Microvesicles and exosomes in metabolic diseases and inflammation

Journal Pre-proof Microvesicles and exosomes in metabolic diseases and inflammation L Dini, S. Tacconi, E. Carata, A.M. Tata, C. Vergallo, E. Panzarini

PII:

S1359-6101(19)30147-9

DOI:

https://doi.org/10.1016/j.cytogfr.2019.12.008

Reference:

CGFR 1128

To appear in:

Cytokine and Growth Factor Reviews

Received Date:

17 November 2019

Revised Date:

22 December 2019

Accepted Date:

30 December 2019

Please cite this article as: Dini L, Tacconi S, Carata E, Tata AM, Vergallo C, Panzarini E, Microvesicles and exosomes in metabolic diseases and inflammation, Cytokine and Growth Factor Reviews (2020), doi: https://doi.org/10.1016/j.cytogfr.2019.12.008

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Microvesicles and exosomes in metabolic diseases and inflammation Dini L.1,2,*, Tacconi S.3, Carata E.3, Tata A.M.1,4, Vergallo C.5, Panzarini E3,* 1

Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, Rome, Italy;

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CNR-Nanotec, Lecce, Italy;

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Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University

of Salento, Lecce, Italy; Research Center in Neurobiology “Daniel Bovet, Sapienza University of Rome, Rome, Italy;

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Department of Pharmacy, University of Chieti-Pescara “G. d’Annunzio”, Chieti, Italy;

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*corresponding authors at: Department of Biology and Biotechnology "C. Darwin", Sapienza University of Rome, piazzale Aldo Moro 5 00185, Rome, Italy; Department of Biological and Environmental Sciences and Technologies (Di.S.Te.B.A.), University of Salento, via Prov.le LecceMonteroni, Centro Ecotekne, 73100 Lecce, Italy

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E-mail addresses: [email protected] (Dini L.), [email protected] (Panzarini E.).

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Graphical abstract

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Highlights

 Extracellular vesicles (EVs) are mainly distinguished in exosomes and microvesicles

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 Recent findings show that EVs are involved in cell-to-cell communication  Both EVs derivation and cargo influence the responsivity of target cells  Macrophage-derived EVs play a key role in MetS-associated inflammation  EVs could be deemed as biomarkers and bioeffectors of metabolic syndrome (MetS)

Abstract Metabolic diseases are based on a dysregulated crosstalk between various cells such as adipocytes, hepatocytes and immune cells. Generally, hormones and metabolites mediate this crosstalk that becomes alterated in metabolic syndrome including obesity and diabetes. Recently, Extracellular Vesicles (EVs) are emerging as a novel way of cell-to-cell communication and represent an attractive strategy to transfer fundamental informations between the cells through the transport of proteins and nucleic acids. EVs, released in the extracellular space, circulate via the various body fluids and modulate the cellular responses following their interaction with the near and far target cells. Clinical and experimental data support their role as biomarkers and bioeffectors in several diseases includimg

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also the metabolic syndrome. Despite numerous studies on the role of macrophages in the development of metabolic diseases, to date, there are little informations about the influence of metabolic stress on the EVs produced by macrophages and about the role of the released vesicles in the organism. Here, we review current understanding about the role of EVs in metabolic diseases, mainly in inflammation status burst. This knowledge may play a relevant role in health monitoring,

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medical diagnosis and personalized medicine.

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Keywords: metabolic disorders; inflammation; extracellular vesicles; M1 macrophages; M2

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macrophages

Abbreviations

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MetS, Metabolic Syndrome; EVs, Extracellular Vesicles; T2D, Type 2 Diabetes; NAFLD, NonAlcoholic Fatty Liver Disease; NASH, Non-Alcoholic Steatohepatitis; ATMs, Adipose Tissue Macrophages; TNF-α, Tumor Necrosis Factor-alpha; IL-6, Interleukin-6; IL-8, Inteleukin-8; IL-15, Inteleukin-15; LPS, LipoPolySaccharide; IL-1, Inteleukin-1beta; IFN, Interferon alpha; IFN, Interferon gamma; CXCL9, Chemokine (C-X-C motif) Ligand 9; CXCL10, Chemokine (C-X-C motif) Ligand 10; CXCL11, Chemokine (C-X-C motif) Ligand 11; IL-4, Inteleukin-4; TGF, Transforming Growth Factor beta; CCL17, Chemokine (C-C motif) Ligand 17; IL-10, Inteleukin-10; CD86, Cluster of Differentiation 86; MHC-II, Major Histocompatibility Complex class II; TLR4, Toll-Like Receptor 4; CCR7, C-C Chemokine Receptor type 7; CD11c, Cluster of Differentiation 11c; iNOS, inducible NOS; NF-kB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; STAT1, Signal Transducer and Activator of Transcription 1; CD163, Cluster of Differentiation 163; CD206, Cluster of Differentiation 2016; Arg1, Arginase 1; STAT6, Signal Transducer and Activator of Transcription 6; WAT, White Adipose Tissue; CCL3, Chemokine (C-C motif) Ligand 3; DCs, Dendritic Cells; VAT, Visceral Adipose Tissue; SAT, Subcutaneous Adipose Tissue; G-CSF, Granulocyte-Colony Stimulating Factor; NLRP3, NOD-Like Receptors 3; MIP, Macrophage Inflammatory Protein; IR, Insulin Resistance; IKK, Inhibitor of nuclear factor Kappa-B Kinase; JNK1, c-Jun N-terminal Kinase; FFAs, Free Fatty Acids; ROS, Reactive Oxygen Species; AGEs, Advanced Glycation End-products; AMPK, AMP-Activated Protein Kinase; PBMCs, Peripheral Blood Mononuclear Cells; TLRs, Toll-Like Receptors family; UPR, Unfolded Protein Response; DAG, Diacylglycerol; FABP, Fatty Acid Binding Protein Activation; PPAR-, Peroxisome Proliferator-Activated Receptor-; NO, Nitric Oxide; EXOs, Exosomes; MVs, MicroVesicles; ILVs, Intraluminal Vesicles; MVBs, MultiVesicular Bodies; ESCRT, Endosomal Sorting Complex Required for Transport; PS, PhosphatidylSerine; Tsg101, Tumor susceptibility 101; Alix, ALG-2 interacting protein X; HSP, Heat Shock Proteins; miR, microRNAs; LDL, Low Density Lipoproteins; HDL, High Density Lipoproteins; VLDL, Very Low Density Lipoproteins; VEGF, Vascular Endothelial Growth Factor; SPRED1, Sprouty-Related EVH1 Domain Containing 1; ICAM-1, InterCellular Adhesion Molecule-1; WHO, World Health Organization

Introduction A wide variety of diseases, including metabolic syndrome (MetS), obesity and diabetes mellitus, are clinically classified as metabolic disorders whose pathogenesisis is caused by fat toxicity, chronic inflammation and oxidative stress and it is based on altered communication between different organs, such as liver, pancreas, adipose tissue and immune system. In this context, a very high integration exists between immune system and metabolism, and macrophages are the key players in the occurrence and progression of metabolic diseases, such as type 2 diabetes (T2D) [1]. Recently, Extracellular Vesicles (EVs) the vesicles are acquiring a growing interest for their ability to transfer biological components between cells. So, they contribute to transform the traditional vision

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of intercellular communication and represent an alternative and versatile cell-to-cell communication modality based on molecules cargo, i.e., proteins, lipids, nucleic acids and membrane receptors. In fact, EVs once released in the extracellular space, circulate via the different fluids of the body and modulate metabolism of both neighboring and distant cells. Clinical data and experimental studies support their role in the MetS: for example, EVs are involved in insulin resistance, dyslipidemia,

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endocrine disorders, lipid toxicity and chronic inflammation [2]. Despite numerous studies on both the role of macrophages in the development of metabolic diseases and the influence of metabolic

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stress on the EVs production by macrophages, the role of EVs in the metabolic pathologies is still poor investigated and been poor understood. Moreover, the knowledge about the influence of EVs on

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inflammatory reactions also could be relevant. To answer to these questions, the accuracy of techniques to be used in analyising EVs is pivotal. Unfortunately, few and confusing informations still exist about the morphology, composition and size distribution of EVs in biological fluids because

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of the limitations related to both analytical methods of the content and EVs preparation and storage. Herein, we discuss the relationship between metabolic disorders and inflammation, and the emerging

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roles of EVs in metabolic disorders and related inflammation status.

Inflammation and MetS

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The MetS refers to the association of a set of physiological (excess of weight, high blood pressure) and biochemical (alterations carbohydrates and lipids) disorders which increase the risk of T2D, cardiovascular disease and cancer. Despite the efforts to define international criteria qualifying the MetS, discrepancies about reference values to diagnose the disease still exist. Several factors influence the occurrence of MetS, such as sex, sedentary lifestyle, alcoholism, smoking, etc. [2]. Obesity has been implicated in the genesis of various metabolic abnormalities including insulin resistance and T2D, dyslipidemia, muscular damage, ectopic fat accumulation, cardiovascular disease and a spectrum of non-cancerous liver diseases, such as Non-Alcoholic Fatty Liver Disease (NAFLD)

and non-alcoholic steatohepatitis (NASH). In recent years, several studies have shown the importance of the inflammatory process in the development and progression of these various metabolic diseases, which are associated with a low-grade systemic chronic inflammation, called “metainflammation”, that affects important metabolic tissues such as adipose tissue, pancreas, liver, skeletal muscle and brain.

Inflammation in obesity and related diseases. In obesity, the first event leading to the development of inflammation in adipose tissue is the recruitment, infiltration and polarization of different leukocyte populations, including neutrophils,

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macrophages, dendritic cells and mast cells, and different subtypes of B and T lymphocytes. Different stimuli, such as adipocyte hypertrophy, release of chemokines, cytokines and adipokines from adipose tissue, adipocytes death, hypoxia and lipotoxicity, are involved in the recruitment of immune cells in adipose tissue [3-5].

Macrophages are the most abundant population of immune cells in obese adipose tissue (Adipose

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Tissue Macrophages, ATMs) and they represent 40-60% of adipose tissue immune cells in obese mice model [6]. In obesity, the pro-inflammatory activity of ATMs stimulates the adipocytes to

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secrete pro-inflammatory mediators, such as TNF-α and IL-6 which, in turn, activate and recruit other immune cells. Adipose tissue is characterized by high endocrine activity and it releases several

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hormones and cytokines, including leptin, adiponectin, resistin, TNF-α, and IL-6, that lead to the burst of a highly pro-inflammatory environment. Similarly, skeletal muscle myocytes can secrete a large number of cytokines and pro-inflammatory mediators during obesity. In particular, myocytes

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can release different inflammatory cytokines, such as IL-6, IL-8, and IL-15, and different other molecules called myokines, i.e., irisin, myonectin and myostatin [7]. These molecules can locally affect myocytes or resident immune cells by paracrine or endocrine activity. As in adipose tissue,

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infiltration and recruitment of immune cells, mainly macrophages and T lymphocytes, also occur in skeletal muscle during obesity-related inflammation [8].

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Macrophages (M0) are a heterogenous population and, depending on their activation status, are classified in M1 or M2 macrophages. M1, or classical activated macrophages, are induced by proinflammatory mediators, such as lipopolysaccharide (LPS) and IFN-, and release different proinflammatory cytokines and chemokines, e.g., IL-1, IL-6, TNF-, IFN-, IFN-, CXCL9, CXCL10 and CXCL11. Conversely, M2 macrophages (called alternatively activated macrophages) are activated by anti-inflammatory stimuli, such as IL-4 and IL-13, and are involved in the resolution of inflammation, angiogenesis and tissue repair by releasing anti-inflammatory mediators, e.g., IL-10, IL-4, TGF- and CCL17. In addition, M1 and M2 macrophages express a different pattern of surface

and intracellular proteins: M1 macrophages present high levels of CD86, MHCI-II, TLR4, CCR7, CD11c and a high activity of inducible NOS (iNOS) and the pro-inflammatory transcription factors Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-kB) and STAT1; M2 macrophages are characterized by high levels of CD163, CD206, FcR, scavenger receptor A-B and intracellular protein such as Arginase 1 (Arg1) and the anti-inflammatory Signal Transducer and Activator of Transcription 6 STAT6 [9]. In Figure 1 the main features characterizing M0, M1 and M2 macrophages are reported. As above stated, macrophages can change their activation status depending on different stimuli; in particular, obesity condition polarizes the macrophages towards a pro-inflammatory M1 phenotype.

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In addition, in obese mice or high-fat diet-fed mice, adipose tissue resident macrophages switch from the anti-inflammatory M2 phenotype to pro-inflammatory M1 one [10]. Conversely, M1 markers are absent in ATMs isolated from obese human; moreover, in vitro treatment of macrophages with glucose, insulin and palmitate triggers their activation called metabolic activation without promote neither M1 nor M2 phenotype [11]. A different spatial distribution of macrophages in obese adipose

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tissue was observed compared to lean adipose tissue. In obese White Adipose Tissue (WAT), macrophages accumulate in clusters around hypertrophic and apoptotic adipocytes and generate the

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so-called crown-like structures; conversely, in lean adipose tissue, ATMs remain in interstitial spaces [12]. ATMs in subcutaneous adipose tissue express high levels of pro-inflammatory mediators, such

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as IL1-, IL-6, IL-8, TNF- and CCL3 [13]. An important role in macrophages recruitment and infiltration is played by dendritic cells (DCs). An increase in circulating levels of DCs was observed in obese patients [14]. In addition, the obese high-fat diet-fed mice show lymphonodes hyperplasia

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in WAT with accumulation of macrophages, cytotoxic T and dendritic cells [15]. In obesity, macrophage infiltration in muscle tissue is followed by their activation towards the proinflammatory M1 phenotype. According to this, there are several studies that have found an increase

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of pro-inflammatory markers and cytokines, such as IL-6, IL-1, TNF- and IFN-γ, and a decrease of anti-inflammatory effectors, such as IL-10 [7,16,17]. In addition, there is an important cross-talk

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between adipose tissue and skeletal muscle one in chronic obesity via chemokines and adipokines released into the blood. Pellegrini et al [18] demonstated a decrease of skeletal muscle contractility and myogenesis genes expression by using a 3D coculture approach of human myotubes and adipocytes derived from visceral (VAT) and subcutaneous (SAT) adipose tissue from lean and obese subjects. Furthermore, the coculture of myotubes and obese VAT adipocytes determines an increase of pro-inflammatory mediators such as G-CSF, IL-6 and CCL7. Another important contribution in the development of obesity-related inflammation is given by neutrophils, that first come in the

inflammatory site and attract other immune cells, including macrophages, DCs and lymphocytes, by releasing chemoattractant molecules, such as TNF-α, IL1-β, IL-8, and CCL3 [19-21]. Obesity-related hyperlipidemia can saturate the lipid storage capacity in adipocytes, leading to an ectopic lipid accumulation in non-adipose tissues and organs, such as the liver, muscle, heart, kidney and pancreas. In the liver, this process leads to a pathological condition known as NAFLD and, particularly its inflammatory form known as NASH, can progress towards fibrosis, cirrhosis and NASH-induced hepatocellular carcinoma. Under physiological conditions, the resident Kupffer cells (KCs) are the most abundant population of macrophages in the liver. In high fat diet-fed mice, KCs accumulate lipids into the liver and display an altered expression of various pro- or anti-inflammatory

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genes, suggesting that also KCs can polarize towards a pro-inflammatory phenotype M1 [22]. In fact, circulating cytokines and adipokines released from inflamed obese adipose tissue activate KCs that, in turn, contribute to the alteration of the liver homeostasis and hepatic inflammation by secreting chemoattracting molecules (i.e., CCL2), pro-inflammatory cytokines (i.e., TNF-α, IL-1β, and IL-6),

Insulin-resistance, type 2 diabetes and inflammation

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macrophage inflammatory protein (i.e., MIP1a and MIP1b) and prostaglandins [23].

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Several evidences have characterized the role of immune cells in the development of obesity-related diseases, such as insulin resistance (IR) and T2D. Different experimental studies, performed on both

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animal and human models, show the close correlation between inflammation and IR in the development of T2D. Several pro-inflammatory effectors, such as IL-1β, IL-6, TNF-α, CRP and many chemokines, have been associated with the development of insulin-resistance and T2D. Various pro-

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inflammatory stimuli can impair insulin signaling by activation of Inhibitor of nuclear factor KappaB Kinase (IKK) and c-Jun N-terminal Kinase (JNK1), that phosphorylate both the insulin receptor and insulin receptor substrates, and modulate different metabolic genes [24]. Recently, it has been

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found that serum level of TNF-α is positively correlated with the pathophysiology of IR and antiTNF-α treatment strategies protect against insulin-resistance and development of T2D [25]. In the

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same manner, various evidences show that IL-1 is associated with impaired insulin-sensitivity and -cells failure: the treatment of T2D patients with anakinra (a recombinant human IL-1 receptor antagonist) reduces the inflammation and improves the secretory function of β-cells [26]. As previously mentioned, during obesity, the infiltration of macrophages in the adipose tissue, muscle and liver increases, and this process is closely associated with the development of obesity-related IR. The reduction in macrophages infiltration, the increase in insulin responsiveness in adipose tissue and skeletal muscle, and the reduction of liver steatosis are observed by blocking the expression of chemoattracting chemokines, such as MCP-1 and CCL2 [27]. The polarization phenotype of ATMs

influences the insulin-sensitivity of adipose tissue. For example, in lean WAT, M2 macrophages are involved in maintenaning the insulin sensitivity by releasing IL-10; conversely, in obese adipose tissue, M1 macrophages enhance IR by secretion of pro-inflammatory mediators, such as IL-6 and TNF- [28]. The upregulation of several inflammatory and macrophage-specific genes in WAT is associated with a dramatic increase of circulating-insulin levels; and the treatment of mice with the antidiabetic drug rosiglitazone induces the downregulation of these genes [29]. In liver, the chemical depletion of KCs improves glucose tolerance and hepatic insulin signaling [30]. On the other hand, new innovative studies have demonstrated that insulin modulates the metabolic and inflammatory activity of macrophages and, in T2D, macrophages can develop an insulin insensitivity. Insulin

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resistant macrophages show impaired immune and inflammatory activity, reduced phagocytosis ability and a M2-like inflammatory phenotype [31].

T2D is also characterized by progressive damage and failure of pancreatic -cells, associated with inflammation in the Langerhans islets [32-34]. Also in the pancreatic islets, the recruitment of immune cells is promoted by several inflammatory mediators [35,36]. In a mouse T2D models, Cucak

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et al. showed increased levels of both resident and recruited macrophages polarized towards proinflammatory M1 phenotype in the pancreatic islets [37]. One of the main effectors for -cells damage

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is IL-1, which is produced by both -cells and infiltrated immune cells. Transcriptome studies in human islets of T2D patients have demonstrated an increase in the expression of IL-1 related genes,

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e.g., IL-11, IL-33, IL-24, IL-6, IL-18R1, IL-1R1, IL-1RL1 and IL-1R2, which are associated with impaired insulin secretion, suggesting that the activation of the IL-1 pathway in islets orchestrates

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immune cell infiltration and β-cell dysfunction [38].

Nutrient excess and metabolic disorders-related inflammation

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In obesity, diabetes or hyperglycemic/hyperlipidemic diet, the organism is exposed to a massive quantity of nutrients, such as glucose and Free Fatty Acids (FFAs), leading to pathological processes of gluco- and lipotoxicity, characterized by the activation or inactivation of immune system that

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amplify and regulate the low-grade inflammation of metabolic disorders. Hyperglycemia and glucotoxicity. Immune activation and inflammatory response induced by high glucose levels depend on several processes, such as increased release of pro-inflammatory effectors, oxidative stress and increased production of Reactive Oxygen Species (ROS), production of Advanced Glycation End-products (AGEs) and reduced activity of AMP-Activated Protein Kinase (AMPK). The treatment of THP-1 monocytes or Peripheral Blood Mononuclear Cells (PBMCs) with high doses of glucose stimulates the production of pro-inflammatory mediators, such as TNF-, IL1, MCP-1, IL-6 and COX2 [39-41]. Further, hyperglycemia promotes monocytes adhesion,

migration and transmigration and increased filopodia formation [42]. Finally, high-glucose levels and glucotoxic stress induce both monocytes differentiation in macrophages and polarization. A study performed by Torres-Castro and coworkers [43] suggests that high glucose concentration-treated in vitro macrophages and hyperglycemic patients-derived monocytes exhibit high levels of proinflammatory markers, such as CD-11c and iNOS, and a down-regulation of anti-inflammatory markers, e.g.,Arg1 and CD206. Furthermore, the exposure of THP1-derived macrophages to glucose promotes M1 polarization in a dose-dependent manner [44]. According to this, our unpublished data show that short-term hyperglycemia, obtained treating THP1-derived macrophages with 15 and 30 mM glucose for 24h, increases the protein expression of canonical M1 markers (i.e., iNOS, CD-86 and MHCI) and mRNA levels of pro-inflammatory cytokines polarization (i.e., IL-1 and IFN-),

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suggesting the induction towards M1 phenotype.

Hyperglycemic stress can affect macrophages activation also indirectly via action of intermediate glucose products or activation of specific inflammatory proteins and pathways. For example, high glucose-treated murine RAW 264.7 macrophages and diabetic mice peritoneal macrophages show

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impaired autophagic system and mitochondrial function, and high ROS levels promoting M1 macrophages polarization [45]. Moreover, M1 phenotype can be obtained upon formation of AGEs

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and their precursors by glycosilation of several circulating proteins and lipoproteins, such as insulin, hemoglobin, albumin, LDL, HDL and VLDL [46].

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It is known that glucose overload affects the energy balance of cells, their metabolic profile and biochemical activity. At the intracellular level, there are several protein sensors, such as AMPActivated Protein Kinase (AMPK), that detect energy variations associated with changes in the

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ADP/ATP ratio. In immune cells the activation or inactivation of AMPK is involved in the modulation of hyperglycemia or obesity-related inflammation. In vivo, AMPK inhibition in macrophages increases obesity-associated liver and adipose tissue inflammation by macrophages

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recruitment and their M1 polarization [47]. In contrast, transfection of macrophages with a constitutively active form of AMPK results in reduction of TNF- and IL-6 production and in

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increase of IL-10 levels [48]. Furthermore, AMPK activation is essential for IL-10-induced JAK/STAT3 signaling, a pathway involved in M2 macrophage activation [49]. Hyperlipidemia and lipotoxicity. A correlation between circulating saturated FFAs and increase of expression levels of different inflammatory markers, such as CRP and IL-6, was found [50,51]. Lipid overload can promote metabolic inflammation by several mechanism, including organelles stress, ROS production, diacylglycerol and ceramides accumulation, mitochondrial dysfunction, and activation of pro-inflammatory pathways. Impaired fatty acids and lipids flux causes the activation of Endoplasmic Reticulum (ER) stress and the Unfolded Protein Response (UPR) signalling that, in

turn, promotes the inflammatory response via the activation of different kinases, inflammatory effectors and the NOD-Like Receptors 3 (NLRP3) inflammasome. Moreover, Toll-Like Receptors (TLRs) family, in particular TLR4 are activated by saturated fatty acids. The activation of TLR4 promotes the release of pro-inflammatory cytokines and chemokines via IKK/NF-kB pathway that, in turn, improves IR and inflammation. In the same manner, palmitate-dependent TLR4 activation increases the expression of different cytokines in monocytes and macrophages, such as IL-1, TNF, MCP-1 and COX-2 [52-55]. Conversely, -3 polyunsaturated fatty acids (-3 PUFA) inhibit the TLR4-induced signalling pathways and downstream target gene expression [56]. In immune cells, FFAs can improve ROS formation through a direct interaction with mitochondrial

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respiratory complexes, or by stimulating NADPH oxidase activity and by interfering with ROS detoxification systems. In addition, FFAs can be incorporated into phospholipids and alter mitochondrial membrane fluidity, electron transport chain and superoxide anion (O2-.) formation [57]. Alteration of lipids metabolism causes accumulation of dangerous lipid species, such as diacylglycerol (DAG) and ceramides. Saturated fatty acids promote ceramide formation in

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macrophages via Fatty Acid Binding Protein activation (FABP), positively correlated with cell death and inflammation [58]. The treatment of macrophages with high levels of palmitate promotes

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ceramide production and release of IL-1 and TNF- [59]. Palmitate exposure markedly alters the phospholipids saturation status and glycerophospholipids, sphingolipids and neutral lipids (i.e.,

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triglycerides and diacylglycerols) amounts [60]. In the contrast, our unpublished data shown that the treatment of THP-1-derived macrophages with palmitate for 24h does not affect the triglycerides content but considerably increase the diacylglycerol levels. Moreover, the effects of saturated FFAs,

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such as palmitate or oleate, on macrophage polarization and activation were not well understood. In fact, recent studies shown that the exposure of macrophages to high levels of different metabolic stimuli (i.e., saturated FFAs, glucose or insulin) determines a different activation of macrophages

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called Metabolically Activated Macrophages (MMe). This alternative phenotype includes many features of M1 and M2 activation and presents a different surface protein pattern from classically

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activated macrophages. As explained by Kratz and colleagues [11], this occurs because the release of pro-inflammatory cytokines and the expression of surface markers are two independent processes. MMe activation is mediated by two different mechanisms: 1) palmitate binds TLRs and promotes pro-inflammatory cytokines production and 2) palmitate is internalized and promotes antiinflammatory action via Peroxisome Proliferator-Activated Receptor- (PPAR-) activation.

Extracellular vesicles Extracellular vesicles have long been considered as waste conveyors used by cells to get rid of harmful or useless molecules. Today, it has been demonstrated that they are carriers of molecules, including proteins, lipids and nucleic acids, between cells; in this manner, EVs are capable to interact and modify target cells [2,61]. EVs are nanosized vesicles derived from cell membranes and secreted in the extracellular environment. The cargo defines both the functions but also the morphological parameters (shape and size) of vesicles. The cargo is extensively analysed by using integrated highthroughput OMICS techniques; the recent results about EVs cargo molecules are summarized in EVpedia (http://evpedia.info) and ExoCarta (http://www.exocarta.org).They are released by almost

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all cell types and naturally circulate in many fluids of the body (blood, lymph, urine, milk, etc.) [62]. These vesicles are heterogeneous in size and reflect the condition of their parent tissue representing a potential diagnostic and therapeutic tool of various diseases, including metabolic and cancer ones [63,64]. On the basis on the status of releasing cells and on the origin modality, various types of EVs are recognized in relation to their possible functions: apoptotic bodies, microvesicles, exosomes,

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oncosomes, and prostasomes [65]. Recently, the international scientific community has suggested a combination of different techniques (e.g., ultracentrifugation, exclusion chromatography size,

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immunocapture, etc.) to determine morphological, biophysical and biochemical criteria to characterize and categorize isolated EVs subtypes and the carried molecules [66] and has proposed

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to essentially distinguish two subtypes of EVs: exosomes (EXOs) and microvesicles (MVs) (Figure 2).

EXOs (with a diameter ranging between 30 and 100 nm) denote EVs deriving from Intraluminal

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Vesicles (ILVs) formed during the maturation of MultiVesicular Bodies (MVBs). They are secreted in the extracellular environment after fusion of MVBs with the plasma membrane [67]. Their formation involves the assembly of four ESCRT complexes (Endosomal Sorting Complex Required

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for Transport) [67]. Biogenesis of EXOs starts upon the accumulation of tetraspanins (CD9 and CD63) in the endosomal membrane, followed by recruitment of ESCRT-0 and ESCRT-I, which

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segregate the transmembrane ubiquitinated cargo. Recruitment of the sub-complexes ESCRT-III, via ESCRT-II, induces the curvature and fission of ILVs (the source of future EXOs). Moreover, a pathway of EXOs biogenesis involving syndecans and syntenin proteins exists, but the role of ESCRTs in the mechanism carrying the formation of vesicles remains still to be determined. Also, an ESCRT complex-independent mechanism of EXOs biogenesis was described; it involves regulation of tetraspanins (CD9, CD63 or CD81) and some lipids (i.e, ceramides) [68]. Membrane targeting of EXOs towards the plasma membrane and their secretion engage membrane trafficking proteins of the family of small G proteins (GTPases), such as Rab11, Rab35 and Rab27. Rab11 and Rab35 regulate

vesicle trafficking of early endosomes towards the plasma membrane; conversely, Rab27 is involved in addressing the late endosomes (lysosomes) to the cell membrane [69]. EXOs participate in many physiopathological responses. Several studies have demonstrated their ability to regulate immune and anti-cancer response, due to their ability to transfer molecules of the Major Histocompatibility Complex (MHC) between immune cells [70]. MVs, 50 nm-1 μm sized, origin by budding of the plasma membrane caused by remodeling of the cytoskeleton upon the rise of cytoplasmic calcium concentration, that stimulates an enzymatic machinery leading to the exposure of PhosphatidylSerine (PS) on outer leaflet of the plasma membrane [71]. The loss of plasma membrane asymmetry causes a transient overload of

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phospholipids resulting in the formation of MVs. However, some MVs do not present PSs residues, suggesting that other still poorly characterized mechanisms would contribute to the release of MVs. The secretion of MVs depends on both the Rho GTPase and Rho-associated kinases signaling pathways. Like EXOs, MVs carry biological messengers between many cells types and are associated with the development of various pathologies [72].

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EVs are formed by a lipid bilayer containing soluble or membrane proteins, lipids, metabolites and nucleic acids. The proteomic, lipidomic and genomic analysis (grouped in the EVpedia database [73])

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have defined the molecules carried by the EVs. EXOs are enriched in the proteins involved in endosomal trafficking, such as tetraspanins Tsg101 (tumor susceptibility 101), CD9, CD63 or Alix

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(ALG-2 interacting protein X). These markers are used to characterize this population of vesicles and/or isolate them by immunoaffinity. However, different subtypes of exosomes may co-exist, probably reflecting the pathways of biogenesis [74]. MVs display on their membrane functional

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antigens and/or adhesion molecules, a property used to determine their cell origin by flow cytometry. Moreover, cytoskeletal (actin, tubulin), MHC, chaperones (Heat Shock Proteins-HSP), and nucleic acid binding proteins (proteins ribosomal 40S and 60S, histones, elongation factors), metabolic

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enzymes (often mitochondrial), and proteins involved in membrane trafficking (annexins, Rab GTPases) are present [75]. Different types of nucleic acids (DNA, RNA, mRNA, interfering RNA or

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microRNAs) are also carried by EVs. However, mechanisms governing the role of gene information associated with EVs remain to be elucidated [76]. The interaction of MVs and EXOs with the target cells and the transfer of their content modulate the cell response. EVs use a variety of mechanisms to interact with cells and induce the activation of signaling cascades. The modality of action of EVs on target cells depends on both proteins and glycoproteins present on the surface of EVs and those expressed on surface of target cells. This interaction can either stimulate intracellular signaling cascades either be followed by an internalization of vesicles clathrin-coated [77]. The EVs membrane can also directly merge with the

membrane of the target cells with or without endocytosis (macropinocytosis, phagocytosis), release its contents into the cytosol or fuse with endosomes present in the cytoplasm.

Extracellular vesicles, biomarkers of different components of MetS All the risk factors contributing to the genesis and maintenance of the MetS influence the production of EVs and it is difficult to determine the contribution of each separately. However, it is clearly established that patients with MetS show elevated platelets, endothelial cells and erythrocytes derived EVs into the blood compared to healthy subjects [78,79] (Table 1).

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Visceral obesity is considered the most dangerous factor, because it predisposes to the development of IR, which, in turn, promotes the accumulation of lipid, atherosclerosis and hypertension. In obese patients, adipose tissue is infiltrated with pro-inflammatory macrophages that cause chronic lowgrade inflammation associated with obesity [86]. During obesity, the concentration of circulating EVs originating from platelets, endothelial cells, adipocytes and leukocytes [75] increases [80].

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High plasma concentrations of Low Density Lipoproteins (LDL), cholesterol and triglycerides reflect a diet rich in fat and carbohydrates. These conditions induce apoptosis of liver cells in vitro and

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hepatic steatosis in vivo [87]. LDL accumulation in monocytes-macrophages infiltrated into the vascular walls facilitates their transformation into foam cells leading to the formation of atheroma

derived from monocytes [81,82].

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plaque. Overload of lipids causes both in animals model and in humans an increase in circulating EVs

The increase in blood pressure reflects the complex interaction between different regulation systems

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involved in the MetS. The secretion of angiotensin II by adipose tissue regulates the production of aldosterone which plays a crucial role in blood pressure control [88]. The concentration of circulating EVs, mainly MVs, increases in hypertensive animal models [83] and they could be used as a

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prognostic/diagnostic marker of atherothrombotic pathologies [72]. In physiological condition, the activation of the insulin receptor leads to the absorption of glucose

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and increases glycogen and protein synthesis. Obesity is associated with development of IR, which negatively affects both the absorption of glucose and the lipolysis in the insulin dependent tissues [89], causing hyperlipidemia. In the liver, the lack of regulation of glycogenolysis and gluconeogenesis in IR favors hyperglycemia, which affects the functionality of several tissues and organs, such as eyes, nervous system, heart, vessels, kidneys, etc. In the vascular endothelium, IR reduces the production of nitric oxide (NO) and increases the vasoconstriction, resulting in hypertension. IR is the main cause of T2D. The amount of circulating MVs, derived from platelets, monocytes or endothelial cells, significantly increases in T2D patients, compared to healthy subjects

[84]. A positive correlation between monocytes-derived EVs and insulin sensitivity in diabetic patients has been recently suggested [85].

Extracellular vesicles, bioeffectors on target tissues. EVs are key players in cell-to-cell communication and could play a major role in the MetS by their ability to transfer their content to target cells. EVs-induced cellular response are mainly influenced by EVs type, that depends on the cell origin and their composition, rather than by their amount (Figure 3). Vascular effects. The effect of EVs on endothelial dysfunction associated to the MetS has been

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evaluated in experiments in which EVs isolated from patients plasma with or without MetS were injected in mice and it has been observed that EVs induce alterations of the response of the aorta to acetylcholine [90,91]. This causes the production of ROS dependent activation of neutral sphingomyelinase [90] via activation of Fas/Fas Ligand signaling pathway [91]. In addition, high concentrations of glucose or angiotensin-II increase MVs production by endothelial cells leading to

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prooxidant, pro-coagulant and pro-inflammatory responses [92,93]. Injection of these MVs into mice causes the formation of atheromatous plaques through the induction of the production of ROS by

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endothelial cells [93]. Obesity significantly reduces the pro-angiogenic potential of EVs derived from adipocyte mesenchymal cells, in particular by negatively modulating the content of pro-angiogenic

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factors, such as Vascular Endothelial Growth Factor (VEGF), or microRNAs, in particular miR-126 [93,94]. miR-126 is one of the most abundant microRNAs loaded in MVs derived from endothelial cells. It mainly affects the expression of Sprouty-related EVH1 Domain Containing 1 (SPRED1)

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protein and thereby it modulates the angiogenic signaling pathways [93]. Metabolic effects. EXOs derived from adipose tissue of obese mice induce IR associated with macrophage activation that increases secretion of pro-inflammatory cytokines [95]. In addition to

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adipokines, which can modulate insulin sensitivity [75,96,97], EXOs derived from adipose tissue also transfer microRNAs that specifically target transcripts of insulin signaling pathways pro-

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inflammatory and fibrotic signaling proteins [98], suggesting their involvement in the development of T2D. By silencing the gene encoding TLR4, mice are protected against the deleterious effects of adipose tissue-derived EXOs suggesting their role in the inflammatory response [95]. In the hyperlipidemic condition, the saturated fatty acids induce the production of EVs by hepatocytes, muscle cells and adipocytes [75,99,100] that are capable to transfer lipids, mainly ceramides, to muscle cells or macrophages [99,100]. EVs secreted upon lipid stimulation possess also fibrotic and inflammatory properties in hepatocytes [101]. This highlights that EVs could be involved

in the development of NASH and suggests their use for prognostic purposes in order to evaluate the progression of this disease [100]. Recent studies have suggested the pivotal role of EXOs-associated miRNAs in different kinds of metabolic disorders, such as diabetes, fatty liver, obesity and atherosclerosis. Previous studies focused mainly on proteins carried by EVs as key players in cell-to-cell communication, but the new technologies for isolating EXOs and studying their content have highlighted the clinical value, both in diagnosis and treatment, of miRNAs carried by EXOs for the diagnosis and treatment of metabolic disorders (rewieved in Table 2).

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Effects on the immuno-inflammatory system. Atherosclerosis, one of the major cardiovascular complications of MetS, is characterized by chronic inflammation of the arteria tonaca intima that causes accumulation of LDL and apoptotic cells. The oxidation of LDL and the secondary necrosis of apoptotic cells make the environment highly inflammatory and immunogenic. Autophagy in endothelial cells by cleaning and recycling components has a protective role against the development

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of the atheromatous plaques. Recently, it has been highlighted that EVs derived from aortic smooth muscle induce an alteration in autophagic endothelial machinery by transfer of microRNA [112]. EVs

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derived from immune cells could also contribute to maintain and amplify the inflammatory response associated with atherosclerosis. EXOs released by macrophages, dendritic cells or foamy cells induce

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in endothelial cells an inflammatory response [113,114] associated with activation of NFκB, production of pro-inflammatory signals and expression of adhesion molecules, e.g., Inter-Cellular Adhesion molecule-1 (ICAM-1). These effects can be initiated by TNF-α carried by EVs, mainly

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MVs, released by inflammatory macrophages [114]. MVs can transport pro-apoptotic signals to cardiomyocytes by interacting with cells via their TNF-α receptors [115]. Thus, EVs are bioeffectors that contribute to the spread of pro-inflammatory signals, that promote the instability of the

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atheromatous plaques leading to myocardial infarction. Host/microbiota interactions via extracellular vesicles. In recent years, many studies have revealed

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the importance of intestinal microbiota in the development of MetS. In fact, dysbiosis is associated with obesity and liver diseases. EVs represent a link between the host and its microbiota. Intestinal epithelial cells produce EXOs enriched in certain small RNAs that are internalized by bacteria, modulating their growth and their profile genes expression [116]. Similarly, EXOs isolated from the intestinal microbiota of obese mice induce IR in healthy mice [117]. Therefore, EVs are considered as a new actors in the complex communication between the host and its microbiota. Perspectives in EVs involvement in metabolic disorders and conclusions

Today, about 500 million people around the world are affected by metabolic disorders and their complications. World Health Organization (WHO) estimates that this number will increase to about 700 million by the 2045 as the result of uncorrected lifestyle that causes obesity and its related metabolic disorders. At the moment, great efforts are make to prevent and cure metabolic complications and new discoveries in the EVs field encourage the researchers in exploiting these naturally constructed nanovesicles in clinical applications. In fact, EVs are considered very appealing as nanovesicle drug delivery vectors or biomarkers in liquid biopsy because of carried molecules protected by lipid bilayer membrane [118]. This makes EVs particularly exploitable as liquid biopsy source in several disease, including matabolic disorders and related inflammation [119,120].

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However, it is still difficult to use EVs in a wide range of diagnostic applications due to the difficulty in separating pure fraction of EVs. A new methodology integrating acoustics and microfluidics tecniques has been recently suggested to isolate EVs from blood [121].

In summary, associated with several metabolic risk factors, EVs represent new interesting tools in understanding the role of inflammation in MetS. In this manner, EVs could be new biomarkers

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predictive of metabolic pathologies and new exploitable structures in therapy. In fact, the knowledge of the composition of EVs and the understanding their possible activities in cellular targets along with

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the new analytical technologies could help to design innovative strategies to modulate their biogenesis and their physiological and/or pathological actions. This could lead to a customization of treatments

Conflict of interests

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Declarations of interest: none

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according to subtypes of EVs identified in patients, their compositions and their biological activities.

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[121] M. Wu, Y. Ouyang, Z. Wang, R. Zhang, P.H. Huang, C. Chen, H. Li, P. Li, D. Quinn, M. Dao, S. Suresh, Y. Sadovsky, T.J. Huang, Isolation of exosomes from whole blood by integrating acoustics and microfluidics, Proc. Natl. Acad. Sci. U. S. A. 114 (40) (2017) 1058410589. https://doi.org/10.1073/pnas.1709210114.

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Ada Maria Tata is currently associate professor of Comparative Anatomy and Cytology at the Department of Biology and Biotechnology Charles Darwin- University of Rome Sapienza. From 1999 she teaches for undergraduate courses of Biological Sciences and Biotechnology. She holds a PhD in Evolutionary Biology. After, she covered the Post-doc position at the Dept of Neurochemistry, CSIC, Barcelona (Spain) where she studied 1) the cholinergic control of the cell growth and differentiation of normal and pathological myelinating glial cells and 2) the acetylcholine as modulator of the inflammatory state in nervous system disorders. The recent interests are focused on the normal and altered functions of muscarinic and nicotinic cholinergic receptors in neurological disorders (i.e., glioblastoma, multiple sclerosis; neurodegenerative diseases) and the identification of new agonists and antagonists useful as new molecular therapeutic tools. Dr. Tata has published numerous articles as well as book chapters. Her work has been presented at many national and international conferences. She is member of National and International Scientific Societies.

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Cristian Vergallo achieved the degree in Biology and the Ph.D. in Biology and Biotechnology at the University of Salento, Lecce (Italy). From 2011 until now he has held several academic appointments both as Research Assistant at University of Salento, Department of Biological and Environmental Science and Technology (Di.S.Te.B.A.) and as Postdoctoral Fellow (Research Associate) at University of Chieti-Pescara “G. d’Annunzio”, Department of Pharmacy (Chieti, Italy), with responsibilities on research projects, teaching and degree thesis co-supervision.

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His research concerns the study of the underlying mechanisms involved in the interaction between biological systems and i) static/pulsed magnetic fields, ii) nanomaterials or iii) plant-based natural compounds, as well as the molecules expressed by phagocytes and dying cells under different external stimuli. Main investigating methods include optical/electron microscopy (LM, CLSM, TEM and SEM) and proteomics. He has got collaborations with other foreign research groups working in the same fields of application (Hungary, Iran).

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Cristian Vergallo has co-authored about 30 scientific papers, published in peer-reviewed international journals since 2009. He serves both as reviewer and as editorial board member several scientific journals, and has participated, as auditor, speaker or member/coordinator of local organizing committee, in numerous national/international training courses, seminars, workshops and conferences.

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Elisa Panzarini is currently researcher of Comparative Anatomy and Cytology at the Department of Biological Sciences and Technology (Di.S.Te.B.A.), University of Salento, Lecce, where she teaches Cytology, Histology and Embryology (Biotechnology degree) and Developmental Biology (Medical Biotechnology and Nanobiotechnology master degree). She holds a PhD in Biology and Biotechnology from University of Salento. During the PhD, she studied the efficacy of photodynamic therapy as anticancer approach. She collaborates on projects (National Research Council-CNR of Lecce) focused in the design of nanoparticles for drug delivery. She was member of the Lecce section of the National Institute of Nuclear Physics (INFN) and collaborated in the design of a proton therapy protocol based on use of metal nanoparticles. She is currently working on 1) the study of extracellular vesicles released in cancer and metabolic diseases exploitable to design diagnosis devices and new personalized therapeutic approaches; 2) the study of interaction between nanomaterials and living organisms; 3) the involvement of extracellular vesicles in cell-to-cell communication in inflammatory modulation. Dr. Panzarini has published numerous articles as well as book chapters. Her work has been presented at many national and international conferences. She is member of National and International Scientific Societies.

Luciana Dini is currently Full Professor of Comparative Anatomy and Cytology, Department of Biology and Biotechnology Charles Darwin University of Rome Sapienza.

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From 2000 to 2018, she is Full Professor of Comparative Anatomy and Cytology at University of Salento, where she covered several management position: member of the PhD School in Biology and Biotechnology, responsible for the Socrates/ Erasmus/LLP program for the Faculty of Science, component of the Senato Accademico, Director of the Master I level DATA MANAGER IN ONCOLOGY: expert in the design and management of a clinical study (2010-2015) and Director of the international Master ISUFI- Electron Microscopy: a tool for industrial quality and environmental monitoring (2000-2001).

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She has co-authored about 200 scientific papers, published in peer-reviewed international journals. She serves both as reviewer and as editorial board member several scientific journals, and has participated, as auditor, speaker or member/coordinator of local organizing committee, in numerous national/international training courses, seminars, workshops and conferences.

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Her research concerns the 1) NANOSCIENCE AND NANOTECHNOLOGY (Characterization of nanomaterials. Biomedical and agri/food applications of engineered nanoparticles. Nanoparticles and their interaction with biological system), 2) CELL DIFFERENTIATION and CELL DEATH, 3) CELL COMUNICATION based on microvescicles and exosomes release.

Stefano Tacconi, after his master's degree in 2016, he carried out research in Biochemistry and Molecular Biology in the field of mitochondrial function in hepatic steatosis. In 2017 he received a PhD position at the Laboratory of Comparative Anatomy and Cytology of the Department of Biological and Environmental Sciences of the University of Salento. Currently, he works on metabolic disorders, extracellular vesicles and immune function.

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Dr. Elisabetta Carata received a PhD in Biology and Biotechnology from the University of Salento and continued to study as a post-doctoral fellow at the laboratory of Comparative Anatomy and Cytology of University of Salento 2011-2013. She is currently researcher at the Department of Biological and Environmental Sciences and Technologies of University of Salento, Italy, where her work focuses on the effects of TRIM family proteins on autophagy and inflammation in the glioblastoma cells. Knowledge and skills about the following research topics: microscopy (LM, CLSM, TEM and SEM), cell culture, cell biology, autophagy, proteomics, nanomaterials.

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Figures captions

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Figure 1. Main features of MØ, M1 and M2 macrophages. A: Scheme showing up the macrophage polarization from the phenotype MØ (naïve, non-polarized) toward the M1 (pro-inflammatory) or M2 (anti-inflammatory) one. M1 activation is associated with inflammation, tumor resistance, and

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graft rejection, whereas M2 activation is associated with immune regulation, matrix deposition, tissue remodelling, and graft acceptance. B: Schematic representation of the interactive regulation between M1 and M2 signalling. STAT-1 is activated by IFN-γ receptor. IRF-5, NF-kB, and AP-1 are activated

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by TLR-4. Enhanced AP-1 expression is mediated by cytokine receptors. IL-4 receptor signalling activates STAT-6 and IRF-4. The fatty acid receptor activates PPAR-γ. TLR-4 enhances CREB and

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C/EBP levels. The other transcription factors and cytokines listed in Figure 2A also regulate the interaction between M1 and M2 signalling. Abbreviations (in alphabetical order): AP-1 = activator protein 1; Arg-1 = arginase 1; C/EBPs = CCAAT-enhancer-binding proteins; CCL = C-C motif chemokine Ligand; CCR-7 = C-C chemokine receptor type 7; CD = Cluster of Differentiation; CREB = cAMP response element-binding protein; CXCL = chemokine (C-X-C motif) Ligand; FcγR = Fc region gamma receptor; Fizz-1 = found in inflammatory zone protein 1; IFN = Interferon; IGF-1 = insulin-like growth factor 1; IL = Inteleukin; IL-4Rα = Inteleukin 4 receptor alpha; IL-R1= Inteleukin 1 receptor; iNOS = inducible nitric oxide synthase; IRF = Interferon regulatory factor; MCH-II =

major histocompatibility complex class II; MMP-9 = matrix metallopeptidase 9; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; P 50 and 65 = protein of 50 or 60 kDa; PDGF = platelet-derived growth factor; PPAR-γ = peroxisome proliferator-activated receptor gamma; PTX3 = pentraxin 3; ScavengerR-A/B = scavenger receptor class A or B; STAT = signal transducer and activator of transcription; TGF-β1 = transforming growth factor beta 1; TLR-4 = toll-like receptor 4;

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VEGF = vascular endothelial growth factor; Ym1/2 = Ym1 and Ym2 chitinase-like proteins.

ro of -p re lP na ur Jo Figure 2. Main features of extracellular vesicles. A: Schematic representation of the mechanisms of formation of microvesicles and exosomes, highlighting the origin of vesicle proteins. Extracellular vesicles are formed either by budding of the plasma membrane, in which case they are referred to as

microvesicles, or as intraluminal vesicles within the lumen of multivesicular endosomes. Multivesicular endosomes fuse with the plasma membrane to release intraluminal vesicles that are then called exosomes. B: Molecular profiling of microvesicles and exosomes. Extracellular vesicles can carry various cargoes, including cell adhesion molecules, cell-type specific antigens, cytoskeletal molecules, enzymes, lipids, membrane organisers, nucleic acids and other cytoplasmic molecules. This content can widely vary between cells and conditions. C-E: Electron micrographs of EV subtypes (exosomes and microvesicles) released by human THP-1 cells-derived macrophages into cell-culture media. C: cryo-electron microscopy micrographs of EVs harvested from cell-culture media by sequential centrifugal ultracentrifugation; D: transmission electron microscopy

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micrographs of multivesicular bodies containing exosomes; E: scanning electron microscopy micrographs of microvesicles released from cell surface. Abbreviations (in alphabetical order): 14-33 = 14-3-3 proteins; ALIX = asparagine-linked-glycosylation-protein-2-interacting protein X; APP = amyloid precursor protein; ARF-6 = adenosine diphosphate-ribosylation factor 6; CD = Cluster of Differentiation; CXCR-4 = C-X-C chemokine receptor type 4; DNA = deoxyribonucleic acid; ERKs

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= extracellular signal–regulated kinases; FasL = apoptosis-stimulating-fragment Ligand; GPDH = glycerol-3-phosphate dehydrogenase; GTPases = guanosine triphosphate phosphohydrolases; HSP =

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heat shock protein; HSPG = heparan sulfate proteoglycan; ICAMs = intercellular adhesion molecules; LFA-1 = lymphocyte function-associated antigen 1; MHC I and II = major histocompatibility

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complex class I and class II; mRNA = messenger ribonucleic acid; PECAM-1 = platelet endothelial cell adhesion molecule 1; PLD = phospholipase D; PMEL = premelanosome protein; PrP = prion protein; RAB(s) = rat-sarcoma-viral-oncogene-homolog (Ras)-associated binding proteins; RNA(s)

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= ribonucleic acid(s); ROCK = Ras homologous-associated protein kinase; Tau = tau protein; TCR = T cell receptor; TDP-43 = trans-activation-response-element DNA binding protein 43; TFR = transferrin receptor; TSG-101 = tumor susceptibility gene 101; TSPAN = tetraspanin; VPS = vacuolar

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protein sorting-associated protein 4; WNTs = wingless-type mouse-mammary-tumor-virus (MMTV)

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integration site protein family.

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Figure 3. Extracellular vesicles as bioeffectors of metabolic complications. The production of extracellular vesicles of various cellular origins increases during metabolic syndrome. These

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extracellular vesicles affect different target tissues by inducing vascular, metabolic or immunoinflammatory effects. These effects are induced by different extracellular vesicle constituents, such as proteins, microRNA, lipids, etc. Abbreviations (in alphabetical order): Fas(L) = apoptosisstimulating-fragment (Ligand); ICAM-1 = intercellular adhesion molecule 1; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; TNF-α = tumor necrosis factor alpha; VEGF = vascular endothelial growth factor.

Table 1. Extracellular vesicles as biomarkers for prognosis/diagnosis of metabolic disorders.

Obesity Dyslipidemia High blood pressure

References [75,80] [81,82] [83] [84,85]

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Insulin resistance e type 2 diabetes

Features Increase of circulating EVs Release of EVs by adipocytes Increase of circulating EVs Increase of circulating EVs in rat model Increase of circulating EVs in humans Positive correlation between monocytes-derived EVs and insulin sensitivity

Table 2. EXOs-associated microRNAs, their target and function

miR33a/b miR-92a miR-193b/365 miR-103/107 miR-133a miR-199 miR-214 miR-29 miR-122 miR-143

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miR-155

Reference [102] [103] [104] [105] [54,106]

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miR-29a/b

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miR-320c

Function Marker for diabetic nephropathy Marker for type II Kidney diabetic nephropathy Pancreas Insulin secretion Cholesterol metabolism Liver macrophages and hepatocellular carcinoma Marker of brown Brown adipose tissue adipose tissue activity Liver Adipogenesis Marker of Myocardium cardiomyocyte death Liver Marker of NAFLD Endothelial cells Angiogenesis Heart Myocardial fibrosis Cholesterol metabolism Liver and hepatocellular carcinoma Insulin resistance Liver angiogenesis Intercellular Macrophages communication

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miR-130a/145

Target Glomerular mesangial cells

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miRNAs

[107] [108] [109] [105] [110] [105] [105] [111]