Extracellular vesicles in neuroinflammation: Pathogenesis, diagnosis, and therapy

Abstract

Extracellular vesicles (EVs) are bilayer membrane vesicles and act as key messengers in intercellular communication. EVs can be secreted by both neurons and glial cells in the central nervous system (CNS). Under physiological conditions, EVs contribute to CNS homeostasis by facilitating omnidirectional communication among CNS cell populations. In response to CNS injury, EVs mediate neuroinflammatory responses and regulate tissue damage and repair, thereby influencing the pathogenesis, development, and/or recovery of neuroinflammatory diseases, including CNS autoimmune diseases, neurodegenerative diseases, stroke, CNS traumatic injury, and CNS infectious diseases. The unique ability of EVs to pass through the blood-brain barrier further confers them an important role in the bidirectional communication between the CNS and periphery, and application of EVs enables the diagnosis, prognosis, and therapy of neuroinflammatory diseases in a minimally invasive manner.

Graphical abstract

Extracellular vesicles (EVs) are nano-sized particles that are produced and released by various cells. Functionally, EVs carry and transport a broad range of bioactive molecules for intercellular communication. In this review, Wang and colleagues discuss the key roles of EVs in the pathogenesis, diagnosis, and therapy of neuroinflammatory diseases.

Introduction

Extracellular vesicles (EVs) are membrane-enclosed particles that are produced and released by prokaryotic and eukaryotic cells under both physiological and pathological conditions. In general, EVs can be divided into three categories: exosomes (40–150 nm), ectosomes (also known as microvesicles or microparticles, 150–1,000 nm), and larger apoptotic bodies (>1,000 nm). The inward budding and fission of endosomal membranes produce intraluminal vesicles (ILVs) in cytosolic multivesicular bodies (MVBs), which fuse with the plasma membrane to release exosomes. In contrast, ectosomes and apoptotic bodies are generated by the direct blebbing of plasma membrane.¹

As novel messengers of intercellular communications, EVs carry and transport a broad range of bioactive molecules, including lipids, proteins, metabolites, DNA fragments, and various RNA species (mRNAs, microRNAs, and long non-coding RNAs).² Contents of the exosome may be delivered to the recipient cell either by interaction at the cell surface or by internalization of EVs through clathrin-mediated endocytosis, caveolae, lipid rafts, micropinocytosis, and phagocytosis (Figure 1).³ Importantly, EVs can not only mediate communication locally within tissues but they also cross organ systems via liberation into peripheral blood. In addition to blood, EVs are also detected in other biofluids, including saliva, breast milk, urine, and cerebral spinal fluid (CSF).⁴ Functionally, EVs are critical for various physiological processes, including organ development, immune responses, reproductive activities, and neuronal communications.5, 6, 7, 8 In addition, they also participate in a broad range of diseases, including cancer, cardiovascular diseases, metabolic diseases, autoimmune diseases, and infectious diseases.9, 10, 11, 12, 13

Figure 1.

Biogenesis, composition, and uptake of EVs

Ectosomes (microvesicles) and apoptotic bodies arise from the outward budding of the plasma membrane. In contrast, exosomes are generated through invagination and fission of the endosomal membrane. Multivesicular bodies containing intraluminal vesicles, which are future exosomes, then fuse with the plasma membrane to release exosomes into the extracellular milieu. All types of EVs share a common composition of an outer lipid bilayer and intravesicular contents composed of nucleic acids, proteins, and metabolites. Protein cargos (e.g., TSG101, Alix) and tetraspanins (e.g., CD63, CD81) are common markers used to characterize exosomes. Once released, exosomes deliver signals to recipient cells through the interaction of ligand/receptor molecules on their respective surfaces. In addition, exosomes and their cargos can be taken up by recipient cells via multiple manners such as direct fusion of the membrane, endocytosis, micropinocytosis, and phagocytosis.

EVs are secreted by all major cell populations of the central nervous system (CNS), including neurons, astrocytes, oligodendrocytes, and microglia, and contribute to their intercellular communication.14, 15, 16 The communication between neurons and glial cells, known as neuroimmune interactions, mediates both CNS homeostasis and neuroinflammation. Under physiological conditions, EVs are key messengers for glia-neuron communication and essential regulators of CNS homeostasis, such as maintenance of myelin, elimination of waste, and establishment of synaptic activity.17, 18, 19 For example, during an intense firing, neurons release a large amount of EVs, which induce the upregulation of the excitatory amino acid transporter 2 (EAAT2, also called GLT1) in astrocytes to remove excess glutamate from the synaptic cleft.²⁰ Under pathological conditions, cellular stress triggers an enhanced release of EVs. Depending on the content, EVs can promote either disease progression or tissue repair in CNS insults.²¹ Although the etiology and pathogenesis of CNS disorders are diverse, CNS autoimmune/degenerative/infectious diseases, stroke, and CNS traumatic injury are associated with neuroinflammatory responses such as glial activation, recruitment of leukocytes to the CNS, and enhanced proinflammatory cytokine production in the CNS.22, 23, 24 As key mediators of neuroimmune crosstalk, EVs have emerged as important regulators of neuroinflammatory responses. In addition to glial cells, both CNS-resident leukocytes, such as perivascular macrophages, and CNS-infiltrating leukocytes produce EVs during neuroinflammatory diseases and, thereby, regulate each other and impact the function of CNS-resident cells. Understanding the functional role and underlying mechanisms of EVs in the onset and resolution of these neuroinflammatory diseases is gaining intense interest.

Given that EVs can cross the blood-brain barrier (BBB) and serve as cargo carriers for the bidirectional communication between the CNS and the periphery, the function of EVs in assessment and therapy of CNS diseases is attracting increasing attention.²⁵ On the one hand, EVs are an intriguing source of biomarkers for the diagnosis and prognosis of CNS inflammatory disorders through minimally invasive procedures. Since their cargo is a complex selection of bioactive molecules from the cell of origin, EVs may reflect the status of those cells at the time of release. In different CNS pathologies, EVs may change in size, number, and contents.²⁶ In addition, CNS-derived EVs can cross the BBB and enter peripheral blood, making them easily accessible for the diagnosis and prognosis of CNS diseases. On the other hand, EVs are also considered as promising drug-delivering tools for treating CNS inflammatory diseases, since (1) EVs are biocompatible, (2) EVs are able to cross blood-tissue barriers, (3) EVs can be attracted to the site of inflammation through the interaction between their surface ligands and receptors on target cells, and (4) EVs can be manipulated by bioengineering and loaded with therapeutic agents.^27,28 In this review, we summarize the function of EVs in the pathogenesis and development of inflammation-related CNS disorders as well as their application in the diagnosis, prognosis, and therapy of these diseases.

EVs in neuroinflammatory diseases

EVs in CNS autoimmune diseases: Multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the CNS affecting more than 2 million people globally.²⁹ MS patients show a wide variety of neurological and psychic symptoms, such as visual disturbances, paresthesias, paresis, ataxia, and depression.³⁰ Although the etiology of MS is still unclear, it is widely accepted that a complex interaction between genetic, epigenetic, and environmental factors triggers an autoimmune attack against myelin.²²

EVs detected in the serum/plasma and CSF have been shown to be a potential source of biomarkers of disease status and treatment effects in MS.31, 32, 33, 34, 35 In MS patients, the circulating EVs show significant alterations in myelin-associated proteins and microRNA profiles.^36,37 Specifically, levels of miR-122-5p, miR-196b-5p, miR-301a-3p, and miR-532-5p were decreased whereas levels of miR-Let-7i were increased in serum exosomes of MS patients as compared with those of healthy controls.^36,37 Due to its proximity to the CNS, CSF is rich in MS biomarkers, including those sequestered in EVs.³⁴ EVs detected in CSF display neuronal, microglial/macrophagic, oligodendroglial, and astrocytic markers, implying that they originate from all of these CNS-resident cell populations. In addition, CSF concentrations of myeloid cell-derived ectosomes are increased in patients with clinically isolated syndrome or relapsing MS as compared with patients in a stable phase of MS or healthy controls, suggesting microglial/macrophagic EVs as favorable biomarkers of MS.³³ Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model to study MS. Consistently, higher levels of myeloid ectosomes are also detected in the CSF of EAE mice, and mice impaired in ectosome shedding are partially protected from EAE, indicating a pathogenic role of microglia-derived EVs in CNS autoimmune diseases.³³ Indeed, EVs secreted by microglia are rich in proinflammatory cytokines such as interleukin (IL)-1β and microRNAs that are associated with synaptic dysfunction.^38,39 Of note, microglia-derived EVs play both detrimental and protective roles in remyelination, which is decided by the phenotype of microglia. EVs secreted by proinflammatory microglia block remyelination and induce synaptic defects whereas EVs containing anandamide and sphingosine 1 phosphate (S1P) that are produced by pro-regenerative microglia promote myelin repair.^39,40

Neuropathology in MS and EAE is considered to be induced by CNS-infiltrating immune cells, particularly T cells and myeloid cells. Endothelial cell- and platelet-derived EVs isolated from the serum of MS patients have been shown to promote leakage of the BBB, allowing the invasion of T cells and myeloid cells into the CNS.^31,41,42 In addition to transmigration of T cells, EVs have been shown to influence T cell activation (Figure 2). A recent study showed that myelin oligodendrocyte glycoprotein (MOG)_35–55 immunized C57BL/6 mice receiving EVs from blood plasma of naive C57BL/6 mice developed a spontaneous relapsing-remitting EAE phenotype that is primarily driven by CD8⁺ T cells instead of CD4⁺ T cells.⁴³ This modified EAE phenotype and accompanying CD8⁺ T cell immunity are induced by the plasmatic EV cargo fibrinogen, which is also found to be accumulated in plasmatic EVs from relapsing-remitting MS patients.⁴³ Although autoreactive T cells contribute to the pathogenesis and development of MS and EAE, CNS autoimmunity can be ameliorated by regulatory T cells (Tregs). Exosomes circulating in MS patients are able to reduce the frequency of Tregs via miR-Let-7i, which is increased in MS exosomes and can inhibit Treg differentiation from naive T cells.³⁷

Figure 2.

Role of EVs in CNS autoimmune diseases

Neuroinflammation in the human CNS autoimmune disease MS and its animal model EAE is mediated by CNS-infiltrating leukocytes, especially autoreactive T cells and B cells. In CNS autoimmunity, circulating EVs have been shown to induce the differentiation of autoreactive T cells and inhibit the differentiation of regulatory T cells ①. EVs derived from platelets and vascular endothelial cells induce the leakage of the BBB, allowing the invasion of autoreactive leukocytes into the CNS parenchyma ②. In the CNS parenchyma, EVs produced by microglia, the CNS-resident immune cells, contain proinflammatory cytokines and microRNAs, which promote demyelination and synaptic defects in CNS autoimmunity ③.

Emerging evidence implicates engineered exogenous exosomes as favorable therapeutic approaches to treat CNS autoimmune diseases. BV2 cells, which are immortalized microglial cells, have been engineered to produce EVs overexpressing the endogenous “eat me” signal lactadherin (Mfg-e8) and the anti-inflammatory cytokine IL-4. A single injection of Mfg-e8⁺ IL-4⁺ EVs into the cisterna magna ameliorates established EAE by inducing anti-inflammatory marker expression in recipient phagocytes.⁴⁴ Another study showed that intranasal administration of engineered exosomes overexpressing miR-219a-5p improved remyelination in EAE mice, thereby promoting disease recovery.⁴⁵ Mesenchymal stem cells (MSCs) display bystander immunomodulatory and neuroprotective activities, and they represent an attractive candidate for cell-based therapies for a wide range of neuroinflammatory disorders, including MS. Exosomes produced by MSCs have been shown to ameliorate EAE by inducing the anti-inflammatory polarization of T cells and microglia.^46,47 In a recent study, Hosseini Shamili et al.⁴⁸ showed that MSC exosomes conjugated with the LJM-3064 aptamer, which shows affinity toward myelin and can induce remyelination, could suppress inflammation and demyelination in EAE mice. Taken together, these results show that exosomes are not only valuable diagnostic biomarkers of MS/EAE but also potential therapeutic reagents for CNS autoimmune diseases.

EVs in neurodegenerative diseases

Neurodegenerative disorders, including Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD), frontotemporal dementia (FTD), Huntington’s disease (HD), and Parkinson’s disease (PD), are characterized by a progressive and irreversible loss of neurons and widespread neuroinflammation.⁴⁹ Neurodegenerative diseases are also called “proteinopathies” because they show aberrantly accumulated proteins in the CNS. These misfolded and aggregated proteins, such as β-amyloid (Aβ), α-synuclein, prion protein (PrP), fused in sarcoma (FUS), huntingtin (Htt), tau, and transactive response DNA-binding protein 43 kDa (TDP-43), lose their normal physiological functions, form insoluble aggregates (or inclusions), and become neurotoxic. Interestingly, Aβ, α-synuclein, FUS, tau, and TDP-43 are present in exosomes in the brain, CSF, and plasma of patients suffering from corresponding neurodegenerative diseases, implying a potential role of EVs in neurodegenerative diseases.50, 51, 52, 53 Analysis of blood exosomal Aβ and synaptic proteins, such as neurogranin, GAP43, and SNAP25, may provide useful information for the assessment of neurodegenerative diseases.^54,55

Another common feature of neurodegenerative diseases is that the misfolded proteins “spread” to certain brain regions, indicating that the intercellular movement of these proteins contributes to the disease process.⁵⁶ Indeed, PrP^Sc, the misfolded and transmissible form of PrP, is the essential component of the proteinaceous infectious particle termed “prion” and causes the infectious neurodegenerative disease CJD in humans. Unlike the disease-associated PrP^Sc, the normal PrP^C is a cell surface glycosylphosphatidylinositol-anchored protein and is not transmissible. However, both PrP^Sc and PrP^C can be packaged into exosomes for intercellular spread.^57,58 Apart from PrP, exosomes from neuronal cells have also been shown to carry tau, Aβ, α-synuclein, FUS, and TDP-43.51, 52, 53^,59, 60, 61, 62 The exosomes containing toxic proteins are taken up by local and remote neurons and contribute to neuronal loss, implying a mechanism that underlies the spread of toxic proteins in the neurodegenerative brain (Figure 3). In addition to neurons, propagation of toxic proteins can also be mediated by microglia, whose activation is a hallmark of neurodegenerative diseases, and they have been shown to contribute to the spread of Aβ, tau, and α-synuclein via exosome secretion.63, 64, 65, 66 Blockage of exosome synthesis in vivo with the neutral sphingomyelinase-2 inhibitor GW4869 or P2X purinoceptor 7 (P2RX7) inhibitor GSK1482160 decreases amyloid plaque formation and tau propagation, respectively, in corresponding mouse models.^63,67,68 Consistently and interestingly, tau-enriched exosomes have been shown to seed misfolded tau aggregates and induce tauopathies in recipient mice, consolidating a disease-propagating role of EVs in neurodegeneration.⁶⁹

Figure 3.

EVs facilitate the dissemination of viruses and neurodegenerative proteins

Viruses are able to hijack the EV biogenesis machinery to promote viral dissemination. In CNS infectious diseases, EVs protect viruses from antiviral immunity and enhance viral entry into target cells ①. In addition to viruses, EVs can also transport and spread toxic proteins in neurodegenerative diseases. In the CNS, EVs containing pathogenic proteins are taken up by neurons, which introduce seeding of pathogenic proteins, resulting in the spread of disease ②. EVs containing toxic proteins can be taken up by homeostatic microglia and cleared ③. However, during neurodegenerative diseases, microglia alter their phenotype from homeostatic to neurodegenerative. These neurodegenerative microglia can take up EVs but lose the ability to clear pathogenic proteins such as Tau. The uncleared Tau is secreted by neurodegenerative microglia via EVs, which are subsequently taken up by recipient neurons and contribute to disease spread ④.

In addition to facilitating the propagation of pathological proteins, EVs can also play beneficial roles via clearing toxic cellular content and supporting neuronal function. Neuronal glycosphingolipids allow the sequestration of Aβ in exosomes, and the Aβ content is subsequently degraded by microglia through phagocytosis, indicating a synergistic mechanism applied by neurons and microglia to clear Aβ using exosomes.⁷⁰ TDP-43 is encapsulated in exosomes shed by neurons, and inhibition of exosome secretion by inactivating sphingomyelinase 2 exacerbates the disease of transgenic mice expressing the human TDP-43^A315T mutant, suggesting that exosome secretion plays a beneficial role in neuronal clearance of aberrant TDP-43.⁶⁰ Neuron degeneration can be ameliorated by exosomes originating from glial cells. Upon exposure to Aβ, astrocytes release exosomes containing the heat shock protein HspB1, which potentially binds and sequesters extracellular Aβ.⁷¹

Collectively, exosomes play both protective and detrimental roles in neurodegenerative diseases. It seems that microglia, which are key modulators of neurodegenerative diseases, act as a crucial switch of exosome function.⁷² Exosomes facilitate the clearance of aggregated proteins by homeostatic microglia that are capable of clearing proteins. However, under conditions when microglia are impaired in protein clearance ability, neurodegenerative microglia tend to incorporate aggregated proteins into exosomes and spread them to promote disease propagation (Figure 3).²⁵ Therefore, enhancing the protein clearance ability of microglia might be a favorable strategy to treat neurodegenerative diseases by skewing the phenotype of exosomes.

EVs in CNS infection

EVs have been shown to facilitate the dissemination and infection of various viruses, especially RNA viruses (Figure 3).^73,74 Enterovirus 71 (EV71) is a non-enveloped single-stranded RNA virus that can infect the CNS and cause various neurological complications. In addition to the traditional cytolytic manner, EV71 was recently found to spread non-lytically between cells via exosomes.⁷⁵ Exosomes containing viral RNA can also be detected in plasma of children with EV71 encephalitis.⁷⁵ Tick-borne Langat virus (LGTV), a model virus closely related to tick-borne encephalitis virus (TBEV), uses exosomes for viral RNA and protein transmission, which is critical for the final neuroinvasion and neuropathogenesis.⁹ JC polyomavirus (JCPyV), a non-enveloped double-stranded DNA virus, is the major cause of progressive multifocal leukoencephalopathy (PML). An interesting study by Morris-Love et al.⁷⁶ found that JCPyV could be released from and transmitted among glial cells in EVs. The mode of transmission appears to be highly infectious, and the EV membrane protects the JCPyV cargo from neutralizing antibodies. These studies show that EVs contribute to the spread of various CNS-infecting viruses (Figure 3). In addition, EVs also exacerbate virus-induced neuropathology. Upon Japanese encephalitis virus (JEV) infection, microglia secrete exosomes containing microRNAs let-7a and let-7b, and these exosomes further induce death of uninfected neuronal cells by activating caspase.⁷⁷

In addition to viruses, the CNS is highly vulnerable to protozoan parasites such as Toxoplasma and Plasmodium, which possess the ability to shed EVs.^78,79 As compared to non-infected individuals, patients with cerebral or gestational toxoplasmosis show elevated concentrations of EVs in the serum.⁸⁰ EVs are implicated in the pathogenesis of cerebral malaria (CM), which is the most severe neurological complication of malaria, and blocking the production of EVs is protective in animal models of CM.⁸¹ The abovementioned findings indicate that EVs can be applied as diagnostic markers or therapeutic targets for CNS infectious diseases. These studies implicate that EVs mainly play a detrimental role in CNS infectious diseases and, therefore, blocking EV production may present a suitable strategy to treat these diseases. However, the complex role and the multiple sources of EVs in the infected CNS need further clarification to improve our understanding of the role of EVs in the pathogenesis of CNS infectious diseases and to develop tailored EVs precisely inhibiting the disease process or to restore brain functions.

EVs in stroke

Stroke is the leading cause of acquired disability and the second leading cause of fatality, affecting more than 40 million patients worldwide.⁸² A plethora of evidence shows that neuroinflammation is involved in both the pathological mechanisms and secondary injuries of stroke.⁸³ Generally, stroke can be divided into ischemic and hemorrhagic types, with ischemic stroke accounting for 80% of total cases. Consistent with the relative disease incidence of these two types of stroke, most of the relevant studies have focused on exosomes in ischemic stroke. Under hypoxic and ischemic conditions, astrocytes produce exosomes rich in PrP, and these PrP-containing exosomes improve the survival of stressed neurons.⁸⁴ Semaphoring 3A (Sema3A) is an extracellular matrix molecule that hinders axonal outgrowth, and administration of a Sema3A inhibitor promotes functional recovery from middle cerebral artery occlusion (MCAO), an animal model of ischemic stroke. In addition to directly acting on neurons, the Sema3A inhibitor can induce ischemic astrocytes to produce exosomes that are beneficial for axonal elongation.⁸⁵

Stem cell therapy is emerging as an effective therapeutic approach for both ischemic and hemorrhagic stroke.^86,87 Interestingly, accumulative evidence demonstrates that the positive effects of stem cell therapy are mediated to a large extent by their paracrine factors, in particular, exosomes.⁸⁸ Exosomes produced by multipotent MSCs have been shown to restore white matter integrity and promote functional recovery in mice suffering subcortical hemorrhage, which is an animal model of hemorrhagic stroke.⁸⁹ In addition, MSC exosomes can also attenuate brain injury and neuroinflammation after MCAO by inhibiting the differentiation of proinflammatory M1 microglia.⁹⁰ Intriguingly, the therapeutic effect of MSC-derived EVs in patients with acute ischemic stroke is being studied in a phase 2 clinical trial (ClinicalTrials.gov: NCT03384433). The stroke-alleviating efficacy of MSC exosomes can be further enhanced by the overexpression of neuroprotective cargos, including microRNAs. MSC exosomes rich in miR-17-92 cluster and miR-133b are effective in promoting neural plasticity and functional recovery in rats subjected to stroke.91, 92, 93 In addition to manipulating the cargo, another strategy to increase the therapeutic effect of MSC EVs is to increase the targeting to the stroke lesion. Kim et al.⁹⁴ found that magnetic EVs derived from iron oxide nanoparticle (IONP)-harboring MSCs showed enhanced targeting toward the ischemic region and improved the therapeutic outcome in MCAO rats. In another study, the c(RGDyK) peptide, which has a high affinity to cerebral vascular endothelial cells, was conjugated on the surface of MSC-derived exosomes. After intravenous administration, c(RGDyK) exosomes show a strong targeting specificity toward the lesion region of the MCAO brain. Furthermore, c(RGDyK) exosomes loaded with curcumin are effective in inhibiting neuroinflammation and cellular apoptosis in the ischemic lesion.⁹⁵

Engineered exosomes overexpressing the exosomal membrane protein LAMP2b fused to the neuron-specific RVG peptide (LAMP2b-RVG) are able to selectively deliver RNA and protein cargos to various cell populations, including neurons, microglia, astrocytes, and oligodendrocytes, in the brain.⁹⁶ LAMP2b-RVG exosomes loaded with miR-124 or nerve growth factor (NGF), both of which are neuroprotective and neurorestorative, have been shown to alleviate cerebral ischemic injury.^97,98 Collectively, these studies demonstrate that EVs generally play a protective role in stroke and they may become potential and efficacious therapeutic agents for the treatment of stroke.

EVs in CNS traumatic injury

CNS traumatic injury, particularly traumatic brain injury (TBI) and spinal cord injury (SCI), causes nonreciprocal motor-sensory dysfunction and has long-lasting effects on the patient’s physical, psychological, emotional, and spiritual well-being.⁹⁹ Mechanical damage to the CNS induces the death of local neurons and glia at the lesion site shortly after injury. This acute injury phase is followed by a secondary damage phase accompanied by a complex neuroinflammatory response, which further incites neuronal loss, demyelination, and glial scar formation.¹⁰⁰

In the acute but not chronic phase of TBI, plasma concentrations of neuron-derived EVs are significantly decreased, reflecting the phase-specific mechanism of TBI.¹⁰¹ In addition to the number of serum EVs, TBI and SCI alter the expression spectrum of microRNAs and proteomic signature in serum EVs.101, 102, 103, 104, 105, 106 By analyzing microRNAs in brain-derived EVs with next-generation sequencing and machine learning, Ko et al.¹⁰⁶ defined a panel of microRNA biomarkers to classify specific states of TBI. In a recent study, longitudinal trajectories of serum exosomal levels of glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), neurofilament light chain (NFL), and total-tau (t-tau) were analyzed in moderate-to-severe TBI patients.¹⁰⁷ Exosomal levels of t-tau and UCH-L1 are substantially increased immediately after TBI. In addition, higher levels of exosomal NFL and GFAP are observed in patients with diffuse injury as compared with those with focal lesions.¹⁰⁷ In another study, levels of neuropathological proteins, including Aβ, P-T181-tau, P-S396-tau, prion cellular protein, and IL-6, were found to be increased in neuron-derived exosomes (NDEs) in the plasma of TBI patients.¹⁰¹ TBI is considered as a risk factor for neurodegenerative disorders such as AD. As compared with patients with TBI but no cognitive impairment (CI), patients with TBI and CI show higher levels of Aβ42, P-T181-tau, P-S396-tau, and IL-6 in plasma NDEs, indicating that Aβ42 and tau in EVs may serve as biomarkers for cognitive alterations and neurodegenerative changes after TBI.¹⁰⁸

In neuroinflammatory diseases, neurons, glia, and infiltrated immune cells form an integrative network to regulate inflammatory processes in the CNS. Upon SCI, infiltrated macrophages produce exosomes containing NADPH oxidase 2 (NOX2), which are subsequently incorporated into injured axons via endocytosis. In neurons, endocytosed NOX2 oxidizes and inactivates PTEN, thereby stimulating phosphatidylinositol 3-kinase (PI3K)-Akt signaling and regenerative outgrowth.¹⁰⁹ Apart from infiltrating leukocytes, CNS-resident glial cells, which possess immune-regulatory functions, are also activated during CNS traumatic injury. Upon TBI, exosomes secreted by microglia contain high levels of miR-124-3p, which inhibits neuronal inflammation and promotes neurite outgrowth by downregulating mTOR signaling.¹¹⁰ Interestingly, upon CNS traumatic injury, microglia and neurons regulate each other through exosomal miR-124-3p. Neuron-derived, miR-124-3p-containing exosomes have been shown to promote recovery from SCI by suppressing the activation of proinflammatory M1 microglia and A1 astrocytes.¹¹¹ Repetitive TBI (rTBI) is closely associated with neurodegenerative disorders. In rTBI, levels of miR-124-3p are significantly altered in microglial exosomes from the injured brain.¹¹² In vitro, neurodegeneration in repetitive scratch-injured neurons can be alleviated by microglial exosomes with upregulated miR-124-3p. Mechanistically, miR-124-3p inhibits RelA, which is the inhibitory transcription factor of apolipoprotein E (ApoE) that facilitates the proteolytic breakdown of Aβ. Consistent with the in vitro finding, miR-124-3p-enriched microglial exosomes alleviate neurodegeneration in mice with rTBI.¹¹² These studies suggest that miR-124-3p-containing exosomes are beneficial not only for treating acute CNS traumatic injury but also for alleviating TBI-associated neurodegeneration and cognitive deficits.

The transplantation of stem cells is an efficacious therapeutic approach for treating TBI and SCI.113, 114, 115 Due to the safety concerns and poor engraftment of MSC implantation, MSC-originated EVs have emerged as an effective alternative therapy for CNS traumatic injury.^116,117 Exosomes from human placenta-derived MSCs have been shown to improve neurologic function in animal models of SCI by promoting angiogenesis.¹¹⁸ To improve the therapeutic efficacy, Guo et al.¹¹⁹ loaded MSC exosomes with PTEN siRNA. Following intranasal administration, these engineered exosomes reduce the expression of PTEN in the injured spinal cord region and considerably enhance axonal growth and neovascularization, thereby eliciting functional recovery in rats with SCI. Therefore, as compared with MSCs, MSC-derived exosomes might be more suitable for treating CNS traumatic injury in terms of safety and efficacy.

Application of EVs in treating neuroinflammatory diseases

Accumulative data show that EVs not only influence the pathogenesis and development of CNS inflammatory diseases but also serve as key diagnostic, prognostic, and therapeutic agents for these diseases (Figure 4).

Figure 4.

EVs in the diagnosis, prognosis, and therapy of neuroinflammatory diseases

In CNS inflammatory diseases, exosomes produced by CNS-resident cells pass through the BBB and enter the peripheral circulation. CNS-derived EVs in the peripheral blood carry important information about the disease status and they can be collected, separated, and analyzed, enabling the diagnosis and prognosis of CNS diseases in a minimally invasive way. Upon intravenous injection, bioengineered EVs carrying targeting ligands and therapeutic cargos can pass through the BBB and deliver therapeutic agents to the site of damage in the CNS, making them favorable for the treatment of neuroinflammatory diseases.

EVs isolated from CSF contain potential biomarkers for various neuroinflammatory diseases, including MS, AD, and PD.^34,120,121 In addition to biomarkers from the CSF, proteins and microRNAs in the free circulation can also reflect the status of CNS inflammatory diseases.^122,123 However, the low concentration of proteins and microRNAs in peripheral body fluids prevents their application as effective biomarkers for the diagnosis and prognosis of CNS diseases. Low levels of CNS-derived biomarkers in the blood should be attributed to factors including enzymatic degradation (e.g., proteases and ribonucleases) and low permeability of the BBB. Interestingly, these problems can be circumvented by EVs. The content of EVs reflects the internal status of their cell of origin, and EV cargos are protected from enzymatic degradation by the EV membrane. As EVs have the ability to cross the BBB, CNS-derived EVs can transport biomarkers to the peripheral circulation, qualifying them as minimally invasive liquid biopsies with the potential for longitudinal evaluation of disease progression. Furthermore, antibodies against proteins located on the EV membrane can be used to isolate EVs secreted by specific cell populations from peripheral fluids such as serum and plasma. For example, neural cell adhesion molecules NCAM and CD171 (L1CAM) are used as markers to select neuron-derived EVs, and the glutamine aspartate transporter (GLAST) antibody is used to enrich astrocyte-derived EVs.^104,124,125 These features render EVs as an attractive source of biomarkers for detecting and monitoring CNS diseases in a minimally invasive manner (Figure 4).^118,126,127 However, currently used CNS markers cannot determine whether EVs are derived from cells of the central or peripheral nervous system. New markers identified by techniques such as single-cell sequencing may circumvent this problem.

EVs by themselves or as nano-vehicles for the delivery of drugs are being investigated as therapeutic agents.^128,129 Compelling evidence shows that MSC-derived exosomes are effective in mitigating multiple neuroinflammatory diseases using animal models.^{46,47,89,90,118} The good biocompatibility, low immunogenicity, and high target specificity of EVs render them as optimal carriers of therapeutic cargos, including proteins, nucleic acids, or small-molecule drugs (Figure 4). Intriguingly, with the unique BBB-crossing ability, EVs are particularly favorable for the treatment of CNS diseases, in which the BBB presents a great wall preventing the entry of most drugs. Existing evidence indicates that EVs with neurorestorative and anti-inflammatory properties hold translational potential and promise as therapeutic agents for neuroinflammatory diseases such as MS, stroke, and SCI. However, most findings have come from animal experiments and they cannot be simply extrapolated to the clinical situation, which is still a current gap in knowledge. Additional experiments and preclinical studies are necessary to properly translate EV therapeutic approaches from bench to the clinic. Most importantly, the safety and efficacy of EVs in the treatment of neuroinflammatory diseases need to be confirmed by clinical trials.

Conclusion and perspectives

As a unique mode of intercellular communication, EVs mediate the omnidirectional communication among various cell populations in the CNS and periphery. In addition to CNS homeostasis, EVs have been shown to be key players in neuroinflammatory diseases as described in this review. Although these findings have largely deepened our understanding of the underlying mechanism of neuroimmune crosstalk and neuroinflammation, it is clear that EV biology, especially in the context of neuroinflammation, is still in its infancy. Several critical aspects concerning EVs and neuroinflammatory diseases have yet to be addressed. First, the technique of EV isolation should be improved to efficiently isolate the low amounts of CNS-derived EVs from the peripheral blood and separate them from contaminating proteins and lipoprotein aggregates. In addition, techniques enabling the specific separation of exosomes, ectosomes, and apoptotic bodies should be developed and refined. Second, since EVs can traffic through the BBB, it is unknown whether inflammation that happened outside of the CNS affects CNS function via EVs. Similarly, it is unclear whether CNS-derived EVs in neuroinflammation affect cell function in other organs. Third, the pathogenesis of neuroinflammatory diseases is associated with genetic and environmental risk factors, and therefore it is interesting to identify genetic and environmental factors that are linked with EV biogenesis, secretion, or uptake. Fourth, it is unknown whether the biogenesis, transport, or uptake of CNS EVs can be targeted for treating neuroinflammatory diseases. Finally, for the clinical application of EVs, standards concerning EV production, storage, safety, activity, and dosage should be made. Addressing these questions will not only shed light on the underlying mechanism of neuroinflammation but also be favorable for the treatment of neuroinflammatory diseases.