and Reviews 51 (2020) 49–60

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Cytokine and Growth Factor Reviews

journal homepage: www.elsevier.com/locate/cytogfr

An emerging interplay between extracellular vesicles and T Alessandra Aiello1, Flavia Giannessi1, Zulema A. Percario, Eisabetta Affabris*

Department of Sciences, University Roma Tre, Rome, Italy

ARTICLE INFO ABSTRACT

Keywords: Extracellular vesicles (EVs) are small membrane-bound particles that are naturally released from cells. They are Extracellular vesicles recognized as potent vehicles of intercellular communication both in prokaryotes and eukaryotes. Because of Cytokines their capacity to carry biological macromolecules such as , and nucleic acids, EVs influence dif- TNFα ferent physiological and pathological functions of both parental and recipient cells. Although multiple pathways IL-1β have been proposed for cytokine secretion beyond the classical ER/Golgi route, EVs have recently recognized as IFN an alternative secretory mechanism. Interestingly, cytokines/ exploit these vesicles to be released into the extracellular milieu, and also appear to modulate their release, trafficking and/or content. In this re- view, we provide an overview of the cytokines/chemokines that are known to be associated with EVs or their regulation with a focus on TNFα, IL-1β and IFNs.

1. Introduction data have indicated that these vesicles have different origin, size and composition and, based on their biogenesis, have been divided in three During the course of evolution, both prokaryotes and eukaryotes main subgroups: , exosomes and apoptotic bodies [8] (see have developed -to-cell communication strategies that play a vital Fig.1). Although the nomenclature is still a matter of debate because role in the systemic function of multicellular organisms. Classically, there is not a standard method of isolation and analysis of the EVs [9, intercellular communication is mediated through direct cell-cell contact 10], the term microvesicle generally refers to vesicles (150−1000 nm) (juxtacrine signalling) and/or by secreting a diverse array of soluble that bud directly from the plasma membrane. Before their shedding, factors such as , growth factors, cytokines and chemokines. cytoplasmic protrusions are generated by the cell, which undergoes These soluble molecules can act both on the cell itself (autocrine sig- fission events, finally resulting in the budding of microvesicles fromthe nalling) and on neighbouring (paracrine signalling) and distant cells cellular membrane [11]. Although microvesicles can be generated by (endocrine signalling). In the last two decades, transfer of extracellular resting cells, stimulating events, leading to increased intracellular cal- vesicles (EVs) has emerged as a third mechanism of intercellular com- cium levels, result in cellular membrane remodelling and enhance mi- munication. EVs are heterogeneous membrane-enclosed structures re- crovesicles secretion [12]. The term refers to smaller vesicles leased by cells into the extracellular milieu in an evolutionally con- (30−150 nm) that are generated intracellularly as intraluminal vesicles served manner, ranging from organisms such as prokaryotes to higher (ILVs) by inward invagination of endosome membranes, giving rise to eukaryotes and . The first observation of EVs in the extracellular multivesicular bodies (MVBs). These endosomal compartments then milieu dates back to the late 1940s when Chargaff and West observed fuse with lysosomes for ILV degradation, or with the plasma membrane EVs as procoagulant platelet-derived particles in normal plasma [1]. releasing the ILVs into the extracellular milieu where they are called Since then, EVs have been isolated from most cell types and biolo- exosomes (reviewed by ref [9,10]). This type of vesicle is the most in- gical fluids, including blood, urine, saliva, breast milk, amniotic fluid, vestigated class of EVs, and their name was first proposed in 1987 when ascites, cerebrospinal fluid, bile and semen [2–7]. The accumulating Johnstone and colleagues observed by ultrastructural studies the

Abbreviations: EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles; ER, endoplasmic reticulum; DCs, dendritic cells; IFN, Interferon; ISG, interferon-stimulated genes; NK, Natural Killer; TLRs, Toll-like-receptors; LPS, lipopolysaccharide; UPS, unconventional secretion; ABC, ATP binding cassette; TNF, tumour necrosis factor; CD40L, CD40 ; FasL, Fas ligand; TRAIL, TNF-related inducing ligand; TACE, TNFα converting enzyme; TEVs, tumor-derived EVs ⁎ Corresponding author at: Department of Sciences, Roma Tre University, Viale Guglielmo Marconi, Rome, Italy. E-mail addresses: [email protected] (A. Aiello), [email protected] (F. Giannessi), [email protected] (Z.A. Percario), [email protected] (E. Affabris). 1 A.A. and F.G. equally contributed to the work. https://doi.org/10.1016/j.cytogfr.2019.12.003 Received 20 November 2019; Received in revised form 17 December 2019; Accepted 17 December 2019 Available online 18 December 2019 1359-6101/ © 2020 Elsevier Ltd. All rights reserved. A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60 presence of vesicles released by MVBs fusing with the response, it is increasingly important to understand the secretory during the differentiation of reticulocytes in red blood cells [13]. Fi- (exocytic) pathways and the endocytic compartments involved in cy- nally, apoptotic bodies are released when plasma membrane blebbing tokine transport, the regulatory molecules and cellular machinery that occurs during apoptosis. dictate the levels and the timing of each cytokine released (reviewed in Initially, it was believed that EVs were simply removing un- ref. [19,20]). Even though cytokines were earlier known as “soluble necessary proteins and other molecules from the releasing cells, and factors”, today it is recognized that they can also function as integral were therefore considered the “garbage bins” of the cells [14]. In the membrane proteins and some are never released from the cell. mid-1990s, EV were shown to have an immunological function in an- Many secreted proteins, including cytokines, and proteins integral tigen presentation and as vesicles able to induce T cell responses [15]. to the plasma membrane, reach their destination using the classical Recently, EVs were shown to be also alternative carriers for the delivery secretory pathway (see bottom section of Fig. 2). These proteins usually of cytokines/chemokines. In this review we provide an overview of the carry a signal peptide at the N terminus and/or a transmembrane do- cytokines/chemokines that are known to be transported into extra- main that directs their insertion into the endoplasmic reticulum (ER) cellular vesicles and/or regulate the vesicular secretion, content or for synthesis as either soluble or transmembrane precursors. They are target with a focus on three major cytokines: TNFα, IL-1β and inter- then trafficked in COPII-coated vesicles to the Golgi apparatus forfur- ferons (IFNs). ther processing. Finally, at the trans-Golgi network (TGN) they are loaded into vesicles or carriers for constitutive delivery to the plasma membrane from which they can be secreted, or not, into the extra- 2. Cytokines and their release through an unconventional cellular space or delivered to other organelles (for a comprehensive pathway: the extracellular vesicles review see ref. [21]). Although all innate immune cells have the capacity for constitutive Cytokines are small, non-structural proteins with low molecular exocytosis, the release of cytokines and other proteins can occur weights (< 30 kDa) that act as intercellular messengers by exhibiting through regulated secretory pathways in some specialized cell types autocrine, paracrine and endocrine actions [16–18]. Pleiotropism re- such as granulocytes, cytotoxic T cells, natural killer (NK) cells, en- presents the hallmark of a cytokine [19]. Beyond their role in the innate dothelial cells and platelet [22]. In these cases, cytokines can be tightly and adaptive immunity where they integrate function of several cell packed and stored in secretory granules, lysosome-related organelles or types, cytokines have a major role in various functions that affect every secretory lysosomes and later released at the cell surface. The “regu- organ system in the body including cell differentiation, inflammation, lated” or “granule-mediated” pathways for exocytosis or secretion can angiogenesis, tumorigenesis and microbial pathogenesis. use a combination of granules and smaller secretory vesicles to release In all likelihood, cytokines evolved from a primordial form re- stored cytokines. Unlike constitutive secretion that provides a con- presented by intracellular proteins interacting with DNA, acting as tinuous release of newly made proteins through a stream of small-vo- transcription and repair factors but poorly, if at all, secreted. Only later lume carriers, regulated secretion has the ability to orchestrate the in evolution did their active secretion appear by allowing cytokines to rapid delivery of a large bolus of cytokines to a specific site and in act as extracellular ligands for specific membrane receptors present on response to a specific signal [20]. Therefore, the secretory pathway is responsive target cells [18]. Cytokines and their receptors possess a often chosen according to the final scope: enhancing the rapidity or high affinity for each other with dissociation constants ranging from efficiency of cytokine release, directing cytokines to a target ormax- 10−10 to 10-12 M, thus explaining why cytokines exert their biological imizing their dispersal. effects in picomolar concentrations. Not surprisingly, multiple me- The molecules identified as part of the trafficking machinery com- chanisms have evolved for cellular control of cytokine secretion by prise the SNAREs (reviewed by ref. [19]). They include subfamilies of highlighting the potency of these mediators and the fine tuning re- vesicle-associated membrane proteins (VAMPs) and syntaxins that are quired to mount an effective but limited response, in order to prevent classified as R-SNAREs and Q-SNAREs, according to the amino acid an excessive and dysregulated release that could be responsible of a composition of their core SNARE domains. The final event necessary for wide range of diseases, such as acute and chronic inflammatory and the delivery and release of cytokines at the cell surface is the fusion of autoimmune diseases. Since the secretion of cytokines by immune cells granules, vesicles or endosomes with the plasma membrane, a plays a significant role in determining the course of an immune

Fig. 1. Extracellular vesicles: biogenesis and release. EVs are mainly classified in three subgroups: a) microvesicles/microparticles that bud from the plasma membrane; b) exosomes that are gen- erated as ILVs by inward invagination of en- dosomes membranes giving rise to MVBs and then released into the upon fusion of MVBs with the plasma membrane; c) Apoptotic bodies that blebs from cells under- going apoptosis. N, nucleus; ER, endoplasmic reticulum; G, Golgi complex; MVB, multi- vesicular bodies; ILVs, intraluminal vesicles; Ly, lysosome; EE, early endosome.

50 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

Fig. 2. Different secretory pathways are re- sponsible for the release of cytokines. Secretory pathways can be divided in two main types: the classical and the unconventional one. In the classical pathway, proteins (e.g. cyto- kines) are synthesized in the ER and trans- ported to Golgi complex, from where they can be loaded into membrane-bound vesicles and released, or packaged and stored into secretory granules (regulated exocytosis) and later re- leased. The unconventional pathways involve the release of cytokines bypassing the classical ER/Golgi route. In this case, different me- chanisms have been proposed for crossing the plasma membrane: the use of membrane transporters, exosome release and microvesicle shedding. N, nucleus; ER, endoplasmic re- ticulum; G, Golgi complex; MVB, multi- vesicular bodies; ILVs, intraluminal vesicles; Ly, lysosome; Exo, exosomes; MV, micro- vesicles.

phenomenon that requires the involvement of other trafficking ma- also the result of specific physiological needs, depending on whether chinery components, including Rab GTPase [20]. the cytokine acts near the secreting cell or at a distance. In support of In addition to the conventional membrane-based transport path- this concept, Fitzgerald and colleagues reported that tissue explants, ways, other distinct exit routes have been described, collectively called where cells are sustained in close proximity by neighbouring cells, re- unconventional protein secretion (UPS) [23] (see upper section of leased more cytokines in a soluble form than T cells or monocyte sus- Fig. 2). Two kinds of proteins are unconventionally secreted. The first pensions, where a greater amount of cytokines are found associated category includes leaderless proteins, i.e. proteins lacking a signal with EVs [24]. peptide, including different growth factors and cytokines such as IL-1β Since EV-associated cytokines are biologically active upon inter- and IL-18, which cannot be targeted to the ER and, thus are translated acting with sensitive cells, and the small size and flexibility of EVs on free ribosomes in the cytoplasm. These proteins can be secreted via enable them to cross major biological barriers such as blood-brain three pathways mediated by distinct mechanisms: Type I or pore- barrier, they represent an important system of cell-cell communication mediated translocation across the plasma membrane; Type II or ABC in both health and disease. EV-associated cytokines demonstrate a wide transporter-based secretion and Type III or autophagosome/endosome- range of pleiotropic functions in several biological processes. For in- based secretion. Instead, the second category comprises proteins that, stance, in HIV-positive individuals most cytokines were markedly en- despite having signal peptide and/or transmembrane domain, enter the riched in exosomes and the exposure of naïve peripheral blood mono- ER but bypass the Golgi apparatus to reach the plasma membrane. This nuclear cells to these exosomes resulted in the induction of CD38 is the Type IV, or Golgi-bypass, pathway [23]. expression on naïve and central memory CD4+ and CD8+ T cells, In addition to the UPS pathways, secreted proteins such as cytokines probably contributing to the viral propagation via bystander cell acti- and chemokines can reach the extracellular space also through EVs. In vation [27]. Moreover, plasma exosomes from HIV-positive subjects particular, they are packaged into microvesicles shedding from the correlated with increased oxidative stress markers and induced the plasma membrane or in MVBs and secreted in membrane-bound ve- expression of genes related to interferon responses and immune acti- sicles like exosomes. In this regard, Fitzgerald and colleagues showed vation [29]. Also, tumour-derived EVs containing transforming growth that cytokine encapsulation into EVs was a general biological phe- factor β (TGFβ) are implicated in the induction of myeloid-derived nomenon observed in vitro, ex vivo and in vivo; essentially all cytokines suppressor cells that promote tumour progression [30] by stimulating can be encapsulated into EVs [24]. Interestingly, the pattern of en- the migration of cancer cells [31], as well as tumour immune escape by capsulated cytokines changes in response to stimulation, suggesting inhibiting the lymphocyte response to IL-2 in favour of regulatory T that the association with EVs is not necessarily a property of a parti- cells [32]. Furthermore, proteomic analysis revealed that exosomes also cular cytokine. Indeed, Kandere-Grzybowska and colleagues showed contained other proteins, including vascular endothelial growth factor that IL-6 was excluded from the secretory granules, but was secreted in (VEGF), epidermal growth factor (EGF), MCP-1 and IL-4 that promote 40–80 nm vesicular structures upon stimulation of human mast cells tumour cell survival, proliferation and migration [32]. In another study, with IL-1 [25]. Kodidela and co-workers analysed the cytokine profiling cancer-derived exosomes containing TGFβ were shown to induce dif- of exosomes deriving from the plasma of HIV-infected alcohol drinkers ferentiation of fibroblasts into myofibroblasts that support tumour and cigarette smokers and observed that some cytokines (TNFα, IL-1β, growth, vascularization and metastasis [33]. Interestingly, this effect IL-8, IL-6, IL-1ra, IL-10) and chemokines (MCP-1 and RANTES) were was observed only when exosomes expressed sufficient TGFβ levels on present in exosomes of healthy subjects, but their levels varied between their surface, together with expression of the transmembrane pro- the study groups [26]. These data indicate that external stimuli, such as teoglycan betaglycan on the exosomal surface. Betaglycan was shown the alcohol and tobacco, may induce changes in cytokines/chemokines to have a key role in tethering TGFβ to the vesicle membrane and in secreted into exosomes. Additionally, pathological conditions can affect transferring TGFβ to recipient cells by increasing the affinity of TGFβ the profile of cytokines associated with EVs. For instance, in HIV-po- for binding to type II [33]. sitive individuals or in diabetic patients the profile of cytokines in EVs is EV-associated cytokines are important in physiological functions; significantly increased [27,28]. In patients with diabetes, the associa- macrophage-derived exosomes containing IL-6 and IL-8 are actively tion of specific cytokines with EVs was strongly influenced by disease transported into the human placenta and stimulate placental cytokine duration and successful treatment [28]. release, suggesting a novel mechanism by which immune cells can The release of a cytokine in a free or EV-associated form might be signal to the placenta and regulate the immune response against the

51 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

fetus during pregnancy [34]. Another example is represented by the association of macrophage migration inhibitory factor (MIF) with [ 35 ] [ 57 ] [ 54 ] [ 53 ] [ 52 ] [ 50 ] [ 31 ] [ 42 ] [ 25 ] [ 41 ] [ 40 ] exosomes released by the epididymal epithelium; these exosomes are [ 34 , 51 ] [ 45–49 ] [ 36–39 ]

[ 43 , 44 71 ] transferred to spermatozoa and modulate sperm motility during the [ 30–33 , 55 56 ] Ref. transit along the male reproductive tract [35]. Many other examples of EV-associated cytokines and their effects on recipient cells are reported in Table 1. In conclusion, it should be noted that the choice of a particular secretory pathway does not necessarily exclude other mechanisms: some proteins exploit both conventional and unconventional pathways for secretion, and the use of different exit routes may be dictated bythe stimulus received or the cell environment. The secretion of IL-1β for example makes use of various molecular mechanisms and pathways (for review, see Sitia and Rubartelli [58]. In the following paragraphs the attention will be focused on three main cytokines: TNFα, IL-1β, and IFNs.

3. Tumour necrosis factors

The term tumour necrosis factor (TNF) identifies a superfamily that comprises 19 ligands, including TNFα, TNFβ, CD40 ligand (CD40 L), to neighbouring cells. Fas ligand (FasL), TNF-related apoptosis inducing ligand (TRAIL) and

trans 29 receptors. Its members regulate several complex signalling pathways leading to apoptosis, survival, inflammation, and antiviral state [59]. The first member of TNF superfamily discovered and the mostex- tensively studied is TNFα (old name cachectin). Its discovery dates back to 1970s and it was named “tumour necrosis factor” because re- Modulation of sperm motility during the epididymal transit. Differentiation of monocytes into immature DCs. Induction of regulatory T cells,myeloid-derived regulation suppressor of cells cancer and cell differentiation migration, inductionin myofribroblasts. of Angiogenesis Monocyte recruitment Antitumor immune response by chemoattractingcells. and activating dendritic cells and T production (IL-8, MCP-1, MIP-1β, MIP-1α) by human monocytes. Macrophage chemotaxis Activate T cells making themMCP-1, MIP-1β, resistant RANTES to and apoptosis, TNFα induceredox through in signalling a epithelial in TNFα-mediated cells pathway IL-8, Caspase and activation transmit and apoptosis in tumor cells and lymphoid cells. Regulation of cancer cell migration. Induction of neutrophilic chemokines (MCP-1 and IL-8). Effects on recipient cells sponsible for the necrosis of some tumours in vivo and in vitro [60]. TNFα is a potent pro-inflammatory mediator that has a pivotal role in the inflammatory action of the innate immune system. This cytokine can stimulate the proliferation of normal cells, exerts cytolytic or cy- tostatic activity against tumour cells and to cause inflammatory, anti- viral and immunoregulatory effects [61–64]. TNFα exists in two forms: a type II (mTNFα, also

raft-dependent sometimes referred to as pro-TNFα) and a soluble form (sTNFα) derived

via from the membrane form by proteolytic cleavage. TNFα is initially synthesized as a transmembrane precursor protein of 25 kDa, after which it is transported via the rough endoplasmic reticulum (RER), Golgi apparatus and the recycling endosomes to the cell surface [65]. The monomers of TNFα associate at the plasma membrane as non- covalent trimers, prior being cleaved by the metalloprotease called TNFα converting enzyme (TACE or ADAM17) [66]. As result, the 17 kDa soluble TNFα, a trimeric soluble cytokine, is released and found in blood plasma, where it circulates throughout the body and acts at a distance from the site of its synthesis. The molecular actions of both soluble and membrane TNFα typically occur through binding to one of two receptors: TNFR1 (also sometimes referred to as p55/p60), and TNFR2 (also known as p75/p80) [67]. Importantly, the two receptors differ by the presence of an intracellular death domain (DD) atthe Associated to exosomes released byto the spermatozoa. epididymal epithelium and transferred Detected on the surface of DC-derived EVs. Associated with thymus exosomes-like particlesand and microvesicles. tumor-derived exosomes Associated with platelet microparticles. Detected in platelet microparticles. Exosomes derived from heat-stressed tumorpathway. cells Associated to macrophage-derived exosomes and tumor-derived microvesicles Induction of placental inflammatory cytokines and modulation of Exosomes produced by synovial fibroblastsrheumatoid obtained arthritis, from exovesicles patients released with from(DCs) LPS-activated or dendritic tumour cells cells. derived exosomes and microvesicles released from melanoma cells. Associated with tumor-derived exosomes. Native form is released from intestinal epithelial cells. Released in vesicular structures (40–80 nm) by mast cells upon IL-1 stimulation. Microvesicles shedding from human macrophages. Microvesicles shedding from mature DCsfrom and murine microglial macrophages. cells andEndothelial exosomes cell-derived apoptotic bodies. carboxyl end of TNFR1 that is able to drive either apoptosis or in- flammation through interaction with associated adaptor molecules. Unlike TNFR1, TNFR2 lacks a DD, but it possesses an intracellular TRAF-binding motif, by which it also has the potential to modulate the inflammatory status [66]. Activation of TNFR1 was found to stimulate NF-κB expression to a significantly greater extent than TNFR2 [68]. Furthermore, the two receptors are expressed on different cell types: while most cells express constitutive but low levels of TNFR1, only MIF GM-CSF TGF-β VEGF CCL5 CCL2, CCL3, CCL4, CCL5 and CCL20 IL-8 (CXCL8) CX3CL1 (fractalkine) Associated with microparticles released from apoptotic lymphocytes. Membrane bound or soluble TNFα Fas ligand and TRAIL Microvesicles released during activation-induced death of human T cells, or DC- IL-10 IL-32 IL-6 IL-18 IL-1β IL-1α Cytokines/chemokines Secreting cells some cells, such as leukocytes and endothelial cells, express detectable surface levels of TNFR2 [67]. The expression levels of TNFR proteins can also be regulated by cytokines, such as IFNγ. Although both TNFα forms can bind to each of the two receptors, they display a dichotomous effect: the membrane TNFα preferentially binds to the TNFR2 with high

factors affinity, whereas the soluble form interacts with TNFR1 with high specificity [69,70]. Others Chemokines Tumor necrosis Interleukins

Table 1 Examples of EV-associated cytokines and chemokines. The nomenclature used to define the specific extracellular vesicles in which cytokines/chemokines were detected is the same adopted in the correspondent articles. TNFα usually exploits the conventional ER-Golgi route and the

52 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

Fig. 3. The connection between Type I IFN, TNFα, IL-1β and EV secretion. a) Type I IFN induces the expression of ISG15, an ubiquitin like-modifier that binds MVB proteins, such as TSG101, through a process referred to as ISGylation. ISG15 conjugation triggers MVB fusion with lysosomes and de- gradation, thus inhibiting exosome secretion. b) Schematic representation of classical and unconventional secretion of TNFα. In the clas- sical pathway (black arrows), new synthesized pro-TNFα is transported from ER through Golgi complex to recycling endosomes and then at the plasma membrane where it is cleaved by TACE and the soluble form is released into the extracellular space. TNFα can also un- conventionally (red arrows) secreted in exo- somes following the rerouting of TACE into Rab4+ endosomes where it converged with its substrate pro-TNFα. For instance, in presence of HIV Nef protein, the NAKC (Nef-associated kinase complex) forms a complex with the in- tegrin-associated adaptor protein paxillin leading to the phosphorylation of paxillin and TACE, finally resulting in the association of paxillin with activated TACE. The latter is transferred into lipid rafts and from here shuttled into EVs. c) IL-1β is released into EVs through an inflammasome-dependent mechanism. A first signal induces the expression of NLRP3 and the precursor form of IL-1β (indicated as pro-IL-1β). The second signal activates the inflammasome, which catalyses the processing of pro- IL-1β and gasdermin D. The latter forms a pore allowing the direct secretion of IL-1β and enhancing ionic fluxes across the membrane, which amplify thein- flammasome activation triggering a cascade of events that affect the membrane curvature causing the shedding of microvesicles containing IL-1β. MatureIL-1βinthe cell can be also recruited into ILVs of MVBs and thus released into exosomes. N, nucleus; ER, endoplasmic reticulum; G, Golgi complex; MVB, multivesicular bodies; ILVs, intraluminal vesicles; Ly, lysosome; Exo, exosomes; MV, microvesicles. endosomal network for delivery to the plasma membrane, although infection in the routing of TACE and the secretion of vesicular TNFα differences in the exocytic pathway responsible for TNFα release have endosomes [72]. In detail, membrane-associated Nef recruits the been observed. In mast cells, TNFα can be secreted both via secretory Polycomb protein Eed and other kinases and adaptor proteins, which granules and by constitutive vesicle trafficking. Mature eosinophils also constitute the so-called NAKC (Nef-associated kinase complex), and contain a population of specific granules responsible for the storage of mimics an receptor signal. Since Eed binds integrin subunits, many cytokines, including preformed TNFα. In contrast, in cytotoxic T the NAKC forms a complex with Paxillin, the integrin-associated and NK cells, TNFα bypasses the granules and is secreted by con- adaptor protein, leading to the phosphorylation of Paxillin and TACE, stitutive carriers or by recycling endosomes [20]. respectively by Lck, Pak2 and Erk1/2. These events change the complex In addition to the mechanisms mentioned above, the secretion of contributing to the activation of TACE and its association with Paxillin vesicular TNF endosomes has been recently described as an alternative followed by their transfer into lipid rafts. Once transferred to lipid rafts, pathway for TNFα secretion (see Fig. 3b). In this regard, Obregon and activated TACE is shuttled into EVs via pre-existing Rab4+ and pro- colleagues reported that exovesicles derived from lipopolysaccharide TNF-containing compartments [72]. Additionally, Ostalecki and col- (LPS)-activated dendritic cells (DCs) carry large amounts of soluble 17 leagues showed that the neurogenic locus notch homolog protein 1 kDa TNFα [44]. DC-derived exovesicles containing major histo- (Notch1) is a crucial co-factor in the endosomal trafficking of TACE compatibility complex II (MHC-II), CD40 and CD83 molecules in ad- [73]. In another study recently conducted by Zhao and co-workers it dition to TNFR1, TNFR2 and TNFα are internalized by epithelial cells was verified whether the role played by Lck and Hck in regulating the and stimulate the release of chemokines and cytokines such as IL-8, HIV Nef-induced uploading of TACE into EVs was a general property of MCP-1, macrophage inflammatory protein 1β (MIP-1β), RANTES and Src family tyrosine kinases (SFK) [74]. In this regard, they identified a TNFα itself, suggesting the potential role of EVs in amplifying the tyrosine phosphorylation-regulated secretion pathway mediated by Hck magnitude of the innate immune response [44]. On the other hand, a that enables vesicle secretion of TACE. This alternative pathway turned membrane bound form of TNFα was observed by Zhang and co-workers out to be independent of iRhom2, a member of rhomboid-like super- in exosomes produced by synovial fibroblasts obtained from individuals family that acts as chaperon or regulatory subunit controlling the with rheumatoid arthritis (RASF) [43]. These exosomes activate Akt transport of TACE pro-domain from the ER to the Golgi network [74]. and NF-κB pathways and render T cells resistant to apoptosis, probably Since in the latter TACE usually undergoes maturation following the contributing to the T-cell mediated pathogenesis of rheumatoid arthritis removal of its pro-domain by the pro-protein convertase Furin, TACE [43]. Instead, Söderberg and co-workers reported that melanoma-de- must converge with Furin into post-Golgi MVB-like endosomal com- rived TNFα-containing exosomes transmit redox signalling in trans to partment from where EVs, containing a mature but hypoglycosylated neighbouring cells by inducing high levels of reactive oxygen species in TACE, originate for proteolytic cleavage to occur in the Golgi-bypass T cells, thus suggesting a possible role of “immune counterattack” for pathway [74]. Altogether these events suggest that TNFα vesicular tumour-derived exosomes [71]. In order to have a larger pool of pro- secretion mechanism is spatially separated and not necessarily con- TNFα cleaved intracellularly and secreted through vesicular endo- nected to TNFα surface shedding. In this respect, Ostalecki and col- somes, it is required the translocation of the TNF-converting enzyme leagues speculate that activation stimuli that include internalization TACE into Rab4+ early endosomes, where the protease converged with signals for TACE (e.g., Nef or PMA) induce endosomal pro-TNFα clea- its substrate pro-TNFα. Lee and colleagues demonstrated a specific role vage and secretion, whereas signals confined to the plasma membrane played by the multifunctional accessory protein Nef during HIV cause TNFα shedding from the cell surface [73].

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Although the role of vesicular TNFα has been less studied than its associated with vesicles [90–95]. One of the best-known proteins that soluble counterpart, different EVs also stimulate the activation of target forms the inflammasome is the NOD-like receptor protein 3 (NLRP3). cells through TNFα-signalling pathway. Lee and colleagues showed that Once activated, NLRP3 undergoes a conformational change that allows peripheral blood mononuclear cells that ingested Nef/ADAM17-con- its oligomerization through the association with other cytoplasmic taining plasma extracellular vesicles (HIV-pEVs) release TNFα [72]. proteins such as the adapter apoptosis-associated speck-like protein Moreover, exosomes from HIV-1-infected cells enhance T-cell activa- containing a C-terminal caspase recruitment domain (ASC). As result, a tion, viral replication and viral reactivation from HIV reservoir through structure is formed that, in turn, acts as a scaffold for pro-caspase-1 a Nef- and TNF-dependent mechanism [75,76]. maturation. Once mature, caspase-1 cuts and activates its target pro- In addition to TNFα, other TNF superfamily members including teins, including pro-IL-1β (for a comprehensive review see ref [96]). FasL, TRAIL and CD40 L are sorted into EV membrane. Different studies Two signals are required for NLRP3 activation: the first one, known reported as cytotoxic T lymphocytes (CTLs), NK cells and DCs kill target as priming signal, is triggered by the ligand-binding interactions of dif- cells through the release of FasL-carrying EVs [46–48]. DC-derived ferent Toll-like receptors (TLRs) or other pattern recognition receptors exosomes also express on their surface TNFα, FasL and TRAIL by which (PRRs) and induces the activation of the NF-κB pathway, leading to the they can trigger caspase activation and apoptosis in tumour cells. These transcription and transduction of pro-IL-1β, as well as of inflammasome vesicles activate and stimulate NK cells to secrete interferon-γ (IFNγ) components. The second signal allows the activation of the inflamma- through a TNF-dependent signalling [47]. Moreover, tumour cells have some that then cleaves the immature form of caspase-1 constitutively been observed to release FasL-bearing EVs, which maintain their expressed in cells. The active form of caspase-1 induces the maturation of functional activity in triggering Fas-dependent apoptosis of lymphoid different pro-inflammatory cytokines such as IL-1β, IL-18 or other pro- cells [49]. Such vesicles may hinder lymphocytes and other im- teins like gasdermin D. Mature gasdermin D proteins are involved in the munocompetent cells from entering neoplastic lesions and exert their formation of pores by which cytokines are released. These channels are antitumor activity. On the other hand, mast cells and platelet release also able to facilitate the perturbation of the transmembrane calcium/ CD40L-containing EVs [77,78]. Not only the ligands but also the TNF potassium equilibrium, another strong signal that stimulates the in- receptors appear to be associated with EVs. In this respect, Hawari and flammasome formation [96], [97]. The first signal seems not to be en- colleagues identified the release of vesicles that range in diameter from ough for a significant release of EVs [98], whereas the second one ap- 20 nm to 50 nm as a mechanism for the generation of soluble, full- pears to stimulate a robust EV secretion. Different types of stimuli have length 55 kDa TNFR1 [79]. been reported to promote inflammasome activation finally resulting in In conclusion, the release of TNF superfamily molecules into EVs IL-1β secretion via vesicles: endogenous danger signal such as extra- decreases the risk of their degradation by surface protease, increases cellular ATP that leads to the strong activation of NLRP3 inflammasome their local concentration into the extracellular milieu and favours their increasing the release of EVs. In this respect, LPS- stimulated THP-1 cells aggregation into trimers, thus augmenting their biological activity [48]. shed pro-IL-1β containing microvesicles after activation of P2 × 7 re- ceptors by exogenous ATP stimulation [36]. Similarly, ATP activates 4. Interleukin-1 vesicle-mediated unconventional protein secretion from human macro- phages [92]. Another stimulus is represented by the ionic fluxes that IL-1 family comprises 11 members including IL-1α, which is con- cause modifications of membrane polarization. For instance,+ K efflux + sidered the ancient ancestor of this family, and IL-1β [80,81]. Initially, simultaneously to Ca2 influx causes inflammasome activation and hasa they were considered interchangeable because both are produced by well-established effect on vesicular production. Calcium influx causes inflammatory cells and bind to the same receptor (IL-1R) on target cells also the activation of different calcium-dependent proteins such as cal- with similar affinity. Subsequently, it became clear that these twoin- pains, floppase and scramblase involved in membrane and cytoskeletal terleukins presented important differences. IL-1α is produced by dif- modifications, thus facilitating the release of EVs[99]. Calpain is a cy- ferent cell types and it is directly synthesized as an active molecule, tosolic enzyme involved in the processing of different cytoskeletal pro- which is mainly localized in the nucleus and poorly secreted into the teins and its inhibition appears to reduce vesicle shedding [100]. Calpain extracellular space. In contrast, IL-1β is produced by specific cell types, inhibition, following the activation of ionic fluxes mediated by P2 × 7- mainly as cytoplasmic protein, and is synthesized as a precursor that receptor, reduces the secretion of EVs, and also inhibits the inflamma- requires a maturation process to be converted into its active form some activation, suggesting the existence of a connection between these [82,83]. In light of this, necrosis causes the release of a functional IL-1α two processes [92]. Other two signals that induce NLRP3 activation al- and a pro-, but not active, IL-1β into the extracellular milieu [84,85]. lowing IL-1β secretion are lysosomal damage and the release of cathe- IL-1β is a potent pro-inflammatory cytokine that was originally psine B in the cytosol. These events can be triggered by both endogenous identified as endogenous pyrogen. Additionally, it has stimulatory ef- and exogenous particulates (Monosodium urate crystals, CPPD crystals, fects on CD4+T cells and promotes differentiation into the T helper cell cholesterol crystals, amyloid b, silica crystals, asbestos, and alum, β- lineages, particularly Th17 cells and a non-classically derived Th1 cell glucans) (reviewed by ref [96]). Cathepsine B can be involved in the lineage [86]. secretion of EV-associated proteins after inflammasome activation trig- The release of the active form of IL-1β follows a finely regulated gered by dsRNA recognition in human macrophages [101]. process [87]. The cleavage is performed by the IL-1-converting enzyme In addition to the pathways mentioned above, there is a non-cano- (ICE), renamed caspase-1, which is contained within a specialized in- nical route for the activation of the inflammasome and the maturation tracellular complex called inflammasome [88]. Since IL-1β lacks the of IL-1β that involves the caspase-4/5, which directly recognize in- leader sequence, it is mostly secreted through an active pathway that tracellular LPS. The caspase-4/5-mediated activation of the inflamma- does not follow the classical ER-Golgi route [89]. In this respect, var- some strongly stimulates the release of IL-1β, IL-18 and other EV-as- ious secretory pathways have been proposed: i) passive release by dying sociated proteins. Accordingly, a caspase-4 inhibitor blocks the EV cells; ii) translocation across the plasma membrane; iii) translocation secretion of LPS-stimulated human macrophages [94]. into intracellular vesicles followed by exocytosis; iv) release via exo- To date, few articles described the release of both IL-1β and its somes or microvesicles (for a comprehensive review see ref. [58]). precursor into the vesicles upon inflammasome activation and the se- Regarding the last mechanism, it is known that the production of EVs is cretory mechanism involved has not yet well defined [37,41,91,102]. related to many inflammatory processes, including the maturation Altogether these data show the relationship between EVs and the ma- process of IL-1β (see Fig. 3c). The latter is dependent on the formation turation process of IL-1β, suggesting that EVs can represent an alter- of the inflammasome, a multiprotein complex of innate immunity that native exit route for this cytokine. can also be involved in the secretion and loading process of proteins

54 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

5. Interferons (IFNs) 5.2. IFNs and EV secretion

5.1. Introduction to the IFN system As previously mentioned, EVs originate through a budding process from the cellular membrane (microvesicles), or upon fusion of MVBs IFNs, discovered by Isaacs and Lindenmann in 1957, are funda- with the plasma membrane (exosomes). Exosomes generation can occur mental effectors of antimicrobial and antitumor innate immunity being in ESCRT-dependent or -independent manner using molecules and important regulators of the adaptive immune response. They act in a mechanisms that all enveloped viruses have hijacked from the infected specie-specific manner and are divided in three antigenically unrelated cells to assembly and release viral particles [129]. In this respect, en- groups: type I IFNs (IFN-I, i.e., a group of structurally similar cytokines veloped viruses, and in particular retroviruses, have been proposed to including 13–14 subtypes of IFN-α along with IFN-β, IFN-ε, IFN-κ, IFN- be Trojan exosomes [130–132]. Before EVs were recognized as phy- ω, IFN-δ, IFN-ζ, and IFN-τ), type II IFN (IFN-II, i.e., IFN-γ) and type III siological entities, it was well known that IFNs were able to affect en- IFNs (IFN-III, i.e. IFN-λ1 or IL-29, IFN-λ2 or IL-28A, IFN-λ3 or IL-28B veloped virus budding, release and infectivity by increasing the ex- and IFN-λ4 in humans) [103]. The three IFN types bind to three dif- pression of many genes codifying restriction factors [133], such as ferent specific receptors in mammals and use JAK-STAT as the main ISG15, which appear to have regulatory functions on EV production and pathway. They induce common but also type-spe- content. cific effects with intersections of the signal transduction pathways de- ISG15, is an ubiquitin-like modifier involved in the regulation of pending on the cell type and the environment present in the different many cellular pathways [134–136]. Although it was first identified tissues and organs [104,105]. Although existing as multiple isoforms, studying type I IFNs-treated cells [137], ISG15 is induced also by type type I IFNs bind to the same protein complex (IFNAR) consisting of two III IFNs, viral and bacterial infections, but not by type II IFN. It exists as receptor chains (IFNAR1 and IFNAR2) and activating a spectrum of a 17 kDa precursor protein that is rapidly processed into its mature 15 activities [106]. Similarly, type II IFN (IFN-γ) signals through a receptor kDa form via protease cleavage to expose a carboxy-terminal motif, (IFNGR) composed of two subunits (IFN-γR1 and IFN-γR2) and all type which allows the covalent binding of ISG15 to target proteins by a III IFNs share the same receptor complex (IFNLR) composed of IFN-λR1 three-step process referred to as ISGylation [135]. ISG15 exists also as (IL-28R1) and IL-10R2 subunits. The receptors for type I and II IFNs, an unconjugated protein that mainly localizes in the cytoplasmic frac- and the IL10R2, involved in IFN-λ signalling, are widely distributed on tion, and, interestingly, it is also released into the extracellular milieu the surface of most cell types with few exceptions. Instead, the cell via non-conventional secretion because lacks a secretory signal peptide surface expression of the high‐affinity receptor subunit for type III IFNs, [138]. ISG15 has been found in neutrophil granules, microvesicles IFNLR1, is more restricted, thereby limiting cell responsiveness to these [139], and in exosomes originated from TLR3-activated human brain cytokines [103,104,107]. IFNs regulate the expression of hundreds of microvascular endothelial cells [140] or released via apoptosis [134]. genes. A useful on-line open access database (Interferome, version 2.01, Regarding its activity as antiviral restriction factor, ISG15 inhibits the update of 2015, http://interferome.its.monash.edu.au/interferome/ HIV budding process; it ISGylates TSG101, a component of the ESCRT-I home.jspx) enables a comprehensive vision of IFN-regulated processes complex, thus inhibiting its ability to target Gag to favour the viral and pathways by a compilation of microarray datasets derived from budding process from the cellular membrane [129]. Recently, ISGyla- various cell types and tissues stimulated with type I, II and III IFNs tion has been reported to influence also exosome secretion. Villarroya- [108–110]. Beltri and colleagues observed that ISGylation of TSG101 triggers MVB IFN-αs, IFN-β and type III IFNs are involved in the multi-level reg- co-localization with lysosomes, thus promoting the aggregation and ulation of antiviral and antitumoral responses, whereas type II IFN, i.e. degradation of MVB proteins finally causing the decrease of MVB IFN-γ, is more linked to activation and perpetuation of inflammation. In numbers and the impairment of exosome secretion (see Fig. 3a). In- particular, type I IFNs induce differentiation, maturation and activation stead, inhibition of lysosomal functions or autophagy restored exosome of myeloid dendritic cells (mDCs) that, in turn, promote antiviral T cell secretion [141,142]. immunity [111]. Moreover, type I IFN released by plasmacytoid den- Another IFNs-induced factor that appears to have a regulatory dritic cells (pDCs), a dendritic cell subset specialized in IFN production, function on EV production is interferon regulatory factor 1 (IRF-1), a stimulates the activation of NK cells, biases the immune system toward well-known that is markedly and persistently in- a Th1 response, primes CD8+ T cells, induces memory CD8+ T cells and creased by IFN-γ, but transiently increased by type I IFN [143]. IRF-1 promotes the development of regulatory T (Tregs) cells and differ- plays important roles in the transcriptional regulation of type I IFN entiation of B cells into antibody-secreting plasma cells [112]. Type I genes and IFNs-induced ISGs, but it also regulates some GTPases, such IFN also possesses inflammatory properties through the activation of as Rab27a that is known to control EV release. In this respect, Yang and NLRP3 inflammasome that leads to the production of IL-1β andIL-18 colleagues observed an induction of IRF-1 and Rab27a both in vitro in and finally to pro-inflammatory pyroptotic cell death[112]. Like other hypoxic hepatocytes and in vivo in warm ischemia/reperfusion (IR) and multifunctional cytokines, their excessive or inappropriate activity can orthotopic liver transplantations [144]. They also reported that IFN-γ cause toxicity and even death [113–116]. In this respect, an IFN sig- stimulation, IRF-1 transduction, or ischemia/reperfusion (IR) promoted nature, i.e. increased or constitutive production of IFN-α and interferon Rab27a expression and EV secretion, whereas silencing of IRF-1 de- stimulated genes (ISGs), has been observed in different human creased them and Rab27a silencing decreased both EV secretion and pathologies. An emblematic example is the HIV infection, where IFN-α liver IR injury. IR-induced EVs expressed on the surface higher oxidized is chronically produced and appears to be a double-edged sword. Al- phospholipids (OxPL) that activated neutrophils through TLR4 though it exerts potent antiviral properties by reducing viral replication pathway, and the use of OxPL-neutralizing E06 antibody blocked the and inducing apoptosis in HIV-infected cells, both human and effect of these EVs and decreased liver IR injury. Therefore, these studies support a role of IFN-α in the immune activation and in- findings suggest a novel mechanism by which IRF-1 regulates Rab27a flammation during HIV chronic infection [117–119]. Kinetics of the transcription and EV secretion, leading to OxPL activation of neu- early innate immune activation during HIV-1 infection of humanized trophils and subsequent hepatic IR injury [144]. mice have further shed light on the complexity of the interaction with the IFN system during the eclipse, burst and chronic phase of the in- 5.3. The EV content and its effects on target cells fection [120]. To learn more about the IFN system we refer the reader to the many In addition to regulate the release of EVs, IFNs appear to affect also reviews available [e.g.,[104,121–128]. their content. This effect seems to concern in particular RNAs, whose extracellular transport and function have been recently reviewed (see

55 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60 ref. [145]). For instance, De Marino and colleagues showed that the exosomes more effective in promoting the differentiation of PBMCs into treatment with IFNα-2a of HIV-infected monocytes elicits an increase in Tregs, which can then exert their immune suppression effect possibly the packaging of all types of measured viral RNAs (i.e., TAR, TAR-gag providing a novel strategy to treat graft-versus-host disease (GVHD) as and genomic) into EVs [146]. Therefore, IFN-α-stimulated EVs might be well as other immune-associated disorders. a potential mechanism for the chronic activation observed in long term Recently, tumor immunoevasion strategies that involve EVs have cART (combination antiretroviral therapy) patients, with possible im- also been reported. In this respect, it was observed that metastatic plications also in other viral infections. Moreover, an IFN-α dose-de- melanoma releases a high level of EVs, mostly exosomes, carrying PD- pendent alteration of ESCRT-I, -II and Alix was observed by the authors L1, whose expression on EV surface is up-regulated by IFN-γ. Since PD- suggesting that the RNA packaging in EVs might be performed by L1 interacts with PD-1 on T cells to elicit the immune checkpoint re- ESCRT-independent machineries. sponse, these vesicles suppress the function of CD8+ T cells facilitating Another interesting example regards miRNAs. For instance, human tumor growth [153]. The immunoevasion mediated by PD-L1 is also monocytes activated by IFN-α, LPS or a combination of both, generate induced by glioblastoma-derived EVs that show some PD-L1–dependent exosomes carrying significantly altered microRNA profiles compared to inhibition of T cell activation. Also, PD-L1 DNA resulted to be present in non-activated monocytes [147]. Indeed, an enhanced expression of circulating EVs from glioblastoma patients [154]. These results suggest miR-155, miR-146a, miR-146b and miR-125a-5p was observed in both a possible use of the PD-L1 content in cancer-derived exosomes as a treated monocytes and in their released exosomes. In contrast, miR- predictor for positive or negative clinical response to anti-PD-1 therapy. 222, which is involved in the maintenance of the brain homeostasis and In addition, tumor-derived EVs (TEVs) can also "educate" healthy cells in the protection of the endothelium, was decreased in exosomes de- to promote metastases [155]. TEVs downregulate type I IFN receptor rived from treated monocytes, whereas its expression was increased in and the expression of IFN-inducible cholesterol 25-hydroxylase (CH25 treated cells. These exosomes originating from LPS- and IFNα-treated H). The latter produces 25-hydroxycholesterol that inhibits TEV uptake. monocytes activate human brain microvascular endothelial cells to Accordingly, mice incapable of downregulating the IFN receptor and stimulate some adhesion molecules (CCL2, ICAM1 and VCAM1) and CH25H are resistant to TEV uptake, TEV-induced pre-metastatic niche cytokines (IL-1β and IL-6) through a TLR4/ MyD88 (myeloid differ- and melanoma lung metastases. Therefore, low CH25H levels in leu- entiation primary response gene 88) pathway. This pathway activates kocytes from melanoma patients correlate with poor prognosis. An anti- NF-κB and increases monocyte chemotaxis, suggesting that monocytes hypertensive drug, reserpine, suppresses TEV uptake and disrupted have an impact on brain vascular function through intercellular exo- TEV-induced formation of the pre-metastatic niche and melanoma lung some signalling [147]. Indeed, blocking exosome release mitigates the metastases. These results suggest the importance of CH25H in defense inflammatory response. Furthermore, it was observed that MRC-5 cells, against education of normal cells by TEVs and argue for the possible use often used to prepare viral vaccines, are poorly susceptible to rabies of reserpine in adjuvant melanoma therapy [155]. virus infection because the infection induces the release of IFN-β and exosomes with an up-regulated content of miR-423-5p. This miRNA 6. Concluding remarks inhibits SOCS-3 (suppressor of cytokine signaling-3), a negative feed- back inhibitor of JAKs activity in the JAK/STAT signal transduction, EVs constitute a large and diversified family of membrane-bound thus resulting in the increase of IFN action [148]. vesicles that represent a third system of intercellular communication Concerning IFN-γ, a recent study showed that a robust pool of IFN-γ complementing the two already known systems: the cell-cell contacts was present within secretory granules and cellular vesicular compart- and the communication mediated by soluble factors, such as cytokines. ments in both CCL11 (Eotaxin-1)- and TNFα-activated and control Multiple pathways have been identified for the cytokine secretion human eosinophils [149]. In stimulated cells IFN-γ was found also at that include both the classical ER/Golgi route and unconventional the cell surface, including on extracellular vesicles, indicating a possible pathways. Interestingly, studies over the last few years have identified mechanism by which IFN-γ is trafficked and secreted during in- EVs as another non-canonical mechanism by which cytokines can be flammatory responses [149]. secreted into the extracellular milieu and affect physiological and pa- The alteration of the EV content induced by IFNs affects the activity thological functions of target cells. Moreover, cytokines have been re- and/or phenotype of recipient cells. In this respect, it has been recently ported to influence EV biogenesis and cargo. Different biological ex- observed that IFN-α induces the transfer of resistance to hepatitis B planations have been hypothesised to justify the loading of cytokines virus (HBV) from liver non-parenchymal cells to hepatocytes via exo- into EVs. Their encapsulation may be a mechanism to dispose of somes [150]. These exosomes exploit the hepatitis A virus receptor overproduced products by simultaneously protecting the cells from an (TIM-1) and the two primary endocytic routes, clathrin-mediated en- autocrine effect or a way to protect the cytokines from environmental docytosis and macropinocytosis, to enter hepatocytes. Subsequently, degradation. Moreover, the association of cytokines with EVs may fa- lysobisphosphatidic acid (LBPA), an anionic lipid closely related to cilitate their delivery at distant target cells, the cytokine concentration endosome penetration of viruses, facilitates membrane fusion of exo- at the cell surface and their uptake by cells that otherwise might not be somes in late endosomes/multivesicular bodies (LEs/MVBs) and the targeted by cytokines in solution. Indeed, it is reasonable to think that accompanying exosomal cargo uncoating. These results highlight the even if the amount of cytokines associated with EVs might be small, similarities between the entry mechanisms of exosomes and viruses they can exert effects on recipient cells. To date, no systematic studies [150]. Moreover, it was reported that EVs released by HSV-1-infected have been yet carried out to determine the complete spectrum of EVs- cells could control HSV-1 dissemination promoting its persistence in the associated cytokines. Since EVs are increasingly involved in a wide host through type I IFN production [151]. Indeed, the released EVs range of pleiotropic functions both in physiological and pathological carry innate immune components such as STING and other host and conditions, such as antigen presentation, neuronal communication and viral factors that activate innate immune responses in recipient cells protection, sperm maturation, but also acute and chronic inflammatory and inhibit HSV-1 replication. and autoimmune diseases or cancer, their production needs to be highly Interestingly, another study showed as exosomes from mesenchymal regulated. In this respect, a possible regulatory role could be exerted by stem cells (MSCs) of human umbilical cord treated with IFN-γ or a restriction factors that cells also use to control enveloped virus combination of TGF-β plus IFN-γ increased the proportion of Tregs ori- spreading such as Tetherin/BST-2, APOBEC3G, TRIM5a and IFITMs ginated from PBMCs. This effect was associated with the TGF-β, IDO [156–162]. In particular, Tetherin/BST-2 traps newly formed virions at (Indoleamine-pyrrole 2,3-dioxygenase), IL-10 and IFN-γ content in the plasma membrane [156], whereas IFITMs inhibit fusion of influenza exosomes [152]. Considered these results, the combination of TGF-β A virus particles, possibly blocking the transition from membrane and IFN-γ might be used to stimulate MSCs in vitro in order to obtain hemifusion to the opening of a fusion pore [158,161,162].

56 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

To date, what we know on EVs represents only the emerging tip of [19] P. Lacy, J.L. Stow, Cytokine release from innate immune cells: association with an iceberg and advances in basic and applied research are required. diverse membrane trafficking pathways, Blood 118 (2011) 9–18, https://doi.org/ 10.1182/blood-2010-08-265892. Importantly, the cargo and membrane composition of EVs reflect the [20] J.L. Stow, R.Z. Murray, Intracellular trafficking and secretion of inflammatory physiological state of the cells from which they originate, therefore cytokines, Cytokine Growth Factor Rev. 24 (2013) 227–239, https://doi.org/10. identification of differences in circulating EVs originating from normal 1016/j.cytogfr.2013.04.001. [21] S. Ferro-Novick, N. Brose, Nobel 2013 Physiology or medicine: Traffic control and “abnormal” cells (i.e., infected, tumour or damaged cells) will system within cells, Nature 504 (2013) 98, https://doi.org/10.1038/504098a. certainly result in the development of new and not invasive therapeutic [22] R.D. Burgoyne, A. Morgan, Secretory granule exocytosis, Physiol. Rev. 83 (2003) and diagnostic applications. 581–632. [23] C. Rabouille, Pathways of unconventional protein secretion, Trends Cell Biol. 27 (2017) 230–240, https://doi.org/10.1016/j.tcb.2016.11.007. Author statement [24] W. Fitzgerald, M.L. Freeman, M.M. Lederman, E. Vasilieva, R. Romero, L. Margolis, A system of cytokines encapsulated in extra cellular vesicles, Sci. Rep. A.A. and F.G. equally contributed to the work. Each author wrote a 8 (2018) 8973, https://doi.org/10.1038/s41598-018-27190-x. [25] K. Kandere-Grzybowska, R. Letourneau, D. Kempuraj, J. Donelan, S. Poplawski, part of the manuscript that was then completely revised and edited by W. Boucher, A. Athanassiou, T.C. Theoharides, IL-1 induces vesicular secretion of all of them. A.A. and F.G. take care of all the figures. Since the available IL-6 without degranulation from human mast cells, J. Immunol. 171 (2003) literature concerning this topic is extensive and touches the most dis- 4830–4836. [26] S. Kodidela, S. Ranjit, N. Sinha, C. McArthur, A. Kumar, S. Kumar, Cytokine parate fields, from viral infections to cancer, we apologize whether profiling of exosomes derived from the plasma of HIV-infected alcohol drinkers some articles have not been cited. and cigarette smokers, PLoS One 13 (2018) e0201144, https://doi.org/10.1371/ journal.pone.0201144. [27] K.A. Konadu, J. Chu, M.B. Huang, P.K. Amancha, W. Armstrong, M.D. Powell, Funding F. Villinger, V.C. Bond, Association of cytokines with exosomes in the plasma of HIV-1-Seropositive individuals, J. Infect. Dis. 211 (2015) 1712–1716, https://doi. This work was supported by the grant “Excellence Departments, org/10.1093/infdis/jiu676. [28] A. Tokarz, I. Szuścik, B. Kuśnierz-Cabala, M. Kapusta, M. Konkolewska, MIUR-Italy (ARTICOLO 1, COMMI 314 – 337 LEGGE 232/2016)”. A. Żurakowski, A. Georgescu, E. Stępień, Extracellular vesicles participate in the transport of cytokines and angiogenic factors in diabetic patients with ocular Declaration of Competing Interest complications, Folia Med. Cracov. 55 (2015) 35–48. [29] S. Chettimada, D.R. Lorenz, V. Misra, S.T. Dillon, R.K. Reeves, C. Manickam, S. Morgello, G.D. Kirk, S.H. Mehta, D. Gabuzda, Exosome markers associated with None. immune activation and oxidative stress in HIV patients on antiretroviral therapy, Sci. Rep. 8 (1) (2018) 7227. References [30] X. Xiang, A. Poliakov, C. Liu, Y. Liu, Z.B. Deng, J. Wang, Z. Cheng, S.V. Shah, G.J. Wang, L. Zhang, W.E. Grizzle, J. Mobley, H.G. Zhang, Induction of myeloid- derived suppressor cells by tumor exosomes, Int. J. Cancer 124 (2009) 2621–2633, [1] E. Chargaff, R. West, The biological significance of the thromboplastic proteinof https://doi.org/10.1002/ijc.24249. blood, J. Biol. Chem. 166 (1946) 189–197. [31] Y. Wang, J. Yi, X. Chen, Y. Zhang, M. Xu, Z. Yang, The regulation of cancer cell [2] T. Pisitkun, R.F. Shen, M.A. Knepper, Identification and proteomic profiling of migration by lung cancer cell-derived exosomes through TGF-β and IL-10, Oncol. exosomes in human urine, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 13368–13373. Lett. 11 (2016) 1527–1530. [3] C. Lässer, M. Eldh, J. Lötvall, Isolation and characterization of RNA-containing [32] A. Clayton, J.P. Mitchell, J. Court, M.D. Mason, Z. Tabi, Human tumor-derived exosomes, J. Vis. Exp. 9 (2012) e3037, https://doi.org/10.3791/3037. exosomes selectively impair lymphocyte responses to interleukin-2, Cancer Res. 67 [4] S. Keller, J. Ridinger, A.K. Rupp, J.W. Janssen, P. Altevogt, Body fluid derived (2007) 7458–7466. exosomes as a novel template for clinical diagnostics, J. Transl. Med. 9 (2011) 86, [33] J. Webber, R. Steadman, M.D. Mason, Z. Tabi, A. Clayton, Cancer exosomes trigger https://doi.org/10.1186/1479-5876-9-86. fibroblast to myofibroblast differentiation, Cancer Res. 70 (2010) 9621–9630, [5] M.P. Caby, D. Lankar, C. Vincendeau-Scherrer, G. Raposo, C. Bonnerot, Exosomal- https://doi.org/10.1158/0008-5472.CAN-10-1722. like vesicles are present in human blood plasma, Int. Immunol. 17 (2005) [34] B. Holder, T. Jones, V. Sancho Shimizu, T.F. Rice, B. Donaldson, M. Bouqueau, 879–887. K. Forbes, B. Kampmann, Macrophage exosomes induce placental inflammatory [6] A. Poliakov, M. Spilman, T. Dokland, C.L. Amling, J.A. Mobley, Structural het- cytokines: a novel mode of maternal-placental messaging, Traffic 17 (2016) erogeneity and protein composition of exosome-like vesicles (prostasomes) in 168–178, https://doi.org/10.1111/tra.12352. human semen, Prostate 69 (2009) 159–167, https://doi.org/10.1002/pros.20860. [35] R. Sullivan, F. Saez, J. Girouard, G. Frenette, Role of exosomes in sperm ma- [7] C. Lässer, S.E. O’Neil, L. Ekerljung, K. Ekström, M. Sjöstrand, J. Lötvall, RNA- turation during the transit along the male reproductive tract, Blood Cells Mol. Dis. containing exosomes in human nasal secretions, Am. J. Rhinol. Allergy 25 (2011) 35 (2005) 1–10. 89–93, https://doi.org/10.2500/ajra.2011.25.3573. [36] A. MacKenzie, H.L. Wilson, E. Kiss-Toth, S.K. Dower, R.A. North, A. Surprenant, [8] S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for Rapid secretion of interleukin-1beta by microvesicle shedding, Immunity 15 extracellular vesicles, J. Extracell. Vesicles 2 (2013), https://doi.org/10.3402/jev. (2001) 825–835. v2i0.20389. [37] C. Pizzirani, D. Ferrari, P. Chiozzi, E. Adinolfi, D. Sandonà, E. Savaglio, F.Di [9] M. Colombo, G. Raposo, C. Théry, Biogenesis, secretion, and intercellular inter- Virgilio, Stimulation of P2 receptors causes release of IL-1beta-loaded micro- actions of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30 vesicles from human dendritic cells, Blood 109 (2007) 3856–3864. (2014) 255–289, https://doi.org/10.1146/annurev-cellbio-101512-122326. [38] F. Bianco, E. Pravettoni, A. Colombo, U. Schenk, T. Möller, M. Matteoli, [10] G. van Niel, G. D’Angelo, G. Raposo, Shedding light on the cell of extra- C. Verderio, Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release cellular vesicles, Nat. Rev. Mol. Cell Biol. 19 (2018) 213–228, https://doi.org/10. from microglia, J. Immunol. 174 (2005) 7268–7277. 1038/nrm.2017.125. [39] Y. Qu, L. Franchi, G. Nunez, G.R. Dubyak, Nonclassical IL-1 beta secretion sti- [11] F. Dreyer, A. Baur, Biogenesis and functions of exosomes and extracellular ve- mulated by P2X7 receptors is dependent on inflammasome activation and corre- sicles, Methods Mol. Biol. 1448 (2016) 201–216, https://doi.org/10.1007/978-1- lated with exosome release in murine macrophages, J. Immunol. 179 (2007) 4939-3753-0_15. 1913–1925. [12] E. Cocucci, G. Racchetti, J. Meldolesi, Shedding microvesicles: artefacts no more, [40] Y. Berda-Haddad, S. Robert, P. Salers, L. Zekraoui, C. Farnarier, C.A. Dinarello, Trends Cell Biol. 19 (2009) 43–51, https://doi.org/10.1016/j.tcb.2008.11.003. F. Dignat-George, G. Kaplanski, Sterile inflammation of endothelial cell-derived [13] R.M. Johnstone, M. Adam, J.R. Hammond, L. Orr, C. Turbide, Vesicle formation apoptotic bodies is mediated by interleukin-1α, Proc. Natl. Acad. Sci. U. S. A. 108 during reticulocyte maturation. Association of plasma membrane activities with (2011) 20684–20689, https://doi.org/10.1073/pnas.1116848108. released vesicles (exosomes), J. Biol. Chem. 262 (1987) 9412–9420. [41] S. Gulinelli, E. Salaro, M. Vuerich, D. Bozzato, C. Pizzirani, G. Bolognesi, M. Idzko, [14] R.M. Johnstone, The Jeanne Manery-Fisher Memorial Lecture 1991. Maturation of F. Di Virgilio, D. Ferrari, IL-18 associates to microvesicles shed from human reticulocytes: formation of exosomes as a mechanism for shedding membrane macrophages by a LPS/TLR-4 independent mechanism in response to P2X receptor proteins, Biochem. Cell Biol. 70 (1992) 179–190. stimulation, Eur. J. Immunol. 42 (2012) 3334–3345, https://doi.org/10.1002/eji. [15] G. Raposo, H.W. Nijman, W. Stoorvogel, R. Liejendekker, C.V. Harding, 201142268. C.J. Melief, H.J. Geuze, B lymphocytes secrete antigen-presenting vesicles, J. Exp. [42] H. Hasegawa, H.J. Thomas, K. Schooley, T.L. Born, Native IL-32 is released from Med. 183 (1996) 1161–1172. intestinal epithelial cells via a non-classical secretory pathway as a membrane- [16] A. Isaacs, J. Lindenmann, Virus interference. I. The interferon, Proc. R. Soc. Lond., associated protein, Cytokine 53 (2011) 74–83, https://doi.org/10.1016/j.cyto. B, Biol. Sci. 147 (1957) 258–267. 2010.09.002. [17] K. Gulati, S. Guhathakurta, J. Joshi, N. Rai, A. Ray, Cytokines and their role in [43] H.G. Zhang, C. Liu, K. Su, S. Yu, L. Zhang, S. Zhang, J. Wang, X. Cao, W. Grizzle, health and disease: A brief overview, MOJ Immunol. 4 (2016) 121, https://doi. R.P. Kimberly, A membrane form of TNF-alpha presented by exosomes delays T org/10.15406/moji.2016.04.00121. cell activation-induced cell death, J. Immunol. 176 (2006) 7385–7393 Erratum in: [18] C.A. Dinarello, Historical insights into cytokines, Eur. J. Immunol. 37 (2007) J. Immunol. 177 (2006) 2025. 34–45, https://doi.org/10.1002/eji.200737772. [44] C. Obregon, B. Rothen-Rutishauser, P. Gerber, P. Gehr, L.P. Nicod, Active uptake

57 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

of dendritic cell-derived exovesicles by epithelial cells induces the release of in- 80 kDa tumor necrosis factor receptor, Cell 83 (1995) 793–802. flammatory mediators through a TNF-alpha-mediated pathway, Am. J. Pathol. 175 [70] M. Grell, H. Wajant, G. Zimmermann, P. Scheurich, The type 1 receptor (CD120a) (2009) 696–705, https://doi.org/10.2353/ajpath.2009.080716. is the high-affinity receptor for soluble tumor necrosis factor, Proc. Natl. Acad.Sci. [45] M.J. Martínez-Lorenzo, A. Anel, S. Gamen, I. Monleón, P. Lasierra, L. Larrad, U. S. A. 95 (1998) 570–575. A. Piñeiro, M.A. Alava, J. Naval, Activated human T cells release bioactive Fas [71] A. Söderberg, A.M. Barral, M. Söderström, B. Sander, A. Rosén, Redox-signaling ligand and APO2 ligand in microvesicles, J. Immunol. 163 (1999) 1274–1281. transmitted in trans to neighboring cells by melanoma-derived TNF-containing [46] I. Monleón, M.J. Martínez-Lorenzo, L. Monteagudo, P. Lasierra, M. Taulés, exosomes, Free Radic. Biol. Med. 43 (2007) 90–99. M. Iturralde, A. Piñeiro, L. Larrad, M.A. Alava, J. Naval, A. Anel, Differential se- [72] J.H. Lee, S. Wittki, T. Bräu, F.S. Dreyer, K. Krätzel, J. Dindorf, I.C. Johnston, cretion of Fas ligand- or APO2 ligand/TNF-related apoptosis-inducing ligand- S. Gross, E. Kremmer, R. Zeidler, U. Schlötzer-Schrehardt, M. Lichtenheld, carrying microvesicles during activation-induced death of human T cells, J. K. Saksela, T. Harrer, G. Schuler, M. Federico, A.S. Baur, HIV Nef, paxillin, and Immunol. 167 (2001) 6736–6744. Pak1/2 regulate activation and secretion of TACE/ADAM10 proteases, Mol. Cell [47] S. Munich, A. Sobo-Vujanovic, W.J. Buchser, D. Beer-Stolz, N.L. Vujanovic, 49 (2013) 668–679, https://doi.org/10.1016/j.molcel.2012.12.004. Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via [73] C. Ostalecki, S. Wittki, J.H. Lee, M.M. Geist, N. Tibroni, T. Harrer, G. Schuler, TNF superfamily ligands, Oncoimmunology 1 (2012) 1074–1083. O.T. Fackler, A.S. Baur, HIV nef- and Notch1-dependent endocytosis of ADAM17 [48] E. Zuccato, E.J. Blott, O. Holt, S. Sigismund, M. Shaw, G. Bossi, G.M. Griffiths, induces vesicular TNF secretion in chronic HIV infection, EBioMedicine 13 (2016) Sorting of Fas ligand to secretory lysosomes is regulated by mono-ubiquitylation 294–304, https://doi.org/10.1016/j.ebiom.2016.10.027. and phosphorylation, J. Cell. Sci. 120 (2007) 191–199. [74] Z. Zhao, T. Kesti, H. Uğurlu, A.S. Baur, R. Fagerlund, K. Saksela, Tyrosine phos- [49] G. Andreola, L. Rivoltini, C. Castelli, V. Huber, P. Perego, P. Deho, P. Squarcina, phorylation directs TACE into extracellular vesicles via unconventional secretion, P. Accornero, F. Lozupone, L. Lugini, A. Stringaro, A. Molinari, G. Arancia, Traffic 20 (2019) 202–212, https://doi.org/10.1111/tra.12630. M. Gentile, G. Parmiani, S. Fais, Induction of lymphocyte apoptosis by tumor cell [75] C. Arenaccio, C. Chiozzini, S. Columba-Cabezas, F. Manfredi, E. Affabris, A. Baur, secretion of FasL-bearing microvesicles, J. Exp. Med. 195 (2002) 1303–1316. M. Federico, Exosomes from human immunodeficiency virus type 1 (HIV-1)-in- [50] L.A. Truman, C.A. Ford, M. Pasikowska, J.D. Pound, S.J. Wilkinson, I.E. Dumitriu, fected cells license quiescent CD4+ T lymphocytes to replicate HIV-1 through a L. Melville, L.A. Melrose, C.A. Ogden, R. Nibbs, G. Graham, C. Combadiere, Nef- and ADAM17-dependent mechanism, J. Virol. 88 (2014) 11529–11539, C.D. Gregory, CX3CL1/fractalkine is released from apoptotic lymphocytes to sti- https://doi.org/10.1128/JVI.01712-14. mulate macrophage chemotaxis, Blood 112 (2008) 5026–5036, https://doi.org/ [76] C. Arenaccio, C. Chiozzini, S. Columba-Cabezas, F. Manfredi, M. Federico, Cell 10.1182/blood-2008-06-162404. activation and HIV-1 replication in unstimulated CD4+ T lymphocytes ingesting [51] M. Baj-Krzyworzeka, K. Weglarczyk, B. Mytar, R. Szatanek, J. Baran, M. Zembala, exosomes from cells expressing defective HIV-1, Retrovirology 11 (2014) 46, Tumour-derived microvesicles contain interleukin-8 and modulate production of https://doi.org/10.1186/1742-4690-11-46. chemokines by human monocytes, Anticancer Res. 31 (2011) 1329–1335. [77] D. Skokos, H.G. Botros, C. Demeure, J. Morin, R. Peronet, G. Birkenmeier, [52] T. Chen, J. Guo, M. Yang, X. Zhu, X. Cao, Chemokine-containing exosomes are S. Boudaly, S. Mécheri, Mast cell-derived exosomes induce phenotypic and func- released from heat-stressed tumor cells via lipid raft-dependent pathway and act as tional maturation of dendritic cells and elicit specific immune responses in vivo, J. efficient tumor vaccine, J. Immunol. 186 (2011) 2219–2228, https://doi.org/10. Immunol. 170 (2003) 3037–3045. 4049/jimmunol.1002991. [78] D.L. Sprague, B.D. Elzey, S.A. Crist, T.J. Waldschmidt, R.J. Jensen, T.L. Ratliff, [53] S.F. Mause, P. von Hundelshausen, A. Zernecke, R.R. Koenen, C. Weber, Platelet Platelet-mediated modulation of adaptive immunity: unique delivery of CD154 microparticles: a transcellular delivery system for RANTES promoting monocyte signal by platelet-derived membrane vesicles, Blood 111 (2008) 5028–5036, recruitment on endothelium, Arterioscler. Thromb. Vasc. Biol. 25 (2005) https://doi.org/10.1182/blood-2007-06-097410. 1512–1518. [79] F.I. Hawari, F.N. Rouhani, X. Cui, Z.X. Yu, C. Buckley, M. Kaler, S.J. Levine, [54] H.K. Kim, K.S. Song, J.H. Chung, K.R. Lee, S.N. Lee, Platelet microparticles induce Release of full-length 55-kDa TNF receptor 1 in exosome-like vesicles: a me- angiogenesis in vitro, Br. J. Haematol. 124 (2004) 376–384. chanism for generation of soluble cytokine receptors, Proc. Natl. Acad. Sci. U. S. A. [55] G.J. Wang, Y. Liu, A. Qin, S.V. Shah, Z.B. Deng, X. Xiang, Z. Cheng, C. Liu, 101 (2004) 1297–1302. J. Wang, L. Zhang, W.E. Grizzle, H.G. Zhang, Thymus exosomes-like particles in- [80] D. Boraschi, D. Lucchesi, S. Hainzl, M. Leitner, E. Maier, D. Mangelberger, duce regulatory T cells, J. Immunol. 181 (2008) 5242–5248. G.J. Oostingh, T. Pfaller, C. Pixner, G. Posselt, P. Italiani, M.F. Nold, C.A. Nold- [56] M. Szajnik, M. Czystowska, M.J. Szczepanski, M. Mandapathil, T.L. Whiteside, Petry, P. Bufler, C.A. Dinarello, IL-37: a new anti-inflammatory cytokine oftheIL- Tumor-derived microvesicles induce, expand and up-regulate biological activities 1 family, Eur. Cytokine Netw. 3 (2011) 127–147, https://doi.org/10.1684/ecn. of human regulatory T cells (Treg), PLoS One 5 (2010) e11469, https://doi.org/ 2011.0288. 10.1371/journal.pone.0011469. [81] M.J.H. Nicklin, J.L. Barton, M. Nguyen, M.G. FitzGerald, G.W. Duff, K. Kornman, A [57] S. Schierer, C. Ostalecki, E. Zinser, R. Lamprecht, B. Plosnita, L. Stich, J. Dörrie, sequence-based map of the nine genes of the human Interleukin-1 cluster, M.B. Lutz, G. Schuler, A.S. Baur, Extracellular vesicles from mature dendritic cells Genomics 79 (2002) 718–725, https://doi.org/10.1006/geno.2002.6751. (DC) differentiate monocytes into immature DC, . Sci. Alliance 1(2018) [82] C.A. Dinarello, IL‐1: Discoveries, controversies and future directions, Eur. J. e201800093, , https://doi.org/10.26508/lsa.201800093. Immunol. 40 (2010) 599–606, https://doi.org/10.1002/eji.201040319. [58] R. Sitia, A. Rubartelli, The unconventional secretion of IL-1β: handling a dan- [83] A. Werman, R. Werman-Venkert, R. White, J.K. Lee, B. Werman, Y. Krelin, gerous weapon to optimize inflammatory responses, Semin. Cell Dev. Biol. 83 R.N. Apte, The precursor form of IL-1alpha is an proinflammatory ac- (2018) 12–21, https://doi.org/10.1016/j.semcdb.2018.03.011. tivator of transcription, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 2434–2439, [59] A. Kumar, W. Abbas, G. Herbein, TNF and TNF receptor superfamily members in https://doi.org/10.1073/pnas.0308705101. HIV infection: new cellular targets for therapy? Mediators Inflamm. 2013 (2013) [84] C.J. Chen, H. Kono, D. Golenbock, G. Reed, S. Akira, K.L. Rock, Identification of a 484378, https://doi.org/10.1155/2013/484378. key pathway required for the sterile inflammatory response triggered by dying [60] E.A. Carswell, L.J. Old, R.L. Kassel, S. Green, N. Fiore, B. Williamson, An en- cells, Nat. Med. 13 (2007) 851–856. dotoxin-induced serum factor that causes necrosis of tumors, Proc. Natl. Acad. Sci. [85] I. Cohen, P. Rider, Y. Carmi, A. Braiman, S. Dotan, M.R. White, R.N. Apte, U. S. A. 72 (1975) 3666–3670. Differential release of chromatin-bound IL-1alpha discriminates between necrotic [61] D. Tracey, L. Klareskog, E.H. Sasso, J.G. Salfeld, P.P. Tak, Tumor necrosis factor and apoptotic cell death by the ability to induce sterile inflammation, Proc. Natl. antagonist mechanisms of action: a comprehensive review, Pharmacol. Ther. 117 Acad. Sci. U.S.A. 107 (2010) 2574–2579, https://doi.org/10.1073/pnas. (2008) 244–279. 0915018107. [62] C.A. Smith, T. Farrah, R.G. Goodwin, The TNF receptor superfamily of cellular and [86] V. Santarlasci, L. Cosmi, L. Maggi, F. Liotta, F. Annunziato, IL-1 and T Helper viral proteins: activation, costimulation, and death, Cell 76 (1994) 959–962. Immune Responses, Front. Immunol. 4 (2013) 182, https://doi.org/10.3389/ [63] R.M. Locksley, N. Killeen, M.J. Lenardo, The TNF and TNF receptor superfamilies: fimmu.2013.00182. integrating mammalian biology, Cell 104 (2001) 487–501. [87] J.L. Casanova, L. Abel, L. Quintana-Murci, Human TLRs and IL-1Rs in host de- [64] S. Gupta, Tumor necrosis factor-alpha-induced apoptosis in T cells from aged fense: natural insights from evolutionary, epidemiological, and clinical genetics, humans: a role of TNFR-I and downstream signaling molecules, Exp. Gerontol. 37 Annu. Rev. Immunol. 29 (2011) 447–491. (2002) 293–299. [88] C.A. Dinarello, Immunological and inflammatory functions of the Interleukin-1 [65] J.L. Stow, P.C. Low, C. Offenhäuser, D. Sangermani, Cytokine secretion in mac- family, Annu. Rev. Immunol. 27 (2009) 519–550. rophages and other cells: pathways and mediators, Immunobiology 214 (2009) [89] A. Rubartelli, F. Cozzolino, M. Talio, R. Sitia, A novel secretory pathway for in- 601–612, https://doi.org/10.1016/j.imbio.2008.11.005. terleukin-1 beta, a protein lacking a signal sequence, EMBO J. 9 (1990) [66] M.D. Turner, B. Nedjai, T. Hurst, D.J. Pennington, Cytokines and chemokines: At 1503–1510. the crossroads of cell signalling and inflammatory disease, Biochim. Biophys. Acta [90] E. Välimäki, J.J. Miettinen, N. Lietzén, S. Matikainen, T.A. Nyman, Monosodium 1843 (2014) 2563–2582, https://doi.org/10.1016/j.bbamcr.2014.05.014. urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional [67] L.M. Sedger, M.F. McDermott, TNF and TNF-receptors: From mediators of cell protein secretion from human primary macrophages, Mol. Cell Proteomics 12 death and inflammation to therapeutic giants - past, present and future, Cytokine (2013) 749–763, https://doi.org/10.1074/mcp.M112.024661. Growth Factor Rev. 25 (2014) 453–472, https://doi.org/10.1016/j.cytogfr.2014. [91] W. Cypryk, T. Öhman, E. Eskelinen, S. Matikainen, T.A. Nyman, Quantitative 07.016. proteomics of extracellular vesicles released from human monocyte-derived [68] S.M. McFarlane, G. Pashmi, M.C. Connell, A.F. Littlejohn, S.J. Tucker, macrophages upon β-Glucan stimulation, J. Proteome Res. 13 (2014) 2468–2477, P. Vandenabeele, D.J. MacEwan, Differential activation of nuclear factor-kappaB https://doi.org/10.1021/pr4012552. by tumour necrosis factor receptor subtypes. TNFR1 predominates whereas TNFR2 [92] E. Välimäki, W. Cypryk, J. Virkanen, K. Nurmi, P.M. Turunen, K.K. Eklund, activates transcription poorly, FEBS. Lett. 515 (2002) 119–126. K.E. Åkerman, T.A. Nyman, S. Sampsa Matikainen, Calpain activity is essential for [69] M. Grell, E. Douni, H. Wajant, M. Löhden, M. Clauss, B. Maxeiner, ATP-Driven unconventional vesicle-mediated protein secretion and inflammasome S. Georgopoulos, W. Lesslauer, G. Kollias, K. Pfizenmaier, P. Scheurich, The activation in human macrophages, J. Immunol. 197 (2016) 3315–3325, https:// transmembrane form of tumor necrosis factor is the prime activating ligand of the doi.org/10.4049/jimmunol.1501840.

58 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

[93] W. Cypryk, M. Lorey, A. Puustinen, T.A. Nyman, S. Matikainen, Proteomic and M.H. Malim, G.J. Towers, M. Dorner, Kinetics of early innate immune activation bioinformatic characterization of extracellular vesicles released from human during HIV-1 infection of humanized mice, J. Virol. 93 (2019) e02123–18, macrophages upon influenza A virus infection, J. Proteome Res. 16 (2017) https://doi.org/10.1128/JVI.02123-18. 217–227, https://doi.org/10.1021/acs.jproteome.6b00596. [121] A.J. Lee, A.A. Ashkar, The dual nature of type I and II interferons, Front. Immunol. [94] M.B. Lorey, K. Rossi, K.K. Eklund, T.A. Nyman, S. Matikainen, Global character- 9 (2018) 2061, https://doi.org/10.3389/fimmu.2018.02061. ization of protein secretion from human macrophages following non-canonical [122] L.B. Ivashkiv, L.T. Donlin, Regulation of type I interferon responses, Nat. Rev. Caspase-4/5 inflammasome activation, Mol. Cell Proteomics 16 (2017) 187–199, Immunol. 14 (2014) 36–49, https://doi.org/10.1038/nri3581. https://doi.org/10.1074/mcp.M116.064840. [123] R.A. Porritt, P.J. Hertzog, Dynamic control of type I IFN signalling by an in- [95] N. Singhto, R. Kanlaya, A. Nilnumkhum, V. Thongboonkerd, Roles of macrophage tegrated network of negative regulators, Trends Immunol. 36 (2015) 150–160, exosomes in immune response to calcium oxalate monohydrate crystals, Front. https://doi.org/10.1016/j.it.2015.02.002. Immunol. 9 (2018) 316, https://doi.org/10.3389/fimmu.2018.00316. [124] C.J. Secombes, J. Zou, Evolution of interferons and interferon receptors, Front. [96] E.K. Jo, J.K. Kim, D.M. Shin, C. Sasakawa, Molecular mechanisms regulating Immunol. 8 (2017) 209, https://doi.org/10.3389/fimmu.2017.00209. NLRP3 inflammasome activation, Cell. Mol. Immunol. 13 (2016) 148–159, [125] P.J. Hertzog, N.A. de Weerd, A structural “star” in interferon gamma signaling, https://doi.org/10.1038/cmi.2015.95. Immunol. Cell Biol. 97 (2019) 442–444, https://doi.org/10.1111/imcb.12255. [97] W. Cypryk, T.A. Nyman, S. Matikainen, From Inflammasome to Exosome-Does [126] E.A. Hemann, M. Gale Jr., R. Savan, Interferon lambda genetics and biology in Extracellular Vesicle Secretion Constitute an Inflammasome-Dependent Immune regulation of viral control, Front. Immunol. 8 (2017) 1707, https://doi.org/10. Response? Front. Immunol. 9 (2018) 2188, https://doi.org/10.3389/fimmu.2018. 3389/fimmu.2017.01707. 02188. [127] R. Savan, Post-transcriptional regulation of interferons and their signaling path- [98] Y. Zhang, F. Liu, Y. Yuan, C. Jin, C. Chang, Y. Zhu, X. Xiuyuan Zhang, C. Chunyan ways, J. Interferon Cytokine Res. 34 (2014) 318–329, https://doi.org/10.1089/jir. Tian, F. Fuchu He, J. Jian Wang, Inflammasome-derived exosomes activate NF-κB 2013.0117. signaling in macrophages, J. Proteome Res. 16 (2017) 170–178, https://doi.org/ [128] M.J. Gale, R. Savan, Introduction to the Special Issue on Interferon Lambda: 10.1021/acs.jproteome.6b00599. Disease Impact and Therapeutic Potential, J. Interferon Cytokine Res. 39 (2019) [99] H. Kalra, G.P. Drummen, S. Mathivanan, Focus on extracellular vesicles: in- 585. troducing the next small big thing, Int. J. Mol. Sci. 17 (2016) 170, https://doi.org/ [129] D.G. Demirov, E.O. Freed, Retrovirus budding, Virus Res. 106 (2004) 87–102. 10.3390/ijms17020170. [130] S.J. Gould, A.M. Booth, J.E.K. Hildreth, The Trojan exosome hypothesis, Proc. [100] M. Crespin, C. Vidal, F. Picard, C. Lacombe, M. Fontenay, Activation of PAK1/2 Natl. Acad. Sci. 100 (2003) 10592–10597, https://doi.org/10.1073/pnas. during the shedding of platelet microvesicles, Blood Coagul. Fibrinolysis 20 (2009) 1831413100. 63–70. [131] N. Izquierdo-Useros, M.C. Puertas, F.E. Borràs, J. Blanco, J. Martinez-Picado, [101] J. Rintahaka, N. Lietzén, T. Öhman, T.A. Nyman, S. Matikainen, Recognition of Exosomes and retroviruses: the chicken or the egg? Cell. Microbiol. 13 (2011) cytoplasmic RNA results in cathepsin-dependent inflammasome activation and 10–17, https://doi.org/10.1111/j.1462-5822.2010.01542.x. apoptosis in human macrophages, J. Immunol. 186 (2011) 3085–3092, https:// [132] N. Izquierdo-Useros, M. Naranjo-Gómez, I. Erkizia, M.C. Puertas, F.E. Borràs, doi.org/10.4049/jimmunol.1002051. J. Blanco, J. Martinez-Picado, HIV and mature dendritic cells: Trojan exosomes [102] S. Yoon, A. Kovalenko, K. Bogdanov, D. Wallach, MLKL, the protein that mediates riding the Trojan horse? PLoS Pathog. 6 (2010) e1000740, , https://doi.org/10. necroptosis, also regulates endosomal trafficking and extracellular vesicle gen- 1371/journal.ppat.1000740. eration, Immunity 47 (2017) 51–65, https://doi.org/10.1016/j.immuni.2017.06. [133] D. Hotter, F. Kirchhoff, Interferons and beyond: Induction of antiretroviral re- 001. striction factors, J. Leukoc. Biol. 103 (2018) 465–477, https://doi.org/10.1002/ [103] H.M. Lazear, T.J. Nice, M.S. Diamond, Interferon-λ: Immune Functions at Barrier JLB.3MR0717-307R. Surfaces and Beyond, Immunity 43 (2015) 15–28, https://doi.org/10.1016/j. [134] P.F. Dos Santos, D.S. Mansur, Beyond ISGlylation: Functions of Free Intracellular immuni.2015.07.001. and Extracellular ISG15, J. Interferon Cytokine Res. 37 (2017) 246–253, https:// [104] N.A. de Weerd, T. Nguyen, The interferons and their receptors–distribution and doi.org/10.1089/jir.2016.0103. regulation, Immunol. Cell Biol. 90 (2012) 483–491, https://doi.org/10.1038/icb. [135] Y.C. Perng, D.J. Lenschow, ISG15 in antiviral immunity and beyond, Nat. Rev. 2012.9. Microbiol. 16 (2018) 423–439, https://doi.org/10.1038/s41579-018-0020-5. [105] R.L. Casazza, H.M. Lazear, Why is IFN-λ less inflammatory? One IRF decides, [136] J.V. Dzimianski, F.E.M. Scholte, É. Bergeron, S.D. Pegan, ISG15: it’s complicated, Immunity 51 (2019) 415–417, https://doi.org/10.1016/j.immuni.2019.08.019. J. Mol. Biol. (2019) 30136–30146, https://doi.org/10.1016/j.jmb.2019.03.013 [106] G. Schreiber, The molecular basis for differential type I interferon signaling, J. S0022-2836. Biol. Chem. 292 (2017) 7285–7294, https://doi.org/10.1074/jbc.R116.774562. [137] A.L. Haas, P. Ahrens, P.M. Bright, H. Ankel, Interferon induces a 15-kilodalton [107] E.A. Hemann, M. Gale Jr., R. Savan, Interferon lambda genetics and biology in protein exhibiting marked homology to ubiquitin, J. Biol. Chem. 262 (1987) regulation of viral control, Front. Immunol. 8 (2017) 1707, https://doi.org/10. 11315–11323. 3389/fimmu.2017.01707. [138] H.G. Han, H.W. Moon, Y.J. Jeon, ISG15 in cancer: beyond ubiquitin-like protein, [108] S.A. Samarajiwa, S. Forster, K. Auchettl, P.J. Hertzog, Interferome: the database of Cancer Lett. 438 (2018) 52–62, https://doi.org/10.1016/j.canlet.2018.09.007. interferon regulated genes, Nucleic Acids Res. 37 (2009) 852–857. [139] D. Bogunovic, M. Byun, L.A. Durfee, A. Abhyankar, O. Sanal, D. Mansouri, [109] P. Hertzog, S. Forster, S. Samarajiwa, Systems biology of interferon responses, J. S. Salem, I. Radovanovic, A.V. Grant, P. Adimi, Mycobacterial disease and im- Interferon Cytokine Res. 31 (2011) 5–11, https://doi.org/10.1089/jir.2010.0126. paired IFN-γ immunity in humans with inherited ISG15 deficiency, Science 337 [110] I. Rusinova, S. Forster, S. Yu, A. Kannan, M. Masse, H. Cumming, R. Chapman, (2012) 1684–1688. P.J. Hertzog, Interferome v2.0: an updated database of annotated interferon- [140] L. Sun, X. Wang, Y. Zhou, R.H. Zhou, W.Z. Ho, J.L. Li, Exosomes contribute to the regulated genes, Nucleic Acids Res. 41 (2013) 1040–1046. transmission of anti-HIV activity from TLR3-activated brain microvascular en- [111] J.F. Fonteneau, M. Larsson, A.S. Beignon, K. McKenna, I. Dasilva, A. Amara, dothelial cells to macrophages, Antivir. Res. 134 (2016) 167–171. Y.J. Liu, J.D. Lifson, D.R. Littman, N. Bhardwaj, Human immunodeficiency virus [141] C. Villarroya-Beltri, F. Baixauli, M. Mittelbrunn, I. Fernández-Delgado, type 1 activates plasmacytoid dendritic cells and concomitantly induces the by- D. Torralba, O. Moreno-Gonzalo, S. Baldanta, C. Enrich, S. Guerra, F. Sánchez- stander maturation of myeloid dendritic cells, J. Virol. 78 (2004) 5223–5232. Madrid, ISGylation controls exosome secretion by promoting lysosomal degrada- [112] A. Aiello, F. Giannessi, Z.A. Percario, E. Affabris, The involvement of plasmacytoid tion of MVB proteins, Nat. Commun. 7 (2016) 13588, https://doi.org/10.1038/ cells in HIV infection and pathogenesis, Cytokine Growth Factor Rev. 40 (2018) ncomms13588. 77–89, https://doi.org/10.1016/j.cytogfr.2018.03.009. [142] C. Villarroya-Beltri, S. Guerra, F. Sánchez-Madrid, ISGylation - a key to lock the [113] L. Ronnblom, M.L. Eloranta, The interferon signature in autoimmune diseases, cell gates for preventing the spread of threats, J. Cell. Sci. 130 (2017) 2961–2969, Curr. Opin. Rheumatol. 25 (2013) 248–253. https://doi.org/10.1242/jcs.205468. [114] S. Kretschmer, M.A. Lee-Kirsch, Type I interferon-mediated autoinflammation and [143] T. Taniguchi, K. Ogasawara, A. Takaoka, N. Tanaka, IRF family of transcription autoimmunity, Curr. Opin. Immunol. 49 (2017) 96–102. factors as regulators of host defense, Annu. Rev. Immunol. 19 (2001) 623–655. [115] A. Soper, I. Kimura, S. Nagaoka, Y. Konno, K. Yamamoto, Y. Koyanagi, K. Sato, [144] M.Q. Yang, Q. Du, J. Goswami, P.R. Varley, B. Chen, R.H. Wang, A.E. Morelli, Type I interferon responses by HIV-1 infection: association with disease progres- D.B. Stolz, T.R. Billiar, J. Li, D.A. Geller, Interferon regulatory factor 1-Rab27a sion and control, Front. Immunol. 8 (2018) 1823, https://doi.org/10.3389/ regulated extracellular vesicles promote liver ischemia/reperfusion injury, fimmu.2017.01823. Hepatology 67 (2018) 1056–1070, https://doi.org/10.1002/hep.29605. [116] C.S. Ng, H. Kato, T. Fujita, Fueling Type I Interferonopathies: Regulation and [145] S. Ressel, A. Rosca, K. Gordon, A.H. Buck, Extracellular RNA in viral-host inter- Function of Type I Interferon Antiviral Responses, J. Interferon Cytokine Res. 39 actions: thinking outside the cell, Wiley Interdiscip. Rev. RNA. 10 (2019) e1535, (2019) 383–392, https://doi.org/10.1089/jir.2019.0037. https://doi.org/10.1002/wrna.1535. [117] J.P. Herbeuval, J.C. Grivel, A. Boasso, A.W. Hardy, C. Chougnet, M.J. Dolan, et al., [146] C. DeMarino, M.L. Pleet, M. Cowen, R.A. Barclay, Y. Akpamagbo, J. Erickson, CD4+ T-cell death induced by infectious and noninfectious HIV-1: role of type 1 N. Ndembi, M. Charurat, J. Jumare, S. Bwala, P. Alabi, M. Hogan, A. Gupta, interferon-dependent, TRAIL/DR5-mediated apoptosis, Blood 106 (2005) N. Noren Hooten, M.K. Evans, B. Lepene, W. Zhou, M. Caputi, F. Romerio, 3524–3531. W. Royal 3rd, N. El-Hage, L.A. Liotta, F. Kashanchi, Antiretroviral drugs alter the [118] T. Doyle, C. Goujon, M.H. Malim, HIV-1 and interferons: who’s interfering with content of extracellular vesicles from HIV-1-Infected cells, Sci. Rep. 8 (2018) 7653, whom? Nat. Rev. Microbiol. 13 (2015) 403–413, https://doi.org/10.1038/ https://doi.org/10.1038/s41598-018-25943-2. nrmicro3449. [147] P. Dalvi, B. Sun, N. Tang, L. Pulliam, Immune activated monocyte exosomes alter [119] T. Démoulins, A. Abdallah, N. Kettaf, M.L. Baron, C. Gerarduzzi, D. Gauchat, microRNAs in brain endothelial cells and initiate an inflammatory response S. Gratton, R.P. Sékaly, Reversible blockade of thymic output: an inherent part of through the TLR4/MyD88 pathway, Sci. Rep. 7 (2017) 9954. TLR ligand-mediated immune response, J. Immunol. 181 (2008) 6757–6769, [148] J. Wang, Y. Teng, G. Zhao, F. Li, A. Hou, B. Sun, W. Kong, F. Gao, L. Cai, C. Jiang, https://doi.org/10.4049/jimmunol.181.10.6757. Exosome-mediated delivery of inducible miR-423-5p enhances resistance of MRC- [120] J.K. Skelton, A.M. Ortega-Prieto, S. Kaye, J.M. Jimenez-Guardeño, J. Turner, 5 cells to rabies virus infection, Int. J. Mol. Sci. 20 (2019), https://doi.org/10.

59 A. Aiello, et al. Cytokine and Growth Factor Reviews 51 (2020) 49–60

3390/ijms20071537 pii: E1537. Alessandra Aiello is PhD student in the lab of Professor [149] L.A.S. Carmo, K. Bonjour, L.A. Spencer, P.F. Weller, R.C.N. Melo, Single-cell Elisabetta Affabris at the Department of Sciences, Roma Tre analyses of human eosinophils at high resolution to understand compartmentali- University, Rome since 2016. She completed her master zation and vesicular trafficking of Interferon-Gamma, Front. Immunol. 9 (2018) degree in Biology for the Molecular, Cellular and Patho- 1542, https://doi.org/10.3389/fimmu.2018.01542. physiological Research at Roma Tre University in 2016. For [150] Z. Yao, Y. Qiao, X. Li, J. Chen, J. Ding, L. Bai, F. Shen, B. Shi, J. Liu, L. Peng, J. Li, her PhD, she is investigating virus–host interactions Z. Yuan, Exosomes exploit the virus entry machinery and pathway to transmit studying effects of extracellular HIV-1 Nef protein in plas- alpha interferon-induced antiviral activity, J. Virol. 92 (2018) e01578–1618, macytoid dendritic cells and exosome production. https://doi.org/10.1128/JVI.01578-18. [151] T. Deschamps, M. Kalamvoki, Extracellular vesicles released by herpes simplex virus 1-Infected cells block virus replication in recipient cells in a STING- Dependent manner, J. Virol. 92 (2018) e01102–18, https://doi.org/10.1128/JVI. 01102-18. [152] Q. Zhang, L. Fu, Y. Liang, Z. Guo, L. Wang, C. Ma, H. Wang, Exosomes originating from MSCs stimulated with TGF-β and IFN-γ promote Treg differentiation, J. Cell. Flavia Giannessi is PhD student in the lab of Professor Physiol. 233 (2018) 6832–6840, https://doi.org/10.1002/jcp.26436. Elisabetta Affabris at the Department of Sciences, Roma Tre [153] G. Chen, A.C. Huang, W. Zhang, G. Zhang, M. Wu, W. Xu, Z. Yu, J. Yang, B. Wang, University, Rome since 2017. She completed her master H. Sun, H. Xia, Q. Man, W. Zhong, L.F. Antelo, B. Wu, X. Xiong, X. Liu, L. Guan, degree in Biology for the Molecular, Cellular and Patho- T. Li, S. Liu, R. Yang, Y. Lu, L. Dong, S. McGettigan, R. Somasundaram, physiological Research at University Roma Tre in 2017. For R. Radhakrishnan, G. Mills, Y. Lu, J. Kim, Y.H. Chen, H. Dong, Y. Zhao, her PhD, she is investigating exosome production induced G.C. Karakousis, T.C. Mitchell, L.M. Schuchter, M. Herlyn, E.J. Wherry, X. Xu, by extracellular HIV-1 Nef in human monocyte-derived W. Guo, Exosomal PD-L1 contributes to immunosuppression and is associated with macrophages. anti-PD-1 response, Nature 560 (2018) 382–386, https://doi.org/10.1038/ s41586-018-0392-8. [154] F.L. Ricklefs, Q. Alayo, H. Krenzlin, A.B. Mahmoud, M.C. Speranza, H. Nakashima, J.L. Hayes, K. Lee, L. Balaj, C. Passaro, A.K. Rooj, S. Krasemann, B.S. Carter, C.C. Chen, T. Steed, J. Treiber, S. Rodig, K. Yang, I. Nakano, H. Lee, R. Weissleder, X.O. Breakefield, J. Godlewski, M. Westphal, K. Lamszus, G.J. Freeman, A. Bronisz, S.E. Lawler, E.A. Chiocca, Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles, Sci. Adv. 4 (2018), https://doi.org/10. Zulema Antonia Percario obtained her master degree in 1126/sciadv.aar2766 eaar2766. Biochemistry at “Universidad Nacional de Cordoba” [155] A. Ortiz, J. Gui, F. Zahedi, P. Yu, C. Cho, S. Bhattacharya, C.J. Carbone, Q. Yu, (Argentine) in 1981 and completed her PhD in K.V. Katlinski, Y.V. Katlinskaya, S. Handa, V. Haas, S.W. Volk, A.K. Brice, K. Wals, Biomolecular and Cellular Sciences at Roma Tre University, N.J. Matheson, R. Antrobus, S. Ludwig, T.L. Whiteside, C. Sander, A.A. Tarhini, Rome (Italy) in 2009. She was working on Interferons J.M. Kirkwood, P.J. Lehner, W. Guo, H. Rui, A.J. Minn, C. Koumenis, J.A. Diehl, signal transduction at the Virology Lab of the Italian S.Y. Fuchs, An interferon-driven oxysterol-based defense against tumor-derived National Institute of Health (Istituto Superiore di Sanità) in extracellular vesicles, Cancer Cell 35 (2019) 33–45, https://doi.org/10.1016/j. Rome since 1989 and at University Roma Tre since 1976. At ccell.2018.12.001. present she is working at Department of Sciences, [156] P.W. Ramirez, S. Sharma, R. Singh, C.A. Stoneham, T. Vollbrecht, J. Guatelli, University Roma Tre as Responsible of Confocal Microscopy Plasma membrane-associated restriction factors and their counteraction by HIV-1 and Collaborator for Scientific Research in the Laboratory accessory proteins, Cells 8 (2019), https://doi.org/10.3390/cells8091020 pii: directed by Professor Elisabetta Affabris. She is currently E1020. interested in cytokine signal transduction pathways and [157] M.H. Malim, D. Pollpeter, APOBEC restriction goes nuclear, Nat. Microbiol. 4 extracellular vesicles. (2019) 6–7, https://doi.org/10.1038/s41564-018-0323-3. [158] M. Chemudupati, A.D. Kenney, S. Bonifati, A. Zani, T.M. McMichael, L. Wu, J.S. Yount, From APOBEC to ZAP: diverse mechanisms used by cellular restriction Elisabetta Affabris is Full Professor of Virology and Head factors to inhibit virus infections, Biochim. Biophys. Acta Mol. Cell. Res. 1866 of Molecular Virology and Antimicrobial Immunity Lab at (2019) 382–394, https://doi.org/10.1016/j.bbamcr.2018.09.012. Department of Sciences, University Roma Tre, Rome, Italy. [159] T. Pertel, S. Hausmann, D. Morger, S. Züger, J. Guerra, J. Lascano, C. Reinhard, She completed her master degree in Biological Science at La F.A. Santoni, P.D. Uchil, L. Chatel, A. Bisiaux, M.L. Albert, C. Strambio-De- Sapienza University of Rome in 1976 and soon afterward Castillia, W. Mothes, M. Pizzato, M.G. Grütter, J. Luban, TRIM5 is an innate im- she started to work with a fellowship of CNR-Progetto mune sensor for the retrovirus capsid lattice, Nature 472 (2011) 361–365, https:// Finalizzato Virus at Istituto Superiore di Sanità, Laboratory doi.org/10.1038/nature09976. of Virology under the supervision of Professor Giovanni [160] J.M. Jimenez-Guardeño, L. Apolonia, G. Betancor, M.H. Malim, Battista Rossi. She became Staff Investigator at University Immunoproteasome activation enables human TRIM5α restriction of HIV-1, Nat. of Rome La Sapienza in 1981 and Associate Professor of Microbiol. 4 (2019) 933–940, https://doi.org/10.1038/s41564-019-0402-0. Microbiology at University of Messina in 1988. Her main [161] F. Siegrist, M. Ebeling, U. Certa, The small interferon-induced transmembrane research interests are virus–cell interactions in retroviral genes and proteins, J. Interferon Cytokine Res. 31 (2011) 183–197, https://doi. infections, HIV Nef protein functions, antiviral innate im- org/10.1089/jir.2010.0112. munity and viral immunoevasion, interferons regulation of signal transduction. [162] J.M. Perreira, C.R. Chin, E.M. Feeley, A.L. Brass, IFITMs restrict the replication of multiple pathogenic viruses, J. Mol. Biol. 425 (2013) 4937–4955, https://doi.org/ 10.1016/j.jmb.2013.09.024.

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