Extracellular Vesicles in Pathogenic Infection, Transmission, and Immunity

Junyao Xiong; Usama Ashraf; Jing Ye; Shengbo Cao

doi:10.1016/j.eng.2024.06.011

Engineering ›› 2024, Vol. 43 ›› Issue (12) :238 -252. DOI: 10.1016/j.eng.2024.06.011

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Extracellular Vesicles in Pathogenic Infection, Transmission, and Immunity

Junyao Xiong ^a^,^b^,^c^,^d^,^#
, Usama Ashraf ^e^,^#
, Jing Ye ^a^,^b^,^c^,^d^,^*
, Shengbo Cao ^a^,^b^,^c^,^d^,^*

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Abstract

Extracellular vesicles (EVs) are secreted by cells and widely exist in body fluids, serving as an essential vehicle of intercellular communication. In recent years, EVs have gained significant attention owing to their ability to carry nucleic acids, lipids, and proteins of host and pathogen origins. A distinct composition of EVs during pathogenic infection contributes to mediating pathogenic infection, transmission, and immunity to target cells. Therefore, studying the role of EVs in pathogenic infection is crucial for understanding pathogen-host interactions and developing new anti-pathogenic therapies. This review offers an overview of current knowledge of EVs in the context of infection, transmission, and immunity to pathogens. Harnessing EVs’ biology during pathogenic infection may lay a foundation for the mitigation of pathogenic infection and associated disease outcomes.

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Extracellular vesicles / Pathogen / Infection / Transmission / Immunity

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Junyao Xiong, Usama Ashraf, Jing Ye, Shengbo Cao. Extracellular Vesicles in Pathogenic Infection, Transmission, and Immunity. Engineering, 2024, 43(12): 238-252 DOI:10.1016/j.eng.2024.06.011

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1. Introduction

Extracellular vesicles (EVs) are small, vesicle-like structures with a bilayer membrane that are secreted from cells [1], [2], [3], [4]. In eukaryotes, EVs mainly include exosomes and microvesicles. Exosomes are formed by the fusion of intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) with the cell membrane, and typically range in diameter from 30 to 150 nm, while microvesicles ranging from 100 to 1000 nm in diameter are directly released from the cell membrane into extracellular spaces [1], [5], [6]. Thus, a small fraction of microvesicles share a similar diameter with exosomes. The simultaneous release of exosomes and microvesicles also complicates the distinction of their respective roles. In prokaryotes, both Gram-negative and Gram-positive bacteria have the ability to secrete vesicles, which originate from the bacterial outer membrane during their propagation. These vesicles include outer membrane vesicles (OMVs) from Gram-negative bacteria and cytoplasmic membrane vesicles (CMVs) from Gram-positive bacteria, both of which are collectively referred to as EVs [7], [8].

Since most studies on EVs focus on eukaryotes, we mainly describe the formation process of EVs in eukaryotes. Exosomes are mostly generated through the endosomal sorting complex required for transport (ESCRT) pathway [9], although their biogenesis can also be independent of the ESCRT [10]. In the ESCRT-dependent mechanism, the ESCRT 0 complex identifies ubiquitylated proteins on the early endosome, separates the proteins into microdomains, and binds the ESCRT I complex. This complex then recruits ESCRT II subunits, which initiate reverse budding of the nascent ILVs. Cytosolic nucleic acids, proteins, and lipids have direct access to the interior of ILVs. The ESCRT II complex then recruits ESCRT III subunits inside the neck of the nascent ILVs, resulting in their cleavage into mature late endosomes, also known as MVBs. Based on different biochemical characteristics, certain MVBs are transported to lysosomes for protein degradation, while others are transported to cell membranes for fusion and the release of their ILVs into the extracellular space; these ILVs are then defined as exosomes [4], [9]. Studies have shown that exosomes containing the cluster of differentiation 63 (CD63) marker can still be released even when the ESCRT is knocked out, indicating that exosomes can also be formed independently of the ESCRT [10]. Recent research has discovered an ESCRT-independent mechanism for the production of ceramides by means of neutral sphingomyelinase. This mechanism enables the generation of membrane subdomains that modify the curvature of the membranes, allowing for the formation of independent vesicles [10], [11].

The formation mechanisms of microvesicles are less studied, but it is known that microvesicles can originate directly from the plasma membrane (PM) and bud outward to form vesicles [1], [6], [12]. In general, EVs are identified based on their cup-shaped appearance, specific membrane proteins (CD9, CD63, and CD81), and intracellular proteins (Alix and TSG101) [1], [9], [12]. A schematic of EV biogenesis is provided in Fig. 1.

EVs secreted by different cells share a similar morphology and diameter. They can encapsulate multiple pathogen- and host-derived nucleic acids, proteins, and lipids that perform integral functions after transfer to recipient cells. However, the type and abundance of nucleic acids, proteins, and lipids incorporated in EVs are variable, depending on the cell type and its physiological or pathological state [2], [3], [5], [6], [13], [14], [15]. Of the specific markers of EVs, CD81 is the most highly enriched protein in EVs, even though it is primarily localized to the PM, whereas the endosome-enriched CD63 protein is the least enriched of these proteins [6], [9], [16]. EVs contain RNAs that can be transferred to other cells and tissues. Studies on RNAs inside EVs have shown that these vesicles are particularly enriched in small noncoding RNAs (ncRNAs), such as small nuclear RNAs (snRNAs), microRNAs (miRNAs), transfer RNAs (tRNAs), and Y RNAs [15], [17]. EVs derived from the inner membrane have a lipid bilayer membrane structure and are rich in lipid rafts and membrane-related components, such as sphingomyelin and phosphatidylserine [6].

2. Isolation of EVs

The available methods for EV isolation are summarized in Fig. 2; these include ultracentrifugation (UC), density gradient centrifugation (DG), polymer precipitation (PP), size exclusion chromatography (SEC), ultrafiltration (UF), and immunocapture (IC) [14], [18], [19], [20], [21]. Among these, the gold-standard method for isolating EVs is UC, which involves the removal of cells, cell debris, and apoptotic debris, followed by UC to isolate the EVs [20]. DG involves the formation of specific mediums with varying densities in a centrifugal tube, such as sucrose or iodixanol. Subsequently, UC is used to stagnate particles of different densities in their corresponding areas of equal density [19], [20]. Another method, PP, involves changing the solubility and dispersion of EVs using a highly hydrophobic polymer, such as polyethylene glycol [21]. According to the principle of SEC, it is necessary to use columns filled with porous polymer microspheres. This is because EVs can be eluted quickly due to their large diameter, while proteins with small diameters require a longer elution time [22]. UF requires a membrane with a fixed pore size that allows small molecules to pass through while trapping larger molecules [23]. The principle of IC involves the use of magnetic beads to selectively enrich samples of EVs based on the presence of specific membrane proteins such as CD9, CD63, and CD81 [12], [24].

During viral infection, virions are released into cell supernatants along with EVs. Due to the similar particle size of virions and EVs, it is challenging to distinguish EVs from virions by means of conventional methods. Although there are no standard methods for purifying EVs at present, existing techniques offer two comprehensive methods for separating EVs from virus-infected cell supernatants. One commonly used method for purifying EVs is UC combined with the DG method (UC-DG), which involves a two-step process. Firstly, cell debris and apoptotic debris are removed from cell supernatants through multiple low-speed centrifugation steps. Next, the EVs are concentrated using UC. Finally, the purified EVs are harvested through DG by using either sucrose or iodoxanol, where EVs and virions stagnate in their corresponding density areas owing to their different densities under UC. The UC-DG method is simple and allows for the purification of EVs from a large volume of different species. Nevertheless, it has disadvantages such as being time consuming, leading to impurity pollution, and causing structural damage to EVs. Another approach, UC in combination with the IC method (UC-IC), provides an alternative for the purification of EVs. This approach also involves initially removing cell and apoptotic debris via differential centrifugation and concentrating the EVs by UC. The most important step is the purification of EVs through the IC method, which requires the use of magnetic beads to selectively enrich EVs based on the membrane proteins CD9, CD63, and CD81. Finally, the purified EVs are eluted for downstream analysis. EVs obtained from the UC-IC method have high purity, and their original morphology is preserved. However, this method requires antibodies specific to the corresponding species [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]. While most commercially available antibodies are suitable for human and mouse, there is a lack of antibodies for certain species such as pigs, cattle, sheep, and poultry, which makes it challenging to isolate EVs from these animals. Moreover, even if lab-made antibodies for these species were to be produced, their production and purity might not be stable and convenient for other researchers. Therefore, further exploration is needed regarding a method to isolate EVs from virus-infected cells.

3. Quantification of EVs

Currently, several methods for quantifying EVs are available, including nanoparticle tracking analysis (NTA), bicinchoninic acid (BCA), quantitative real-time polymerase chain reaction (qRT-PCR), digital polymerase chain reaction (dPCR), flow cytometry, and microfluidic technology (Fig. 3). NTA is a light-scattering technique that can be used to determine the number and distribution of nanoparticles [40]. BCA is a widely used method for protein quantification that can be used to determine the concentration of total EV protein. qRT-PCR is the commonly used method to quantify RNA, which requires a probe, standard plasmid, and commercial reagents. At present, no standard internal reference genes have been identified in EVs, making it difficult to quantify the EVs directly using the qRT-PCR method. However, in the case of a pathogen infection or nucleic acid delivery, researchers can indirectly quantify the EVs by measuring the abundance of the pathogen genome or nucleic acid cargoes via qRT-PCR [31], [41]. dPCR is an absolute quantitative method that enables large-scale parallel polymerase chain reaction (PCR) analysis at the single-molecule level, resulting in higher sensitivity, accuracy, and the ability to determine the absolute concentration of target copies per microliter without the need for a standard curve or reference [42], [43], [44]. Flow cytometry is a mature technique that can be used to quantitatively analyze EVs based on their membrane proteins (CD9, CD63, and CD81 protein) [45]. Studies have also developed fluorescence-based and high-resolution flow cytometric methods that allow for both quantitative and qualitative analysis of EVs in multiple parameters [46], [47]. Currently, there are several microfluidic-based methods used for quantifying EVs, including surface plasmon resonance (SPR) [48], electrochemical biosensors [49], and fluorescence labeling [50], [51].

4. Roles of EVs in viral infection, transmission, and immunity

In recent years, there has been significant interest in EVs, as they have been linked to the mediation of viral infection, transmission, and immunity. Viruses can hijack EVs to aid their propagation by building a secretory mechanism that facilitates their budding processes and producing disseminators that can transfer viral contents to other target cells [2], [3], [14]. In contrast, host cells can exploit EV biogenesis to prevent viral infection by stimulating immune responses [5], [52]. Given the crucial roles of EVs in the interplay between viruses and host cells, this review aims to provide an overview of the roles of EVs in regulating viral infection and transmission. We also highlight several mechanisms by which viruses evade the host immunity via EVs. A schematic showing the role of EVs in regulating viral infection, transmission, and immunity is presented in Fig. 4.

4.1. Viral components in EVs

EVs are released by cells in all body organs and fluids. The EVs secreted during viral infection may contain viral and cellular elements (i.e., nucleic acids, lipids, and proteins). The roles of EVs in viral infection are currently categorized into “promotion” and “inhibition.” The former involves the promotion of viral infection by delivering viral or cellular components, inducing immune evasion, and suppressing the immune response [53], while the latter indirectly regulates the interferon signaling pathway, mainly through miRNAs or proteins in EVs, inhibiting viral infection [32], [37], [54]. An overview of EVs—including their methods of isolation, source of secretion, critical molecular components, and biological roles in DNA and RNA viral infection—is outlined in Tables 1 [25], [55], [56], [57], [58], [59], [60], [61], [62], [63] and 2 [54], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], respectively. All molecular components inside EVs from virus-infected body fluids or cells are summarized in Fig. 5.

Among DNA viruses, EVs isolated from the serum of African swine fever virus (ASFV)-infected pigs have been found to harbor only a few viral proteins but be enriched in hundreds of host proteins involved in the coagulation pathway [55]. EVs from HepAD38 cells infected with hepatitis B virus (HBV) contain intact virions, and the presence of HBV surface antigen on the surface of EVs has also been observed [56]. EVs from the sera of chronic hepatitis B (CHB) patients contain both HBV nucleic acids and HBV proteins [87]. Furthermore, when fibroblasts are infected with human cytomegalovirus (HCMV), they release more vesicles and alter the particle size of vesicles compared with those from uninfected cells. In addition, EVs from HCMV-infected cells contain viral proteins with late domain sequences [57]. Both viral RNA and cellular elements (miRNAs and innate immune sensor protein stimulator of interferon genes (STING)) can be exported to uninfected cells via EVs from herpes simplex virus 1 (HSV-1)-infected cells [25]. Moreover, JC polyomavirus (JCPyV) infection is associated with the release of EVs that drive the infection of target cells, favoring viral dissemination to and within the central nervous system. JCPyV virions can either attach to or be enclosed within the EVs derived from infected cells [58].

The generation of EVs is also associated with infections caused by RNA viruses, including double-strand RNA (dsRNA) viruses, simple-strand and positive-sense RNA ((+) ssRNA)) viruses, and simple-strand and negative-sense RNA ((-) ssRNA)) viruses. A study on the rice dwarf virus (RDV), a dsRNA plant arbovirus that causes a severe rice disease in Asia [88], demonstrated that the purified EVs secreted from RDV-infected leafhopper cells are enriched with virions [64]. Among (+) ssRNA viruses, the biogenesis of EVs has mainly been reported in viruses belonging to the Coronaviridae, Flaviviridae, Picornaviridae, and Retroviridae families.

In Coronaviridae, EVs extracted from porcine epidemic diarrhea virus (PEDV)-infected cells show a dysregulated pattern of cellular miRNAs [89]. EVs derived from PEDV-infected newborn piglet serum display significantly decreased expression levels of complements C3, C6, and complement factor B (CFB), in comparison with serum samples from uninfected piglets [54]. Furthermore, EVs obtained from porcine hemagglutinating encephalomyelitis virus (PHEV)-infected N2a cells incorporate a variety of cargoes, including viral elements (PHEV N protein and virions) and host elements (pattern recognition receptors (PRRs) and interferon-stimulated genes (ISGs)) [34]. EVs secreted upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of African green monkey kidney cells (Vero E6), human lung adenocarcinoma cells (Calu-3), and human pulmonary alveolar epithelial cells (HPAEpiC) contain large amounts of virions [65].

In Flaviviridae, intact classical swine fever virus (CSFV) virions are enclosed within EVs secreted from CSFV-infected porcine kidney cells (PK-15) and can be transferred to uninfected cells through the translocation of EVs [66]. EVs purified from dengue virus (DENV)-infected mosquito cells, including Aedes albopictus (C6/36) and Aedes aegypti cells, contain DENV-2 infectious RNA and proteins [67]. Moreover, EVs from DENV-2 infected C6/36 cells are larger than those secreted from uninfected cells, and virus-like particles are packaged into them, which can infect naïve C6/36 cells [30]. Furthermore, DENV-2 infection of Aedes aegypti mosquitoes enhances the accumulation of proviral proteins in the EVs secreted into the mosquito saliva, which has been found to promote viral transmission [68]. EVs isolated from hepatitis C virus (HCV)-infected human hepatoma cells have been found to contain full-length viral RNA, viral core protein, and virions, which in turn can transmit HCV and establish a productive infection in naïve cells [69]. Japanese encephalitis virus (JEV) infection of mouse microglial cells (N9) stimulates the production of EVs carrying miRNA let-7a/b, and the co-incubation of EVs secreted from JEV-infected N9 cells with cultured mouse neuronal cells or primary cortical neurons causes neuronal cell damage due to excessive caspase activation [70].

The tick-borne Langat virus (LGTV), a relative of the tick-borne encephalitis virus, employs arthropod EVs for the enhanced transmission of viral RNA and proteins (NS1 and E) to human skin keratinocytes and blood endothelial cells. EVs derived from infected blood endothelial cells subsequently facilitate viral RNA and protein transmission to neuronal cells across the blood-brain barrier [71]. West Nile virus (WNV) infection significantly alters the levels of certain host miRNAs, sncRNAs, and mRNAs incorporated into EVs in an interferon (IFN)-dependent and -independent manner [72]. EVs released from Zika virus (ZIKV)-infected C6/36 cells carry viral RNA and E protein, and have the ability to infect and activate naïve mosquito and mammalian cells [73], [90]. In addition, human glioblastoma cells (SNB-19) infected with ZIKV secrete unique subpopulations of EVs that have specific viral protein profiles and infectious genomes, and the release of these EVs, viral genomes, and capsid protein following infection is regulated by the EV-enriched tetraspanin CD63 [74].

In Picornaviridae, the EVs from coxsackievirus B3 (CVB3)-infected human colon adenocarcinoma cells (Caco-2) contain viral structural proteins (VP1 and VP2) and virions, and can initiate efficient infection in receptor-negative host cells [29]. Enterovirus 71 (EV71) infection of human colorectal (HT-29) and human monocytic cells (THP-1) increases the secretion of EVs and the differential packaging of viral genomic RNA and miR-146a into EVs [75]. EVs derived from EV71-infected rhabdomyosarcoma cells contain EV71 RNA and capsid protein VP1, and can also establish a productive infection in human neuroblastoma cells (SK-N-SH) [76]. Furthermore, full-length genomic RNA and viral proteins of foot-and-mouth disease virus (FMDV) have been identified in purified EVs isolated from FMDV-infected PK-15 cells, which are capable of transmitting infection to naive PK-15 cells and suckling mice [31]. Enveloped virions (eHAV) cloaked in host-derived EVs are the dominant form of virus released from hepatitis A virus (HAV)-infected cells [77]. The HAV structural protein pX is crucial in the membrane envelopment of HAV, and its domain can guide exogenous proteins into EVs [78]. In addition, EVs isolated from Seneca Valley virus (SVV)-infected porcine kidney cells (IBRS-2) harbor viral genome and VP1 and VP3 protein, and enable the virus to proliferate in both susceptible and non-susceptible cells [38].

In Retroviridae, semen EVs purified from subgroup J avian leukosis virus (ALV-J)-infected rooster seminal plasma contain viral genomic RNA and partial viral proteins, and can transmit ALV-J infection to host cells and establish productive infection [26]. Furthermore, EVs secreted from human immunodeficiency virus (HIV)-infected human monocytic cells (U937) are enriched in miRNAs that potentially target innate immune responses [79]. The latent HIV could be activated by EVs purified from U937 cells chronically infected with HIV [91]. EVs purified from reticuloendotheliosis virus (REV)-positive semen contain viral genomic RNA and viral proteins, which can establish productive infections both in vivo and in vitro [36].

The EVs generated by the viruses of the Arteriviridae, Hepeviridae, and Togaviridae families present a similar composition and aid in facilitating viral infection to uninfected cells. For example, EVs obtained from porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells contain viral genomic RNA and partial viral proteins (nsp2, GP4, GP5, M, and N protein), which are capable of infecting both PRRSV-susceptible and non-susceptible cells [35]. Membrane-associated rat hepatitis E virus (HEV) particles are present within the MVB, and the EV biogenesis pathway is required for viral egress [92]. Additionally, EVs derived from Chikungunya virus (CHIKV)-infected cells contain CHIKV RNA and are considered to be infectious [27].

Among (-) ssRNA viruses, infection with caprine parainfluenza virus type 3 (CPIV3) leads to enhanced release of EVs from host cells, and the secreted EVs carry viral F protein, F and M gene, and miRNAs [28]. The infection of chicken fibroblasts (DF-1) by Newcastle disease virus (NDV) causes the release of EVs carrying viral structural proteins NP and F, which can further be transferred to uninfected cells through these EVs [33]. EVs from the saliva of rice stripe virus (RSV)-infected planthopper contain all four viral genomic RNAs and virions, and these packed virions show a tendency to replicate and cause disease in rice plants [80]. EVs produced by severe fever with thrombocytopenia syndrome virus (SFTSV)-infected cells harbor infectious virions and viral proteins (NSs and NP); these packed EVs efficiently transport the carried virions to uninfected cells, thus assisting in achieving efficient replication [39].

The relationship between the characteristics of a virus and its presence in EVs is an intriguing area of study. Virion size is believed to be a potential factor influencing the presence of virions in EVs. However, due to the limitations of the various isolation and detection methods used in different studies, it remains unclear whether the viral packaging into EVs is dependent on the size of virions. Since the particle sizes of EVs range from 30 to 1000 nm [1], [6], [12], small virions may tend to have a greater probability of being enclosed in EVs, which would be intriguing to examine by comparing both smaller and larger viruses in parallel. For example, virions with both a small particle size (CVB3, EMCV, HAV, and RSV, 0-35 nm) and a medium particle size (CSFV, HBV, HCV, JCPyV, and RDV, 35-80 nm) can be detected inside EVs. However, the presence of large virions (larger than 80 nm) within EVs has rarely been reported [39], [65]. Moreover, various characteristics of viruses—such as their DNA or RNA composition and envelope presence—may affect their packaging into EVs. Studies have shown that both DNA viruses (HBV and JCPyV) and RNA viruses (RDV, CVB3, CSFV, EMCV, HAV, HCV, and RSV), or both enveloped viruses (CSFV, HBV, and HCV) and nonenveloped viruses (CVB3, EMCV, HAV, and JCPyV), can be encapsulated in EVs. To draw more definitive conclusions, future studies on EVs derived from a wider range of viruses are warranted.

The above studies suggest that EVs secreted by virus-infected cells can encapsulate viral components (e.g., virions, viral proteins, and viral genomes) or host components (host proteins and miRNAs, etc.). However, most EV isolation methods such as UC, UF, and SEC are simplistic and may not effectively separate EVs from free viral particles, leading to uncertainties regarding the components of EVs. Although some studies have purified EVs by means of density gradient centrifugation and IC methods and have identified virus-like particles within EVs through electron microscopy, further confirmation through immunoelectron microscopy is needed to verify whether these particles are indeed virions.

4.2. Roles of EVs in viral infection

During viral infection, the viral components in EVs usually aid the virus in establishing efficient infection. CPIV3 infection of bovine kidney cells shows enhanced release of EVs harboring viral elements and host miRNAs, among which the enrichment of miR-126-3p is associated with inhibition of the cellular autophagy response to promote viral replication [28]. EVs carrying CVB3 virions exhibit the high infection efficiency of receptor-negative cells through various entry routes, and the inhibition of EVs coupling with virions attenuates CVB3-induced immunological system dysfunction and pathogenicity in vivo [29]. Encephalomyocarditis virus (EMCV) infection triggers the release of several subpopulations of EVs that differ in terms of their molecular composition, releasing time, and ability to transfer viral infection [81], and the release of EVs has been found to be controlled by the EMCV Leader protein via stimulating a secretory autophagy [93]. EVs secreted from EV71-infected cells contain EV71 RNA and miR-146a. The viral RNA can be transferred to new target cells with full replication potential, while the miR-146a suppresses the IFN-I response to facilitate viral replication [75]. The NP protein of NDV packed into EVs from infected chicken fibroblast cells (DF-1) can be transferred to uninfected cells, which consequently induces proviral and cytokine-suppressing effects [33]. RDV hijacks the release pathway of EV biogenesis to traverse the apical plasmalemma into saliva-stored cavities in the salivary glands of leafhopper vectors, ultimately allowing the horizontal transfer of EVs into the phloem of rice plants to establish initial viral infection as leafhopper vectors feed [64]. The virions enclosed in EVs from REV-positive chicken serum show higher transmission efficacy and replication in chicken cells, causing attenuation of the host innate immune response [36].

The presence of angiotensin-converting enzyme 2 (ACE2)-exposing EVs enhances SARS-CoV-2 infection in Vero E6 cells, and inhibition of EV uptake diminishes the infection efficiency of SARS-CoV-2 [94]. Conversely, another study has claimed that ACE2-exposing EVs—either alone or in conjunction with TMPRSS2—are more effective than soluble ACE2 in inhibiting SARS-CoV-2 spike-dependent infection [95]. Therefore, further studies are needed to establish a universally accepted narrative about the effectiveness of the actual role of ACE2-exposing EVs in SARS-CoV-2 infection. SVV infection of porcine kidney cells and ZIKV infection of human astrocytes induce a significant release of viral-element-enriched EVs, and the treatment of SVV- and ZIKV-infected cells with the EV inhibitor GW4869 results in significantly reduced viral replication [38], [96].

Rather than enhancing viral infection, the host components inside EVs generally aid in the inhibition of viral infection. The EVs released by DENV-infected cells restrict DENV infection at an early stage in mosquito cells by limiting membrane fusion, but not cell attachment or entry [82]. FMDV infection leads to a significant increase in the abundance of miRNA-136 in EVs, which in turn inhibits the proliferation of FMDV in infected cells [32]. EVs obtained from HBV-infected hepatocytes increase the mRNA expression of NKG2D ligands (i.e., elicit IFN-γ production from natural killer (NK) cells) in THP-1 macrophages or hepatic F4/80+ cells, promoting viral RNA degradation in hepatocytes [59]. HSV-1-infected cells release EVs that incorporate STING protein, which exerts antiviral activity while maintaining cell viability [25], [60], [61]. Rabies virus (RV) infection of human embryonic lung fibroblast cells (MRC-5) triggers the expression of miR-423-5p in EVs, which—by targeting SOCS3 promotes the type-I IFN response to repress RV replication [37].

In addition, several studies have shown that EVs secreted from body fluids can help the host to resist viral infection and maintain its equilibrium state. For example, EVs derived from goat milk have a significant impact on reducing the replication of DENV-2 and the secretion of mature virions [97]. Serum EVs isolated from uninfected newborn piglets demonstrate a strong antiviral effect against PEDV infection due to enrichment with complement proteins, particularly C3, C6, and CFB [54]. The host miRNAs, miR-let-7e and miR-27b, in the EVs from porcine milk inhibit PEDV replication in vivo and in vitro by targeting PEDV N and host HMGB1 proteins [98]. EVs released from the semen inhibit ZIKV infection in genital cells by compromising viral membrane integrity and preventing their attachment to the host cell surface; this has further been found to be associated with the lipid contents—rather than protein or RNA cargo—of EVs [83]. In a similar context, EVs secreted from human saliva cause anti-ZIKV activity by impeding viral attachment to monkey and human cells, and this viral inhibitory effect is independent of the protein contents of the EVs [99].

Considering the abovementioned roles played by EVs in viral infection, it is worth noting that most studies have primarily focused on the phenomenon, rather than elucidating the molecular mechanisms by which EVs induce such effects; thus, there is still a need to investigate the molecular mechanisms in future studies.

4.3. Roles of EVs in viral transmission

Studies have shown that EVs are a source of dissemination for a variety of viruses, and some viruses utilize EVs to manipulate environments favoring their transmission. EVs from ALV-J-infected rooster seminal plasma can transmit ALV-J infection to specific pathogen-free (SPF) hens and subsequently mediate the vertical transmission of ALV-J from the SPF hens to their progeny chicks [26]. EVs derived from CHIKV-infected cells are infectious, and the infectious viral elements can be transmitted to neighboring naïve cells by the EV pathway [27]. EVs released from DENV2-infected cells mediate viral transmission from arthropod to mammalian cells through interaction with the tetraspanin-domain-containing glycoprotein Tsp29Fb, and a further study revealed that the inhibition of Tsp29Fb protein or EV release are both potential therapeutics to block the transmission of DENV2 [67].

EVs function as efficiently as free virus in transmitting HBV infection to uninfected hepatoma cells [87]. HCMV enhances EV biogenesis by upregulating the ESCRT level in human fibroblasts, and the resultant secreted EVs have been found to enhance viral spread to uninfected cells, indicating that HMCV utilizes EV biogenesis to transfer proviral signals to neighboring uninfected cells, thereby priming them to be permissive for enhanced spread efficiency [57]. LGTV uses tick-derived EVs as the means for viral content transmission to vertebrate cells and vertebrate-derived EVs for dissemination within a vertebrate host to induce neuroinvasion [71]. This EV-mediated transmission of viral cargo between and within the hosts can be altered by modulating the EVs with changes in salt, pH, and temperature [100]. EVs from PRRSV-infected cells favor a productive infection in both PRRSV-permissive and -nonpermissive cells by compromising the host response; the inhibition of EV release impairs EV-mediated PRRSV replication and transmission [35]. Interference with each phase of the insect EV pathway affects the transmission of RSV from insect vectors to rice plants [80]. EVs produced during SARS-CoV-2 infection mediate viral entry into host cells independent of the known receptors of recipient cells, supporting the EV-mediated cell-to-cell transmission of SARS-CoV-2 [65].

Thus far, there is a paucity of studies on the roles played by EVs in viral transmission. The majority of published studies shed light on intercellular transmission but lack a comprehensive understanding of EVs’ function in cross-species transmission.

4.4. Roles of EVs in viral immunity

HAV can acquire an envelope by hijacking the host membrane, which protects the virus from antibody-mediated neutralization by giving it an EV-like morphology and promotes viral spread within the liver [77]. EVs released from HBV-infected hepatocytes contain viral nucleic acids, which increase the expression of natural killer group 2 member D (NKG2D) ligands in macrophages by stimulating myeloid differentiation primary response protein 88 (MyD88), toll/IL-1 receptor (TIR) domain-containing adaptor molecule 1 (TICAM-1), and mitochondrial antiviral signaling (MAVS) protein-dependent pathways [59]. In contrast, the HBV infection of hepatocytes increases immunoregulatory miRNA abundance in EVs; upon transferring to macrophages, this suppresses IL-12p35 mRNA expression to dampen the host innate immune response [59]. Furthermore, these hepatocyte-derived HBV-EVs can also be endocytosed into monocytes, wherein they stimulate PD-L1 expression to confer immune suppression [101]. EVs derived from the sera of chronic hepatitis B patients directly interfere with NK cell function in a retinoic acid-inducible gene I (RIG-I)-dependent manner, which dampens the activation of both the nuclear factor κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) pathways [87]. EVs of HCV-infected cells have demonstrated potential to transmit the infection to naïve cells independent of viral structural proteins, and this transmission is partly resistant to neutralizing antibodies [69]. The transmission of HCV-RNA-incorporating EVs to plasmacytoid dendritic cells (pDCs) triggers IFN-α production, inducing an unopposed innate response in replication-nonpermissive bystander cells [84].

Nef-containing EVs from HIV-infected cells are also known to potentiate immune response by perturbing cholesterol metabolism and reorganizing lipid rafts in bystander cells. The HIV-Nef-EVs are taken up by macrophages, leading to down-regulation of ATP binding cassette transporter type A1 (ABCA1) expression, reduction of cholesterol efflux, and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42. Moreover, the alteration in lipid rafts is associated with the re-localization of toll-like receptor 4 (TLR4) and triggering receptor expressed on myeloid cells 1 (TREM1) to rafts, ERK1/2 phosphorylation, NLRP3 inflammasome activation, and inflammatory cytokine secretion [85]. Another study has demonstrated that EVs released during the HIV infection of CD4+ T cells, but not CD42 T cells, efficiently inhibit HIV infection in vitro [86]. EVs isolated from the plasma of HIV-infected patients are known to associate with immune activation and oxidative stress in HIV patients. In addition, proteomic analysis of these plasma EVs has detected the presence of immune activation markers (CD14, CRP, HLA-A, and HLA-B), oxidative stress markers (CAT, PRDX1, PRDX2, and TXN), and Notch4 molecules in them [102].

EVs derived from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected lymphoma cells activate the canonical MEK/ERK pathway without affecting the other innate immune regulators, which allows the virus to exert these changes independently of pathogen recognition [62]. However, KSHV-EVs harboring mitochondrial DNA on their surface have shown the tendency to trigger an antiviral response by stimulating the cGAS-STING signaling pathway [63]. ZIKV-EVs are known to modulate immunity by targeting the two major cell populations, monocytes and endothelial vascular cells. EVs produced from ZIKV-infected C6/36 cells, carrying ZIKV RNA and proteins, induce the differentiation of host naïve monocytes to a proinflammatory state by augmenting tumor necrosis factor-alpha (TNF-α) production. In addition, these EVs promote vascular endothelial cell damage by inducing the expression of coagulation (TF) and inflammation (PAR-1) receptors onto the endothelial cell surface, resulting in increased endothelial permeability [73]. A similar cell-to-cell EV-mediated immune modulation has been reported for DENV. DENV activates the release of EVs from platelets through CLEC2 and further initiates the expression of CLEC5A and TLR2 on neutrophils and macrophages, resulting in the formation of neutrophil extracellular trap (NET) and the release of proinflammatory cytokines [103]. Moreover, blocking both CLEC5A and TLR2 simultaneously can effectively reduce the DENV-induced inflammatory response, which in turn confers protection to infected mice [103].

EVs produced during the infections of several other viruses, including CSFV [66], FMDV [31], [32], JCPyV [58], PRRSV [35], REV [36], SARS-CoV-2 [65], and SVV [38], confer immune protection to the viruses by preventing their recognition by neutralizing antibodies. Whether or not the EV-mediated perturbation of host immunity occurs during other viral infections remains largely unknown and requires investigation in future studies.

In addition to the functions mentioned above, EVs play other roles during viral infection. Several studies have shown that RNAs and proteins can serve as biomarkers for cancer diagnosis [104], [105]. RNA and protein levels within EVs, which are influenced by pathogenic infections, could potentially be utilized as biomarkers for diagnosing infections. For example, the CD81 levels of serum-derived EVs in patients with chronic hepatitis C are correlated with inflammatory activity and fibrosis severity, which helps to elucidate the role of CD81 in HCV pathogenesis [106]. Moreover, several pieces of evidence demonstrate that EVs have the potential to serve as molecule-delivery vehicles for disease treatment. Interferon-induced transmembrane protein 3 (IFITM3)-containing EVs can effectively deliver IFITM3 protein across the placental barrier and suppress ZIKV infection in the fetus [107]. EV-mediated mRNA delivery in vivo is safe and can be used to induce SARS-CoV-2 immunity [108]. However, while many studies have focused on the components and functions of EVs in pathogenic infections, there is little research on the role of EVs in diagnosing and treating pathogens. Therefore, the potential for pathogen diagnosis and treatment via EVs needs to be deeply explored in the future.

5. Roles of EVs in other pathogenic infection

An overview of EVs, including their methods of isolation, source of secretion, critical molecular components, and biological roles in bacterial, fungal, and parasitic infections, is outlined in Table 3 [109], [110], [111], [112], [113], [114], [115], [116], [117], [118], [119], [120], [121], [122], [123].

5.1. Roles of EVs in bacterial infection

Bartonella henselae (B. henselae) is a blood-borne, Gram-negative bacterial pathogen for which the natural reservoir host is the domestic cat. B. henselae-derived EVs contain hemin-binding protein C (HbpC), which increases bacterial resistance to haem toxicity [109]. Gram-negative bacteria, including Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa), release EVs that can be internalized by cells through endocytosis, delivering lipopolysaccharide (LPS) into the cytosol to trigger the caspase-11-dependent effector response [110]. Furthermore, EVs secreted by Moraxella catarrhalis (M. catarrhalis) contain the Moraxella immunoglobulin (Ig) D binding protein (MID), and these MID-containing EVs act as bait to avoid direct interaction with host B cells by redirecting the adaptive humoral immune response [111].

Methicillin-resistant Staphylococcus aureus (MRSA) is a type of Gram-positive bacteria whose common virulence strategy is to produce pore-forming toxins that damage the PM of host cells. The bacterial DNA induces the secretion of a disintegrin and metalloprotease 10 (ADAM10)-bearing EVs from human cells and mice tissues. These EVs protect the host cells by srving as scavengers that can bind multiple toxins, improving the survival of infected mice [112]. Neisseria gonorrhoeae (N. gonorrhoeae), which can cause suppurative infection of the urogenital system, secrets the bacterial outer membrane porin PorB via EVs. The porin PorB targets the mitochondrial membrane of macrophages, which leads to the loss of mitochondrial membrane potential, release of cytochrome, and activation of apoptotic cascade [113]. In addition, EVs derived from Porphyromonas gingivalis (P. gingivalis), a periodontal Gram-negative anaerobe, contain gingipains on the surface of EVs that increase vascular permeability, leading to disease both in vitro and in vivo [114]. EVs produced by Streptococcus pneumoniae (S. pneumoniae) are internalized into host cells and consequently promote host defense by triggering immune cell recruitment and cytokine response. On the other hand, these EVs also provide an anti-inflammatory environment for bacterial survival [115].

5.2. Roles of EVs in fungal and parasitic infection

Cryptococcus gattii (C. gattii) is the most significant cluster of life-threatening fungal infection in healthy human hosts. The EVs released by virulent C. gattii strains act as an accelerator of intracellular proliferation and virulence. These fungal EVs are taken up by infected host macrophages and transported to the phagosome, where they trigger the rapid intracellular growth of fungal cells [116]. Leishmania parasites are responsible for a group of tropical and sub-tropical infections known as leishmaniases. EVs released by Leishmania at elevated temperature and low pH are taken up by naïve cells from the extracellular environment, facilitating pathogen-host communication [117]. Trypanosoma brucei is a blood disease prevalent in Africa that causes low spirit and decreased appetite in animals, with a significant impact on livestock production and life. Trypanosoma brucei rhodesiense (T. b. rhodesiense)-derived EVs incorporate the serum resistance-associated protein (SRA) that is essential for infectivity in humans. Furthermore, these EVs transfer SRA to non-human infectious trypanosomes to evade human innate immune responses. Trypanosome EVs can also target mammalian erythrocytes, causing rapid erythrocyte clearance and anemia [118].

5.3. Effects of EVs derived from plants on pathogenic infection

Arabidopsis thaliana secretes EVs that deliver sRNAs to the Botrytis cinerea; these sRNA-containing vesicles accumulate at the infection sites and are taken up by fungal cells, resulting in silencing of the fungal genes critical for pathogenicity [119]. Another study showed that mRNA-containing EVs released from Arabidopsis thaliana are transported into fungal cells, where the delivered host mRNAs are translated, resulting in decreased infection of the fungal pathogen Botrytis cinerea [120]. P. gingivalis, a periodontal pathogen, selectively takes up ginger exosome-like nanoparticles (GELNs) in a phosphatidic acid (PA)-dependent manner through interactions with hemin-binding protein 35 (HBP35) on its surface, resulting in a significant reduction in the pathogenicity of P. gingivalis [121]. Furthermore, GELN-derived miRNA (miRNA aly-miR396a-5p) inhibits the SRAS-CoV-2-induced cytopathic effect in Vero E6 cells and lung inflammation by inhibiting the expression of the viral S and Nsp12 proteins [122]. Moreover, EVs isolated from the extracellular fluid of sunflower seedlings are incorporated by Sclerotinia sclerotiorum (S. sclerotiorum), exhibiting the function of growth inhibition, morphological changes, and cell death [123]. Edible plant-derived exosome-like nanoparticles (EPDENs) from grapefruit, grapes, ginger, and carrots contain proteins, lipids, and microRNA, which exhibit distinct biological effects by inducing the expression of anti-inflammatory genes and antioxidation genes [124].

6. Tracing of EVs

Despite the rapid development of EV studies, ranging from disease pathophysiology to therapeutic drug delivery, there is still a need for improved molecular tools to track them. Currently, EVs are mainly labeled with the lipophilic dyes PKH-67, DiO, or DiR; the permeable dyes CFSE or CFDA; the physical marker ⁹⁹mTc-HMPAO; and quantum dots [125]. However, studies have developed novel tracer methods for imaging EVs in vivo and in vitro. For example, labeling EVs with different luciferase enzymes tethered to CD63 allows for highly sensitive tracking of EVs both in vitro and in vivo [51]. Moreover, CD63 fusion with NanoLuc or ThermoLuc permits the cost-effective in vivo quantification of EVs or real-time imaging in a semi-high-throughput manner [51]. EV labeling with a nano luciferase (NanoLuc)-CD63 marker has facilitated the spatial and temporal tracking of cardiac EVs in a mouse model [126].

7. Concluding remarks

In summary, EVs regulate pathogenic infections, which can lead to the exacerbation or improvement of disease outcomes, depending on the pathogenic type and the signals encapsulated in the EVs. Given the fact that EV biogenesis and several cell responses are similar across pathogenic infections, utilizing knowledge of one pathogen and applying it to other pathogens may help in establishing molecules with broad-spectrum anti-pathogenic activities. Since most studies lack a deeper analysis of EV cargo molecules, in-depth EV profiling using high-throughput technological platforms may offer opportunities for personalized medicine and the treatment or control of diseases.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2022YFD1801500 and 2022YFD1800105), the National Natural Science Foundation of China (32030107 and 32372993), and the Fundamental Research Funds for the Central Universities (2662023PY005).

Compliance with ethics guidelines

Junyao Xiong, Usama Ashraf, Jing Ye, and Shengbo Cao declare that they have no conflict of interest or financial conflicts to disclose.

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