Bacterial Extracellular Vesicles in the Regulation of Inflammatory Response and Host-Microbe Interactions

Abstract

Extracellular vesicles (EVs) are a new revelation in cross-kingdom communication, with increasing evidence showing the diverse roles of bacterial EVs (BEVs) in mammalian cells and host-microbe interactions. BEVs include outer membrane vesicles (OMVs) released by gram-negative bacteria and membrane vesicles (MVs) generated from gram-positive bacteria. Recently, BEVs have drawn attention for their potential as biomarkers and therapeutic tools because they are nano-sized and can deliver bacterial cargo into host cells. Importantly, exposure to BEVs significantly affects various physiological and pathological responses in mammalian cells. Herein, we provide a comprehensive overview of the various effects of BEVs on host cells (i.e., immune cells, endothelial cells, and epithelial cells) and inflammatory/infectious diseases. First, the biogenesis and purification methods of BEVs are summarized. Next, the mechanisms and pathways identified by BEVs that stimulate either pro-inflammatory or anti-inflammatory responses are highlighted. In addition, we discuss the mechanisms by which BEVs regulate host-microbe interactions and their effects on the immune system. Finally, this review focuses on the contribution of BEVs to the pathogenesis of sepsis/septic shock and their therapeutic potential for the treatment of sepsis.

1. Introduction

Extracellular vesicles (EVs) are non-replicative nano-sized particles with a diameter range of 20–300 nm, released by eukaryotic cells, bacteria, and archaea from their outer membranes into the surrounding environment^1,2. EVs can also be referred to as membrane vesicles (MVs), outer membrane vesicles (OMVs), microvesicles, exosomes, or ectosomes, depending on their parent cells and characteristics^3,4. In 1966, bacterial EVs (BEVs) were first observed under electron microscopy as lipid-like structures in Escherichia coli culture supernatants⁵. Both Gram-positive and Gram-negative bacteria produce EVs. However, EVs from Gram-positive bacteria were not reported until 2009⁶. This is likely because Gram-positive bacteria lack an outer membrane and instead have a thick peptidoglycan layer, which hinders EV production.

Since their discovery, BEVs have been found to contain various types of molecular and surface cargo, including proteins, nucleic acids, lipids, and various other metabolites, such as virulence factors⁷. Importantly, these BEV cargoes can elicit diverse effects on bacterial surveillance, including nutrient uptake, biofilm formation, horizontal gene transfer, antimicrobial activity, virulence factor secretion, and toxin release⁷. In addition, BEVs can transport their contents to recipient cells either extracellularly through receptor-ligand interactions or can be absorbed via membrane fusion or receptor-mediated endocytosis into host cells^{8 9}. BEVs also function as bridges for cross-kingdom communication. For example, BEVs have been demonstrated to interact with mammalian cells and thereby affect their behavior, including cell proliferation, angiogenesis, and immunoregulation, from triggering proinflammatory responses in immune cells to several other immunomodulatory effects in non-immune cells^10–12. Interestingly, many studies have indicated that BEVs can play either pro-inflammatory or anti-inflammatory roles, depending on their cargoes^13–16.

The immunomodulatory qualities and abilities of BEVs to deliver cargo to host cells have made them desirable tools for future therapies and disease prevention. Additionally, the non-replicative nature of BEVs renders them noninfectious and, therefore, relatively safe for various biomedical applications, such as targeted drug delivery and vaccine development¹². In this review, we summarize the current knowledge about the role of BEVs in (i) pro-inflammatory responses, (ii) anti-inflammatory effects, (iii) alteration of host-microbe interactions, and (iv) sepsis development and prevention/treatment.

2. Biogenesis of bacterial extracellular vesicles (BEVs)

BEV synthesis varies between gram-negative and gram-positive bacteria because of differences in their outer cell structures. Gram-negative bacteria contain an outer membrane underlying a thin peptidoglycan layer, whereas gram-positive bacteria lack an outer membrane and contain a thick peptidoglycan layer. The detailed mechanisms of BEV synthesis in gram-negative bacteria have been summarized in our previous review⁷. Briefly, four models have been proposed: (i) reduced cross-links between the outer membrane and peptidoglycan layer lying underneath^17,18; (ii) accumulation of some envelope proteins, peptidoglycan fragments, and membrane lipids that exert pressure on the membrane, resulting in membrane budding off in the form of BEVs¹⁹; (iii) accumulation of lipopolysaccharide (LPS) and phospholipids causing BEV biogenesis²⁰ (iv) extracellular stimulation for local curvature formation in the bacterial outer membrane. For example, through extracellular stimulation, the Pseudomonas quinolone signal (PQS) can induce membrane blebbing in Pseudomonas aeruginosa (P. aeruginosa)²¹.

In addition, several factors (i.e. as growth conditions, environmental stress, and genetic factors) have been reported to affect BEV synthesis⁷. Notably, high growth temperatures²², oxygen stress²³, chemical stress, and other forms of physical stress have been shown to increase EV production in various bacterial species^24,25. Furthermore, pathogenic bacteria have been found to produce more EVs than their counterpart non-pathogenic strains²⁶. This suggests that BEV production may have been adopted by pathogens to increase their virulence. Recent studies have demonstrated that BEV biogenesis does not occur randomly; rather, it is regulated by genetic factors. For example, PSMα3 can promote EV secretion, and deletion of PSMα3 results in significant reductions in EV secretion from Staphylococcus aureus²⁷.

It is important to note that manipulation of BEV biogenesis by various means is species-specific in terms of their effects on bacterial growth and BEV production^28–31. Interestingly, one effective approach is to alter culture media nutrients^{29, 30}. For instance, depletion of sulfate from the culture system could increase EV production from N. meningitides, because the VacJ/Yrb ABC (ATP-binding cassette, a phospholipid transporter) is utilized to generate BEVs through removing excess phospholipid content caused by sulfate deletion²⁹. A recent study by Hirayama et al.³⁰ identified that glycine, a known disruptor of peptidoglycan, significantly promoted BEV yields from E. coli. In addition, nearly trifold amounts of Propionibacterium freudenreichii EVs are produced when cultured in milk ultrafiltrate (UF) medium compared with yeast extract lactate (YEL)³². Besides the media nutrient composition, pH also affects BEV production from Lactobacillus casei and Lactobacillus plantarum³¹.

Furthermore, genetic modifications of bacteria by altering the expression of PGase, a protein that interferes with peptidoglycan layer integrity, could favor BEV biogenesis³³. With future in-depth mechanistic findings of BEV biogenesis, an increasing number of bioengineering tools will be developed to promote or block BEV generation.

3. Isolation and detection of BEVs in laboratory settings

Recently, BEVs have been used in advanced nanotherapeutics for the delivery of anti-microbials, chemotherapy, drugs, and vaccine developments^34–36. However, the small- or large-scale production of BEVs is often challenging and requires an additional purification method to collect enough BEVs³⁷. Therefore, an ideal approach for isolation is expected to yield a large number of BEVs with intact structure³⁸. The most widely used techniques to isolate and concentrate BEVs are ultracentrifugation (UC) and ultrafiltration (UF)³⁸. Notably, depending on the method used, the physical properties (morphology, size, and total yield) of BEVs differ. For instance, Reimer et al. (2021) reported that although UC and UF are both useful techniques for BEV extraction, UF is much faster and yields a higher concentration of small-sized vehicles than UC³⁸. Similarly, upon isolation and purification, BEVs were further subjected to functional assays to test their ability to enter host cells and stimulate cytokine and chemokine production³⁹. However, different methodologies used to extract BEVs can impact the following experimental analysis³⁸. In this regard, we briefly summarize the methodologies below: An overall schematic of BEV isolation and quantification is outlined in Figure 1.

Figure1:

Overview of BEV preparations. (1) Bacterial Culture: properties of BEVs are affected by several factors including growth medium, temperature, oxidative stress, hyperosmotic shock, iron depletion etc. (2) Isolation of BEVs from culture filtrates: achieved by low-speed centrifugation. (3) Concentration: Additional steps to ensure the high yields and purity of BEVs are obtained by ultracentrifugation and ultra-diafiltration. (4) Purification: density gradient centrifugation or gel filtration allows the separation of BEVs from other interfering extracellular matrix (i.e., flagella, pili, and fimbria). (5) Qualitative and quantitative analysis of BEVs: different approaches either measure the BEV cargo content (i.e., protein, lipids, DNA, RNA, or other particles), or BEV morphology.

3.1. Isolation and purification of BEVs

Usually, BEVs can be harvested from the cell-free supernatants of bacterial culture³⁷. Compared with gram-positive bacteria, more attention has been focused on the isolation and purification of gram-negative BEVs^{34, 40–44}. Nevertheless, the isolation and purification steps for both gram-negative and gram-positive bacteria are similar. The isolation of BEVs begins by separating the supernatant containing vesicles from the bacterial cell debris, large proteins, and membrane aggregates, which is achieved by high-speed centrifugation or several differential centrifugation steps. The supernatant was further filtered to remove residual bacterial components. However, various BEVs may have differential size distributions and form large aggregates that can lead to an almost 50% loss of isolated EVs. Considering the low yield of BEVs, pre-concentration steps are crucial, which are usually achieved by ultracentrifugation or ultrafiltration⁴⁰. Unfortunately, ultracentrifugation can also fail to separate BEVs from interfering with extracellular materials, such as flagella, fimbria, pills, and large protein complexes³⁸. In this scenario, density gradient centrifugation or gel filtration played a significant role in the purification of BEVs (Fig. 1). For example, iodixanol is a commonly used density-gradient medium for the purification of BEVs⁴⁰.

3.2. Qualitative and quantitative detection of BEVs.

Purified BEVs are further identified (qualitative) and quantified using various laboratory approaches³⁷. The particulate structure of BEVs contains several proteins, lipids, DNA, RNA, and other biomolecules. The quantitative values of these biomolecules act as proxies for the quantification of isolated BEVs³⁶. Several different protein tests, including Bradford, protein bicinchoninic acid (BCA), Lowry, and immunoblotting (tracking specific proteins) are usually performed for BEV quantification^37,39. Likewise, quantification of BEV proteins using Qubit fluorometric analysis has been the method of choice in multiple studies^34,37. Similarly, previous studies have shown that the quantification of lipids, particularly 3-deoxy-D-manno-octulosonic acid, a specific component of LPS, can reflect the number of BEVs isolated (Malachite Green)^38,40. Quantification of lipids can be achieved by using a lipophilic dye (FM-64) or enzyme-linked immunosorbent assay (ELISA) targeting LPS⁴⁰. Similarly, with the advancement of cutting-edge technologies (e.g., mass spectrometry), the absolute quantification of BEVs, including proteins and lipids (phospholipids and fatty acids) is possible⁴⁰. In addition to absolute quantification of BEV properties, visualization of BEVs can be performed using transmission electron microscopy (TEM). This approach can provide critical information such as shape, size, and the presence of interfering non-EV components, mainly flagella and large protein aggregates⁴⁰.

Growing research has shown that BEV components such as protein, DNA, RNA, and lipids vary between different species, and these components may not directly reflect BEV concentrations³⁴. Thus, there is a dire need for a standardized approach to address the factors that may interfere with BEV isolation and quantification, such as bacterial growth conditions, bacterial stages at which BEVs are isolated, their size, and the content of BEVs. Considering these limitations, NanoSight nanoparticle tracking analysis (NAT) is gaining popularity because of its ability to quantify BEVs irrespective of BEV contents³⁴.

Taken together, variation in the quantitative value of BEVs not only exists between different bacterial species and strains, but also between different types of quantitative assays used. Bitto et al.^{34, 39} reported that the BEV quantification method could alter the biological functions of BEVs, which was evident by the significantly different experimental outcomes. For example, they observed higher production of interleukin-8 (IL-8) in cells stimulated by an equivalent BEV concentration that was quantified using Bradford when compared to cells stimulated with BEVs quantified by BCA or Qubit protein assay³⁹. This disparity in response is possible due to the lack of a standardized protocol for the isolation, purification, and quantification of BEVs. Thus, future studies will be paramount to establish a standardized method for isolating and quantifying BEVs by addressing interfering factors.

4. The effects of BEVs in pro-inflammatory responses

Over the past decade, numerous studies have indicated that BEVs elicit various effects on host cells, and many studies have further explored the functional role of BEVs in their parent bacteria^45–47. Recently, multiple groups have demonstrated that BEVs stimulate diverse pro-inflammatory responses^48,49, as different composition/contents encased in BEVs could affect distinct receptors of targeting cells and the intracellular signaling pathways^50–52, as summarized below and depicted in Figure 2.

Figure 2:

Model of BEV-mediated pro-inflammation mechanisms. Gram-positive/gram-negative bacteria produce BEVs with MAMPs from cell membrane components LTA/lipoproteins and LPS respectively. LPS and LTA/lipoproteins activate membrane receptors TLR2 and TLR4 respectively, which results in intracellular signaling with MyD88. Further intracellular signaling activates NF-κB, a transcription factor that causes the production of pro-inflammatory cytokines and chemokines that contribute to various immune responses, as well as inactive cytokines. Intracellular receptors such as NOD1 can also contribute to NF-κB expression via activation from peptidoglycan of BEVs transported into the cell and aid from RIPK2 signaling. Endosomal TLR7, TLR8, TLR9, all activated by RNA, can also increase NF-κB expression, while other bacterial ligands such as gingipains can activate MAPK and NF-κB signaling pathways. Alternatively, intracellularly transported BEVs utilize other proteins such as GBPs, SNX-10, and Hemolysin for exposing LPS (or directly exposing bacterial toxins such as flagellum) to inflammasomes NLRP3/NLRC4, which activate caspase-1 and caspase-11 by transforming into inflammasome complexes. Then, caspase-1 can reactivate inactive cytokines, produce more pro-inflammatory cytokines, or can even induce pyroptosis via GSDMD. Caspase-11 is also capable of increasing pro-inflammatory cytokine production and inducing pyroptosis. Some BEVs can activate inflammasome pathways by K⁺ efflux or mitochondrial dysfunction, which can cause pyroptosis by reducing anti-apoptotic factor MCL-11.

4.1. Activation of pattern recognition receptors

Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) present on mammalian cells that regulate the host immune system by responding to microbe-associated molecular patterns (MAMPs)⁵³. TLRs belong to the type 1 transmembrane glycoprotein family, which is composed of a transmembrane, intracellular Toll-like/interleukin-1 receptor (TIR), and an extracellular domain⁵⁴. The extracellular domain comprises 20 leucine-rich repeat (LRR) modules that determine how TLRs differentiate between different MAMPs⁵⁴. On the other hand, the intracellular TIR domain is pivotal in controlling downstream signaling cascades and levels of signaling molecules and transcription factors, such as myeloid differentiation primary response 88 (MyD88) and nuclear factor kappa B (NF-κB), which subsequently promote the secretion of pro-inflammatory cytokines⁵⁵. TLRs allow the immune system to identify unknown particles and protect against invading foreign particles⁵³.

Ten different TLR types were identified based on location and ligand recognition. Of these, six TLRs were related to bacterial components and four were related to nucleic acids. Recent studies have demonstrated the ability of BEVs to interact with TLR2 and TLR4 and induce pro-inflammatory responses (Fig. 2)^55–59. The BEV-mediated activation of TLRs can be attributed to the membrane composition of their parent bacteria. For example, BEVs from gram-positive bacteria contain lipoteichoic acid (LTA), and gram-negative BEVs contain LPS, which can activate TLR2 and TLR4⁵⁶. This concept of TLR activation by BEV-sourced ligands is supported by the fact that inhibition of TLR4-LPS binding suppresses the proinflammatory response elicited by LPS-containing EVs from gram-negative E. coli⁶⁰. The role of LPS in BEV-activated TLR4 has been further documented, as Pep19–2.5, an inhibitor of several MAMPs, including LPS, can reduce pro-inflammatory cytokines stimulated by BEV-LPS and TLR4 activation⁵⁸. Moreover, the importance of BEV-TLR interactions in immunomodulation is also demonstrated by how E. coli EVs struggle to invoke pro-inflammatory responses in immune cells (e.g., macrophages) when TLR4 is absent from the system⁵⁹.

Despite the multitude of studies supporting the TLR4 and TLR2 pathways as the main mechanisms of BEV pathogenesis, recent studies have implicated that BEV pathogenesis is not fully dependent on LPS/LTA-activated receptors^61–64. For example, several groups have highlighted that various MAMPs found in BEVs are involved in other TLR-associated pro-inflammatory responses^61–64. In addition, S. aureus EVs containing RNA and DNA activate TLR7, TLR8, and TLR9, whereas S. aureus EV peptidoglycans activate TLR2⁶¹. As a matter of fact, TLR2, TLR4, and TLR9, each has various protein- and lipid-based ligands, and with the aid of other molecules (e.g., superantigens and phenol-soluble modulins), they play a critical role in developing an inflammatory response. Thus, different BEVs may activate different inflammation pathways⁶⁵. However, some studies suggest that S. aureus EVs produce a pro-inflammatory response preferably through the lipoprotein/TLR2 pathway, where lipoproteins act as the primary MAMPs compared to other MAMPs for TLR2, such as nucleic acids and peptidoglycan⁶². For example, lipoproteins have been identified as the main MAMP in Fillifactor alocis EVs for TLR2. Importantly, lipoprotein/TLR2-induced pro-inflammatory cytokines are responsible for the increase in osteoclasts and decrease in osteoblasts, leading to bone loss⁶⁴. BEVs can interact with various TLRs, leading to the production of pro-inflammatory cytokines that are not typically induced by TLR2/4 pathways⁶³. Furthermore, the function of a BEV acting as a MAMP may vary depending on its size, resulting in a variety of pro-inflammatory cytokines being produced from different signaling pathways⁶³.

In addition to TLRs, NOD-like receptors (NLRs), such as NOD1 (nucleotide-binding oligomerization domain-containing protein 1), are intracellular receptors with a leucine-rich repeat (LRR) domain that is activated by BEV-peptidoglycan (Fig. 2)⁶⁶. Once NLRs are activated, they recruit signaling molecules such as RIPK2 (receptor-interacting serine/threonine kinase 2) to induce inflammatory pathways such as NF-κB and MAPK (Fig. 2)⁶⁶. Interestingly, E. coli EVs can activate NOD1 receptors to induce IL-8 and IL-6 in epithelial cells, which is EV source-dependent⁶⁷. This may be ascribed to the varying amounts of peptidoglycan processing enzymes in various EVs released by different strains of E. coli⁶⁷. Taken together, the ability of BEVs to induce a pro-inflammatory reaction is linked towards BEV-packaged ligands that activate PRRs, which stimulate distinct cell-signaling pathways responsible for host immune responses.

4.2. Regulation of cytokine release pathways

Various BEVs have been reported to invoke pro-inflammatory cytokines, such as interleukin (IL) and interferon (IFN) family cytokines, through different intracellular pathways that lead to a variety of inflammatory responses in host immune cells^60,68–74. For example, Soult et al. (2015) showed that E. coli EVs could increase the secretion of IL-6 in endothelial cells⁶⁰. This could potentially be explained by the increased LPS/TLR4-mediated activation of NF-κB⁶⁰. In addition, P. aeruginosa EVs can induce pro-inflammatory chemokines, such as CXCL1 and CCL2, in neutrophils and macrophages through several receptors and MAMPs that combine to form a more potent inflammatory response⁷⁵. Similarly, Helicobacter pylori (H. pylori) EVs stimulate the production of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α in epithelial and macrophage cells⁶⁸. Notably, H. Pylori EVs also induced IL-17 and IFN-γ, which regulate immune responses by T helper cells TH₁₇ and TH₁ respectively⁶⁸. Furthermore, H. Pylori EV-induced IL-6, which is integral towards host immune responses in gastric environments, may be attributed to the activation of either the LPS/TLR4 pathway or the PG (peptidoglycan)/NOD1 pathway⁷⁶. Likewise, S. aureus EVs can cause skin inflammation via pro-inflammatory cytokines such as IFN-γ, but mainly IL-17, which stimulates complementary Th₁₇ cells to produce IgE antibodies⁷¹. In keratinocytes, S. aureus EVs can induce CXCL8 to activate neutrophils towards skin inflammation. Additionally, lipoproteins and protein A found in S. aureus EVs also contribute to initiating an inflammatory pathway involving TLR2 and NF-κB⁷².

Given that E. coli EVs can induce more pro-inflammatory cytokine production than E. coli LPS alone⁷³, other components of BEVs besides LPS may also contribute to the pro-inflammatory response. Indeed, Gingipains, found on the surface of Porphyromonas gingivalis (P. gingivalis) EVs, act as virulence factors to induce pro-inflammatory cytokines (i.e., IL-6, IL-1β, IL-8, and TNF-α) that are potentially regulated by the NF-κB, MAPK, and STING (stimulator of interferon genes) pathways^69,70. Aggregatibacter actinomycetemcomitans EVs encase small RNAs (sRNAs) that control the production of TNF-α from immune cells and blood cells through the TLR8 and NF-κB pathways⁷⁴.

4.3. Initiation of inflammasome pathways

Recent studies have also demonstrated that BEVs can initiate inflammasome pathways to recognize/eliminate pathogens and induce specific actions such as pyroptosis^77–80. Inflammasomes are intracellular PRRs induced by various MAMPs found in BEV cargo, and most are NLRs such as NLRP3 and NLRC4 (Fig. 2)^66,77,78,80. Inflammasomes are initiated by the production of pro-inflammatory cytokines, such as inactive IL-1β and IL-18, through the NF-κB pathway involving TLR4/TLR2. Activation of inflammasomes by MAMPs leads to caspase-1 cleavage, which activates inactive cytokines and results in the release of pro-inflammatory cytokines with the help of ASC (apoptosis-associated speck-like protein containing CARD)^77–79,81. Responses involving caspase-1 are categorized as canonical, while non-canonical inflammasome responses involve either caspase-11 from mice or caspase 4/5 from humans⁷⁹. In addition to the production of pro-inflammatory cytokines, inflammasome-activated caspase-1 can initiate pore formation in the cellular membrane via gasdermin-D (GSDMD), leading to inflammasome-induced pyroptosis, a type of immunoregulatory cell death that allows the removal of bacteria-infected host cells^78,79.

Given that inflammasomes are cytosolic protein complexes, the mechanisms by which BEVs transfer their virulence factors from the extracellular space into the host cell cytosol have been of great interest, as there are several molecules involved in this process that perform entirely different tasks depending on their environment^77–83. BEV-LPS has been linked to these inflammasome pathways, as enterohemorrhagic (EHEC) E. coli EVs can transport LPS into host cells, inducing a caspase-11 pathway that causes pyroptosis and IL-1 secretion⁸⁰. Similarly, P. aeruginosa EVs contain LPS, which can induce a caspase-11 NLRP3 inflammasome pathway and a caspase-5 non-canonical inflammasome pathway, suggesting that LPS is a favored MAMP in the activation of most inflammasome-induced pathways⁷⁷. However, some BEVs derived from H. pylori or periodontal pathogenic bacteria do not contain LPS, which can activate caspases. This suggests that several different MAMPs besides LPS may induce inflammasome responses^77,78. For example, BEVs from the periodontal bacterium P. gingivalis seem to induce pro-inflammatory responses through inflammasomes, as evidenced by the appearance of IL-1β, NF-κB activation, and ASC requirement. However, they possess a different version of LPS that lacks hexa-acylated A, which plays a significant role in inflammasome activation and subsequent pro-inflammatory responses⁷⁸. Since they lack LPS, B. pertussis is thought that Bordetella pertussis prioritize K+ efflux to activate the NLRP3 inflammasome complex through ASC assembly and IL-1β secretion (Fig. 2)⁷⁹. In addition, S. aureus EVs combine the effects of lipoproteins and pore-forming toxins (PFT) to activate TLR2 and the NLRP3 inflammasome, respectively, resulting in pro-inflammatory cytokine production and pyroptosis in macrophages⁸¹. Interestingly, flagellin from pathogenic Salmonella typhimurium EVs induces the NLRC4 inflammasome pathway to produce IL-1β⁸³ (Fig. 2). This is completely different from LPS-dependent pathways, as this pathway could trigger a stronger host response to pathogenic bacteria, as evidenced by quicker inflammasome assembly and prevention of excessive pyroptosis observed in LPS-containing BEVs⁸³. In addition to receptor-ligand interactions, BEVs can induce apoptosis to control inflammasome responses, as they can increase IL-1β production through caspase-11 and NLRP3 by interfering with mitochondrial protein production, resulting in a lack of molecules such as MCL-11 that prevent cell death (Fig. 2)⁸².

Another mechanism by which MAMPs interact with inflammasome pathways is by guanylate binding proteins (GBPs), which play an essential role in host defense by destroying pathogen membranes and exposing their substituents to PRRs to induce an immune response⁸⁴. GBPs are vital for inflammasome-mediated cytokine release and pyroptosis. Specific signals involved in inflammasome pathways, such as caspase-11, are activated by LPS carried by E. coli EVs and rely on GBPs, possibly due to lipid A found in LPS, to complete this process^84,85. In addition, B. pertussis EVs can potentially interfere with interferon pathways through lipooligosaccharide (LOS) to induce the caspase-11 inflammasome pathway, resulting in changes in GBP regulation⁷⁹. The mechanisms by which GBPs expose intracellular receptors to LPS are heavily disputed. Some studies suggest that GBPs use their ability to change membrane permeability to release LPS from outer membranes, while others suggest that BEVs transport LPS directly to these receptors^84,85. However, it is important to note that GBP is limited in mouse models as it differs from human GBP. Other proteins, such as hemolysin, which are known for their ability to cause membrane dysfunction, may provide another mechanism for LPS exposure to inflammasomes.⁸⁶. Edwardseilla tarda EVs associated with a significant concentration of hemolysin can induce significant amounts of the pro-inflammatory cytokines IL-1β and IL-18 through caspase-11 activation, suggesting that hemolysin plays a notable role in exposing LPS from BEVs to intracellular receptors⁸⁶. SNX-10 is a protein that regulates endosomes, which are associated with the delivery of LPS to cytosolic receptors. SNX-10 may regulate PIKfyve, a lipid kinase that controls endosomes transporting LPS from BEVs into the cytosol to activate caspase-5⁸⁷.

Interestingly, inflammasomes and other immune interactions differ depending on exposure to bacteria alone versus EVs. P. gingivalis EVs, for example can cause a significantly stronger inflammatory response than P. gingivalis alone by inducing more pro-inflammatory cytokines, activating inflammasomes, and inducing pyroptosis, possibly due to a greater concentration of gingipains found on P. gingivalis EVs compared to bacteria⁸⁸. This difference in responses could indicate how bacteria alter the host response for their benefit. For instance, the pro-inflammatory characteristics of P. gingivalis EVs can be used to extract nutrients from damaged host tissues. In contrast, a reduced inflammatory profile of whole P. gingivalis bacteria can be used to avoid host defenses⁸⁸. In the future, the immunomodulatory effects of BEVs on inflammasome activation should be further investigated, as they may provide new avenues to develop vaccines and pharmaceuticals that protect against bacterial sepsis.

5. Role of BEVs in anti-inflammatory responses

Unlike the BEVs derived from pathogenic bacteria that stimulate pro-inflammatory response described above, recent studies have shown that BEVs released by probiotic/commensal bacteria may elicit anti-inflammatory responses, as highlighted below and depicted in Figure 3.

Figure 3:

Model of BEV-mediated anti-inflammatory mechanisms. BEVs carry ligands LTA, Lp19180, and PSA to activate TLR2, which activates intracellular signals Gadd45α and CD14 that increase NF-κB expression. In contrast, BEVs can inhibit TLR4 activated from LPS stimuli, which subsequently lowers activity of other signaling molecules such as MyD88, p65, and NF-κB. Furthermore, BEVs transported into the host cell can inhibit certain pathways such as NF-κB by SlpB and MAPK by miR-199a-3p, and they can even inhibit pro-inflammatory cytokine production. Regulation of NF-κB expression results in greater concentration of anti-inflammatory cytokines, which provides beneficial action for the immune system. Other environmental factors such as pH and growth medium can enhance the anti-inflammatory effects of BEVs.

5.1. Manipulation of anti-inflammatory TLR pathways and cytokine production

Accumulating evidence has demonstrated that probiotic/commensal bacteria can affect inflammation through TLR pathways to invoke an anti-inflammatory response^89–92. For example, one of the microorganisms prevalent in the human gastrointestinal system is the commensal bacterium, Bacteriodes fragilis (B. fragilis). The released BEVs contain polysaccharide A (PSA), which is known to trigger anti-inflammatory immune responses and aid in the prevention of colitis⁹². This PSA encased in B. fragilis-EVs acts as a MAMP for TLR2 on dendritic cells to induce the production of the anti-inflammatory cytokine, IL-10, through Gadd45α (growth arrest and DNA-damage-inducible protein) (Fig.3). Consequently, this leads to the appearance of regulatory T cells (T_REGS), which further induce IL-10⁹². Similarly, B. vulgatus can release anti-inflammatory BEVs that contain polysaccharides to interact with TLR2/TLR4 on bone marrow-derived dendritic cells (BMDCs), resulting in tolerance towards pathogenic E. coli and preventing future inflammation⁹¹. In addition, EVs released by Lacticaseibacillus rhamnosus (L. rhamnosus) strain JB-1 contain LTA, which modulates anti-inflammatory mechanisms, such as intestinal epithelial cell (IEC) internalization and IL-10 production⁹⁰. Moreover, in response to dextran sodium sulfate (DSS)-induced colitis, L. rhamnosus GG EVs suppressed the production of pro-inflammatory cytokines such as IL-1β and TNF-α through the inhibition of the TLR4/NF-κB pathway, as evidenced by the reduced expression of MyD88, p65, and p-p65⁹³. It is important to note that TLRs seem to play a more important role in pro-inflammatory cytokine production⁸⁹, however, anti-inflammatory BEVs rely on similar signaling mechanisms to enhance their immunomodulatory role in host responses, as TLRs are required to carry out anti-inflammatory responses such as anti-inflammatory cytokine production.

BEVs have been shown to play an important role in producing anti-inflammatory cytokines or blocking the production of pro-inflammatory cytokines^15,94–98. When stimulated with LPS from E. coli, HT-29 epithelial cells can produce the pro-inflammatory cytokine IL-8. However, EVs from P. freudenreichii strain CIRM-BIA 129 can reduce the production of IL-8 by downregulating the NF-κB pathway, potentially owing to the presence of SlpB (surface-layer protein B) in EVs, as SlpB has anti-inflammatory properties⁹⁸. Similarly, H. pylori EVs induced the production of anti-inflammatory cytokines IL-4 and IL-10 in RAW 264.7 macrophages⁹⁴. Furthermore, Bifidobacterium longum EVs from strain AO44 induced IL-10 in murine splenocytes and DC/CD4+ cultures, which is potentially linked towards an increase in CD8+/CD4+ T cell activity⁹⁷. Interestingly, BEVs can reverse the effects induced by pro-inflammatory stimuli, such as DSS-induced colitis¹⁵. For example, EcN (E. coli Nissle 1917) EVs can mitigate the effects of DSS-induced colitis, as they have been linked to a reduction in pro-inflammatory cytokines (i.e., IL-1β, TNF-α, and IL-17) and an increase in the anti-inflammatory cytokine IL-10¹⁵. Additionally, Bacteroides thetaiotaomicron EVs can induce the production of IL-10, which is typically absent in inflammatory bowel disease (IBD), in various types of cells upon DSS-induced colitis. Similarly, bone marrow-derived macrophages (BMDMs) treated with LPS produced more IL-10 and less TNF-α when exposed to B. thetaiotaomicron EVs because of the increased TLR2 activation by coreceptor CD14 or changes in the epigenetic marker H3K4me1 (histone 3 lysine 4 methylation), although this interpretation is highly disputed⁹⁵.

In contrast to S. aureus EVs, Staphylococcus epidermidis ATCC12228 EVs can reverse the production of pro-inflammatory cytokines IL-6, IL-23, and IL-17F in murine skin caused by imiquimod-induced (IMQ) psoriasis, an inflammatory skin disease⁹⁶. The underlying mechanism could potentially be associated with a decrease in IL-36, which is responsible for inflammatory responses from immune cells and other pro-inflammatory cytokines found in psoriasis⁹⁶. Similarly, Clostridium butyricum (C. butyricum) EVs can reduce the production of pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in LPS-treated RAW264.7 cells by blocking the NF-κB and MAPK pathways⁹⁹. In addition, C. butyricum EVs can upregulate the expression of miR-199a-3p in RAW264.7 cells which consequently, suppresses map3k4 gene expression, leading to inactivation of the MAPK signaling pathway⁹⁹.

Lactobacillus, a genus of probiotic bacteria, produces BEVs, which serve as one of the most widely studied anti-inflammatory BEVs. They have been implicated in a multitude of anti-inflammatory responses in the host^100–102. For example, treatment of TNFα-stimulated Caco-2 cell model of IBD with Lactobacillus EVs greatly reduced the expression of phosphorylated p65, an important signal of the NF-κB pathway, and blocked pro-inflammatory IL-8 production¹⁰². Interestingly, while LPS stimulation alone caused an increase in pro-inflammatory cytokines (TNF-α, IL-1β, and IL-8) in broiler cells, Limosilactobacillus reuteri EVs from strain BBC3 combined with LPS resulted in decreased production of these pro-inflammatory cytokines and increased production of the anti-inflammatory cytokine IL-10 in broiler macrophages¹⁰⁰. In addition, when splenic lymphocytes were co-cultured with broiler cells treated with L. reuteri BBC3 EVs, anti-inflammatory cytokines IL-10 and TGF-β were increased, whereas pro-inflammatory cytokines IL-17 and IFN-γ were blocked, indicating the large scope of anti-inflammatory responses induced by BEVs in the entire host system¹⁰⁰.

Lactobacillus may also generate anti-inflammatory responses by negating the effects of pathogenic stimuli. This is exemplified by how L. plantarum EVs cause the decrease of pro-inflammatory cytokine IL-6 induced by S. aureus EVs in immune cells and skin cells¹⁰³. Notably, despite being probiotic and providing benefits to the host, not all Lactobacillus bacteria have anti-inflammatory properties, as L. plantarum EVs induce not only the anti-inflammatory cytokine IL-10 but also the pro-inflammatory cytokines IL-1β and IL-6 to activate the host immune system¹⁰¹. Furthermore, L. plantarum EVs potentially utilize proteomic sections of Lp19180, a lipoprotein found in the cellular membrane, as a ligand of TLR2 to induce the NF-κB pathway¹⁰¹.

Recent studies have also indicated that some anti-inflammatory pathways induced by BEVs may depend on various environmental changes. BEVs from agitated L. casei and L. plantarum cultures grown at pH 5 resulted in the greatest IL-10 production in THP-1 cells compared to other conditions³¹. BEVs produced at these specific conditions may potentially be more apt to interfere with TLR4 and LPS binding interactions³¹. In addition, BEVs from L. casei and L. plantarum grown at pH 5 resulted in the reduction of TNF-α in THP-1 cells; however, it is noteworthy that the various proteomic compositions and particle concentrations caused by differing environmental conditions did not correlate with anti-inflammatory effects³¹. Thus, it is necessary to identify the anti-inflammatory mechanisms of BEVs caused by various BEV cargoes and culture environments, thereby proving beneficial in developing future BEV pharmaceuticals.

5.2. Pathogenic usage of anti-inflammatory mechanisms

Although we generally associate pathogenic bacterial EVs with pro-inflammatory, it is also important to consider some of their anti-inflammatory qualities. Such BEV-mediated anti-inflammatory cytokine pathways could provide possible explanations for how pathogenic bacteria utilize BEVs to cause persistent infection^{78,89,104–108}. For example, although Streptococcus pyogenes EVs can induce pro-inflammatory cytokines (i.e., TNF-α, IL-1β and IL-8) through bacterial toxins such as SLO (streptolysin O) in monocytic cells, BEVs from invasive strain SSI-1 cannot induce pro-inflammatory cytokines that non-invasive strain JRS4 can. This could be interpreted by which BEVs aid in evading host defenses, because anti-inflammatory bacterial toxin C5a peptidase found in SSI-1 may contribute to the reduction of pro-inflammatory cytokines¹⁰⁵. Interestingly, S. aureus EVs were found to induce only a small amount of TNF-α compared to the large amount of pro-inflammatory cytokines induced by S. aureus bacteria¹⁰⁸. This suggests that pathogenic BEVs can deflect immune system alerts during the initial infection of host cells. P. gingivalis EVs also contain sphingolipids (SL) that regulate TLR2/TLR4 pathways, resulting in the reduced production of pro-inflammatory cytokines (i.e., IL-6, IL-8, IL-1β, and TNF-α) in THP-1 cells, allowing pathogenic bacteria to evade the development of host defenses against infection¹⁰⁷. Furthermore, pathogenic BEVs can increase the chances of bacterial infection by producing a combination of pro-inflammatory and anti-inflammatory cytokines⁷⁸. For example, P. gingivalis EVs induce not only the pro-inflammatory cytokines TNF-α and IL-8 through the NF-κB pathway, but also induce anti-inflammatory IL-10 secretion⁷⁸. The presence of IL-10 could indicate inflammatory anergy and a reduction in pro-inflammatory responses, leading to the retention of bacterial populations. In addition, BEVs from P. gingivalis (and other periodontal pathogens) contain sRNAs that may contribute towards avoiding immune defense by blocking the production of inflammatory cytokines (i.e., IL-5 and IL-13) in T cells¹⁰⁴. Indeed, when introduced into antibiotics such as metronidazole, pathogenic B. fragilis EVs cause an increase in both pro-inflammatory cytokines (i.e., IL-8, TNF-α, IL-1α) and anti-inflammatory cytokines (i.e., IL-10)¹⁰⁶. This mixed production of cytokines can prevent excessive host cell damage and dampen the host immune response, thereby creating an opportunity for the pathogen to remain in the host environment. Pathogenic E. coli EVs can produce a significant amount of pro- and anti-inflammatory cytokines⁸⁹, possibly because of the similarities between the two types of cytokine pathways that respond to LPS. The Anti-inflammatory cytokine IL-10 might be able to reduce the production of pro-inflammatory cytokines such as IL-17, allowing pathogenic bacteria to remain in the host by limiting the immune response to inflammation⁸⁹. Taken together, these findings may explain chronic bacterial infections and ineffective antibiotic treatment. Thus, effective drugs targeting pathogenic bacteria should be considered to avoid BEV-mediated immune defense.

6. BEVs in the regulation of host-microbe interactions

Several studies have elucidated host-microbe relationships through the interactions of BEVs with the host system and have further identified how the host invokes diverse immune responses to BEVs^21,109. BEVs play a critical role in the regulation of these cross-kingdom communications^110–112 and gut microbiota homeostasis^113,114, as discussed below.

6.1. Diverse approaches for BEVs to enter host cells/environment.

To better understand how BEVs communicate with host cells, we first introduced how BEVs are transported into host cells (Fig. 3, red arrow). There has been much evidence that BEVs enter intracellular environments, such as periodontal BEVs that can pass through the blood-brain barrier. More specifically, these EVs cross blood vessel barriers into brain monocytes by endothelial-interfering gingipains^69,74,115. However, the manner in which BEVs enter host cells is dependent on the bacterial species and the type of host cells. In addition to phagocytic cells, non-phagocytic cells can absorb BEVs through macropinocytosis or endocytic approaches mediated by clathrin, caveolin, lipid rafts and membrane fusion⁹. Clathrin-mediated endocytosis (CME) utilizes clathrin, which surrounds vesicles that contain extracellular cargo with support from adaptor proteins (i.e., AP2), resulting in further processing of intracellular endosomes and lysosomes¹¹⁶. Caveolin-dependent endocytosis uses caveolin as a membrane coating, a cholesterol-dependent protein capable of manipulating the membrane shape and forming caveolae that carry out endocytosis¹¹⁷. Micropinocytosis is specialized towards intaking cargo of greater size compared to other endocytic methods, whereas lipid raft endocytosis can continue via caveolin or GTPases⁹.

BEV uptake via endocytosis is specific to a specific uptake method. For example, EHEC EVs require AP2 and dynamin to enter the host cells via CME¹¹⁸. Likewise, Cañas et al. (2016) demonstrated that E. coli EVs from the gut microbiome strains ECOR12 and EcN require CME for internalization by HT-29 cells¹¹⁹. In contrast, enterotoxigenic E. coli (ETEC) EVs contain heat-labile enterotoxin (LT), which initiates caveolin-dependent lipid raft endocytosis in HT-29 cells¹²⁰. Moraxella catarrhalis EV uptake by A549 epithelial cells also requires lipid raft endocytosis, a mechanism that is common among other pathogenic gram-negative bacterial EVs¹²¹. Notably, BEV uptake can be blocked by the addition of chlorpromazine (an inhibitor of CME) and dynasore (an inhibitor of dynamin), both of which regulate clathrin-coated vesicle formation¹¹⁹. Similarly, the internalization of ETEC-EVs can be disrupted by the addition of filipin, an inhibitor of lipid raft formation¹²⁰.

In addition, a combination of endocytic pathways can facilitate BEV uptake by host cells. For example, H. pylori EVs use CME as their main method of entry into host cells; however, they also utilize CME-independent endocytosis to a lesser degree¹²². Furthermore, BMDMs mainly utilize macropinocytosis to internalize F. tularensis EVs; however, internalization of F. tularensis EVs can also occur through other endocytic methods, as evidenced by the fact that dansylcadaverine and MβC, inhibitors of claritin-dependent and claritin-independent methods, respectively, caused a significant reduction in BEV entry¹²³. Similarly, the gram-positive bacterium Bacillus cereus produces pro-inflammatory BEVs that enter Caco2 cells via dynamin through lipid raft cytosis or clathrin-mediated endocytosis (CME)¹²⁴.

As described above, while the mechanism underlying the host internalization of BEVs may vary from cell to cell and species to species, it is a great advantage for bacteria to communicate widely between species. BEVs are particularly beneficial when bacteria are inaccessible to the host organism. This phenomenon creates new opportunities for bacteria to cause infections without direct contact with host cells¹²⁵. For example, unlike whole bacteria, B. fragilis EVs not only activate more TLRs because of the increased concentrations of receptor ligands (i.e., LPS and RNA), but also travel inside host cells to induce intracellular receptors¹²⁵. Interestingly, E. coli EVs can carry Cre proteins through the intestinal mucosa and spread among various organs¹²⁶, demonstrating their ability to act as vehicles for protein transport into inaccessible areas.

6.2. BEV-mediated microbe-host interaction in the immune and endo/epithelium systems

As described above, BEVs can regulate both the pro- and anti-inflammatory responses. Hence, a significant portion of BEV-mediated microbe-host interactions are related to the immune system. In addition, the endothelium and epithelial structures act as the first lines to interact with infected bacteria. Accordingly, BEVs have been reported to greatly affect endothelial/epithelial cells. Therefore, the following discussion focuses on the effects of BEV-mediated microbe-host interactions on the host immune and endo/epithelium systems.

First, BEVs have been shown to affect macrophage polarization^{88, 127–130}. For example, P. gingivalis EVs can alter murine macrophages towards an M1-like proinflammatory phenotype by reprogramming their metabolism from oxidative phosphorylation towards glycolysis by altering the expression of genes such as Irg-1, a regulator of TCA (the citric acid) cycle⁸⁸. Similarly, E. coli EVs can increase the expression of reactive oxygen species (ROS) and CD86 in macrophages, which exhibit an M1-like phenotype¹²⁸. In contrast, Pediococcus pentosaceus-derived EVs promote macrophages towards M2-like characteristics by increasing Arg-1, PD-L1, and IL-10 expression and stimulating the development of anti-inflammatory immune cells such as myeloid-derived suppressor cells (MDSCs)¹³⁰. The anti-inflammatory effects of P. pentosaceus EVs were demonstrated by blocking immune cell recruitment and mitigating the harmful effects of DSS-induced colitis¹³⁰. More interestingly, it has been reported that E. coli EVs may manipulate the communication between macrophages. For example, CirA, a BEV protein highly concentrated in lower iron inflammatory environments, has been shown to mediate the transport of inflammatory signals, such as iNOS and COX-2, in exosomes from infected macrophages toward uninfected macrophages¹³¹.

Second, other types of host immune cells are also susceptible to BEVs^{125,132–135}. Lajqi et al. (2022) recently showed that neutrophils stimulated with lower concentrations of gut microbiota EVs activated the TLR2/MyD88 pathway to express more pro-inflammatory and antibacterial mediators, such as ROS, MCP-1, CD11a, and CD32¹³⁴. In contrast, the anti-inflammatory cytokine IL-10 is expressed when neutrophils are stimulated with higher doses of gut microbiota EVs¹³⁴. Regarding dendritic cells (DCs), it has been reported that gut microbiota EVs can augment DC anti-inflammatory reactions¹³⁵. EVs released by different strains of E. coli can stimulate DCs to produce T cell-responsive cytokines, such as IL-12 for Th1 cells from EcN-EVs and TGF-β for Tregs from ECOR12-EVs¹³⁵. These BEVs can also influence crosstalk between DCs and T-helper cells by increasing surface markers (i.e., CD86 for T-cell activation) and miR-155–5p in DC-derived exosomes (miR-155–5p for the regulation of T-cell differentiation)¹³⁵.

Third, some BEVs can be used towards manipulate the immune response to favor bacterial infection/survival. For example, Legionella pneumophila EVs stimulate THP-1 cells to produce pro-inflammatory cytokines; however, THP-1 cells pretreated with L. pneumophila EVs exhibited an immune response beneficial towards L. pneumophila infection¹³⁶. Pre-treatment of THP-1 cells with these BEVs causes the upregulation of miR-146a, an anti-inflammatory miRNA involved in the TLR2/NF-κB pathway, which allows L. pneumophila to avoid bacterial clearance from M1 macrophages while causing persistent infection of other immune cells through pro-inflammatory cytokines and anti-apoptotic factors¹³⁶. Moreover, L. pneumophila EVs also contain the small RNA RsmY, which is similar in function and sequence to eukaryotic miRNAs that attenuate RIG-1 expression in THP-1 cells¹³⁷. This interferes with the TLR pathways responsible for IFN-β production, resulting in enhanced bacterial survival¹³⁷. Another pneumococcal bacterium, S. pneumoniae, also produces BEVs that help bacteria survive in macrophages to avoid host defenses¹³⁸. However, S. pneumoniae EVs can be rapidly recognized by DCs, resulting in a significant increase in TNF-α¹³⁹, demonstrating their inability to process antigens for subsequent immune responses. In addition, P. aeruginosa EVs can participate in immune invasion by controlling neutrophil activation and migration to avoid other immune cells from initiating bacterial killing¹⁴⁰.

Finally, BEVs have been shown to affect endothelial and epithelial cell survival^141–145. Bielaszewska et al. reported that BEVs from EHEC can initiate apoptosis by transporting enterohemorrhagic E. coli hemolysin (EHEC-Hly), a pore-forming toxin, into endothelial/epithelial cells¹⁴¹. In addition, Acinetobacter baumanii EVs play an important role in transporting outer membrane protein A (OmpA) into epithelial cells to induce mitochondrial dysfunction through the DRP1 pathway, resulting in cell death¹⁴². Lee et al. recently showed that E. coli EVs can induce cytokine IL-8 through the NF-κB pathway and cell adhesion molecules, such as ICAM-1, on human endothelial cells to encourage neutrophil recruitment¹⁴³. Furthermore, E. coli EVs can increase leukocyte migration into mouse lungs via the upregulation of pro-inflammatory cytokines/chemokines in epithelial cells¹⁴⁴. Likewise, pathogenic S. aureus and S. epidermis EVs can also induce the release of cytokines IL-8 and chemokine MCP-1 (monocyte chemoattractant protein 1) in epithelial cells, leading to the migration of leukocytes, neutrophils, and macrophages¹⁴⁵.

7. BEVs in the pathogenesis and prevention/treatment of sepsis

Sepsis is defined as life-threatening multiorgan dysfunction caused by a dysregulated host response to pathogen infection. Bacterial sepsis occurs when the persistence of bacteria becomes uncontrolled and host proinflammatory immune responses are abnormally elevated. Given that BEVs play a crucial role in controlling either pro- or anti-inflammatory responses, it is well appreciated that BEVs can significantly affect the development of sepsis and septic shock. Additionally, BEVs can be used as vaccines or antibiotic delivery tools to prevent or treat sepsis.

7.1. BEVs contribute to the pathogenesis of sepsis

As mentioned above, BEVs can stimulate pro-inflammatory responses with a much higher potency than the parent bacteria themselves. This finding suggests that BEVs are critical factors in the development of severe sepsis and septic shock. More importantly, accumulating evidence has demonstrated that some bacteria (i.e., Acinetobacter baumannii) can use BEVs to help them survive and resist antibiotics, leading to severe sepsis^{7, 146–148}. Indeed, BEVs can carry antibiotic-degrading enzymes that can act as distractors for antibiotics⁷. Kim et al. (2020) found that when sub-lethal doses of ampicillin were administered to methicillin-resistant S aureus (MRSA), the production of BEVs increased and encased higher levels of β-lactam-degrading enzymes¹⁴⁹. In addition, other bacteria could use BEV cargo to help build up biofilm communities under stress conditions, and even partake in the horizontal transfer of antibiotic resistance genes^7,147–150. For example, exposure of various bacterial species to MRSA-BEVs resulted in protection from death and lower growth rates when treated with ampicillin¹⁴⁹. Interestingly, Ye et al. (2021) observed that the injection of imipenem in mice infected with multidrug-resistant Klebsiella pneumonia caused excretion of K. pneumonia BEVs and excessive inflammatory responses¹⁵¹. They further identified that these BEVs interacted with Lox-1 receptors and caspase-11, resulting in macrophage pyroptosis and a severe inflammatory response¹⁵¹. Taken together, these studies suggest that BEVs can promote and spread antibiotic resistance, thereby greatly contributing to the pathogenesis of sepsis and septic shock.

7.2. BEVs in the prevention and treatment of sepsis

Recently, BEVs isolated from probiotic bacterial strains have been shown to protect against sepsis^{7, 147, 152}. Li et al. (2017) used probiotic L. plantarum EVs to enhance the host immune response against vancomycin-resistant Enterococcus (VRE) infection in C. elegans¹⁵². Likewise, Wang et al. (2020) observed that the antimicrobial compounds in B. thailandensis EVs were able to disrupt biofilm formation in antibiotic-resistant A. baumannii¹⁵³. These studies indicate that natural BEVs can be used to combat antibiotic-resistant bacteria. Furthermore, components of BEV cargo and surface proteins can be further subjected to extraction, purification, and modification to develop vaccines against antibiotic-resistant bacteria^{147, 150}. Additionally, BEVs can be modified to precisely deliver antibiotics to infected cells at lower doses, thereby mitigating the development of new resistant strains^{146, 153}. For example, Gao et al. (2018) wrapped nanoparticles containing vancomycin and rifampicin in S. aureus-BEVs¹⁵⁴. They observed that macrophages would take up these modified BEVs and deliver antibiotics to the internal S. aureus infection, which is more efficient than antibiotics administered alone¹⁵⁵. Hence, these studies open possibilities for new specific and efficient targeting therapies to treat antibiotic-resistant sepsis.

In addition to antibiotic delivery, modification of BEV components is a promising strategy for vaccine development. Surface LPS and other surface markers allow BEVs to act as both antigens and adjuvants to trigger innate and adaptive immune responses^{155, 156}. Kim et al. (2013) showed that, by injecting 0.5–1μg of E. coli EVs in advance, mice had 80–100% survival rates after a lethal infection of E. coli¹⁵⁷. Other studies have used BEVs as adjuvants in vaccines. For instance, Prados-Rosales et al. (2014) found that mice had less viable Mycobacterium tuberculosis in their lungs when mycobacterial EV immunization was coupled with the BCG vaccine¹⁵⁶. Most importantly, modified or unmodified BEVs can activate both humoral and cellular immunity, which are crucial for developing protective memory cells after vaccination^157–159. Particularly, BEVs can increase levels of IFN-γ and thus encourage Th1-cell responses that strengthen macrophage and dendritic cell activity against intracellular infections^{157, 159}. Recently, the European Medicines Agency approved the 4CMenB vaccine, which incorporates BEVs into the vaccine against meningitis B¹⁶⁰. This vaccine was derived when BEVs were isolated and used to control the 2011 meningitis outbreak in New Zealand¹⁶⁰. Therefore, BEVs may be a valid and promising tool for vaccine development to prevent sepsis and septic shock.

8. Conclusions and future perspectives

In this review, we summarize the current literature on: (i) the role of BEVs in pro-inflammatory responses, (ii) BEV-mediated anti-inflammatory effects, (iii) BEV-mediated host-microbe interactions, and (iv) the role of BEVs in sepsis. Specifically, the major characteristics of BEVs are their ability to affect pro- and anti-inflammatory responses, as well as their impact on the regulation of certain MAMPs and signaling molecules, resulting in the alteration of receptor activity, cytokine production, immune cell function, and host-bacterial communication^109,161. This implies that BEVs play multiple roles in the host environment. Indeed, BEV-related immunology is of utmost importance, as it solves some of the current significant challenges in sepsis, including the rise in antibiotic resistance^{146,162, 163}.

Despite overwhelming research addressing the effects of BEVs using animal models, future investigations need to transfer these results towards in vivo experiments for clinical relevance¹⁶⁴. Although BEV cargo acts as a facilitator of cell signaling pathways, it remains unknown whether BEV cargo can perform other mechanisms to control the host response. For example, sRNAs encased in BEVs can regulate the immune response through different mechanisms such as regulation of transcription, epigenetics, cell metabolism, and miRNA activity in host cells^165–170. Recently, Fan et al. (2023) identified sRNA45033 within P. gingivalis EVs that could induce pro-inflammatory and pro-apoptosis effects through CBX5 gene expression via epigenetic regulation¹⁷¹. This is exemplified by the changes in H3K9me3 in response to P. gingivalis EVs, suggesting the involvement of DNA methylation. However, the mechanism requires further investigation¹⁷¹. Additionally, bioinformatic technology may uncover a relationship between BEV-sRNAs and human mRNAs, which could affect gene expression if these interkingdom RNAs can link together¹⁷²; however, the experimental methods proposed to test these theories have yet to be applied practically.

Given the diverse immunological effects of BEVs, it is expected that new discoveries from BEV research could lead to more advanced biomedical applications of BEVs, beyond the above-mentioned roles in antibiotic resistance and vaccine development. Antiviral defenses could become another perspective in the field of BEV research, as several BEVs have been shown to inhibit viral replication and infection through macrophages by inducing pro-inflammatory cytokine pathways associated with MX-1 (a GTPase known to inhibit viruses) or TLR4 to activate the immune system¹⁷³. Moreover, these mechanisms could have therapeutic value, as S. typhimurium EVs were found to attach to the spike receptor-binding domain (RBD) and may act as a SARS-CoV-2 vaccine¹⁷⁴. Nonetheless, further studies are required to utilize the antiviral properties of BEVs in a more flexible manner. Considering the broad spectrum of positive and negative effects on host systems and their potential implications for various therapeutics, BEVs will undoubtedly become a predominant focus of immunology and biomedical research for decades.