Introduction

Severe trauma is one of the disorders with the greatest healthcare and economic impact in society today [1]. Worldwide, it is the leading cause of mortality in young adults, and involves the highest incidence of potential life years lost. In traumatized and poly-traumatized patients, quick, precise, and valuable recognition of presenting injury pattern is of outmost need for proper patient management as delayed diagnosis may cause secondary complications and exaggerate mortality and morbidity. Recently, intensive efforts have been made to identify indicators that are associated with the pathological processes of the disease and organ injury [2]. The heterogeneity of the injury cases makes it difficult to accurately assess the level of trauma, predict the clinical outcome, and optimize the therapy for individual patients; therefore ,the search of certain specific and sensitive biomarkers, which will help to overcome these difficulties is continuing. Exosomes (Exos), one subclass of EVs, which was described long time ago, and which role was re-considered recently, now appeared to be a promising biomarker candidate for the broad range of diseases. Despite the growing number of evidences confirming the role of Exos in physiological intercellular communication and their potential as biomarkers in some diseases and cancer entities [3], less is known about their role as mediators and markers of acute organ injury in the traumatized patients. In the following sections, we aim to provide an overview of existing literature with the focus on the role of Exo/EVs during acute organ injury. We focus on inflammatory diseases including sepsis and systemic inflammatory response syndrome (SIRS), traumatic brain injury, acute cardiac damage, acute respiratory distress syndrome (ARDS), and acute liver and kidney injury. Next to the individual organ damage, we also review studies focused on multiple trauma and Exos.

Extracellular vesicles

Since the first reference of EVs in platelet-free serum in 1946 by Chargaff et al. [4], the understanding of EVs biogenesis, structure, and functions has been significantly improved. According to the “Minimal information for studies of extracellular vesicles 2018” (MISEV2018), EVs is “the generic term for particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, i.e., do not contain a functional nucleus” [5]. Historically EVs were classified to three major classes—Exos, microvesicles (MVs), and apoptotic bodies (ApoEVs) according to their cellular origin (Fig. 1) [3]. Exosomes and MVs are both released by healthy cells, whereas ApoEVs resulted from the apoptotic process, when the cell's cytoskeleton breaks up and causes the membrane to bulge outward. Exos with a size of 30–150 nm are the smallest subpopulation of EVs; they are released upon fusion of multivesicular bodies with the plasma membrane and further exocytosis. Due to their endocytotic origin, Exos are commonly enriched in endosome-associated proteins such as Rab GTPases, SNAREs, Annexins, and Flotillin. Some of these proteins (e.g., Alix and Tsg101) are commonly used as exosome markers. Tetraspanins family of membrane proteins (CD9, CD63, and CD81) is also abundantly present in Exos and considered to be used as a markers [3, 6, 7]. MVs are shed from the plasma membrane by budding; they vary in size between 100 and 800 nm and are enriched in CD63, CD81, and Annexin V proteins. The biggest in size population of EV are ApoEV, ranging in size between 200 and 5000 nm and expressing Annexin V [3, 6, 7]. Despite the big number of study focused on ApoEVs’ characterization, there are still striking discrepancies in the literature in the characterization and isolation of ApoEVs [8]. Since there are no specific markers for different subtypes of EVs, which would distinguish endosome-origin Exos and plasma membrane-derived MVs, International Society of EVs urged to consider use of operational terms for EV subtypes, which refer to physical characteristics of EVs (f.ex. size), or biochemical composition (f.ex. CD63+/CD81+-EVs) or cellular origin (f.ex. platelet EVs), unless authors can establish reliable specific markers of subcellular origin [5].

Fig. 1
figure 1

Extracellular vesicles (EVs): exosomes (Exos), microvesicles (MVs), and apoptotic bodies (ApoEVs). Schematic drawing of biogenesis and uptake of EVs. Exos are the smallest vesicles (30–150 nm in diameter) originating by endocytosis. MVs (also called microparticles) have size of 100–800 nm in diameter and are released from cell membrane by budding. ApoEVs (0.2–5 µm in diameter) are released from cell membrane surface in late stage of apoptosis. Membrane of Exos and MVs beside others contain MHC, tetraspanins CD9, CD63, CD81, and cell-specific receptor proteins. Exosomal cargo is enriched with broad range of RNAs, DNAs, and protein molecules

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EVs have gained widespread interest due to their ability to carry bioactive components such as RNAs, DNA, and proteins. However, besides their luminal cargo, EVs can also carry a significant surface cargo encompassing DNA and especially proteins such as for example CD41+ for platelets, CD144+ for endothelial cells, or CD45+ for leukocytes derived EVs [9]. Both, Exos and MVs, are known to facilitate intercellular communication processes between cells in close proximity as well as distant cells. EVs cargo is actively loaded prior to the release from parental cell [6, 10, 11] and could significantly influence target cells' metabolism, function, and life span [6, 12, 13]. EVs can contain proteins such as cytokines, chemokines, heat shock and major histocompatibility complex (MHC) proteins, lipids, messenger RNA (mRNA), and microRNAs (miRNAs). Since EVs are present in most biological fluids (blood, urine, saliva, semen, bronchoalveolar lavage, bile, ascitic fluid, breast milk, and cerebrospinal fluid (CSF) [14, 15]), they hold promise as a diagnostic tool. They can be isolated from the small amount of biological fluids and clinical samples and their cargo, which represents tissue-specific molecules with higher stability, can serve as disease-specific biomarkers. Furthermore, since their release and composition can be modulated by environmental factors, they can also serve as markers for disease status and treatment outcomes [14, 15].

EVs isolation and characterization methods

Similarity in some of EVs subtype characteristics (overlap** size, biochemistry, surface markers) makes search of disease-specific EVs biomarker technically challenging [3, 5]. The broad range of isolation and characterization methods together with inconsistence in the EVs definition in modern scientific literature provide additional complexity to this search [5]. Among different methods used for EVs isolation, there are four major groups focused on the isolation of the smallest subtype-Exos: ultracentrifugation, ultrafiltration, affinity, and osmotic precipitation-based methods. The most widely used method for exosome isolation is differential (ultra-) centrifugation [16]. The separation of Exos from different samples with this method is based on serial and differentiated centrifugation with g-forces rising up to 100.000g. Although differential centrifugation (ultracentrifugation) is effective for the isolation of Exos, the technique is time-consuming, labor-intensive, and heavily instrument-dependent for both research laboratories and clinical settings alike [17, 18]. Density gradient ultracentrifugation is a modification of this technique aimed at increased purity of isolated Exos. In this method, the sample is added to an inert gradient medium for centrifugal sedimentation and particles are separated on the basis of their buoyant densities by density gradient ultracentrifugation using sucrose or iodixanol [19, 20]. While the purity and quality of Exos isolation is increased with this technique, low yield due to the multiple-step protocols is commonly observed. Ultrafiltration is often used as a purification technique after EV isolation for example by ultracentrifuge. Depending on the size of MVs, this method allows the separation of Exos from proteins and other macromolecules. Nevertheless, micro-/ultrafiltration is also applicable for exosome isolation [11, 21]. This method is a fast, simple technique which does not need any expensive equipment and can concentrate large sample volumes [22,23,24,25]. However, it is characterized by lower exosome quality and suboptimal RNA purity as compared to ultracentrifugation [26]. Affinity-based immunomagnetic beads isolation method is based on the specific binding between monoclonal antibodies, loaded on magnetic beads and certain receptor molecules present on the surface of the Exos. Antibody coated beads against, for example, the tetraspanin proteins CD9, CD63, or CD81 are incubated with samples from which Exos are to be isolated. Then, the exosome–magnetic bead complexes are loaded onto a column, which is placed in a magnetic field. Therefore, the magnetically labeled Exos are retained within the column, while other cell components (unlabelled) run through [27]. The advantage of this approach is that it is target-specific and ensures the integrity of the extracted Exos. The method is also relatively easy to carry out and does not require expensive equipment. Additionally, this method allows selection and extraction of specific exosome fractions. However, the difficulties of exosome elution from the beads, and the need of sophisticated analytical tools to analyze Exos extracted from patient material together with expensive reagents make this method not user-friendly for point-of-care testing [20, 25, 27, 28]. Osmotic precipitation—an alternative technique that is increasingly being applied to isolate Exos—is the use of precipitants such as polyethylene glycol combined with low-speed centrifugation to pellet Exos for subsequent processing [29, 30]. This method is simple and fast and requires only a basic equipment [19, 20]; however, the purity of isolated exosome is compromised impairing downstream analysis. In addition, the polymer substance present in the isolate may interfere with downstream experiments [22, 31].

Exosome characterization methods could be divided into biophysical characterization of exosomal size range; molecular approaches to characterize the surface proteins and methods developed for analysis of exosomal cargo composition. The most common technique to determine the size and concentration of Exos in a sample is the Nanoparticle Tracking Analysis (NTA). The method is based on the Brownian motion of particles, which move rapidly in a liquid sample and act as point refractors when they pass through a laser beam. Videos can be recorded and a detailed differential particle-size distribution graph can be produced using analytical software [32]. A quite similar method is Dynamic Light Scattering (DLS), which is also based om the Brownian motion of small particles [27], but instead of scattered light, DLS uses the fluctuation in the intensity of scattered light to measure exosome size [33,34,35]. Another biophysical approach, commonly used to characterized morphology and size of the exosome is Electron microscopy [transmission (TEM) and scanning (SEM) electron microscopy] [36]. In addition, Flow Cytometry is often used to characterize EVs by mean of size, and absolute number; however, the limited sensitivity and resolution of flow cytometers should be considered. For smaller particles, such as Exos some approaches like the use of latex beads coated with monoclonal antibodies, which can bind and “pull-out” Exos can be introduced to allow their analysis by flow cytometry [37, 38]. Several methods have been developed for analysis of exosomal RNA cargo. Those methods include microarray analysis, next-generation sequencing (NGS), and digital droplet PCR (ddPCR) [36]. With regard to proteins, the protein content of Exos could be analyzed by Western blotting, proteomic technology, and fluorescence-based cell sorting [36].

The role of EVs during systemic inflammation and organ injury

Below, we provide an overview of the in vitro, in vivo and patients’ study, investigating the role of EVs as mediators, biomarkers, and/or therapeutics in traumatic injury. To avoid additional discrepancy in the nomenclature of EVs, we utilize the original author’s nomenclature used in the studies.

EVs in sepsis/SIRS

According to the trauma register of the German Society of Traumatology (DGU-Polytraumaregister), more than 6% of multiple injured patients additionally develop septic complications and 20% of them develop multiple organ failure [39,40,41]. Sepsis is a systemic response of the immune system, in which Exos and MVs, originating from different type of cells, were shown to play diverse roles. Thus, the positive correlation of the increased production of platelet-MVs and poor outcome was shown in endotoxemia pig model [42]. In patients with septic shock, increased levels of platelet- and leukocyte-derived EVs and low level of endothelial cells-specific MVs were correlated with unfavorable outcome. One of the explanations of such correlation could be that in these patients, platelet-derived Exos are enriched with reactive oxygen species, which could induce vascular cell apoptosis [43]. Similarly, monocyte-derived microparticles (tissue factor+ and CD13+) were shown to be significantly increased in patients with trauma and severe sepsis [44]. This increase correlated significantly with the injury severity score (ISS) and acute physiology and chronic health evaluation score (APACHE II) in trauma patients; and with APACHE II and the international society of thrombosis and homeostasis (ISTH) overt disseminated intravascular coagulation (DIC) diagnostic criteria in sepsis patients [44]. In addition, MVs were shown to be important part of host protective mechanism in sepsis. Neutrophil-derived alpha-2-macroglobulin (A2MG)-containing MVs were shown to be elevated in plasma from patients with sepsis and their immunomodulatory role was verified in vivo. Administration of A2MG-enriched microparticles to mice with microbial sepsis provided protection against hypothermia, reduced bacterial titers, elevated immunoresolvent lipid mediator levels in inflammatory exudates, and reduced systemic inflammation [45].

Regardless of the role EVs are playing during the sepsis, it is less known, how different EVs could provide pro- or anti-inflammatory effects. According to the ExoCarta database (http://www.exocarta.org), Exos transfer cytokines, such as interleukins IL-1β, IL1α, IL-18, IL-32, IL-6, IL-8, macrophage migration inhibitory factor, tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), fractalkine, and chemokine ligands CCL2, CCL3, CCL4, CCL5, and CCL20; all known to be associated with the development of different inflammatory diseases [46]. Next to the inflammatory response via cytokines, also the complement system could play important role in EVs-associated effects [12]. It is well known that activation and interaction of the complement system with the other cascades such as coagulation lead to both pro- and anti-inflammatory reactions, which can affect morbidity and mortality in diseases, including trauma injury and sepsis [47,48,49]. MHC-bearing MVs could also function in this way; for example, it was described that non-survivors of septic shock exhibit increased numbers of EVs bearing complement component 5a receptor (C5aR) as compared with sepsis survivors [49].

In addition, EVs cargo miRNAs were shown to play an important role in mediation of sepsis in several in vitro, in vivo and patients’ studies. Recently, it was proposed and confirmed in sepsis mouse model that miRNAs from sepsis plasma Exos promote inflammation by inducing cytokine production via TLR7-MyD88 signaling [50]. The pro-inflammatory role of exosomal miR-155 was shown in LPS-induced sepsis in mice [51]. This was confirmed in in vitro study, where the treatment of RAW cells with miRNA-155 inhibitor results in significant reduction of LPS-induced TNFα production [52]. Also in septic patients, it was shown that miR-155 is associated with a high sepsis-related organ failure assessment (SOFA) score and correlates with the appearance of immunomodulating CD39+Tregs [53]. miR-146a was shown to play an opposite role and reduce the pro-inflammatory response in LPS-induced sepsis in mice [51].

Another view on possible role of EVs during the sepsis could be gained from the studies using EVs as therapeutics. It was shown that overexpressed in MSC-derived EVs, miR-223 mediates cardio-protection during sepsis via downregulation of Semaphorin-3A and Stat3 [54]. Pre-treatment of MSC with Il-1β was shown to enhance the production of miR-146a-enriched Exos, which leads to increase of septic mice survival [55]. LPS pre-treated MSC-derived Exos were suggested to have improved regulatory abilities for macrophage polarization and resolution of chronic inflammation by shuttling let-7b miRNA [56].

Summarizing the above, EVs play an important role in the development of sepsis and septic organ failure. In particular, EVs microRNAs miR-155 and miR-146a are mediators of inflammation, and might be good targets for future therapeutics, targeted on decrease of septic organ damage and mortality.

EVs in injury and trauma

EVs in traumatic brain injury

Traumatic brain injury (TBI) causes 37% of injury-related death in trauma patients [57]. Therefore, TBI is a major cause of mortality and morbidity particular in the younger population and is associated mainly with long-term-disabilities in survivors [58,59,60]. The cell–cell communication in the brain strongly depends on paracrine mechanisms mediated, in particular, via EVs [61], which are known to be released from all brain cells: neurons, astrocytes, microglia, and oligodendrocytes [62,63,64,65].

Neuroinflammation is an important step in TBI development, which was shown to be mediated by EVs [63]. For example, microglia-originated EVs, containing pro-inflammatory molecules, such as miR-155 and IL-1ß, are systemically detectable 24 h after TBI [63]. The role of EVs as a regulator of the immunological response in TBI was proofed in in vivo experiments, showing that transfer of astrocyte-shed EVs from inflammatory brain damaged animal into healthy animal leads to neuroinflammation [63]. An alteration in the permeability of the blood brain barrier is another crucial step in the development of TBI, in which EVs and their cargo could play a role. In case of systemic inflammation, TNF-α increase could lead to increased permeability of the blood–brain barrier that in turn make Exos able to cross the blood–brain barrier and therefore induce inflammatory processes in brain tissue [66]. miR-132-containing neuron-derived Exos were shown to regulate blood–brain barrier permeability by affecting expression of vascular endothelial cadherin [67]. EVs and EVs miRNAs were found to play role in blood–brain communication during peripheral inflammation. It was shown that choroid plexus epithelium cells could sense and transmit information about the peripheral inflammatory status to the central nervous system via the release of EVs into the cerebrospinal fluid, which transfer this pro-inflammatory message (miR-1a, miR-146, miR-9, and miR-155) to recipient brain cells [55].

Exosomal miRNAs play an important role in TBI establishment and usually have specific expression pattern during TBI that make them good candidates as biomarkers of this type of trauma. For example, it was shown that IL-1β-induced acute neuroinflammation and oxidative stress are characterized by the presence of astrocytes-released Exos. These Exos are enriched with specific subset of 22 miRNAs, known to influence inflammation and apoptosis via targeting BCL-1, TLR-4, BCL2L1, Bcl-2-associated X protein (BAX), and caspase 3 proteins [68]. In mice TBI model, the significant difference in the expression of miR-129-5p, miR-212-5p, miR-9-5p (all up-regulated in TBI) and miR-152-5p, miR-21, miR-374b-5p, and miR-664-3p (all down-regulated in TBI) was detected [69]. Moreover, the expression of miR-21 was shown to be increased at different time-points after TBI systemically and in neurons [69, 70]. In rodent TBI model, specific EVs miRNA expression pattern (significantly increased miR-21, miR-146, miR-7a, and miR-7b; decreased miR-212) indicates the presence of an enhancement loop among neuroinflammation and EVs [71]. In another study, around 50 exosomal miRNAs were described to be altered after TBI (31 up-regulated and 19 down-regulated) [72].

Beside miRNAs, also EVs cargo proteins might be considered as TBI biomarkers. The presence in exosomal cargo of classical neurotrauma biomarkers including ubiquitin C-terminal hydrolase L1 (UCHL1), Tau, Occludin, and amyloid β proteins was shown to be associated with a poor outcome and neurological deficit after TBI [73,74,75]. Exosomal Tau protein level was shown to correlate with cognitive, affective, and somatic post-concussive symptoms in US veterans with TBI [74]. Also exosomal IL-10 level correlates with behavioral symptoms after TBI [76]. In patients with severe TBI, MVs were shown to be enriched with glial fibrillary acid protein (GFAP) and aquarporin-4 [77], but not with neuron-specific enolase (NSE), although systemic increase of NSE is well known as a common biomarker of brain injury [77]. Generally, EV-derived markers of neuro-damage hold a great potential as TBI biomarkers as they provide more dynamic view of damage as common systemic biomarkers [76, 78].

MSC-derived exosome is considered as a cell-free therapeutic tool to reduce inflammatory consequences of TBI. Bone marrow stromal cells (BMSCs)-derived Exos were shown to inhibit expression of the pro-apoptotic protein BAX and the pro-inflammatory cytokines TNF-α and IL-1β, while enhancing the expression of the anti-apoptotic protein BCL-2, and thus to ameliorate early inflammatory response following brain injury [79]. Furthermore, BMSCs-Exos could modulate microglia/macrophage polarization by downregulating the expression of inducible nitric oxide synthase (INOS) and upregulating the expression of CD206 and arginase-1 [79]. MSC-derived Exos loaded with neuroprotective miR-216a-5p were shown to inhibit neuroinflammation and promote neuronal regeneration and in particular recovery of sensorimotor function and spatial learning ability [80]. MSC exosome-treated TBI rats show significant improvement in spatial learning as measured by the modified Morris water maze test and sensorimotor functional recovery [191], and it seems to be consistent to expect EVs with similar properties in other diseases and injuries. In case such EVs with specific protein signatures would be identified, they can add to the list of potential good biomarkers. We summarized cell-type specific EVs, detected in circulation in patients with systemic inflammation and with different organ injuries in Table 2. Further studies should enrich this list, proof their specificity, and describe their function.

Table 2 Cell-type specific EVs found in systemic inflammation and organ injury in in vitro, in vivo and patients studies
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Conclusion

EVs with their unique miRNAs and proteins signatures are of great interest as biomarkers for the wide range of diseases and pathologies. We believe that scientific efforts in this field should be focused on development of simple and robust methods for isolation and characterization of circulating EVs; compilation and replenishment of databases, containing information about disease/injury-specific EVs, and such study should use standard and ubiquitous EVs nomenclature. If such combined efforts are made, we will soon receive a set of new biomarkers that will help accurately assess the level of trauma, predict the clinical outcome, and optimize the therapy for individual patients.