The Role of Extracellular Vesicles in Demyelination of the Central Nervous System

It is being increasingly demonstrated that extracellular vesicles (EVs) are deeply involved in the physiology of the central nervous system (CNS). Processes such as synaptic activity, neuron-glia communication, myelination and immune response are modulated by EVs. Likewise, these vesicles may participate in many pathological processes, both as triggers of disease or, on the contrary, as mechanisms of repair. EVs play relevant roles in neurodegenerative disorders such as Alzheimer’s or Parkinson’s diseases, in viral infections of the CNS and in demyelinating pathologies such as multiple sclerosis (MS). This review describes the involvement of these membrane vesicles in major demyelinating diseases, including MS, neuromyelitis optica, progressive multifocal leukoencephalopathy and demyelination associated to herpesviruses.     

Extracellular Vesicles in Diagnosing Chronic Coronary Syndromes the Bumpy Road to Clinical Implementation

Coronary artery disease (CAD), comprising both acute coronary syndromes (ACS) and chronic coronary syndromes (CCS), remains one of the most important killers throughout the entire world. ACS is often quickly diagnosed by either deviation on an electrocardiogram or elevated levels of troponin, but CCS appears to be more complicated. The most used noninvasive strategies to diagnose CCS are coronary computed tomography and perfusion imaging. Although both show reasonable accuracy (80–90%), these modalities are becoming more and more subject of debate due to costs, radiation and increasing inappropriate use in low-risk patients. A reliable, blood-based biomarker is not available for CCS but would be of great clinical importance. Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles containing bioactive contents e.g., proteins, lipids and nucleic acids. EVs are often referred to as the “liquid biopsy” since their contents reflect changes in the condition of the cell they originate from. Although EVs are studied extensively for their role as biomarkers in the cardiovascular field during the last decade, they are still not incorporated into clinical practice in this field. This review provides an overview on EV biomarkers in CCS and discusses the clinical and technological aspects important for successful clinical application of EVs.     

Glioblastoma Extracellular Vesicle-Specific Peptides Inhibit EV-Induced Neuronal Cytotoxicity

WHO Grade 4 IDH-wild type astrocytoma (GBM) is the deadliest brain tumor with a poor prognosis. Meningioma (MMA) is a more common “benign” central nervous system tumor but with significant recurrence rates. There is an urgent need for brain tumor biomarkers for early diagnosis and effective treatment options. Extracellular vesicles (EVs) are tiny membrane-enclosed vesicles that play essential functions in cell-to-cell communications among tumor cells. We aimed to identify epitopes of brain tumor EVs by phage peptide libraries. EVs from GBM plasma, MMA plasma, or brain tumor cell lines were used to screen phage-displayed random peptide libraries to identify high-affinity peptides. We purified EVs from three GBM plasma pools (23 patients), one MMA pool (10 patients), and four brain tumor cell lines. We identified a total of 21 high-affinity phage peptides (12 unique) specific to brain tumor EVs. The peptides shared high sequence homologies among those selected by the same EVs. Dose–response ELISA demonstrated that phage peptides were specific to brain tumor EVs compared to controls. Peptide affinity purification identified unique brain tumor EV subpopulations. Significantly, GBM EV peptides inhibit brain tumor EV-induced complement-dependent cytotoxicity (necrosis) in neurons. We conclude that phage display technology could identify specific peptides to isolate and characterize tumor EVs.     

Extracellular Vesicles and Their Role in the Spatial and Temporal Expansion of Tumor–Immune Interactions

Extracellular vesicles (EVs) serve as trafficking vehicles and intercellular communication tools. Their cargo molecules directly reflect characteristics of their parental cell. This includes information on cell identity and specific cellular conditions, ranging from normal to pathological states. In cancer, the content of EVs derived from tumor cells is altered and can induce oncogenic reprogramming of target cells. As a result, tumor-derived EVs compromise antitumor immunity and promote cancer progression and spreading. However, this pro-oncogenic phenotype is constantly being challenged by EVs derived from the local tumor microenvironment and from remote sources. Here, we summarize the role of EVs in the tumor–immune cross-talk that includes, but is not limited to, immune cells in the tumor microenvironment. We discuss the potential of remotely released EVs from the microbiome and during physical activity to shape the tumor–immune cross-talk, directly or indirectly, and confer antitumor activity. We further discuss the role of proinflammatory EVs in the temporal development of the tumor–immune interactions and their potential use for cancer diagnostics.     

ALCAM/CD166 Is Involved in the Binding and Uptake of Cancer-Derived Extracellular Vesicles

Colorectal cancer (CRC) and ovarian cancer (OvC) patients frequently develop peritoneal metastasis, a condition associated with a very poor prognosis. In these cancers, tumor-derived extracellular vesicles (EVs) cause immunosuppression, facilitate the direct attachment and invasion of cancer cells through the mesothelium, induce the conversion of peritoneal mesothelial cells (PMCs) into cancer-associated fibroblasts (CAFs) and transfer a more aggressive phenotype amongst cancer cells. Although the promoting role of EVs in CRC and OvC peritoneal metastasis is well established, the specific molecules that mediate the interactions between tumor-derived EVs and immune and non-immune target cells remain elusive. Here, we employed the SKOV-3 (ovarian adenocarcinoma) and Colo-320 (colorectal adenocarcinoma) human cell lines as model systems to study the interactions and uptake of EVs produced by ovarian carcinoma and colorectal carcinoma cells, respectively. We established that the adhesion molecule ALCAM/CD166 is involved in the interaction of cancer-derived EVs with recipient cancer cells (a process termed “EV binding” or “EV docking”) and in their subsequent uptake by these cells. The identification of ALCAM/CD166 as a molecule mediating the docking and uptake of CRC and OvC-derived EVs may be potentially exploited to block the peritoneal metastasis cascade promoted by EVs in CRC and OvC patients.     

Reviewing the Significance of Blood–Brain Barrier Disruption in Multiple Sclerosis Pathology and Treatment

The disruption of blood–brain barrier (BBB) for multiple sclerosis (MS) pathogenesis has a double effect: early on during the onset of the immune attack and later for the CNS self-sustained ‘inside-out’ demyelination and neurodegeneration processes. This review presents the characteristics of BBB malfunction in MS but mostly highlights current developments regarding the impairment of the neurovascular unit (NVU) and the metabolic and mitochondrial dysfunctions of the BBB’s endothelial cells. The hypoxic hypothesis is largely studied and agreed upon recently in the pathologic processes in MS. Hypoxia in MS might be produced per se by the NVU malfunction or secondary to mitochondria dysfunction. We present three different but related terms that denominate the ongoing neurodegenerative process in progressive forms of MS that are indirectly related to BBB disruption: progression independent of relapses, no evidence of disease activity and smoldering demyelination or silent progression. Dimethyl fumarate (DMF), modulators of S1P receptor, cladribine and laquinimode are DMTs that are able to cross the BBB and exhibit beneficial direct effects in the CNS with very different mechanisms of action, providing hope that a combined therapy might be effective in treating MS. Detailed mechanisms of action of these DMTs are described and also illustrated in dedicated images. With increasing knowledge about the involvement of BBB in MS pathology, BBB might become a therapeutic target in MS not only to make it impenetrable against activated immune cells but also to allow molecules that have a neuroprotective effect in reaching the cell target inside the CNS.     

MicroRNAs,Multiple Sclerosis,and Depression

Multiple sclerosis (MS) is a chronic disease of the central nervous system that affects the brain and spinal cord. There are several disease courses in MS including relapsing–remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). Up to 50% of MS patients experience depressive disorders. Major depression (MD) is a serious comorbidity of MS. Many dysfunctions including neuroinflammation, peripheral inflammation, gut dysbiosis, chronic oxidative and nitrosative stress, and neuroendocrine and mitochondrial abnormalities may contribute to the comorbidity between MS and MD. In addition to these actions, medical treatment and microRNA (miRNA) regulation may also be involved in the mechanisms of the comorbidity between MS and MD. In the study, I review many common miRNA biomarkers for both diseases. These common miRNA biomarkers may help further explore the association between MS and MD.     

The Role of Extracellular Vesicles from Human Macrophages on Host-Pathogen Interaction

The nano-sized membrane enclosed extracellular vesicles (EVs) released by virtually all cell types play an essential role in intercellular communication via delivering bio-molecules, such as nucleic acids, proteins, lipids, and other molecules to recipient cells. By mediating an active and steady-state cell-to-cell communication, EVs contribute to regulating and preserving cellular homeostasis. On the other hand, EVs can also spread pathogen-derived molecules during infections, subverting the host immune responses during infections and thus worsening pathophysiological processes. In recent years, the biological functioning of EVs has become a widespread research field in basic and clinical branches of medical sciences due to their potential role in therapeutic applications for several diseases. This review aims to summarize the main recent findings regarding the implication of EVs shed by human macrophages (MΦ-EVs) and how they can modulate the host immune response to control or increase the damage caused by infectious agents. We will also present the methods used to describe MΦ-EVs, as well as the potential of these EVs as disease diagnostic tools for some human pathogens. We believe that an in-depth understanding of the host–pathogen interactions mediated by MΦ-EVs may trigger the development of innovative therapeutic strategies against infectious diseases.     

Burn Injury Induces Proinflammatory Plasma Extracellular Vesicles That Associate with Length of Hospital Stay in Women: CRP and SAA1 as Potential Prognostic Indicators

Severe burn injury is a devastating form of trauma that results in persistent immune dysfunction with associated morbidity and mortality. The underlying drivers of this immune dysfunction remain elusive, and there are no prognostic markers to identify at-risk patients. Extracellular vesicles (EVs) are emerging as drivers of immune dysfunction as well as biomarkers. We investigated if EVs after burn injury promote macrophage activation and assessed if EV contents can predict length of hospital stay. EVs isolated early from mice that received a 20% total body surface area (TBSA) burn promoted proinflammatory responses in cultured splenic macrophages. Unbiased LC-MS/MS proteomic analysis of early EVs (<72 h post-injury) from mice and humans showed some similarities including enrichment of acute phase response proteins such as CRP and SAA1. Semi-unbiased assessment of early human burn patient EVs found alterations consistent with increased proinflammatory signaling and loss of inhibition of CRP expression. In a sample of 50 patients with large burn injury, EV SAA1 and CRP were correlated with TBSA injury in both sexes and were correlated with length of hospital stay in women. These findings suggest that EVs are drivers of immune responses after burn injury and their content may predict hospital course.     

A First Phenotypic and Functional Characterization of Placental Extracellular Vesicles from Women with Multiple Sclerosis

Pregnancy is a unique situation of physiological immunomodulation, as well as a strong Multiple Sclerosis (MS) disease modulator whose mechanisms are still unclear. Both maternal (decidua) and fetal (trophoblast) placental cells secrete extracellular vesicles (EVs), which are known to mediate cellular communication and modulate the maternal immune response. Their contribution to the MS disease course during pregnancy, however, is unexplored. Here, we provide a first phenotypic and functional characterization of EVs isolated from cultures of term placenta samples of women with MS, differentiating between decidua and trophoblast. In particular, we analyzed the expression profile of 37 surface proteins and tested the functional role of placental EVs on mono-cultures of CD14⁺ monocytes and co-cultures of CD4⁺ T and regulatory T (Treg) cells. Results indicated that placental EVs are enriched for surface markers typical of stem/progenitor cells, and that conditioning with EVs from samples of women with MS is associated to a moderate decrease in the expression of proinflammatory cytokines by activated monocytes and in the proliferation rate of activated T cells co-cultured with Tregs. Overall, our findings suggest an immunomodulatory potential of placental EVs from women with MS and set the stage for a promising research field aiming at elucidating their role in MS remission.     

Extracellular Vesicles as Biological Indicators and Potential Sources of Autologous Therapeutics in Osteoarthritis

Along with cytokines, extracellular vesicles (EVs) released by immune cells in the joint contribute to osteoarthritis (OA) pathogenesis. By high-resolution flow cytometry, we characterized 18 surface markers and 4 proinflammatory cytokines carried by EVs of various sizes in plasma and synovial fluid (SF) from individuals with knee OA, with a primary focus on immune cells that play a major role in OA pathogenesis. By multiplex immunoassay, we also measured concentrations of cytokines within (endo) and outside (exo) EVs. EVs carrying HLA-DR, -DP and -DQ were the most enriched subpopulations in SF relative to plasma (25–50-fold higher depending on size), suggesting a major contribution to the SF EV pool from infiltrating immune cells in OA joints. In contrast, the CD34⁺ medium and small EVs, reflecting hematopoietic stem cells, progenitor cells, and endothelial cells, were the most significantly enriched subpopulations in plasma relative to SF (7.3- and 7.7-fold higher). Ratios of EVs derived from neutrophils and lymphocytes were highly correlated between SF and plasma, indicating that plasma EVs could reflect OA severity and serve as systemic biomarkers of OA joint pathogenesis. Select subsets of plasma EVs might also provide next generation autologous biological products for intra-articular therapy of OA joints.     

Focus on Extracellular Vesicles: Development of Extracellular Vesicle-Based Therapeutic Systems

Many types of cells release phospholipid membrane vesicles thought to play key roles in cell-cell communication, antigen presentation, and the spread of infectious agents. Extracellular vesicles (EVs) carry various proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), like a “message in a bottle” to cells in remote locations. The encapsulated molecules are protected from multiple types of degradative enzymes in body fluids, making EVs ideal for delivering drugs. This review presents an overview of the potential roles of EVs as natural drugs and novel drug-delivery systems.     

Tumor-Derived Exosomes (TEX) and Their Role in Immuno-Oncology

Extracellular vesicles (EVs) play a key role in health and disease, including cancer. Tumors produce a mix of EVs differing in size, cellular origin, biogenesis and molecular content. Small EVs (sEV) or exosomes are a subset of 30–150 nm (virus–size) vesicles originating from the multivesicular bodies (MVBs) and carrying a cargo that in its content and topography approximates that of a parent cell. Tumor-derived exosomes (TEX) present in all body fluids of cancer patients, are considered promising candidates for a liquid tumor biopsy. TEX also mediate immunoregulatory activities: they maintain a crosstalk between the tumor and various non-malignant cells, including immunocytes. Effects that EVs exert on immune cells may be immunosuppressive or immunostimulatory. Here, we review the available data for TEX interactions with immunocytes, focusing on strategies that allow isolation from plasma and separation of TEX from sEV produced by non-malignant cells. Immune effects mediated by either of the subsets can now be distinguished and measured. The approach has allowed for the comparison of molecular and functional profiles of the two sEV fractions in plasma of cancer patients. While TEX carried an excess of immunosuppressive proteins and inhibited immune cell functions in vitro and in vivo, the sEV derived from non-malignant cells, including CD3(+)T cells, were variably enriched in immunostimulatory proteins and could promote functions of immunocytes. Thus, sEV in plasma of cancer patients are heterogenous, representing a complex molecular network which is not evident in healthy donors’ plasma. Importantly, TEX appear to be able to reprogram functions of non-malignant CD3(+)T cells inducing them to produce CD3(+)sEV enriched in immunosuppressive proteins. Ratios of stimulatory/inhibitory proteins carried by TEX and by CD3(+)sEV derived from reprogrammed non-malignant cells vary broadly in patients and appear to negatively correlate with disease progression. Simultaneous capture from plasma and functional/molecular profiling of TEX and the CD3(+)sEV fractions allows for defining their role as cancer biomarkers and as monitors of cancer patients’ immune competence, respectively.     

Extracellular Vesicles in Alzheimer’s Disease: Friends or Foes? Focus on Aβ-Vesicle Interaction

The intercellular transfer of amyloid-β (Aβ) and tau proteins has received increasing attention in Alzheimer’s disease (AD). Among other transfer modes, Aβ and tau dissemination has been suggested to occur through release of Extracellular Vesicles (EVs), which may facilitate delivery of pathogenic proteins over large distances. Recent evidence indicates that EVs carry on their surface, specific molecules which bind to extracellular Aβ, opening the possibility that EVs may also influence Aβ assembly and synaptotoxicity. In this review we focus on studies which investigated the impact of EVs in Aβ-mediated neurodegeneration and showed either detrimental or protective role for EVs in the pathology.     

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.     

Exposome and Immunity Training: How Pathogen Exposure Order Influences Innate Immune Cell Lineage Commitment and Function

Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined “trained immunity” and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The “exposome” is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.     

Characterization of Extracellular Vesicle Cargo in Sjögren’s Syndrome through a SWATH-MS Proteomics Approach

Primary Sjögren’s syndrome (pSS) is a complex heterogeneous disease characterized by a wide spectrum of glandular and extra-glandular manifestations. In this pilot study, a SWATH-MS approach was used to monitor extracellular vesicles-enriched saliva (EVs) sub-proteome in pSS patients, to compare it with whole saliva (WS) proteome, and assess differential expressed proteins between pSS and healthy control EVs samples. Comparison between EVs and WS led to the characterization of compartment-specific proteins with a moderate degree of overlap. A total of 290 proteins were identified and quantified in EVs from healthy and pSS patients. Among those, 121 proteins were found to be differentially expressed in pSS, 82% were found to be upregulated, and 18% downregulated in pSS samples. The most representative functional pathways associated to the protein networks were related to immune-innate response, including several members of S100 protein family, annexin A2, resistin, serpin peptidase inhibitors, azurocidin, and CD14 monocyte differentiation antigen. Our results highlight the usefulness of EVs for the discovery of novel salivary-omic biomarkers and open novel perspectives in pSS for the identification of proteins of clinical relevance that could be used not only for the disease diagnosis but also to improve patients’ stratification and treatment-monitoring. Data are available via ProteomeXchange with identifier PXD025649.     

Inhibitors of Acetylcholinesterase and Butyrylcholinesterase Meet Immunity

Acetylcholinesterase (AChE) inhibitors are widely used for the symptomatic treatment of Alzheimer’s disease and other dementias. More recent use is for myasthenia gravis. Many of these inhibitors interact with the second known cholinesterase, butyrylcholinesterase (BChE). Further, evidence shows that acetylcholine plays a role in suppression of cytokine release through a “cholinergic anti-inflammatory pathway” which raises questions about the role of these inhibitors in the immune system. This review covers research and discussion of the role of the inhibitors in modulating the immune response using as examples the commonly available drugs, donepezil, galantamine, huperzine, neostigmine and pyridostigmine. Major attention is given to the cholinergic anti-inflammatory pathway, a well-described link between the central nervous system and terminal effector cells in the immune system.     

Current and Future Biomarkers in Multiple Sclerosis

Multiple sclerosis (MS) is a debilitating autoimmune disorder. Currently, there is a lack of effective treatment for the progressive form of MS, partly due to insensitive readout for neurodegeneration. The recent development of sensitive assays for neurofilament light chain (NfL) has made it a potential new biomarker in predicting MS disease activity and progression, providing an additional readout in clinical trials. However, NfL is elevated in other neurodegenerative disorders besides MS, and, furthermore, it is also confounded by age, body mass index (BMI), and blood volume. Additionally, there is considerable overlap in the range of serum NfL (sNfL) levels compared to healthy controls. These confounders demonstrate the limitations of using solely NfL as a marker to monitor disease activity in MS patients. Other blood and cerebrospinal fluid (CSF) biomarkers of axonal damage, neuronal damage, glial dysfunction, demyelination, and inflammation have been studied as actionable biomarkers for MS and have provided insight into the pathology underlying the disease process of MS. However, these other biomarkers may be plagued with similar issues as NfL. Using biomarkers of a bioinformatic approach that includes cellular studies, micro-RNAs (miRNAs), extracellular vesicles (EVs), metabolomics, metabolites and the microbiome may prove to be useful in developing a more comprehensive panel that addresses the limitations of using a single biomarker. Therefore, more research with recent technological and statistical approaches is needed to identify novel and useful diagnostic and prognostic biomarker tools in MS.     

Extracellular Vesicles as Emerging Players in Intercellular Communication: Relevance in Mast Cell-Mediated Pathophysiology

Mast cells are major effector cells in eliciting allergic responses. They also play a significant role in establishing innate and adaptive immune responses, as well as in modulating tumor growth. Mast cells can be activated upon engagement of the high-affinity receptor FcεRI with specific IgE to multivalent antigens or in response to several FcεRI-independent mechanisms. Upon stimulation, mast cells secrete various preformed and newly synthesized mediators. Emerging evidence indicates their ability to be a rich source of secreted extracellular vesicles (EVs), including exosomes and microvesicles, which convey biological functions. Mast cell-derived EVs can interact with and affect other cells located nearby or at distant sites and modulate inflammation, allergic response, and tumor progression. Mast cells are also affected by EVs derived from other cells in the immune system or in the tumor microenvironment, which may activate mast cells to release different mediators. In this review, we summarize the latest data regarding the ability of mast cells to release or respond to EVs and their role in allergic responses, inflammation, and tumor progression. Understanding the release, composition, and uptake of EVs by cells located near to or at sites distant from mast cells in a variety of clinical conditions, such as allergic inflammation, mastocytosis, and lung cancer will contribute to developing novel therapeutic approaches.