non-professional phagocytic cells that uptake particulate matter.However, non-phagocytic cells lack classical receptors to initiate phagocytosis, but if appropriate receptors are expressed on the surface of non-phagocytic cells, phagocytosis can also occur [54].
Lipid-raft-mediated endocytosis is another pathway for the internalization of exosomes carrying cargo into early endosomes.Lipid rafts are microdomains rich in cholesterol, sphingolipids, and protein receptors, whose components are highly organized but can freely float on the plasma membrane [55].Interference with intracellular cholesterol transport can affect lipid-raft-mediated endocytosis, thereby reducing the uptake of exosomes [56].
Membrane fusion is also a mechanism by which exosomes enter recipient cells.During this process, the membrane of the exosome directly contacts and fuses with the plasma membrane of the recipient cell, releasing its contents into the cytoplasm.Exosome membranes exhibit a topology similar to that of the plasma membrane [57,58] and contain more phosphatidylserine molecules exposed to the extracellular environment, which may facilitate their fusion with the membrane of recipient cells [52,59].Additionally, the tetraspanin proteins CD9 and CD81, which are integral to the exosome membrane, have been shown to participate in the membrane fusion process [60].Fusion efficiency of exosome membranes significantly increases under low pH conditions [35].

The internalization of exosomes carrying HBV components exhibits selectivity and universality
The binding of exosomes carrying HBV components to recipient cells exhibits a degree of selectivity, primarily determined by the specificity of membrane surface receptor interactions.For instance, liver cells possessing the NTCP receptor demonstrate significantly higher efficiency in receiving HBV-positive exosomes compared to NTCP-negative liver cells [13].Nevertheless, a substantial amount of exosomes, when incubated with NTCP-negative liver cells, still results in the internalization of viral components into the recipient cells [13].After surface modification of exosomes with leukocyte interleukin-4 receptor (IL4R) fusion protein, they exhibit specific binding to IL4R on M2 tumor-associated macrophages (TAM) [61].The lipid composition of exosomes also influences their targeting.For instance, exosomes derived from glioblastoma cells enriched with phosphatidylethanolamine preferentially target glioblastoma cells, as well as fibrosarcoma and breast cancer cells.
We speculate that this selectivity only affects the efficiency of internalization, whereas exosomes containing HBV-related components secreted by HBVinfected liver cells can be internalized by almost all types of cells within the liver.This is attributed to the fact that hepatocytes, as the most abundant cell type in the hepatic environment, are involved in the synthesis, transportation, and metabolism of products and engage in close exchange of nutrients with various other liver cell types [62].Exosomes originating from liver cells are familiar to other types of liver cells [63].Therefore, theoretically, the HBV components encapsulated within these exosomes can be engulfed by these cells.Despite variations in uptake efficiency, multiple cell types possess nonspecific uptake capabilities [64], with some studies even suggesting that all cell types can nonspecifically internalize exosomes [41].Currently, it has been demonstrated that most cells in the liver environment can receive exosomes containing HBV antigens, microRNAs, and other HBV-related components.These cell types include macrophages [65], NK cells [12], T cells [66], DCs [67], as well as hepatocytes [13] and hepatic stellate cells (HSCs) [68].The phenotypic changes in recipient cells following the binding of exosomes will be discussed in the subsequent section.

HBV modifies exosomes to reshape the host immune microenvironment
Under physiological conditions, exosomes play a crucial role in maintaining the hepatic microenvironment's homeostasis.However, under the persistent influence of HBV, donor cells undergo epigenetic modifications, leading to significant changes in the content of secreted exosomes [69].These exosomes subsequently modulate the epigenetics of recipient cells in the liver [70], controlling their gene expression patterns and levels of key proteins, such as pathway crucial enzymes, membrane surface receptors, and transcription factors.Some exosomes create a host immunosuppressive microenvironment, leading to virus persistence and chronic liver inflammation, as shown in Fig. 3. Others activate immune cells, break immune tolerance, and transfer antiviral capacity to eliminate the virus.Nevertheless, incomplete immune activation may lead to viral explosive replication and inflammatory stress, ultimately leading to liver failure [71].

HBV-positive exosomes remodel the immunosuppressive microenvironment in CHB
HBV evades detection and clearance by immune cells by hijacking exosomes to modify the immune cells' epigenetic inheritance, thus creating an immune tolerance microenvironment.HBV becomes an "invisible virus" with occult replication and transmission, posing high risks of liver fibrosis and liver cancer [29].

Monocytes
Monocytes, the largest blood cells, and an essential immune system component, possess strong phagocytic and deformability abilities [72,73].Changes in their proportion may indicate inflammation or disease, serving as a critical clinical reference index.HBV-infected hepatocytes synthesize and release virus-associated exosomes, including viral proteins, HBsAg, Hepatitis B virus core antigen (HBcAg), viral DNA, and other viral components that upregulate the monocyte PD-L1 expression [39], causing monocyte suppression.However, the mechanism behind exosomes with viral particles upregulating PD-L1 expression remains unclear.Besides directly assembling HBV viral components into exosomes resulting in monocyte dysfunction, HBV can indirectly upregulate exosome proteins and modify monocyte cytokine expression.For instance, Jia et al. discovered several upregulated proteins following HBV infection, including proteasome subunit alpha-7 (PSMA7), PSMD14, PSMC1, PSMD1, and PSMC2, which belong to the ubiquitin-proteasome system (UPS) that degrades and modifies proteins [74].Numerous viruses, including HBV, have been shown to manipulate the UPS [75,76].For example, PSMA7 has been demonstrated to interact specifically with the HBx protein [77].Consequently, HBV utilizes infected hepatocytes to synthesize and load proteasomes into exosomes, thereby inhibiting monocyte Interleukin (IL)-6 secretion and reducing its antiviral effect [74].

Macrophages
Monocytes that migrate into tissues from the blood are called macrophages.These cells process and present antigens to activate other immune cells, participating in pathogen phagocytosis and cytokine secretion, making them crucial defense cells against infection [78,79].To sabotage the macrophages' role as a "damage sensor", HBV secretes various types of exosomes.These exosomes modify the macrophages' epigenetic inheritance, resulting in dysfunction.For example, You et al. found that HBV uses exosomes to deliver miR-27b-3p, directly inhibiting the target genes retinoic acid-inducible gene I (RIG-I) and TANK-binding kinase 1 (TBK1) expression, leading to macrophage dysfunction [80].Interferon upregulates nuclear deaminases such as APOBEC3A and APOBEC3B to exert a direct anti-HBV effect [81,82].Macrophages, as essential cells for endogenous interferon production, can detect viruses through pattern recognition receptors (PRR) such as RIG-I and TBK1, mediate phosphorylation of interferon regulatory factor (IRF)-3 and IRF-7, and promote type I IFN gene transcription [83,84].Thus, exosomes carrying miR-27b-3p secreted by infected hepatocytes inhibit endogenous IFN-α production in macrophages, possibly explaining the persistence of HBV.Moreover, HBV-infected hepatocytes can release miR-21-enriched exosomes that target macrophages and suppress IL-12p35 mRNA levels, leading to diminished IL-12 secretion by macrophages.IL-12 is an important cytokine that activates NK cells, thus further contributing to NK cell dysfunction [65].

NK cells
NK cells are crucial antiviral and antitumor immune cells, capable of exerting direct cytotoxic effects without MHC restriction and antibody dependence.They can also secrete various cytokines to regulate the immune microenvironment and kill target cells [85,86].As mentioned earlier, HBV can indirectly impair NK cell activation through the use of exosomes.While also delivering HBV nucleic acids to directly downregulate RIG-I expression and inactivate NF-κB and p38 pathways, resulting in reduced NK cytotoxicity and IFN-γ production [12], which aligns with multiple studies that have found HBV infection to upregulate representative immunosuppressive factors, block NKG2D and 2B4 activity, and lead to NK cell dysfunction [87,88].In addition, HBV-induced NK cell dysfunction can also evade immunity by regulating OX40L expression on plasmacytoid dendritic cells (pDCs), thereby impairing subsequent NK cell lysis activity [89,90].The expression level of OX40L on pDC was closely correlated with viral load and HBsAg level.In addition to the direct effects of free viral particles and antigenic particles, it has been well documented that exosomes transport and load HBV proteins and viral particles [12,27].Consequently, we hypothesize that exosomes may play a crucial role in pDC and NK dysfunction caused by high viral load, but this requires verification in future studies.

DC cells
DC cells are the most potent antigen-presenting cells, activating various immune cells during the early stage of HBV invasion.As mentioned earlier, they can also activate NK cells to exert antiviral effects, significantly determining the prognosis and outcome of HBV infection [91,92].When immune cells fail to clear HBV entirely, the body enters the chronic hepatitis B infection period, in which DCs determine interferon therapy responsiveness.However, HBV-infected hepatocytes secrete exosomes enriched in transmembrane protein 2 (IFITM2), which acts on DC cells to suppress endogenous IFN-α synthesis pathways.IFITM2 inhibits endogenous IFN-α synthesis in DC cells by inhibiting phosphorylation of extracellular signal-regulated kinase (ERK), TBK, and IRF3 [67].

T cells
T cells are adaptive immune cells with multiple subsets and complex functions.It is widely believed that T cell dysfunction in patients with chronic hepatitis B resulted from a combination of mechanisms [93,94].In line with this, Enomoto et al. [66] discovered that miR-21, miR-192, miR-215, miR-221, and miR-222 were elevated in exosomes secreted by hepatocytes after HBV infection and downregulated the sequence of IL-21 mRNA expression in human T cells, resulting in decreased IL-21 production by T cells.T-cell dysfunction is a key factor in the chronicity of HBV infection, yet unfortunately, the role of exosomes in T-cell dysfunction in hepatitis B is poorly studied.Research has indicated that exosomal delivery of high levels of programmed death-ligand 1 (PD-L1) can lead to macrophage dysfunction, indirectly impairing T-cell function [95].Additionally, exosomes from myeloid-derived suppressor cells (MDSCs) can induce T-cell senescence through the P53 pathway [96].Therefore, we speculate that exosomes may be involved in delivering immunosuppressive cytokines, upregulating inhibitory receptors or ligands, and increasing the proportion of regulatory cells.

The association of exosome-associated immunosuppressive microenvironment and hepatocellular carcinoma development
Tumors should not be regarded as single tumor cells but as organs composed of various tumor cells and nontumor cells with distinct roles constituting the tumor microenvironment (TME), promoting tumor cell proliferation and metastasis, like the relationship between seeds and soil.The HBV-infected hepatocyte exosomes modify recipient cell epigenetic inheritance and create an immunosuppressive microenvironment.It is the precursor of the tumor microenvironment and provides fertile ground for developing hepatocellular carcinoma (HCC).Tumor-associated macrophages (TAMs) are essential components that regulate and constitute the TME [97], with TAMs in tumor tissues predominantly exhibiting an "M2-like" phenotype [98,99].HCC-derived exosomes are rich in long noncoding RNA (lncRNA) TUC339 and can promote HCC proliferation and transmit to adjacent macrophages, regulating macrophage cytokine production, phagocytosis, and M1/M2 polarization [100].
Moreover, hepatitis B virus e antigen (HBeAg)-induced upregulation of MAAS expression in M2 macrophages transfers to HBV-positive HCC via M2 macrophagederived exosomes, promoting HBV-positive HCC proliferation by targeting MAAS promoted the MYC proto-oncogene (c-Myc) [101].Therefore, the chronic infection of HBV and the HBV-associated exosomes disrupt the hepatic microenvironment's homeostasis and suppress favorable immune responses.In contrast, unfavorable immune responses interact with HCC to promote liver cancer progression.
While most studies suggest that exosomes released by HBV-infected liver cells promote immune suppression to facilitate virus replication [12,27,65], there are also findings indicating that HBV-miR-3 exosomes secreted by HBV-infected liver cells promote macrophage IL-6 secretion, thereby limiting virus replication [102].Some research reports that HBV-miR-3 inhibits HBsAg, HBeAg, and HBV replication [103].The apparent contradiction of HBV limiting its own replication might be explained by HBV-miR-3 serving as a negative feedback control mechanism by the virus itself, which contributes to mild liver cell damage and the establishment and maintenance of subsequent persistent infection [103].Additionally, exosomal HBV-miR-3 appears to play a protective role in hepatitis B, but it is positively correlated with HBV DNA, pregenomic RNA (pgRNA), and HBsAg, which are markers of HBV replication [104,105].This indicates that the reshaping of the immune microenvironment by exosomes after HBV infection is extremely complex and cannot be solely attributed to the action of a single factor.

Immune cells activate and secrete exosomes to break HBV-associated immunosuppression
As described above, HBV modified exosomes to create an immunosuppressive hepatic microenvironment.Conversely, HBV can also activate host immune responses.Here, we categorize the immune activation promoted by HBV exosomes into favorable and unfavorable ones.Favorable immune activation can be divided into early and late immune activation.Early immune activation is characterized by complete and thorough clearance of HBV infection during the acute phase, with 15-40% of HBV patients progressing to chronicity, whereas most young and middle-aged adults can achieve self-healing of HBV during the acute phase [106].For example, HBVinfected hepatocytes activate immune cells by releasing exosomes carrying HBV antigens, enabling novel ways of antigen presentation [65].Late immune activation is the stage of chronic infection after the formation of the HBV immunosuppressive microenvironment, and the body breaks this immunosuppression to remove the virus, which can be exemplified by administering interferon to chronically infected patients, where some can provoke autoimmunity and achieve viral suppression through exosome transfer of antiviral capacity [107].And unfavorable immune activation can be divided into incomplete and excessive immune activation.Incomplete immune activation means that the immune system, while limiting HBV replication to some extent, does not achieve complete viral clearance [102,108], which enables continuous occult replication of HBV and prolongs the course of HBV infection, causing persistent infection and chronic inflammation, ultimately resulting in a high incidence of liver cancer.Excessive immune response is manifested as a burst of immune activation caused by large-scale, short-term viral replication, which is most likely to lead to liver failure.

Potential applications of exosomes in hepatitis B
As discussed earlier, exosomes play a vital role in the release, trafficking, and immune microenvironment of HBV, leading to an increasing interest in their functions and potential applications.As depicted in Table 2, they can be utilized as diagnostic markers and progression indicators for hepatitis B virus activity and complications due to their widespread, noninvasive nature and bilayer lipid stability.As shown in Table 3, their carrier capacity, high biological safety, low immunogenicity, and high efficacy make exosomes valuable as immunomodulators, therapeutic targets, carriers for drug delivery, and vaccines [109,110].

Exosomes as potential biomarkers for hepatitis B-related liver disease
Current imaging techniques have limitations for early diagnosis and predicting hepatitis B-related diseases and complications.Liver biopsy is highly accurate but not the first choice for diagnosis due to its invasive nature [111,112].Since exosomes carry mRNA, miRNA, long noncoding RNA, DNA, nucleic acids, and proteins of the hepatitis B virus, they can accurately and specifically reflect the epigenetic status of recipient cells and the liver immune microenvironment [113].Almost all cells can secrete exosomes, which are present in most body fluids, such as blood, cerebrospinal fluid, urine, ascites, lotions, saliva, and others [114][115][116].Exosomes' noninvasive, accurate, specific, and widespread nature makes them more clinically feasible as biomarkers for hepatitis B-related liver disease [117].

Exosomes as potential biomarkers for hepatitis B cirrhosis
Histological evaluation remains the "gold standard" for diagnosing and assessing cirrhosis, and more accurate indicators are urgently needed to assist the diagnosis.Serum exosomes miR-92a-3p and miR-146a-5p are superior to aspartate aminotransferase-to-platelet ratio index (APRI), fibrosis index based on four factors (FIB-4), and liver stiffness measurement (LSM) in diagnosing severe fibrosis in CHB patients, providing a new noninvasive   alternative [118].In addition, prognostic scoring systems to assess patients with cirrhosis include the Child-Pugh score, the Model for End-Stage Liver Disease (MELD), and the MELD-Na score [119].Recent studies have found that serum exosomal AHCY expression is positively correlated with the MELD score, and the predictive power of serum exosomal AHCY mortality in HBV-related liver cirrhosis (HBV-LC) patients is higher than that of the MELD score and Child-Pugh classification, with a sensitivity and specificity of 93.41% and 76.00%, respectively [120].Compensated cirrhosis is asymptomatic and reversible, while decompensated cirrhosis has poor quality of life and severe life-threatening complications.Therefore, early detection and timely intervention are essential for reversing cirrhosis.Although there are still relatively few studies on exosome markers for hepatitis B cirrhosis, we believe that exosome shuttling between hepatitis B-infected hepatocytes, stellate cells, and immune cells may hold the answers we seek.

Exosomes as potential biomarkers for hepatitis B hepatocellular carcinoma
Early detection of HBV-related HCC is directly related to the survival time and prognosis of patients, and in addition to blood tumor markers, markers of exosomes can also collectively constitute predictors.For example, Hsa_ circ_0028861 can detect smaller, early alpha-fetoprotein (AFP)-negative tumors [121].In addition, lipopolysaccharide-binding protein (LBP), kappa variable 3-11 (KV311), and complement component C9 (CO9) can be used in combination as indicators for HCC diagnosis and treatment evaluation [122].Compared with other causes of HCC, HBV-related HCC can regulate exosomal micro-RNA through HBV-related proteins, altering the epigenetic inheritance of target cells and promoting tumor progression.For example, HBc upregulates miR-135a-5p expression in HBV-infected hepatocytes and is transmitted via exosomes to adjacent or distant recipient cells, leading to reduced vesicle-associated membrane protein 2 (VAMP2) transcription, thereby promoting tumor resistance to apoptosis, cell proliferation, and drug resistance in HCC [123].
Moreover, HBX can upregulate the expression of microRNA-155 in exosomes and tumor tissues, which in turn targets and inhibits Phosphatase and tensin homolog deleted on chromosome ten (PTEN) to inhibit apoptosis and promote invasion and migration of HCCs [124].Therefore, the above article suggested that exosomal miR-135a-5p and microRNA-155 could serve as markers and prognostic indicators for hepatitis B-associated HCC.However, exosomal markers on HBV-related HCC must be comprehensively collated and evaluated to improve the sensitivity and accuracy of adjunctively diagnosing early HCC.

Exosomes as potential biomarkers for chronic hepatitis B
It is easy to determine whether there is an HBV infection, such as by checking serum HBsAg and HBV DNA.Recently, it has also been reported that exosomal HBV-miR-3 is positively correlated with HBV DNA, pregenomic RNA (pgRNA), and especially HBsAg [105], and it can be used as an indicator to assist judgment.However, the staging of hepatitis B relies on liver tissue biopsy to confirm, but it is too costly for patients.As such, few patients with hepatitis B undergo liver biopsies to assist staging clinically.Many patients do not belong to the classical four periods of hepatitis B but fall within the indeterminate period, meaning there are no clear antiviral treatment standards for hepatitis B at each stage [129].While exosomes serve as sensitive communication carriers between donor and recipient cells, although there are no exosome markers reported to assist in hepatitis B staging, we believe that exosome markers that respond to viral replication, damage and repair of hepatocytes, liver function status, and non-parenchymal cell stress will be combined in the future to provide a reference for hepatitis B staging.

Exosomes as a potential therapeutic tool for hepatitis B and related liver diseases
As previously mentioned, HBV utilizes exosomes to release, transport, and reshape the immune microenvironment.Furthermore, exosomes play a dual role in the treatment of hepatitis B. The use of exosomes in treating hepatitis B and related liver diseases includes immunomodulators, potential therapeutic targets, modulation of chemotherapy antagonism in hepatitis B liver cancer, prediction of drug responsiveness, carriers for drug delivery, vaccines, or adjuvants, as shown in Fig. 4.

Exosomes as immunomodulators
Exosomes play an important role in communication and delivery between HBV-infected hepatocytes and immune cells, mainly by delivering microRNAs, proteins, and other substances.These components modify the epigenetics of recipient cells and regulate the immune microenvironment, such as immunosuppression and immune activation, as discussed in "HBV modifies exosomes to reshape the host immune microenvironment" section.Exosomes can be employed to suppress unfavorable immune responses and activate favorable ones.According to the currently reported immune responses involved in exosomes associated with hepatitis B-related liver disease, favorable immune responses include: first, exosomes activate immune cells to exert antiviral effects, such as exosomes of HBV-miR-3 secreted by HBV-infected hepatocytes act on macrophages, promote IL-6 secretion, and limit viral replication [102].Second, immune cell-associated exosomes act on infected hepatocytes and transfer their antiviral capacity.For instance, exosomes can transfer IFN-α associated miRNAs from macrophages to HBV-infected hepatocytes and exhibit antiviral activity against HBV replication and expression [107].Exosomes can also transfer TAF treatment-associated lncRNA HOTTIP from macrophages to HBVinfected hepatocytes and exert antiviral effects [130].
In contrast, in the current study of exosomes in hepatitis B-related liver disease, the unfavorable immune responses include: first, exosomes inhibit immune cells and reduce their antiviral capacity.For example, hepatocytes of HBV secrete exosomes containing miR-27-3p, miR-21, and miR-29a acting on macrophages, which inhibit interferon synthesis and IL-12 expression in macrophages, and suppress their antiviral activity [65,80].Second, activation of tumor-associated immune cells promotes tumor proliferation.For example, increased MAAS in M2 macrophages promote HBV-HCC cell proliferation via M2 macrophage-derived exosomes [101].However, it is challenging to isolate exosomes that promote favorable immune responses precisely.However, it is possible to enrich them by providing some cellular stimulation, such as those secreted by immune cells stimulated by antiviral drugs and interferons.Moreover, exosomes secreted by immune cells tend to have a more potent antiviral capacity, such as exosomal lncRNA HOT-TIP, which is more effective than TAF treatment alone and is essential for the antiviral activity of TAF [130].In addition, since the vast majority of exosomes secreted by HBV-infected hepatocytes inhibit the antiviral effects of immune cells, unfavorable immune responses can also be suppressed by inhibiting the secretion of exosomes from HBV-infected hepatocytes.However, exosome inhibitors cannot precisely identify HBV-infected hepatocytes and may inhibit exosomes from all cells.So appropriate medication timing can be selected according to the strength of the interaction between HBV replication and the body's immunity.When HBV infection is dominant and the favorable immune response weakens, hepatocytes secrete more exosomes that inhibit the antiviral effect of immune cells.The administration of exosome inhibitors may have good therapeutic effects in these cases.Nevertheless, further development is needed to improve the type and safety of exosome inhibitors.

Exosomes as a potential therapeutic target for hepatitis B
Exosomes participate in various processes of hepatitis B development, such as HBV replication and secretion, liver fibrosis process, and HCC development.As such, they can also serve as potential therapeutic targets.In addition to modulating the immune cell response, as mentioned in the previous section, exosomes regulate the phenotype of stellate cells and promote the development of liver fibrosis.
For example, in a mouse model of fibrosis, stress-elevated tribbles pseudokinase 3 (TRIB3) interacted with sequestosome 1 (SQSTM1) to promote the secretion of INHBA/Activin A-rich exosomes from hepatocytes and activate HSCs to promote liver fibrosis [131].HBVinfected hepatocytes can also secrete MiR-222 and HBX to promote the activation of HSCs [27,68].In contrast, Kupffer cells produce endogenous miR-690 and transport it via exosomes to hepatocytes, recruited hepatic macrophages (RHMs), and HSCs, directly inhibiting de novo adipogenesis in hepatocytes, inflammation in RHMs, and activation of HSCs [132].Consequently, the progression of hepatitis B fibrosis can potentially be delayed by promoting the secretion of exosomes that inhibit fibrosis, interfering with the secretion of exosomes that promote fibrosis, or producing therapeutic exosome-targeted fibrosis treatments.

Exosomes regulate drug resistance in hepatitis B liver cancer
HBV-positive HCCs can modify the epigenetics of recipient cells through exosomal delivery, leading to resistance to chemotherapeutic agents.For example, HBV-associated exosomes can deliver lysosome-associated membrane protein (Lamp2a) and miR-135a-5p, mediating anti-apoptotic effects and diminished chemotherapeutic responsiveness [123,133].This results in HBV-associated tumors being less sensitive to transcatheter arterial chemoembolization (TACE therapy) than non-HBV-associated liver cancer.Chemotherapy is an option for patients with advanced HCC, but insensitivity to chemotherapeutic agents leads to poor treatment outcomes.Moreover, resistant cells secrete and deliver exosomes to sensitive cells, facilitating angiogenesis and EMT, further inducing multidrug resistance [134].Thus, exosome-based treatments for tumor resistance appear feasible and have achieved preliminary efficacy in colorectal cancer [135][136][137], although such treatments have not yet been reported in hepatitis B-related liver cancer.

Exosomes predict drug responsiveness
Pegylated interferon (Peg-IFN) offers advantages over nucleoside analogs (NAs), including shorter treatment durations, higher HBeAg seroconversion/HBsAg loss rates, and better post-treatment immune control in HBeAg-positive patients [138].However, Peg-IFN therapy often comes with adverse side effects.Therefore, it is essential to identify potential responders and nonresponders before starting Peg-IFN therapy.Exosomal miR-194-5p, miR-22-3p, and IFITM2 can improve prediction accuracy and reduce the treatment exposure for these potential non-responders [67,139].

Exosomes as a vehicle for drug delivery
Exosomes are biologically derived membrane-bound nanovesicles characterized by stability, biocompatibility, low immunogenicity, and the ability to cross biological barriers [140].These attributes make them natural drug delivery carriers.For example, Osteopontin (OPN) plays a crucial role in liver fibrosis, and reducing OPN expression can attenuate oxidative stress and fibrotic processes.Using exosomes as delivery vehicles for small interfering RNA (siRNA) OPN significantly inhibited HSC activation and ECM deposition compared with naked siRNA OPN [141].However, drug delivery via exosomes is inseparable from advances in artificial exosome technology and technology for drug-loaded exosomes [142,143].Although targeted delivery of drug exosomes in hepatitis B-related liver diseases has been rarely reported, they have been utilized in various diseases [144][145][146] and may offer new hope in treating challenging cases such as antiviral treatment for hepatitis B and its complications.

Exosomes as HBV gene editing delivery vehicles
In addition to drug delivery, exosomes can be loaded with clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/ Cas9) systems for genomic intervention in target cells.For instance, exosomes loaded with CRISPR/Cas9 expression plasmid can inhibit the expression of poly (ADP-ribose) polymerase-1 (PARP-1), thereby inducing apoptosis in ovarian cancer cells and enhancing the sensitivity of chemotherapy [147].Similarly, studies leveraging exosomes carrying CRISPR/Cas9 to edit genes and alter the characteristics of cancer cells have been widely reported [148][149][150].Moreover, in treating hepatitis B, endogenous exosomes have shown potential for delivering single guide RNA (gRNA) and Cas9 protein to cleave transfected HBV DNA and disrupt the HBV genome [151].Furthermore, in addition to disrupting the HBV genome, exosomal Cas9 ribonucleoprotein (RNP) delivery by targeting p53 upregulated modulator of apoptosis (PUMA), cell cycle protein E1 (CcnE1) and K (lysine) acetyltransferase 5 (KAT5) in mouse models of acute liver injury, chronic liver fibrosis, and HCC showed powerful therapeutic potential [152].Thus, the exosome delivery CRISPR/Cas9 system is an effective tissue-specific gene therapy route.However, there is still a need to produce stable and efficient engineered exosomes, and challenges related to the off-target effects of gene editing require further optimization.

Exosomes in HBV vaccines
Based on existing reports, exosomal vaccines can be classified into prophylactic and therapeutic vaccines.Due to their ability to efficiently carry and deliver viral nucleic acids and proteins that act as antigens or agonists for innate immune receptors, exosomes have the potential to serve as prophylactic vaccines or adjuvants [153].For example, antigen-loaded exosomal surface antigenic epitopes are expected to be more efficient than their presentation with natural proteins, possibly carrying intact HBsAg within and on their surface, stimulating B cells to induce Th1-type memory cells [154].In addition, activated immune cell exosomes can transfer antiviral effects.Natural exosomes produced after monocyte stimulation can act as adjuvants for the HBsAg vaccine, enhance cellular immune response, and accelerate IgG antibody production compared to free HBsAg [155].
Moreover, due to their immunomodulatory function, exosomes can activate immune cells and break immune tolerance, serving as a therapeutic vaccine for malignant tumors [156,157].Tumor-derived exosomes loaded with tumor antigens can significantly enhance immune cells' recognition and antigen presentation.For example, an exosome-based tumor antigen-assisted co-delivery system effectively induced tumor antigen-specific immune responses and inhibited murine melanoma B16BL6 cell growth in mice compared to simple co-administration [158].In addition, T cell exhaustion triggered against tumors can also be reversed by exosomal vaccines, stimulating a larger cytotoxic T lymphocyte (CTL) antitumor response [159,160].Research with exosome use in other tumors indicates the potential for application in advanced hepatitis B-related liver cancer.

Advantages and challenges of exosome therapy Advantages of exosome therapy
Endogenous nature is a significant advantage of exosomes.Compared to liposomes and other nanoparticles, exosomes are secreted by cells of the organism itself, reflecting the physiological status of the body's microenvironment and possessing low immunogenicity.Therefore, exosomes hold broad and unique advantages in the field of disease diagnosis and treatment.Due to the similarity in structure and composition between the exosomal membrane and the cell membrane, recipient cells exhibit higher efficiency in uptaking exosomes compared to other nanocarriers.Additionally, surface proteins on the exosomal membrane can interact with surface receptors on cell membranes, directly activating signaling pathways in recipient cells and facilitating membrane fusion and internalization processes [161].
Furthermore, exosomes possess innate therapeutic properties.In the absence of cargo loading, natural exosomes can exert therapeutic effects.For example, exosomes secreted by mesenchymal stem cells have been shown to promote macrophage polarization to the M2 phenotype in various diseases, suppressing excessive inflammatory responses and repairing tissue damage [162,163].Exosomes derived from various immune cells can break immune suppression in the tumor microenvironment, promote immune activation, and restrict tumor growth [164,165].Additionally, exosomes from plant sources show promising prospects.For instance, exosome-like nanoparticles derived from artemisinin and ginger can treat cancer [166,167], while vesicles derived from ginger, mulberry bark, and garlic can modulate the gut microenvironment [168][169][170].These exosomes themselves have therapeutic effects, and if their contents are artificially enriched or loaded with drugs, they can exhibit more efficient therapeutic effects than other nanocarriers.

Challenges of exosome therapy
Research on exosome therapy in hepatitis B began approximately 10 years ago, and the therapeutic potential of exosomes in hepatitis B-related liver diseases has only been recently recognized.Exosome therapy in hepatitis B is still in its early stages, mostly consisting of in vitro and animal experiments, with limited identified therapeutic targets.
Firstly, the challenge lies in exosome isolation techniques.Although various methods have been developed for isolating and purifying exosomes, each has its drawbacks.For instance, ultracentrifugation, the most commonly used method, offers significant advantages in separating components with significantly different sedimentation coefficients [171] but is time-consuming and may lead to structural damage and aggregation, hindering downstream analysis [172].Commercial exosome isolation kits offer time-saving, high activity, and good integrity, but are expensive, yield low quantities, and have low purity [173].Some studies propose combining multiple purification methods to capitalize on their respective advantages, such as combining size-exclusion chromatography (SEC) with differential centrifugation [174], but this may extend extraction time and lower activity.Moreover, different exosome isolation methods result in varying particle size distributions, particle numbers, and protein contents [175].Thus, further improvements are needed in exosome purification methods to ensure purity, activity, and standardization.
Secondly, exosome storage poses challenges, as they are difficult to store long-term.Previous studies suggested that the best storage method is freezing at − 80 °C [176].However, research has shown that compared to freshly extracted exosomes, storage at − 80 °C for 4 days alters exosome morphology, and their biological activity decreases after 28 days [177].Recently developed colloidal systems with buffer and optimized parameters for exosome freeze-drying significantly extend the shelf life of exosomes [178].However, there still exists a gap in clinical applications.
Thirdly, difficulties arise in loading drugs into exosomes.Drug loading methods for exosomes can be categorized into pre-separation loading and post-separation loading methods.Pre-separation loading involves loading drugs during exosome biogenesis in parent cells, also known as in vivo or endogenous loading.This method has been successfully reported for the delivery of siRNA and miRNA [179] but still faces challenges such as difficulty in standardizing loading efficiency and susceptibility to parent cell growth conditions.Post-separation loading, also known as ex vivo or exogenous loading techniques, involves loading drugs into exosomes after isolation using passive or active encapsulation techniques.This method offers more controllable loading efficiency and enables manipulation and measurement of encapsulated cargo quantities, addressing the shortcomings of pre-separation loading.However, post-separation loading may lead to disruption of exosomal protein structure, affecting their activity, as well as issues such as exosome aggregation, membrane damage, and low yields [180].Therefore, optimization of exosome drug loading methods is still required to ensure exosome activity and loading efficiency.
Fourthly, the specificity of exosomes needs to be further enhanced.Surface modification is an effective way to improve the specificity of exosome delivery by using exosomal surface proteins as affinity tags and modifying specific proteins onto the exosomal surface [181].Surface modification has been successfully used to enhance exosome targeting to brain cells and improve their ability to cross the blood-brain barrier [182].Similar to other nanomaterials, exosomes can undergo in vitro surface modification.However, as exosomes are endogenous, their surface adhesion molecules, lipids, and ligands are influenced by parent cells, and modifying parent cells can also enhance exosome specificity [183].But this reliance on parent cell surface modification still faces challenges similar to exosome internal drug loading, such as difficulty in ensuring loading efficiency.
Lastly, exosomes have a short half-life in the bloodstream, making it difficult to maintain a safe and effective concentration for exosome therapy, and their tolerability in humans still needs to be determined.Therefore, almost all exosome therapies entering clinical trials currently involve local administration [184], such as subcutaneous injection to promote wound healing [184], aerosol inhalation therapy for pneumonia [185], and exosome gel application to alleviate redness after CO2 laser treatment [186].The path to systemic exosome therapy in humans appears to be long and challenging.

Conclusion and outlook
This review systematically elaborates on the biological processes involving exosomes, covering their biogenesis, release, transport, uptake by recipient cells, and their effects on these recipient cells.We focus on how HBV utilizes exosomes for loading and transportation and propose several hypotheses regarding HBV's involvement in exosomes release, transport, and receptor binding.Subsequently, these HBV-related exosomes alter the characteristics of recipient cells, reshaping the immune microenvironment.Finally, based on the features and functions of exosomes, as well as their applications in other diseases, we summarize and predict the potential applications of exosomes in hepatitis B.
However, research on exosomes in hepatitis B-related diseases is still in its infancy.The mechanism by which HBV affects the sorting of exosome contents remains unclear.The mechanisms by which exosomes regulate non-HBV host cells across cells to reshape the liver's immune microenvironment also require further exploration.The identified targets of exosomes in hepatitis B are still very limited, and many technical challenges in clinical translation need to be overcome.However, exosomes can reveal the status of donor cells and the extracellular microenvironment, influencing the fate of recipient cells.They possess natural advantages in both diagnosis and treatment, making them a highly promising field.

Fig. 2
Fig. 2 hepatitis B virus (HBV) utilizes exosomes for release, transport, and interaction with recipient cells.A After entering host cells, HBV synthesizes viral proteins and genetic material, which are sorted into exosomes, including intact viral particles.B HBV integrates large hepatitis B virus surface antigen (LHB) onto the surface of exosomes, and binds to the LHB/NTCP (Na + /Taurocholate Co-transporting Polypeptide) receptor.CD.There are two ways for exosomes to enter receptor cells after binding to them: membrane fusion and phagocytosis

Fig. 3
Fig. 3 HBV creates an immunosuppressive microenvironment through exosomes.HBV infection changed the cargo of exosomes, resulting in upregulated programmed death ligand 1 (PD-L1) and reduced Interleukin (IL)-6 secretion in monocytes, downregulated interferon (IFN)-α and IL-12 in macrophages, impaired NK cell function, decreased IFN-α in dendritic cells (DCs), and diminished IL-12 secretion in T cells.In addition, the downregulation of IL-12 in macrophages can directly lead to NK cell deactivation and functional impairment.After the development of liver cancer in HBV-infected hepatocytes, the release of tumor-derived exosomes leads to the polarization of M2-type macrophages and stimulating tumor cell proliferation.Infiltrating M2-type macrophages in the tumor tissue also secrete exosomes to enhance tumor cell proliferation

Functions
Immunomodulation HBV-infected hepatocytes HBV nucleic acid NK HBV-positive exosomes impair NK cell function by inhibiting RIG-1 leading to blockage of NF-κB and p38 mitogenactivated protein kinase pathways Enomoto et al. [66] Immunomodulation HBV-infected hepatocytes MiR-21, miR-192, miR-215, miR-221 and miR-222 T cells Expression of IL-21 in T cells is inhibited by transactivation of miR-21, miR-192, miR-215, miR-221 and miR-222 Kakizaki et al. [39] Immunomodulation HBV-infected hepatocytes Viral proteins, HBsAg, HBcAg, viral DNA and other viral components Monocytes Immunosuppression of monocytes through upregulation of programmed PD-L1 expression Shi et al. [67] Immunomodulation HBV-infected hepatocytes IFITM2 DC Exosomal transport of IFITM2 to DC cells inhibits phosphorylation of ERK1/2 and sequentially inhibits phosphorylation of TBK1 and IRF3 to attenuate the synthesis of endogenous IFN-α Zhao et al. [102] Immunomodulation HBV-infected hepatocytes HBV-miR-3 Macrophages HBV-miR-3 in HBV-infected hepatocyte-derived exosomes promotes macrophage IL-6 secretion by downregulating SOCS5 in macrophages and activating the JAK/STAT signaling pathway Table 3 (continued) Kouwaki et al. [65] Immunomodulation HBV-infected hepatocytes HBV nucleic acid Macrophages Hepatitis B-infected hepatocytederived exosomes induce macrophage expression of NKG2D ligands through stimulation of MyD88, TICAM-1 and MAVS-dependent pathways Wu et al. [107] Immunomodulation IFN-α treated Macrophages MiR-574-5p HBV-infected hepatocytes MiR-574-5p reduces pregenomic RNA and polymerase messenger RNA levels by binding to positions 2750-2757 of the HBV genomic sequence Liu et al. [130] Immunomodulation TAF-treated macrophages lncRNA HOTTIP HBV-HCC The antiviral effect of exosomal lncRNA HOTTIP is more potent than that of TAF treatment alone and is essential for the antiviral activity of TAF Tao et al. [101] of HBV-HCC by targeting c-Myc in HCC Zhang et al. [68] Therapeutic Targets HBV-infected hepatocytes MiR-222 HSC Blocking exosomal miR-222 in HBVinfected hepatocytes is a potential therapeutic target for inhibition of iron death-induced HSC activation Kapoor et al. [27] Therapeutic Targets HBV-infected hepatocytes HBX-mRNA, HBX-protein HSC Cause activation of stellate cells Ouyang et al. [188] HBc upregulated the expression of exosomal miR-135a-5p and promoted antiapoptosis, cell proliferation, and chemical resistance through miR-135a-5p/ VAMP2 in HBV-HCCTable 3 (continued) as adjuvants for HBsAg and, when co-injected with antigen, can be used to improve the protective immune response, enhancing the immune response and accelerating the emergence of IgG antibody production compared to free HBsAg Hu et al. [139] Predicting responsiveness to treatment -MiR-194-5p and miR-22-3p -Baseline serum exosomal miR-194-5p and miR-22-3p may serve as novel biomarkers to predict HBeAg seroconversion in CHB patients treated with Peg-IFN Shi et al. [67] Predicting responsiveness to treat-and KAT5 shows potent therapeutic potential in mouse models of acute liver injury, chronic liver fibrosis and HCC Chen et al. [151] delivery of gene editing -gRNA and Cas9 HuH7 cells transfected with 1.2 × HBV expression plasmid The donor CHO cell-produced exosomes could also carry functional gHBV1/Cas9 protein and cleave HBV DNA to smaller fragments in the receptor HuH7 cells transfected with 1.2 × HBV expression plasmid

Table 2
Hepatitis B and related disease exosomal markers

Table 3
Exosome potential applications in hepatitis B and related diseases