Abstract

Introduction

Sepsis was recently redefined as life-threatening organ dysfunction caused by a dysregulated host response to infection. For decades, it has remained a highly lethal condition in which dependable diagnostics and therapeutic decision-making are far from optimal. Currently, sepsis incidence is estimated at 270 cases per 100,000 persons/year followed by an approximate 26% mortality rate ^1,2. Programs such as the Surviving Sepsis Campaign ³ and World Sepsis Day have increased awareness to the global burden of sepsis as well as the related diagnostic/ therapeutic difficulties. Both the Surviving Sepsis Campaign and the most recent Sepsis-3 definitions stress a dire need for reliable biomarkers. The current Surviving Sepsis Guidelines are limited to a weak recommendation regarding the use of procalcitonin for shortening/discontinuation of ongoing antimicrobial treatment only and encourage evaluation of further markers, e.g., for renal dysfunction and coagulopathy ^2,3. An ideal sepsis biomarker should possess a diagnostic and predictive value (high sensitivity, high specificity), display low donor variability, be measured in a rapid and cost-effective bedside assay and withstand validation in multicenter trials ⁴. Establishing a biomarker of sepsis should not be merely limited to correlating its concentration in the blood (and/or other fluids) with a particular disease phase and/or clinical outcome. It should also govern further management, e.g., the initiation of further diagnostics, escalation or de-escalation of a specific antibiotic regimen and initiation or discontinuation of invasive and extracorporeal treatments.

Many compounds with biological impact to the host defense are detectable in body fluids at elevated levels during sepsis and other states accompanied by an acute inflammatory response. Several inflammatory cytokines (circulating peptides) like tumor necrosis factor (TNF)-alpha, interleukin (IL)-1-beta and IL-6 have been identified as markers of sepsis severity and outcome predictors, and were targeted in several clinical trials ^5-8. The failure of the trials targeting the “cytokine storm” was surprising and disappointing; it is now known that their design was based on a false assumption that deaths from sepsis were primarily attributable to the overwhelming synthesis of cytokines (i.e., hyperinflammation) ^9,10. The newest evidence shows that sepsis responses are multifactorial and fluctuate rapidly ⁹. Thus, it is not the abundance of one particular cytokine but rather a specific complex inflammatory “signature” that accurately depicts a patient's immune status as well as the probable prognosis and response to treatment ^9,11.

Only recently, the role of exosomes in critical illness has been summarized ¹². This small population (size range 30-100 nm) of EVs derived from the endosomal system represents only a subset of the extracellular messenger pool. EVs are 30 nm to several micrometers in size, formed either by outward budding of the plasma membrane upon activation, during programmed cell death (i.e., apoptosis), or by exocytosis of multivesicular bodies ¹³. They express epitopes on their membranes that are specific for their parental cells. A review on their function in health and disease has been published recently ¹⁴. Systematic assessment of EVs in sepsis is an area of diagnostics that may support the developing concept of patient-tailored therapy by filling up the wide biomarker gap. Release of EVs represents an immediate cell communication mechanism that fulfills three major functions: a) transfer of an expression pattern to other cells, b) dissemination of the membrane-bound mediators within the liquid phase and c) rapid rearrangement of the cell surface. Like all messengers, EVs can immediately modify a phenotype of neighboring cells in a paracrine fashion but also bring forward an altered micro-milieu to other tissues by transferring the membrane composition and/or expression pattern to other regions of the circulation (endocrine release). Figure ​Figure11 illustrates the different types of EVs and basic modes of cellular release. The existing literature suggests that EV monitoring can effectively aid in the identification and characterization of specific pathophysiological blueprints of a given septic patient as well as in assessing the efficacy of various therapeutic regimens available to that patient. Over the last two decades, a large number of original studies investigating EVs in sepsis have been published. However, more is not always better, and a large number of experiments does not automatically ensure clinical progress.

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Figure 1

Release mechanisms and the extracellular vesicle pool. EVs constitute a dynamic pool with a considerable range in size. The classic term microparticles (100-1000 nm, intermediate size range) contains the EV fraction that is sometimes referred to as microvesicles or ectosomes and is released from the cell surface in response to activation (B). Although larger, apoptotic bodies that emerge during the disintegration of dying cells well range into the size spectrum of apoptotic microparticles, making it impossible to distinguish the underlying biogenesis solely based on dimensions (C). Exosomes emerge from the endosomal network and are considered the smallest fraction of the EV pool. Undergoing active packaging processes, exosomes are eventually liberated by the fusion of the multivesicular body with the surface membrane (A).

This review has several objectives. First, we provide an analysis of the 40 existing studies on EVs in septic patients (summarized in Table ​Table11) with a focus on their study design. This is important given that EVs have been suggested to serve as diagnostic biomarkers ¹⁵, while the methods for their detection and data interpretation in complex diseases such as sepsis are still under development. This translates into large variability in the quality, usefulness and/or comparability among the existing sepsis-related studies. Second, we focus on the role of EVs as biomarkers and mediators in sepsis as well as emphasize their potential utility to guide treatments and their role as potential targets for treatment. Third, we debate whether EVs are a meaningful part of the adequate evolutionary host response or also act as involuntary players of the inflammatory response ¹⁵. Finally, we characterize the influence of EVs in the exemplary, most severe clinical manifestation of sepsis (i.e., disseminated intravascular coagulation, DIC) and common treatment routines (i.e., blood transfusion, mechanical ventilation) used in the intensive care unit (ICU). While a systematic analysis of all existing in vitro and preclinical studies was not the main scope of this review, several preclinical papers relevant to the discussed topics are also included.

Heterogeneity versus standardization: critical assessment of EVs studies

In order to be useful, data from in vitro/ex vivo, preclinical (no patients involved) studies and clinical (patients-based) studies must meet several stringent criteria including: 1) best possible match between the research model and a given clinical disease scenario, 2) an adequate study design, sample sources and sampling time points, 3) adequate statistical power, 4) standardized detection assays and 5) reproducibility. In a complex disorder like sepsis, mediators like EVs dynamically fluctuate and those changes may play a different (if not opposite) role in various body compartments as well as in the local and systemic circulation.

The intensive care unit (ICU) patient populations in general, but especially those fulfilling sepsis criteria ², are extremely heterogeneous. They present with variations in genetic background, age, gender, accompanying diseases, chronically prescribed medications, source of infection (medical or surgical) and pathogens involved. Heterogeneity has been one of the major hindrances in sepsis research, particularly affecting the development of therapies. On the one hand, studies performed in heterogeneous cohorts better reflect the entire population and are easier to power, making any novel findings more robust and applicable to a wider target population. Conversely, large patient cohorts can also mask potential benefits (and harms) of any novel treatment in defined, smaller patient sub-groups. Although retrospective analyses may enable post-hoc detection of such benefits, the investigative value of such findings is weaker. Below, we present several key elements that modify the perception and understanding of EV-based research in the clinical context. The “sepsis” diagnosis encompasses patients with a multitude of scenarios by which the microbial invasion develop. To our knowledge, only one recent study compared the role of EVs among subsets of septic patients based on the site of infection ¹⁶.

For example, at least 19 of the analyzed studies (Table ​Table11) were performed in cohorts with mixed sites of infection (i.e., pneumonia, intra-abdominal infection, urinary tract infection, necrotizing fasciitis, wound infection, meningitis, mediastinitis) and underlying pathologies and injuries (i.e., trauma, cancer, immunosuppression). The relatively small number of existing clinical EVs studies exacerbates the difficulty of any reliable cross-comparisons. It is currently unclear whether the origin of the infection focus in any way modulates the EVs characteristics and their signaling routes and/or diagnostic potential. Although different anticoagulants for blood samples were used, citrate predominated. This is appropriate as citrate is recommended for achieving stable EVs counts throughout the initial processing period ¹⁷. In the context of potential EV utilization, the heterogeneity of the sepsis population remains one of the most significant hurdles, necessitating careful planning (e.g., only including patients with site-specific sepsis focus) and reliance on clear definitions and reporting.

Most recently, sepsis has been redefined as a dysregulated host response to infection and its gradation largely rests upon the state of organ dysfunction present in the patient ². The latter element partly overlaps with the earlier definition of severe sepsis ¹⁸ and can occur in the presence of underlying circulatory and cellular/metabolic abnormalities which are profound enough to additionally increase mortality (defined as “shock”). Given that sepsis and septic shock often develop from an initially non-life-threatening infection, the influence of EVs during this phase may be misinterpreted if patients enrolled for clinical studies are not meticulously selected based on the exact diagnosis and/or disease phenotype. In general, most EV studies were performed in patients suffering from severe sepsis (i.e., including organ dysfunction) according to the former definition, which is referred to as 'sepsis' according to the recent Sepsis-3 definitions ² (see Table ​Table11). In at least 14 studies, the septic patients were in the state of shock. It is unknown whether re-analysis of sepsis studies using the new Sepsis-3 definitions for enrollment (and thus redistributing groups) would change their conclusions; new studies selecting patients based on the new Sepsis-3 have been only recently emerging ¹⁶. Interestingly, redistribution of patients according to the Sepsis-3 definition did not affect conclusions drawn in the recent study by Matsumoto et al. ¹⁹.

In patients, the exact time of the onset of a septic episode is typically unknown. Repetitive blood sampling from intravascular catheters at least once daily is a routine practice in the ICU. In the studies analyzed in Table ​Table11, EVs were characterized based on only one sampling time-point in 23 studies, and on multiple time-points (i.e., time course) in 17 studies. Additionally, marked variation existed regarding the time that elapsed between sepsis diagnosis and the draw of the first blood sample enrolled into the study. These factors related to timing of samples may hinder precise and/or protracted characterization of EV-related events in a rapidly fluctuating disease. Another critical factor is the choice of optimal and defined inclusion/exclusion criteria. EVs are ubiquitously detectable and have pleiotropic effects. Therefore, multiple preexisting disorders may confound the EV concentration in septic patients. The most frequent exclusion criteria were: a) diabetes mellitus ²⁰, b) statin treatment ²¹, c) drugs altering platelet function ^22,23, d) recent blood product transfusion ^22,23, e) any hematologic malignancy, and f) other types of cancer ²⁴. As a result, a substantial part of average ICU septic patients was excluded, potentially rendering the population represented by the existing studies too specific for unbiased comparison with the general ICU population. Future studies could address this problem by maximally reducing exclusion criteria while ensuring a meticulous documentation of patient characteristics. This could be supported by an EV-oriented checklist that helps authors to include the most important patient information and minimal experimental requirements for definition of EVs and their functions ^25,26. Table ​Table11 shows that those studies that described mortality rates in sepsis and/or septic shock reported them within a relatively narrow range. This is appropriate given that it enables cross-comparison among studies. Of note, sepsis mortality was above 50% in four studies ^27-30.

In summary, the analyzed studies (Table ​Table11) enrolled highly heterogeneous septic patient populations with a) diverse exclusion criteria, b) different sites of infection, c) varying disease severity and phenotype, and d) mostly single or double sampling time-points only. Therefore, any future sepsis studies targeting EV sequelae should account for the above discussed elements in their study design. Any improvement of study inter-comparability by amenable standardization procedures will directly enhance their clinical translatability.

Diagnostic and/or therapeutic potential of EVs in sepsis

Once prompted by activation and/or apoptosis, EVs that shed from the cell surface typically retain the membrane composition (or the main features thereof) of the cells they originated from. For example, the release of these vesicles from the endothelium and monocytes can be effectively simulated by incubation with endotoxins such as bacterial lipopolysaccharides (LPS), pro-inflammatory cytokines or TNFα ³¹. Although extracellular vesicles differ in size, composition and biogenesis, some basic characteristics can be summarized. Irrespective of the dimensions, EVs represent spherical, subcellular compartments that are composed of a phospholipid bilayer and various membrane-bound or plasmatic cargo molecules (Figure ​Figure22). Depending on the EV release process, their membrane retains the pattern of the cell surface they originated from. Given that EVs are released from all known tissue types, the retained transmembrane molecules and phospholipids interact with countless cellular processes. The smaller EV fraction (which arises from the early endosome) and the multivesicular bodies display membrane features that resemble organelles of those cells but undergo additional fine-tuned wrapping mechanisms.

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Figure 2

Prototypic vesicle. Although extracellular vesicles differ in size, composition and biogenesis, some basic characteristics can be summarized: Irrespective of the dimensions, EVs represent spherical, subcellular compartments that are composed of a phospholipid bilayer and various membrane-bound or plasmatic cargo molecules. Depending on the process during which they are released, the EV membrane retains the pattern of the cell surface they originated from. Given that EVs are released from all known tissue types, the maintained transmembrane molecules and phospholipids interact with countless cellular processes. The smaller EV fraction (which arises from the early endosome) and the multivesicular bodies display membrane features that resemble these cell organelles but are subject to fine-tuned wrapping mechanisms. In general, the EV content includes proteins/peptides, smaller metabolites, as well as nucleotide sequences.

The released EVs become potential mini-messengers which may function as biomarkers but may also rapidly disseminate, magnify and/or perpetuate the processes triggered by the parent cell. EVs represent cellular shuttles that are capable of transferring a variety of compounds between cells. In general, the EV content includes proteins/peptides, smaller metabolites as well as nucleotide sequences. Upon fusion, vesicles rapidly modify the membrane composition of the receiver cell by transferring phospholipids and transmembrane molecules (Figure ​Figure33A). Meanwhile, a variety of cargo molecules are directly ejected into the plasma of the cell (Figure ​Figure33B). Compounds like growth factors, cytokines or other mediators are able to provoke an immediate metabolic response and to directly interfere with the receiver cell's signal transduction. During states of systemic inflammation and cell activation, tissue factor (TF) and phosphatidylserine (PhtdSer)-bearing vesicles represent microcarriers for the dissemination of a procoagulant phenotype (Figure ​Figure33C). Any component of the vesicle membrane can function as a ligand for receptors at the surface of the receiver cell, thereby triggering multiple responses (Figure ​Figure33D). During endocytosis, vesicles retain their membrane integrity and are engulfed by invagination (Figure ​Figure33E). Aside from proteins and metabolites that directly provoke the transduction of signals, other content like miRNAs are capable of silencing the expression of genes (Figure ​Figure33F). By altering the posttranscriptional processing on an mRNA level, this interference not only affects the synthesis of signaling molecules but secondarily also the expression of transcription factors (Figure ​Figure33G). Finally, EVs also play a role in fundamental mechanisms of immunity, including cytokine synthesis and antigen presentation (Figure ​Figure33H).

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Figure 3

Modes of interaction of EVs with receiver cells. EVs represent cellular shuttles that are capable of transferring a variety of compounds between cells. (A) Upon fusion, vesicles rapidly modify the membrane composition of the receiver cell by transferring phospholipids and transmembrane molecules. (B) Meanwhile, a variety of cargo molecules are directly ejected into the plasma of the cell. Compounds like growth factors, cytokines or other mediators are able to provoke an immediate metabolic response and to directly interfere with the receiver cell's signal transduction. (C) During states of systemic inflammation and cell activation, tissue factor and phosphatidylserine-bearing vesicles represent microcarriers for the dissemination of a procoagulant phenotype. (D) Any component of the vesicle membrane can function as a ligand for receptors at the surface of the receiver cell, thereby triggering a multitude of responses. (E) During endocytosis, vesicles retain their membrane integrity and are engulfed by invagination. (F) Aside from proteins and metabolites that directly provoke the transduction of signals, other content like micro ribonucleic acids (RNA) are capable of silencing the expression of genes. (G) By altering the posttranscriptional processing on a messenger RNA level, this interference not only affects the synthesis of signaling molecules but secondarily also the expression of transcription factors. (H) Finally, EVs also play a role in fundamental mechanisms of immunity, including cytokine synthesis and antigen presentation.

Any compound to serve as a potential biomarker must be stable enough to enable a reproducible detection in the hospital setting. Availability of sensitive assays to quantify cellular and/or circulating compounds and metabolites (including their genomic or transcriptional fingerprints) has enabled us to identify “signatures” of potentially diagnostically relevant biomarkers in virtually any biological sample, such as the blood ³², urine ^33,34, cerebrospinal ³⁵ and nasal ³⁶ fluid.

EVs represent cellular mediators that are released from all known cell types via a number of evolutionarily conserved mechanisms ³⁷. Sepsis triggers a multifaceted cellular activation including massive shedding of membrane vesicles from various cell subtypes ³⁸. Although other methods have been recently attempted ³⁹, flow cytometry is currently the main technology for the high-throughput characterization of EVs. When EVs are used for further investigation, fluorescence-activated cell sorting, a specific form of flow cytometry by which cells are sorted based on their light scattering and fluorescence characteristics can be used. While flow cytometry by itself is reliable and sensitive, the standardization procedures for the detection of EVs are not. Currently, the utility/fidelity of EVs as biomarkers cannot be judged without defining clear standard operation procedures. Specifically, a reproducible detection and in-depth characterization of circulating EVs depends on two key variables: a) sample preparation (e.g., anticoagulation, storage time and temperature, agitation during manipulation or transport) ^17,26 and b) precisely defined flow cytometry settings ⁴⁰. The international societies have been striving to reach an acceptable consensus on standardized sample preparation, documentation and measurement procedures ^26,41-44. Recently, the applicability of size-calibrated beads for the standardization of the EVs count analysis using flow cytometry was tested on platelet-free plasma from healthy volunteers by 44 laboratories using 14 different types of cytometers (with central collection and analysis of raw data). Although the test demonstrated an acceptable variability among most commercial cytometers ⁴⁰, these results need to be verified using clinical samples. Another important limitation of flow cytometry is that a large fraction of EVs remains under the lower limit of detection of the majority of available commercial cytometers (currently around 0.3 µm). This caveat should not be ignored given that the value of EVs as biomarkers may lie in recognizing changes in the ratios of specific EVs subpopulations and/or their presence/absence in a biological material. Such an EV-based approach has shown its value as a diagnostic support in the trauma setting ⁴⁵.

From the technological standpoint, for EVs smaller than 0.3 µm, the method of choice is nanoparticle tracking analysis, which enables detection of specific antigens with fluorescence filter sets ⁴⁶. Although technologically advanced, this method has not yet been verified for EV-based diagnostics in septic patients. Regarding timing, a number of new point-of-care technologies that may deliver results within 2 hours are currently being studied. For example, Herrmann et al. used the aggregation of granulocyte-derived EVs (GEVs) and bacteria for the differentiation of infectious versus non-infectious inflammatory states using a microfluidic chip ^47,48. Another promising method is the lateral flow immunoassay: a one-step chromatographic immunoassay that has undergone continuous performance improvement by the use of modern labels such as gold nanoparticles, magnetic particles, carbon nanoparticles, colored latex beads, quantum dots, organic fluorophores, enzymes and liposomes. Oliviera-Rodriguez et al. have recently tested the value of this assay for the detection of (CD63 and CD9-positive) platelet-derived EVs (PEVs) in human plasma and identified gold nanoparticles as the optimal label ^49,50. Future studies testing the applicability of the lateral flow immunoassay for the early detection of sepsis and its complications using EVs derived from other sources are warranted. Table ​Table22 identifies the main developmental directions needed for closing the current technological gap in EV research.

The main portion of the EV pool under steady-state conditions is platelet-associated (i.e., resting/activated platelets, megakaryocytes) ^51,52. In pathological conditions, however, EVs are mostly derived from activated platelets, endothelial cells (ECs) and leukocytes. These EVs accumulate rapidly in the circulation and are readily detectable by flow cytometry ^53,54. The presence/absence, dynamic changes and/or composition of circulating EVs have a potential to be harnessed as fine-tuned diagnostic descriptors of ongoing pathophysiological inflammatory processes in sepsis (and beyond). In addition to the considerable role of EVs as markers of a systemic inflammatory response, an elevation of TF-bearing vesicles could be considered as an early indicator of a generalized (in some cases even focal) infection. TF-positive EVs could be used to guide further or more aggressive treatment with antimicrobials ^55,56. The mediator/effector role of PhtdSer/ TF-expressing EVs in septic coagulopathy (blood clotting disorder) is detailed in the last subchapter in this review.

Besides analysis of EVs on the basis of vesicle surface antigens (the most important marker molecules displayed in Table S1), identification of cargo like micro-ribonucleic acid (miRNA) also appears to be an interesting research tool ⁵⁷. MiRNAs are short (19-25 nucleotides) noncoding sequences that are both actively and passively incorporated into EVs and that are capable of regulating gene expression by interfering with the translation of messenger RNA. In a preclinical sepsis study, miR-223 containing EVs released by mesenchymal stem cells attenuated cecal ligation and puncture-induced cardiac dysfunction in mice by reducing the Sema3a and Stat3 transcription factors ⁵⁸. Moreover, miR-223 is involved in the modulation of hematopoiesis and is seen as a promising indicator of immune response dysregulation ^59,60. Combined with the expression pattern of other miRNAs, miR-223 could potentially help to distinguish between non-septic and septic patients ⁶¹. Studies in cancer patients have shown that miRNA phenotypes are consistent and reproducible among individuals and serve as a cluster of markers for a number of diseases ^62,63. Other miRNAs, such as miR-15a, miR-30d-5p, miR-30a-5p, miR-192-5p, miR-26a-5p, miR-23a-5p, and miR-191-5p appear to be capable of differentiating septic patients and those with non-infectious inflammation (also called systemic inflammatory response syndrome, SIRS) ^64,65. Some miRNAs are detectable in exosomes and serum only, whereas others are also present in multiple cell types ⁶⁶. Moreover, miR-21 has been shown to be an essential part of the protective effect of remote ischemic preconditioning in sepsis ⁶⁷. Another recent report demonstrated EVs as messengers that can suppress miRNA expression and activate downstream gene expression in the central nervous system during inflammatory states ⁶⁸. However, research on the utility of EV-derived nucleotide sequences is in its infancy. Amplification and detection of miRNAs is usually performed via reverse transcriptase polymerase chain reaction and next-generation sequencing. However, these methods do not adequately discriminate between circulating, Argonaute protein-bound miRNA and miRNA incorporated to EVs. Studies investigating miRNA in sepsis (not focused on EV detection) may in fact have measured EV-associated miRNA (and vice versa). Innovative detection technologies to bypass this limitation are urgently needed (Table ​Table22). Given that there are no clear guidelines regarding EV-RNA analytics, any findings should be interpreted with caution ⁶⁹.

In addition to their role as biomarkers, circulating EVs constitute a dynamic pool of messengers that allow an organism to rapidly respond to altered physiological conditions. Thus, in their mediator-like character, they are capable of conveying a multitude of information to even distant tissues within the body. In a short-range communication format, EVs released by various cells could affect other immuno-competent cells/tissues in their immediate surroundings modulating, for example, a focal inflammatory process. Additionally, EVs may also support cell communication over longer distances. For example, analogous to the soluble endothelial protein C receptor (EPCR), EVs expressing EPCR are able to bind activated protein C (APC) upon external administration of recombinant human protein C in septic patients in order to exert its effects at distant vascular sites ⁷⁰. This mode of mediation appears to be especially relevant in diseases involving endothelial activation and microvascular dysfunction (e.g., sepsis) given that they are accompanied by a robust release of EVs. Sepsis-induced acute kidney injury (AKI) is associated with an impairment of endothelial barrier function and the sequestration of (endothelial cell-derived) EVs is thought to be a substantial causative factor in AKI pathophysiology ⁷¹. In the future, excessive EVs could be eliminated using continuous veno-venous hemofiltration ⁷² or be used for optimizing filtration fractions during hemofiltration ^72,73.

In summary, EVs appear to simultaneously serve as both markers and mediators in sepsis syndromes. This creates a multidirectional opening for their potential utilization: either diagnostic, therapeutic or both (depending on a specific sepsis scenario/ phenotype and objective).

EVs in sepsis: a friend or foe?

EVs are involved in severe pathophysiological events such as infections and/or inflammatory processes. The existing literature ⁷⁴ implies that EVs play an important role in sepsis and septic shock. Given that both the pathogen and the host appear to rely on EVs as their tool of either attack or defense, it needs to be precisely deciphered which EVs populations mediate and promote progression of sepsis and which support its resolution and propel healing. The most intuitive separation can be done based on the source of EV release.

Bacteria, viruses and fungi responsible for septic infections are all well-capable of producing and secreting varieties of membrane vesicles, yet without the specific surface lipid composition seen in eukaryotic microbes ^75-81. Gram-positive and Gram-negative bacteria, as the most frequent cause of sepsis ⁸², can release EVs (in Gram-negative termed outer membrane vesicles) that may carry toxins, adhesins, proteolysins and other virulence factors relevant for the development of the systemic inflammatory response ^83-86 and/or subsequent dysfunction of organs and tissues. Incubation of C. albicans-derived EVs with bone marrow-derived macrophages resulted in synthesis of IL-10, IL-12, transforming growth factor-beta and nitric oxide, as well as increased expression of major histocompatibility class II and cluster of differentiation (CD)-86 on macrophages ⁸⁷ - all suggesting a strong immunomodulating potential for fungal EVs during infections ⁸⁰. Similarly, development of sepsis, a leading cause of death in HIV type 1 patients ⁸⁸, is promoted by exosomes from infected cells containing the accessory extracellular viral protein Nef, which was shown to induce apoptosis of CD4-T-cells ⁸⁹. Viruses are also capable of controlling the release and content of EVs produced by infected cells ⁹⁰ to facilitate the viral spread. In this context, the foes could potentially become friends by protecting vulnerable cohorts of patients against specific hospital pathogens (e.g., S. aureus or E. coli) to prevent secondary infections. For example, bacterial protoplast-derived nanovesicles have been used to vaccinate mice against these germs in models of pneumonia and peritonitis ⁹¹. In the context of the growing antibiotic resistance crisis, this could be a life-saving preventive option (Table ​Table22).

The second major pool of EVs stems from the host organism and can be equally bio-active as the EVs released by microorganisms. Key sources of host EVs during sepsis are circulating cells such as platelets and leukocytes ^92,93, the latter being predominantly innate immune cells such as neutrophilic granulocytes, macrophages, dendritic and natural killer (NK) cells ⁹⁴. In sepsis, especially neutrophilic granulocytes show an increased activation but also higher apoptosis rates ^95,96—the two main conditions that trigger a robust EV release ^97-100. Unlike the microbial EVs with predominantly detrimental effects (as perceived based on the current literature), the action of the host-released EVs appears to be more heterogeneous. A number of protective EV-related mechanisms were recently implied. In a clinical study, GEVs were found in the infectious foci rather than in the circulation ¹⁰¹. Due to the functional relationship to their parent cells, GEVs can exhibit antibacterial effects, support the production of inflammatory mediators ^102-104 and/or protect from vascular dysfunction ^103,105. Selected danger-associated EV subsets (containing alpha-2-macroglobulin excreted by neutrophilic granulocytes) were shown to improve resolution of infection by enhancing bacterial clearance in a mouse sepsis model ^105,106 and by interfering with leukocyte-trafficking in vitro ¹⁰⁷. Similarly, when THP-1 monocytes phagocytized GEVs which were previously isolated from peritoneal and broncho-alveolar lavages of patients with surgical sepsis, their general activity and phagocytic capacity significantly increased in vitro ^101,105. In septic rats, EVs produced by immature dendritic cells reduced mortality by diminishing TNFα and high mobility group box-1 release and supported engulfment of apoptotic cells ¹⁰⁸.

In contrast, the emerging evidence demonstrates that some EVs released by leukocytes in sepsis can be harmful. For example, in a porcine model of endotoxemia, danger-associated populations of PEVs robustly increased in dying but not in surviving animals ¹⁰⁹. Furthermore, lymphocyte apoptosis, one of the major abnormalities observed in both pre-and clinical sepsis ^110,111, was induced by caspase-1 released from circulating EVs in septic patients ¹¹⁰. While the precise EV source was not determined in that study, peripheral blood monocytes are potential candidates given that they were shown to release EVs containing caspase-1 which induced apoptosis of smooth vascular muscle cells in vitro ¹¹². Blocking the release of EVs from bacteria-infected macrophages by pre-treatment with a sphingomyelinase inhibitor in septic mice had cardioprotective effects and prolonged survival ¹¹³. It has also been suggested that EVs contribute to the development of sepsis-induced acute lung injury ⁹⁴. Intratracheal application of EVs released from LPS-primed alveolar macrophages to C57BL/6 mice provoked their alveolar macrophages to release EVs, which caused an increased expression of adhesion molecules, protein and neutrophilia ⁹⁴. In another example, dendritic cells activated by LPS released EVs that stimulated epithelial cells to secrete chemokines (e.g., IL-8 and Chemokine (C-C motif) ligand 5) which are important soluble components of a physiological innate immune response but can simultaneously fuel sepsis by excessive cytokine release ¹¹⁴. Circulating EVs (without classification) containing reactive oxygen species (ROS) from LPS-stimulated mice induced vascular leakage and cardiac dysfunction in C57BL/6 mice ¹¹⁵. The roles of exosomes in septic cardiac dysfunction have recently been reviewed ¹¹⁶.

Many other leukocyte subpopulations are capable of EV release but their role in sepsis has not yet been adequately investigated. NK cells are a suitable example. In contrast to other immune cells, NK cells produce EVs constitutively and independently of their parent cell activation status. NK cells contain so-called killer proteins such as Fas ligand and perforin that influence the tumor growth and immune system homeostasis ¹¹⁷. Interestingly, NK cell-derived vesicles only act against the activated immune cells. This indicates that they could actually modulate septic processes by controlling the expansion and proliferation of stimulated immune cells. Another example is the potential function of EVs in adaptive immunity. Stimulated CD4+ and CD8+ T-cells can induce cytokine production (TNF-alpha, IL-1-beta, soluble IL-1 receptor antagonist) by human monocytes by either direct contact or by releasing EVs as messengers ¹¹⁸.

In summary, the existing evidence strongly suggests that EVs have both protective and detrimental roles, either locally or at distant sites. Whether EVs assume the shape of a friend or foe appears to partly depend on their origin (e.g., microorganism vs. host) and/or specific pathophysiological trait examined.

Role of EVs in specific subsets of septic patients

Conclusions and outlook

Within the last two decades, the multifaceted roles of EVs in inflammation have entered the focus of sepsis research. Given that both sepsis and EVs are two very complex fields and the existing evidence regarding the intricacies of EVs in sepsis is limited, no definitive connecting lines can be currently drawn between those two entities. However, a number of promising speculations pertaining to the role of EVs in sepsis are justified.

First, it is convincing that EVs have a strong role as mediators in sepsis. It has also become apparent that EVs can act both as friends and foes in systemic inflammatory reactions; their friendly or hostile character largely depends on the EV origins and cargo they carry. The latter element makes specific EV subsets potential candidates for drug delivery systems. In the future, the above EV properties could facilitate more precise and individualized treatment approaches.

The identification of specific EVs subsets remains fundamental for the isolation and application in diagnostic and/or therapeutic procedures. At the moment, however, it is clear that the restrictions of characterization (hence our biological understanding of EVs) are limited by the methodological isolation/detection boundaries. The gold standard for high-throughput detection of EVs based on surface antigens is flow cytometry with a current detection limit of well above 0.3 µm. To our knowledge, nanoparticle tracking analysis has not yet been tested in septic patients. Although size transition of EVs is fluent, discrimination of EV subsets is largely based on their dimension, a measure that ignores the biogenesis of the vesicles. Development of new methodologies to rapidly assess circulating miRNA and miRNA incorporated to EVs should have a high priority. Furthermore, quick point-of-care tests such as microfluidic chip tests and lateral flow immunoassays have a great potential to be used with plasma of septic patients, but need additional testing. Given their strong immunomodulatory capabilities and vast diversity of cargo molecules, EVs present a considerable therapeutic potential for the treatment of sepsis. It is not impossible to imagine that EVs could serve in the future ICU as new types of vaccines against secondary infections (Table ​Table22).

Overall, the potential utility points for EVs in sepsis (and other ICU conditions) are multifaceted, reaching from preventive to diagnostic and therapeutic. Yet, before any of these points migrate into the clinic, basic methodological challenges in EVs detection have to be solved and any viable application of EVs must be first a) standardized for detection/description, b) evaluated for clinical applicability, c) validated in multi-center randomized clinical trials and d) thoughtfully adopted for clinical use.

Supplementary Material

Acknowledgments

Abbreviations

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References