Introduction
The human microbiota is a dynamic consortium of microorganisms that play a pivotal role in maintaining homeostasis and overall health within the host. Among these microorganisms, bacterial extracellular vesicles (bEVs) have emerged as novel key players in the pathogenesis of human inflammatory diseases [1]. Traditionally, it was believed that microbial signals primarily exerted their effects at localized sites within the gut or other mucosal surfaces. However, accumulating evidence has revealed that bEVs possess the remarkable ability to travel beyond their site of origin, influencing immune responses at distant organs and tissues throughout the body[2].
Bacterial EVs, typically ranging from 20 to 300 nanometers in size, encapsulate a rich array of biological cargo, including proteins, lipids, nucleic acids, and other bioactive molecules [3]. This diverse composition enables bEVs to engage in complex interactions with host cells, facilitating communication between microbes and the immune system. Importantly, both the surface components of bEVs and their cargo have been shown to possess pro-inflammatory properties, which can modulate immune responses, trigger inflammation, and contribute to the pathogenesis of various diseases [4]. The ability of bEVs to influence distant organ systems underscores their significance in the broader context of host-microbe interactions.
Recent technical advancements, including high-resolution imaging techniques and single-particle tracking methodologies, have greatly enhanced our understanding of bEVs biodistribution within the host[5, 6]. These innovations have provided critical insights into how bEVs traverse biological barriers, such as epithelial layers and the blood-brain barrier, contributing to systemic inflammation, autoimmunity, and even cancer. The capacity of bEVs to penetrate these barriers has profound implications for our understanding of the systemic effects of microbial signals, challenging traditional views of localized immune responses and highlighting the potential for systemic dysregulation.
This review aims to summarize recent advances in our understanding of how bEVs translocate across host barriers to influence distant organ function. By exploring the mechanisms underlying bEVs translocation and their implications in the onset and progression of autoimmune disorders and cancer, we hope to illuminate the intricate interplay between the human microbiota and the host immune system. Understanding these processes may pave the way for novel therapeutic strategies targeting bEVs, potentially offering new avenues for the treatment of autoimmune diseases and other related conditions.
Mechanisms of EV translocation across biological barriers
Crossing the Mucosal Barrier
The gastrointestinal tract serves as the primary interface between the host and its microbiota, playing a crucial role in nutrient absorption, immune regulation, and overall health. This interface is protected by a complex mucosal barrier composed of mucus, epithelial cells, and associated immune cells. Recent studies have demonstrated that bEVs can efficiently navigate this protective layer, a process that is critical for their interaction with the host's immune system [5, 7, 8].
Recent research conducted by Thapa et al [7] highlighted that in ulcerative colitis (UC), bEVs are frequently coated with human IgA and become significantly enriched within the colonic mucosa. These IgA-coated bEVs not only bypass typical mucosal exclusion mechanisms but also promote enhanced uptake by underlying phagocytic immune cells, such as macrophages. This suggests a sophisticated mechanism whereby the host IgA response, rather than being solely protective, inadvertently targets bEVs to immune cells in a manner that fuels colonic inflammation, thereby influencing local mucosal immune dysregulation and contributing to UC pathogenesis.
Moreover, faeces-derived EVs have been implicated in the onset of barrier dysfunction, a mechanism closely linked to various peripheral diseases[9]. The study elucidates how these vesicles can disrupt the integrity of the intestinal barrier. By compromising the epithelial cell layer, faeces-derived EVs may facilitate the translocation of pathogens and inflammatory mediators into the bloodstream, thereby initiating systemic inflammation and potentially contributing to the development of liver-related pathologies. This highlights the dual role of bEVs as both communicators and potential disruptors of the mucosal barrier, illustrating their significance in the pathogenesis of diseases.
Breaching the Blood Barrier
Once bacterial EVs have successfully crossed the mucosal barrier, they can enter systemic circulation, marking a critical transition from localized effects to systemic influence. A recent proof-of-concept study by Bittel et al. utilized an advanced in vivo visualization technique, which demonstrated that bEVs originating from the gut microbiota do not merely remain localized [5]. Instead, they can translocate systemically and were shown to reach distant peripheral organs. Notably, this study provided evidence that these bEVs and their bioactive cargo, such as bacterial enzymes, could be delivered to the liver, spleen, kidney and heart. Even more striking, they showed that these microbial biomolecules cross the blood-brain barrier and being detected in the brain, highlighting the profound capability of bEVs to influence organs far away from their parental cell and suggesting a direct route for microbial influence on central nervous system homeostasis or pathology. This systemic dissemination of microbial signals via bEVs opens avenues for understanding their potential role in a wide array of systemic inflammatory conditions and distant organ pathologies.
Importantly, the ability of bEVs to cross the blood-brain barrier and other systemic barriers has profound implications for understanding how microbiota-derived signals can influence distant organ function. By breaching these barriers, bEVs may play a role in modulating immune responses, contributing to systemic inflammation, and potentially initiating processes involved in the development of autoimmune disorders and other related diseases. This underscores the importance of further exploring the mechanisms through which bacterial EVs translocate across host barriers, as well as their systemic effects, to fully appreciate their role in health and disease.
Target Organ Effects and Disease Associations
Recent studies have provided compelling evidence linking bEVs to liver pathologies, emphasizing their role as critical mediators of liver inflammation. In a recent study by Dorner et al.[4] bEVs released by the gut pathobionts Proteus mirabilis and Klebsiella pneumoniae were shown to breach the intestinal barrier, enter the bloodstream, and selectively accumulate in periportal regions of the liver, despite the absence of intact bacteria in hepatic tissue. In vitro experiments demonstrated that these bEVs engage multiple liver-resident cell types, including Kupffer cells, hepatic stellate cells, and cholangiocytes, where the vesicular lipopolysaccharide component triggers TLR4-dependent assembly of the NLRP3 inflammasome and gasdermin D–mediated pyroptosis, culminating in robust IL-1β release. The resulting pro-inflammatory milieu, enriched in cytokines such as TGF-β1/2, drives stellate-cell activation and collagen synthesis, linking ductal injury directly to fibrogenesis. In vivo, repeated administration of these pathobiont‐derived OMVs to Mdr2-/- mice markedly exacerbated liver pathology, as evidenced by increased periportal inflammatory infiltrates, elevated Kupffer-cell and CD4+ leukocyte recruitment, pronounced α-SMA expression, and enhanced Sirius Red–positive fibrosis—occurring without concurrent colonic inflammation. Finally, these preclinical data were confirmed in a clinical setting. Analyses of PSC-IBD patient samples confirmed an enrichment of pro-inflammatory, LPS-positive bEVs in both serum and liver biopsies, where they co-localize with NLRP3- and GSDMD-positive cells and promote IL-1β production, solidifying their role as key mediators of gut–liver crosstalk and highlighting their potential as diagnostic biomarkers and therapeutic targets in complex hepatobiliary diseases. Such comprehensive evidence positions bEVs as crucial effectors in the gut-liver axis, offering novel avenues for diagnostic and therapeutic interventions in complex hepatobiliary diseases.
Complementing these findings, detailed characterization of lipopolysaccharide-positive (LPS+) bEVs along the gut-hepatic portal vein-liver axis has further elucidated these detrimental pathways. A recent study could show that under conditions of gut dysbiosis, there is an altered profile and increased translocation of pro-inflammatory LPS+ bEVs from the gut lumen, via the portal vein, to the liver [10]. Their work shows that these vesicles carry distinct microbial signatures and that their increased presence in the portal circulation and liver tissue directly contributes to hepatic inflammation. Upon reaching the liver, these LPS+ bEVs have been shown to trigger inflammatory responses through mechanisms including Toll-like receptor 4 (TLR4) activation, leading to the upregulation of pro-inflammatory cytokines and enhanced macrophage activation. This directly implicates gut-derived LPS+ bEVs in the initiation and exacerbation of liver inflammation and associated pathologies. Such comprehensive evidence positions bEVs as crucial effectors in the gut-liver axis, offering novel avenues for diagnostic and therapeutic interventions in complex hepatobiliary diseases.
In addition to influencing the immune system, several studies demonstrate that bEVs from various bacteria can influence bone health. For instance, vesicles from Proteus mirabilis have been shown to inhibit osteoclastogenesis (the formation of bone-resorbing cells) and reduce bone loss by inducing mitochondrial apoptosis through the miR96-5p/Abca1 pathway. 11 Conversely, bEVs from oral pathogens like Filifactor alocis can induce systemic bone loss by activating Toll-like receptor 2 (TLR2) [12, 13]. Similarly, studies on extracellular vesicles derived from general oral bacteria indicate their capacity to affect osteoclast differentiation and activation, suggesting a broader role for the oral microbiome's bEVs in modulating bone turnover. These discoveries indicate that bacterial EVs can influence not only local inflammatory responses but also have systemic effects that may lead to significant changes in bone metabolism. The ability of these vesicles to mediate bone remodelling raises important questions about their role in autoimmune diseases, where bone degradation is often a critical concern. As the interplay between bacterial EVs and the immune system becomes clearer, it is essential to investigate how these vesicles contribute to bone health and disease. Understanding the mechanisms underpinning EV-mediated bone loss could provide valuable insights into the pathogenesis of autoimmune conditions and lead to the development of targeted therapies aimed at mitigating bone degradation associated with chronic inflammation. Indeed, a recent study also implicated bEVs in the pathogenesis of rheumatoid arthritis (RA). This study identified outer membrane vesicles (OMVs) derived from Fusobacterium nucleatum as a significant factor aggravating the severity of RA [14, 15]. The authors could further show an enrichment of Fusobacterium nucleatum in the gut of RA-patients. Fusobacterium derived FadA-containing OMVs translocate into the joints, where FadA engages synovial macrophages by activating the Rab5a GTPase and the transcriptional regulator YB-1, thereby triggering local inflammatory cascades that exacerbate arthritis in both RA patients and collagen-induced arthritis mouse models.
The findings suggest that bacterial EVs can act as systemic immune modulators, potentially triggering or exacerbating autoimmune responses both in the liver and joints. By interacting with the immune system, these vesicles may initiate inflammatory cascades that lead to joint damage and increased disease severity in individuals with RA. This highlights the need for further exploration into how specific bacterial EVs contribute to the immunopathology of autoimmune disorders, as targeting these interactions may open new avenues for therapeutic intervention.
Therapeutic and diagnostic implications
Understanding EV translocation pathways opens new avenues for disease intervention. Blocking the systemic dissemination of pathogenic EVs may provide therapeutic benefits in autoimmune disorders affecting the gut but also peripheral organs. This approach could involve using specific inhibitors to prevent the binding and internalization of these vesicles by host cells, thus reducing their harmful effects on distant organs.
Therapeutic Strategies
Inhibitors of EV uptake: Developing molecules that can block the receptors on host cells responsible for EV uptake, thereby preventing their entry and subsequent inflammatory responses [5].
Neutralizing antibodies: Creating antibodies that can specifically target and neutralize pathogenic EVs, reducing their ability to trigger immune responses [16].
Modulating EV biogenesis: Exploring ways to interfere with the production and release of pathogenic EVs from bacteria, potentially through genetic or pharmacological interventions.
Conversely, bacterial EVs show promise as biomarkers for early disease detection. These vesicles contain specific molecular signatures that can be detected in bodily fluids, providing valuable information about the presence and progression of diseases. For instance, EVs derived from gut pathobionts may indicate early stages of liver inflammation and fibrosis, while those from periodontal pathogens could serve as markers for systemic bone loss [4, 15].
Diagnostic Applications
Biomarker discovery: Identifying unique proteins, lipids, and nucleic acids within bacterial EVs that correlate with specific diseases.
Non-invasive testing: Developing assays to detect disease-associated EVs in blood, saliva, or urine samples, enabling early diagnosis and monitoring.
Personalized medicine: Using EV profiles to tailor treatments based on an individual's specific disease state and microbial interactions.
Given the complexity and versatility of bacterial EVs, continued research is essential to fully understand their roles in health and disease. By elucidating these pathways, researchers can develop novel therapeutic strategies to harness the beneficial effects of bacterial EVs while mitigating their harmful impacts.
Future perspectives
Despite significant progress, several challenges remain. Future studies should address the heterogeneity of bacterial EVs and their specific host interactions. Advanced imaging techniques, coupled with high-resolution muli-omic analyses, will be essential in delineating these complex interactions. These methodologies can provide insights into the spatial and temporal dynamics of EV production, release, and uptake by host cells, allowing for a more precise understanding of their roles in different physiological and pathological contexts.
Moreover, the development of standardized protocols for the isolation and characterization of bacterial EVs is crucial [Langhe]. This would enable reproducible and comparable results across different studies, thereby accelerating the translation of research findings into clinical applications. Investigating the influence of environmental factors, such as diet, lifestyle, and microbiome composition, on EV production and function could also reveal new therapeutic targets and preventive strategies.
Longitudinal clinical studies are needed to validate the diagnostic and therapeutic potential of bacterial EVs in chronic diseases. These studies should involve diverse patient populations and consider factors such as age, sex, genetic background, and comorbidities. By tracking changes in EV profiles over time and correlating them with disease progression and treatment outcomes, researchers can identify biomarkers for early detection, monitor disease activity, and tailor interventions to individual patients.
Collaborative efforts between researchers, clinicians, and industry partners are essential to drive innovation in this field. Such partnerships can facilitate the development of novel EV-based therapies, including engineered vesicles with enhanced therapeutic properties and targeted delivery systems. Regulatory guidelines and ethical considerations must also be addressed to ensure the safety and efficacy of these emerging treatments.
In summary, the future of bacterial EV research holds great promise for transforming our understanding of host–microbe interactions and their impact on health and disease. By overcoming current challenges and leveraging advanced technologies, we can unlock the full potential of bacterial EVs as diagnostic and therapeutic tools.
