Mast cell secretome: Soluble and vesicular components

Accepted Manuscript Title: Mast cell secretome: soluble and vesicular components ´ am Authors: Krisztina V. Vukman, Andr´as F¨ors¨onits, Ad´ ´ T´oth, Edit I. Buz´as Oszvald, Eszter A. PII: DOI: Reference:

S1084-9521(17)30107-6 http://dx.doi.org/doi:10.1016/j.semcdb.2017.02.002 YSCDB 2193

To appear in:

Seminars in Cell & Developmental Biology

Received date: Revised date: Accepted date:

3-10-2016 17-1-2017 7-2-2017

´ am, T´oth Please cite this article as: Vukman Krisztina V, F¨ors¨onits Andr´as, Oszvald Ad´ ´ Buz´as Edit I.Mast cell secretome: soluble and vesicular components.Seminars Eszter A, in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mast cell secretome: soluble and vesicular components Authors name and affiliations Krisztina V. Vukman, András Försönits, Ádám Oszvald, Eszter Á. Tóth, Edit I. Buzás Semmelweis University Department of Genetics, Cell- and Immunobiology, H-1089 Budapest, Hungary

Corresponding author Edit I Buzás Semmelweis University Department of Genetics, Cell- and Immunobiology, Üllői út 26. 1085 Budapest, Hungary e-mail: [email protected]

Graphical abstract

Abstract

Mast cells are multifunctional master cells implicated in both innate and adaptive immune responses. Their role has been best characterized in allergy and anaphylaxis; however, emerging evidences support their contribution to a wide variety of human diseases. Mast cells, being capable of both degranulation and subsequent recovery, have recently attracted substantial attention as also being rich sources of secreted extracellular vesicles (including exosomes and microvesicles). Along with secreted de novo synthesized soluble molecules and secreted preformed granules, the membrane-enclosed extracellular vesicles represent a previously unexplored part of the mast cell secretome. In this review article we summarize available data regarding the different soluble molecules and membrane-enclosed structures secreted by mast cells. Furthermore, we provide an overview of the release mechanisms including degranulation, piecemeal degranulation, transgranulation, and secretion of different types of extracellular vesicles. Finally, we aim to give a summary of the known biological functions associated with the different mast cell-derived secretion products. The increasingly recognized complexity of mast cell secretome may provide important novel clues to processes by which mast cells contribute to the development of different pathologies and are capable of orchestrating immune responses both in health and disease.

Abbreviations CCL CTLR ER EV EXO HSV ICAM IFN Ig IL ITAM LFA LPS LT MCCT MCT MGL MHC MR MV PAF PAI PAP PG PL PMD

chemokine (C-C motif) ligand C-type lectin receptors endoplasmic reticulum extracellular vesicle exosome Herpes simplex virus intercellular adhesion molecule interferon Immunoglobulin interleukin tyrosine-based activation motif lymphocyte function-associated antigen lipopolysaccharide leukotriene chymase- and tryptase-positive mast cell tryptase-positive mast cell macrophage galactose lectin major histocompatibility complex mannose receptor microvesicle platelet activating factor plasminogen activator inhibitor phosphatide phosphatase prostaglandin phospholipase piecemeal degranulation

SNARE TGF Th TLR TNF XCL

soluble NSF attachment protein receptor transforming growth factor T-helper Toll like receptor tumour necrosis factor chemokine (C motif) ligand

Keywords: mast cell, granule, extracellular vesicle, exosome, mediator, cell-to-cell communication

1. Introduction Mast cells are bone marrow-derived hematopoietic innate immune cells. They are resident in various tissues particularly at sites which are exposed to environmental stimuli such as skin, gastrointestinal tract or airways [1]. Mast cells are typically classified into mucosal and connective tissue mast cell categories with different localization, phenotypes, molecular compositions and functions [2]. Mast cells are major components of protective immunity against various infectious agents including bacteria, viruses and parasites, however, they have been most intensively studied in the context of T-helper (Th) 2 immune responses. Mast cells express high affinity receptor FcεRI on their surface. In type I hypersensitivity reaction, following immunoglobulin (Ig) Esensitization, they are activated by specific cross-linking antigens. As a consequence, they secrete various preformed and newly synthesized mediators like histamine, cytokines, and subsequently induce Th2 immune responses [3, 4]. Although it is less known, mast cells also play a key role in the development of Th1 immune responses, and they are involved in Th1 immune diseases such as rheumatoid arthritis [5]. Mast cells express pathogen recognition receptors (PRRs) such as toll like receptors (TLRs) including TLR2, TLR3, TLR4, TLR6, TLR7 and TLR9 that recognize invading pathogens [5]. Following stimulation by a bacterial ligand, they secrete cytokines and express co-stimulatory molecules that promote proinflammatory Th1 immune responses [1, 6-9]. Furthermore, mast cell-induced Th1 immune response is crucial in the clearance of protozoan and bacterial infections for instance by Leishmania major [10], Escherichia coli [11], Pseudomonas aeruginosa [12] or Bordetella pertussis [13, 14]. The broad range of preformed and newly synthesized mediators allow mast cells to interact with both B and T cells and regulate the phenotype of other immune cells by which they can shape the host response. Expression of cell surface markers, adhesion molecules, costimulatory and co-inhibitory molecules are also important in direct cell-to-cell communication [15], although the exact mechanism is unclear yet [16, 17]. Recent studies suggest that mast cells may communicate with other immune cells not only by releasing soluble mediators but also by secreted, membrane enclosed vesicles [18-20]. This review will focus on the possible mechanism of how mast cell-derived vesicles and soluble mediators participate in the orchestration of innate and adaptive immune responses.

2. Content of preformed mast cell granules

Preformed granules of mast cells contain proteoglycans, proteases, biogenic amines, lysosomal enzymes, cytokines and growth factors [21]. Mast cell glycosaminoglycans/proteoglycans include heparin, heparane sulfate [22] and serglycin [23], all responsible for the metachromatic staining of these cells [24]. Granular proteoglycans both mediate storage of granule components such as histamine [25] and regulate the release of secreted molecules [24]. After release into the extracellular space, granular proteoglycans exert both positive and negative effects on the enzymatic properties of mast cell proteases. They are known as anticoagulants regulating haemostasis [24]. Furthermore, serglycin of mast cell granules has been shown to promote apoptosis [26]. Best known examples of mast cell proteases are tryptase and chymase. Murine mucosal mast cells (homologues of human chymase+/tryptase+ mast cells, MCCTs) are characterized by the expression of active tryptase and chymase. In contrast, connective tissue mast cells (corresponding to human tryptase+/chymase- mast cells, MCTs) show tryptase activity only [2]. These enzymes are stored in mast cell granules [27], and may play important roles in inflammation and host defence [28]. In epithelial cells, tryptase upregulates interleukin (IL)-8, intercellular adhesion molecule (ICAM)-1 [29] and it has been suggested to play a role in the immune pathomechanism of psoriasis. Tryptase induces vascular relaxation [30] and activates sensory neurons (promoting secretion of substance P) inducing inflammation [31].This protease also activates other mast cells [32]. Chymase was shown to play a role in gut homeostasis, it regulates intestinal transport and has an effect on gastrointestinal smooth muscle cells [27]. From among lysosomal enzymes found in mast cells, β-hexosaminidase is generally used as a marker of mast cell degranulation [33]. However, the physiological and pathophysiological role of this enzyme remains unclear. It was shown that it can be found both in granules and in lysosomes, and it is essential for glycoprotein metabolism in the maintenance of cell homeostasis [34]. β-hexosaminidase has been also suggested to play a crucial role in eradication of bacterial infections by degrading bacterial cell wall peptidoglycans [34]. Biogenic amines (such as histamine, serotonine and dopamine) are probably the best known bioactive molecules stored in mast cell granules. From among the multitude of known effects of histamine, here we just mention a few examples such as increase in local blood flow and vascular permeability, effect on smooth muscle cells during inflammation and allergy (reviewed recently [21]). Also, it has been established that histamine regulates homeostasis in the body; it controls sleep-wake cycle [35], body temperature [36], gastrointestinal functions [37], and endocrine homeostasis [38]. Of note, studies on either histamine deficient histidine decarboxylase knock-out or serglycin knock-out mice suggest that both histamine and serglycin regulate granule maturation process [19]. Granules also contain cytokines and growth factors which are released during degranulation. These molecules are involved in i) the activation of other cell types, ii) induction of inflammation, iii) development of immune response during infection, iv) pathogenesis of allergy and v) development of autoimmune diseases [9, 39, 40]. 3. Release of preformed granules (degranulation) Mast cells are usually identified by the electron microscopic presence of electron dense lysosome-like secretory granules. These secretory granules are filled with various preformed molecules such as lysosomal proteins, histamine, heparin and β hexosaminidase among others [41]. Upon exposure to various stimuli such exposure to IgE and its antigenic ligands, complement components, peptides/neuropeptides, mast cells release the content of these

granules within minutes by a process called degranulation. Of note, degranulation has been described not only in mast cells but also in eosinophils [42], basophils [43, 44], neutophils [45], neuroendocrine cells and neurons [46]. In the past decades conventional degranulation of mast cells has been studied extensively. Mast cells express the high affinity receptor FcεRI on their surface. Upon cross-linking of the FcεRI-bound IgE by antigen, the receptors aggregate and induce a signalling pathway that involves phosphorylation of tyrosine-based activation motifs (ITAMs) and activation of FYN, LYN and SYK [47]. Soluble NSF attachment protein receptor (SNARE) proteins such as SNAP-23, syntaxin 4, VAMP7 and VAMP8 are involved in translocation of granules from the cytoplasm to the lysosomal compartment or to the internal surface of the plasma membrane. After docking at the plasma membrane, granules fuse with it and release their content to the extracellular milieu [21, 48]. The exocytosis of granules is tightly controlled. It requires mobilization of Ca2+, activation of protein kinase C, hydrolysis of adenosine triphosphate (ATP) and guanosin triphosphate (GTP) and reorganization of the actin cytoskeleton (reviewed recently by Wernersson and Pejler in 2014 [21]) (Figure 1). Once discussing degranulation, we must mention “piecemeal degranulation” (PMD) also (Figure 1.). PMD was first recognized in basophils [49, 50], mast cells [51, 52] and eosinophils [53-55] as a selective release of a part of the granular content. Number of evidences support that it also occurs in neuroendocrine cells and neurons [46, 56]. During PMD, vesicles, containing a given portion of the granular content, bud from the granule’s membrane. This is followed by transportation through the cytoplasm (without granule-togranule fusion) leading to fusion with the plasma membrane and ultimate release of mediators [46, 51]. Similarly to degranulation, the molecular machinery of this process is yet to be identified, although SNAREs seems to play a central role in it [57]. PMD is the most prevalent form of granule loss identified in situ in mast cells during pathological processes in allergy [58], Crohn’s disease [54], urticaria [59], chronic inflammation [60] or malignant tumours [61]. PMD seems to also play a role in communication of mast cells with other cell types. It was shown that during PMD, mediators from mast cells could induce upregulation of chemokine ligand 2(CCL2) in epithelial cells [62], activation of regulatory T-cells [63] and affect the behaviour of tumour cells [64]. In 1983 a novel cell-to-cell interaction has been described between mast cells and other cell types referred to as “transgranulation” or “pseudopod translocation” [65]. When the distance between two cells is small enough, granules are transferred directly from cell to cell by exocytosis and subsequent immediate uptake. Mast cells also extend granule-containing pseudopods towards adjacent cells. In this case the transfer of the granular content to adjacent cells may involve detachment of the pseudopod as an extracellular granule that is subsequently taken up from the extracellular space by other cells. Transgranulation has been observed so far between mast cells and i) fibroblast [66, 67], ii) vascular endothelial cells [65] and iii) neurons [68, 69]. Lipid bodies are roughly spherical, variably osmophilic organelles located in the cytoplasm. They have been described in a wide variety of cells, but were mostly investigated in granulocytes, macrophages and mast cells [70, 71]. Their number has been shown to vary depending on the location of the mast cells, and it correlates with inflammatory conditions such as bacterial or parasitic infections [72] or chronic exposure to metabolic stimuli (insulin) [73, 74]. Lipid bodies were found to contain lipids, variety of proteins [70], RNA [75], tumour necrosis factor-α (TNF-α) [76] and basic fibroblast growth factor (bFGF) [77]. Few cell types (such as enterocytes) were shown to be able to secrete lipid bodies through the endoplasmic reticulum (ER) and Golgi system [78]. Although the biogenesis and exact mechanism of release is yet to be identified in mast cells, lipid bodies are considered to play a role in proinflammatory processes and communication between immune cells [19].

4. Release of de novo synthesized soluble mediators Beside the secretion of pre-formed mediators during degranulation, mast cells are also able to de novo synthesize and release both pro- and anti-inflammatory mediators upon stimulation. As we mentioned earlier, mast cells express PRRs on their surface, and thus, can detect pathogens and respond to them (Figure 1). Mast cell-derived IL-6 and TNF were shown to play roles in the immune response against Herpes simplex virus 2 (HSV2, a TLR2/9 ligand) [79]. Furthermore, poly (I:C), R-848 and CpG oligodeoxynucleotides (ligands of TLR3, TLR7 and TLR9, respectively) were shown to induce TNF, IL-6 but no IL-13 secretion by mast cells. In contrast, stimulation with lipopolysaccharide or peptidoglycan induced the release of IL-13 [80]. Besides TLRs, mast cells also express co-receptors [7]] and C-type lectin receptors (CTLRs) on their surface [14, 81]. Macrophage galactose lectin (MGL), mannose receptor (MR) and dectin1 were shown to play roles in the recognition of Bordatella pertussis by mast cells [14]. Interestingly, although mast cells can be activated by TLR4 ligands, they fail to express CD14, the most important TLR4 co-receptor [82]. Mast cells can be also activated by pathogens through FcεRI, complement receptors and cytokine receptors, and secrete different anti- and pro-inflammatory mediators. There might be cross-talk between these different types of receptors modifying the signalling pathways of each other. This system is very complex and carefully regulated, and results in well-orchestrated immune responses against pathogens. Further mediators reported to be released by mast cells include interferon (IFN)-γ [83], IL-10, IL-12 [84], transforming growth factor (TGF)-β [85], the chemokines CCL1-5, CCL7, CCL18, CCLL2-3, CCLL8, XCL1 [86], lipid mediators such as leukotriene (LT) C4 and LTB4 prostaglandin (PG) D2 [87], and platelet activating factor (PAF) [88]. Lymphocyte-dependent mast cell activation has been investigated extensively during T-cell mediated inflammatory responses. T lymphocytes can induce degranulation, secretion of βhexosaminidase, release of several inflammatory cytokines (tumour necrosis factor (TNF)-α, IL-4 and IL- 6) and chemokines (CXCL2, CCL5) following direct contact with mast cells [89]. Mast cell-derived mediators can also activate T-cells and are known to have immunoregulatory and immune modulating effects on T-cell-dependent responses [90]. Due to their location, they are key cells of early host defence and can act as APCs and induce Tcell activation. Mast cells can be found in most tissues including secondary lymphoid organs, so they have the potential to prime naïve T-cells or directly alter T-cell functions [91] via their cell surface molecules or secreted mediators. Mast cells have been implicated in the regulation of Th2 immune responses where mast cell-derived IL-4 was shown to be important in activation of CD4+ T cells [17]. IL-12 and TNF-α also released by mast cells, induced Th1 polarization, while mast cell-derived TGF-β and IL-6 were shown to be important in Th17 immune responses [91, 92]. In a rodent model of multiple sclerosis, T-cell activation (IFN-γ production, and up-regulation CD44 and CD11a) was shown to be mast cell dependent [93]. Mast cells can regulate T-cell migration directly by producing chemotactic factors (IL-16, CCL2/MCP-1, CCL3/MIP-1a, CCL4/MIP-1b or TGF-b). Mast cells induce the expression of adhesion molecules on endothelial cells and induce T-cell activation indirectly [94]. As far as non-T-cells are concerned, mast cells were shown to communicate with dendritic cells or Bcells also. Mast cell-derived IL-4 initiates the allergic response by inducing IgE production by B-cell-derived plasma cells [8, 17]. Interaction of the CD40 and CD40L co-stimulatory molecules was shown to be involved in mast cell-induced IgE production by B-cell-derived plasma cells [17, 94]. There are many evidences that mast cells can regulate dendritic cell functions by the production of TNF, IL-1, IL-16, IL-18 or PGD2 [94]. Mast cell - dendritic

cell interactions were suggested to affect antigen presentation function of dendritic cells [95, 96]. Both soluble mediators and direct cross-talk were found important in these processes [89]. Mast cells were also shown to induce natural killer cell, eosinophil and neutrophil accumulation and activation in different animal models for bacterial infection, allergy and autoimmune disease [89].

5. Mast cell-derived extracellular vesicles Extracellular vesicles (EVs) are membrane surrounded structures that are secreted by cells and are detectable in biological fluids as well as in conditioned media of cultured cells. EVs are heterogeneous structures. Different subtypes based on biogenesis and size include exosomes (small EVs; sEVs, 30-150 nm), microvesicles (MVs, medium sized EVs, 100-1000 nm) and apoptotic bodies and large oncosomes (large EVs, 1000-5000 nm) [97-99]. Although the above size ranges are not absolute, they seem to identify EV subcategories with distinct biochemical properties possibly reflecting differential biogenic pathways [100, 101]. Exosomes are formed within the endosomal compartment and then released by exocytosis of the cells. In contrast, larger EVs arise through direct budding from the plasma membrane [102] (Figure 1). There is a continuously increasing number of reports on mast cell-derived EVs, although in most studies mast cells are used as non-specific cellular sources of EVs. Of note, in most of these papers there is limited emphasis on mast cell-specific biological functions of EVs[103, 104]. Interestingly, most reports on mast cell-derived EVs focus exclusively on exosomes in spite of the fact that mast cell lines have been shown to secrete larger EVs as well with differential RNA and DNA patterns [105]. Mast cell-derived exosomes carry RNAs including mRNA, miRNA, sRNA in their lumen, and the RNA profile differ between EVs and their donor cells [104, 106]. Importantly, mast cell-derived exosomes are able to transfer functional mRNA and miRNA (exosomal shuttle RNA) to recipient CD34+ progenitor cells and mast cells. These findings suggest that RNA shuttle by exosomes is an important mechanism of cell-to-cell communication. Exosomal RNA may affect different cellular pathways of the recipient cells such as cellular development, migration or biosynthesis of various mediators (Table 1 and Figure 2) [107109]. For example, exosomes derived from mast cells previously exposed to oxidative stress, differ in their RNA content from those secreted by non-exposed cells. These exosomes when added to other cells, could provide resistance against oxidative stress [108]. Major histocompatibility complex (MHC)-II molecules are known to be characteristically present on the surface of exosomes secreted by various different cell types. Strikingly, in the case of mast cells, MHC-II molecules do not only accumulate in the membranes of exosomes, but also inside some special mast cell granules. These MHCII-containing granules show characteristic morphology: type I granules contain small, exosome-like vesicles, while type II granules contain a dense core surrounded by small vesicles [110]. It has been long established that the level of MHC-II molecules increases upon calcium-dependent triggering of mast cells (Table 1) [111]. Calcium triggering induced the release of mast cell exosomes with peptide loaded MHC-II molecules which were suggested to play a role in antigen presentation and Tcell stimulation [111, 112]. Exosomes from mast cells have been reported to carry both of the pan-mast cell markers FcεRI and CD117 (Kit). Kit protein (but not c-Kit mRNA) was transferred by exosomes from mast cells to adenocarcinoma cells, and induced PI3K/AKT signalling, migration and cell proliferation [113], underlining the importance of mast cellderived exosomes in tumour development (Table 1). Furthermore, mast cell-derived

exosomes have been also confirmed by several groups to exhibit typical exosome markers on their surface, such as CD9, CD63 or CD81 (Table 1) [107, 114, 115]. The membrane of mast cell-derived exosomes differs in its lipid composition from the plasma membrane of the parent cells. Interestingly, it is enriched in sphingomyelin, but not in cholesterol. This is contrast from what has been shown for exosomes secreted by other types of cells (in the case of which exosomes were found to be enriched in cholesterol [100, 116]). Mast cell exosomes show a decreased level of phosphatidylcholine, and enrichment in phosphatidylethanolamine [116]. Several lipid-related proteins have been also identified in mast cell exosomes. Such lipid-related proteins include phospholipid scramblase, fatty acid binding protein, phospholipases PLA2, PLC and PLD. Mast cell-derived exosomes were shown to transfer PLA2G4D to neighbouring cells thus, contributing to a CD1-reactive T cell response in psoriasis patients [117] (Table 1). Numerous other mast cell exosome-associated molecules have been also implicated in EVmediated biological functions. TGF-β-carrying exosomes derived from degranulating mast cells were reported to play a role in the pathogenesis of systemic sclerosis possibly by attracting inflammatory cells and by stimulating fibroblasts (Table 1) [118]. Several further proteins or precursors associated with mast cell-derived exosomes, have been described with possible immune-regulatory potential including 60- and 70-kDa heat shock proteins [114, 119], factor V precursor, prothrombin, angiotensinogen and TNF precursors [120]. A distinctive set of mast cell exosome-associated proteins (CD13, ribosomal protein S6 kinase, annexin V, CDC25, γ-actin-like protein, γ-actin and cytoplasmic γ-actin) have been suggested to mediate specific and nonspecific immunostimulatory activities (Table 1) [121]. Mast cellderived exosome-associated molecules such as aldolase, hsp70, phosphatidate phosphatase (PAP) 1, GTPase, free fatty acids and PGE2 have been suggested to play roles in triggering functional maturation of dendritic cells by mast cell-derived exosomes [106, 122]. Furthermore, mast cell-derived exosomes are characterized by immune function-associated membrane proteins such as CD86, CD40L, LFA-1, and ICAM-1 in addition to the previously mentioned MHCII [94]. Mast cell-derived exosomes were reported to i) induce antigenspecific antibody responses in mice in vivo, ii) lead to maturation of dendritic cells (by upregulation of MHCII, CD40, CD80 and CD86) and iii) to induce proliferation and cytokine secretion of B-cells in vitro (Table 1, Figure 2) [110, 114, 115, 119]. Unstimulated mast cells induce proliferation and IgM production of B-cells. This activation is independent of soluble cytokines [123], but seems to involve mast cell exosomes [115]. Moreover, the interaction between mast cells and B-cells appears to be bidirectional as B-cell derived EVs altered mast cell functions as well [124]. Communication between mast cells and T-cells also involves exosomes. Mast cell-derived exosomes stimulate cytotoxic T-cells in vitro, although they require the presence of dendritic cells as well [125] (Figure 2). These exosomes also induce cytokine secretion of splenocytes or CD4 positive T-lymphocytes [110, 115]. Mast cellderived exosomes expressing OX40L ligand, were shown to enhance the proliferation and differentiation of naïve CD4+ cells to Th2 cells via a receptor-ligand interaction (Table 1, Figure 2) [126]. In addition, during inflammation, mast cell-derived exosomes play a key role in the deposition of fibrin. These exosomes carry all proteins required for their attachment to endothelial cells, and they induce plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells in vitro [120] (Figure 2). Increasing number of evidences suggest that mast cell-derived exosomes have distinct functions in tumour progression. They can stimulate antitumour immune responses in early stages of cancer or can also be involved in tumour growth by inducing angiogenesis and recruitment of macrophages and fibroblasts (Figure 2) [20]. Protease-rich mast cell-exosomes were investigated in hyperoxia-induced neonatal lung disease. Although the function of these exosomes remains unknown, they were suggested to alter the phenotype of other cells in both paracrine and endocrine fashions [127]. Mast cell-

derived exosomes were also involved in neuro-immune regulation. They were shown to mediate the exchange of information between mast cells and sensory nerve endings, and resulted in substance P secretion by neurons, which could further activate mast cells [128]. Mast cell-derived exosomes reach neurons through the cerebrospinal fluid as well and may have an effect on neural synapses [129], thus, affecting for instance pain signalling (Table 1, Figure 2) [130].

6. Extracellular vesicle secretion or degranulation? However, it is not an obvious task to distinguish mast cell-derived EV secretion and degranulation from one another, a question overlooked by most of the authors until now. During degranulation, mast cells release membrane surrounded structures with markers similar to those of EVs, such as CD63, CD81 or SNARE proteins [21, 65]. Recently, IgEdependent mast cell degranulation was shown to lead to a rapid release of exosomes. These exosomes expressed regular exosome markers (CD9 and CD63) and their endogenous origin was also confirmed. In addition, they were shown to carry molecules released also in soluble form during steady state conditions and degranulation, such as hsp60, hsp70, Kit and functional carboxypeptidase A3 [131]. Of note, studies investigating mast cell-derived EVs use different conditions to produce and harvest exosomes. In some studies, mast cells are cultured for 2-3 days without any stimulation [106, 115, 123], while in others degranulation is induced in IgE dependent [114, 125] or independent [104, 106, 107] manner, and exosomes are collected either after 30 minutes or after days. This makes the comparison of published results even more difficult. 7. Conclusion Mast cells are innate immune cells located along blood vessels and mucosal surfaces throughout the body. Although their functions are under intense investigation, several authors suggest that their roles are still strongly underestimated. Besides being key players in allergic diseases and anaphylaxis, they have been suggested to play as conductors in certain tumours [132], inflammatory arthritis [133, 134] and inflammatory bowel disease [135]. In addition, mast cell proliferation and accumulation are central features of the human disease, mastocytosis. Because of the heterogeneity of mastocytosis, its diagnosis remains challenging [135, 136]. The discovery of novel components of the mast cell secretome (such as EVs) may shed new light on the functions of mast cells, and may help to identify novel mast cell-derived biomarkers and therapeutic targets in the above diseases. Acknowledgements Founding sources: This work was supported by National Scientific Research Program of Hungary (OTKA) #11958 and #120237; #PD104369, #PD112085; #PD 109051, MEDINPROT Program, BMBS COST Action BM1202 ME HAD, FP7-PEOPLE-2011-ITN– PITN-GA-2011-289033 DYNANO, NVKP_16-1-2016-0017. The first author was also supported by János Bolyai Research Fellowship of the Hungarian Academy of Sciences.

Refereces

[1] S.J. Galli, M. Tsai, Mast cells in allergy and infection: versatile effector and regulatory cells in innate and adaptive immunity, Eur J Immunol 40(7) (2010) 1843-51. [2] A.M. Irani, T.R. Bradford, C.L. Kepley, N.M. Schechter, L.B. Schwartz, Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and antichymase antibodies, J Histochem Cytochem 37(10) (1989) 1509-15. [3] A.J. Melendez, M.M. Harnett, P.N. Pushparaj, W.S. Wong, H.K. Tay, C.P. McSharry, W. Harnett, Inhibition of Fc epsilon RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes, Nat Med 13(11) (2007) 1375-81. [4] E.J. Pearce, Worms tame mast cells, Nat Med 13(11) (2007) 1288-9. [5] J.S. Marshall, C.A. King, J.D. McCurdy, Mast cell cytokine and chemokine responses to bacterial and viral infection, Curr Pharm Des 9(1) (2003) 11-24. [6] S.N. Abraham, A.L. St John, Mast cell-orchestrated immunity to pathogens, Nat Rev Immunol 10(6) (2010) 440-52. [7] W. Dawicki, J.S. Marshall, New and emerging roles for mast cells in host defence, Curr Opin Immunol 19(1) (2007) 31-8. [8] D.D. Metcalfe, D. Baram, Y.A. Mekori, Mast cells, Physiol Rev 77(4) (1997) 1033-79. [9] C.L. Weller, S.J. Collington, T. Williams, J.R. Lamb, Mast cells in health and disease, Clin Sci (Lond) 120(11) (2011) 473-84. [10] M. Maurer, S. Lopez Kostka, F. Siebenhaar, K. Moelle, M. Metz, J. Knop, E. von Stebut, Skin mast cells control T cell-dependent host defense in Leishmania major infections, FASEB J 20(14) (2006) 2460-7. [11] M. Wierzbicki, E. Brzezinska-Blaszczyk, Diverse effects of bacterial cell wall components on mast cell degranulation, cysteinyl leukotriene generation and migration, Microbiol Immunol 53(12) (2009) 694-703. [12] F. Siebenhaar, W. Syska, K. Weller, M. Magerl, T. Zuberbier, M. Metz, M. Maurer, Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent, Am J Pathol 170(6) (2007) 1910-6. [13] N. Mielcarek, E.H. Hornquist, B.R. Johansson, C. Locht, S.N. Abraham, J. Holmgren, Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin, Cell Microbiol 3(3) (2001) 181-8. [14] K.V. Vukman, A. Ravida, A.M. Aldridge, S.M. O'Neill, Mannose receptor and macrophage galactose-type lectin are involved in Bordetella pertussis mast cell interaction, J Leukoc Biol 94(3) (2013) 439-48. [15] T. Kambayashi, E.J. Allenspach, J.T. Chang, T. Zou, J.E. Shoag, S.L. Reiner, A.J. Caton, G.A. Koretzky, Inducible MHC class II expression by mast cells supports effector and regulatory T cell activation, J Immunol 182(8) (2009) 4686-95. [16] J. Kashiwakura, H. Yokoi, H. Saito, Y. Okayama, T cell proliferation by direct cross-talk between OX40 ligand on human mast cells and OX40 on human T cells: comparison of gene expression profiles between human tonsillar and lung-cultured mast cells, J Immunol 173(8) (2004) 5247-57. [17] B.A. Sayed, M.A. Brown, Mast cells as modulators of T-cell responses, Immunol Rev 217 (2007) 53-64. [18] I. Shefler, P. Salamon, A.Y. Hershko, Y.A. Mekori, Mast cells as sources and targets of membrane vesicles, Curr Pharm Des 17(34) (2011) 3797-804. [19] T.C. Moon, A.D. Befus, M. Kulka, Mast cell mediators: their differential release and the secretory pathways involved, Front Immunol 5 (2014) 569. [20] A. Benito-Martin, A. Di Giannatale, S. Ceder, H. Peinado, The new deal: a potential role for secreted vesicles in innate immunity and tumor progression, Front Immunol 6 (2015) 66. [21] S. Wernersson, G. Pejler, Mast cell secretory granules: armed for battle, Nat Rev Immunol 14(7) (2014) 478-94. [22] B. Wang, J. Jia, X. Zhang, E. Zcharia, I. Vlodavsky, G. Pejler, J.P. Li, Heparanase affects secretory granule homeostasis of murine mast cells through degrading heparin, J Allergy Clin Immunol 128(6) (2011) 1310-1317 e8.

[23] E. Ronnberg, G. Pejler, Serglycin: the master of the mast cell, Methods Mol Biol 836 (2012) 20117. [24] E. Ronnberg, F.R. Melo, G. Pejler, Mast cell proteoglycans, J Histochem Cytochem 60(12) (2012) 950-62. [25] L.B. Schwartz, K.F. Austen, Enzymes of the mast cell granule, J Invest Dermatol 74(5) (1980) 34953. [26] F.R. Melo, I. Waern, E. Ronnberg, M. Abrink, D.M. Lee, S.M. Schlenner, T.B. Feyerabend, H.R. Rodewald, B. Turk, S. Wernersson, G. Pejler, A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway, J Biol Chem 286(7) (2011) 5423-33. [27] V. Payne, P.C. Kam, Mast cell tryptase: a review of its physiology and clinical significance, Anaesthesia 59(7) (2004) 695-703. [28] G.H. Caughey, Mast cell tryptases and chymases in inflammation and host defense, Immunol Rev 217 (2007) 141-54. [29] M.R. Namazi, Possible molecular mechanisms to account for the involvement of tryptase in the pathogenesis of psoriasis, Autoimmunity 38(6) (2005) 449-52. [30] E.J. Mackie, C.N. Pagel, R. Smith, M.R. de Niese, S.J. Song, R.N. Pike, Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals, IUBMB Life 53(6) (2002) 277-81. [31] G.H. Caughey, Mast cell proteases as protective and inflammatory mediators, Adv Exp Med Biol 716 (2011) 212-34. [32] S. He, M.D. Gaca, A.F. Walls, A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase, J Pharmacol Exp Ther 286(1) (1998) 289-97. [33] E.Z. da Silva, M.C. Jamur, C. Oliver, Mast cell function: a new vision of an old cell, J Histochem Cytochem 62(10) (2014) 698-738. [34] N. Fukuishi, S. Murakami, A. Ohno, N. Yamanaka, N. Matsui, K. Fukutsuji, S. Yamada, K. Itoh, M. Akagi, Does beta-hexosaminidase function only as a degranulation indicator in mast cells? The primary role of beta-hexosaminidase in mast cell granules, J Immunol 193(4) (2014) 1886-94. [35] K.B. Alstadhaug, Histamine in migraine and brain, Headache 54(2) (2014) 246-59. [36] Y. Makabe-Kobayashi, Y. Hori, T. Adachi, S. Ishigaki-Suzuki, Y. Kikuchi, Y. Kagaya, K. Shirato, A. Nagy, A. Ujike, T. Takai, T. Watanabe, H. Ohtsu, The control effect of histamine on body temperature and respiratory function in IgE-dependent systemic anaphylaxis, J Allergy Clin Immunol 110(2) (2002) 298-303. [37] P.K. Rangachari, Histamine: mercurial messenger in the gut, Am J Physiol 262(1 Pt 1) (1992) G113. [38] F. Roberts, C.R. Calcutt, Histamine and the hypothalamus, Neuroscience 9(4) (1983) 721-39. [39] M. Metz, M. Maurer, Mast cells--key effector cells in immune responses, Trends Immunol 28(5) (2007) 234-41. [40] R.E. Sutherland, X. Xu, S.S. Kim, E.J. Seeley, G.H. Caughey, P.J. Wolters, Parasitic infection improves survival from septic peritonitis by enhancing mast cell responses to bacteria in mice, PLoS One 6(11) (2011) e27564. [41] A.D. Hogan, L.B. Schwartz, Markers of mast cell degranulation, Methods 13(1) (1997) 43-52. [42] M. Spencer, L. Yang, A. Adu, B.S. Finlin, B. Zhu, L.R. Shipp, N. Rasouli, C.A. Peterson, P.A. Kern, Pioglitazone treatment reduces adipose tissue inflammation through reduction of mast cell and macrophage number and by improving vascularity, PLoS One 9(7) (2014) e102190. [43] J.L. Cromheecke, K.T. Nguyen, D.P. Huston, Emerging role of human basophil biology in health and disease, Curr Allergy Asthma Rep 14(1) (2014) 408. [44] R. Nadif, F. Zerimech, E. Bouzigon, R. Matran, The role of eosinophils and basophils in allergic diseases considering genetic findings, Curr Opin Allergy Clin Immunol 13(5) (2013) 507-13. [45] P. Lacy, Mechanisms of degranulation in neutrophils, Allergy Asthma Clin Immunol 2(3) (2006) 98-108.

[46] E. Crivellato, B. Nico, E. Bertelli, G.G. Nussdorfer, D. Ribatti, Dense-core granules in neuroendocrine cells and neurons release their secretory constituents by piecemeal degranulation (review), Int J Mol Med 18(6) (2006) 1037-46. [47] J. Rivera, N.A. Fierro, A. Olivera, R. Suzuki, New insights on mast cell activation via the high affinity receptor for IgE, Adv Immunol 98 (2008) 85-120. [48] J.R. Woska, Jr., M.E. Gillespie, Small-interfering RNA-mediated identification and regulation of the ternary SNARE complex mediating RBL-2H3 mast cell degranulation, Scand J Immunol 73(1) (2011) 8-17. [49] H.F. Dvorak, A.M. Dvorak, Basophilic leucocytes: structure, function and role in disease, Clin Haematol 4(3) (1975) 651-83. [50] A.M. Dvorak, R.A. Monahan-Earley, P. Estrella, S. Kissell, R.E. Donahue, Ultrastructure of monkey peripheral blood basophils stimulated to develop in vivo by recombinant human interleukin 3, Lab Invest 61(6) (1989) 677-90. [51] A.M. Dvorak, S. Kissell, Granule changes of human skin mast cells characteristic of piecemeal degranulation and associated with recovery during wound healing in situ, J Leukoc Biol 49(2) (1991) 197-210. [52] A.M. Dvorak, New aspects of mast cell biology, Int Arch Allergy Immunol 114(1) (1997) 1-9. [53] J.S. Erjefalt, M. Andersson, L. Greiff, M. Korsgren, M. Gizycki, P.K. Jeffery, G.A. Persson, Cytolysis and piecemeal degranulation as distinct modes of activation of airway mucosal eosinophils, J Allergy Clin Immunol 102(2) (1998) 286-94. [54] J.S. Erjefalt, L. Greiff, M. Andersson, E. Adelroth, P.K. Jeffery, C.G. Persson, Degranulation patterns of eosinophil granulocytes as determinants of eosinophil driven disease, Thorax 56(5) (2001) 341-4. [55] M. Karawajczyk, L. Seveus, R. Garcia, E. Bjornsson, C.G. Peterson, G.M. Roomans, P. Venge, Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season, Am J Respir Cell Mol Biol 23(4) (2000) 521-9. [56] E. Crivellato, B. Nico, D. Ribatti, Ultrastructural evidence of piecemeal degranulation in large dense-core vesicles of brain neurons, Anat Embryol (Berl) 210(1) (2005) 25-34. [57] H. Xu, M.G. Arnold, S.V. Kumar, Differential Effects of Munc18s on Multiple DegranulationRelevant Trans-SNARE Complexes, PLoS One 10(9) (2015) e0138683. [58] T. Hugle, Beyond allergy: the role of mast cells in fibrosis, Swiss Med Wkly 144 (2014) w13999. [59] P.R. Criado, R.F. Criado, C.F. Takakura, C. Pagliari, J.F. de Carvalho, M.N. Sotto, C. Vasconcellos, Ultrastructure of vascular permeability in urticaria, Isr Med Assoc J 15(4) (2013) 173-7. [60] E. Crivellato, B. Nico, F. Mallardi, C.A. Beltrami, D. Ribatti, Piecemeal degranulation as a general secretory mechanism?, Anat Rec A Discov Mol Cell Evol Biol 274(1) (2003) 778-84. [61] T. Demitsu, T. Inoue, M. Kakurai, T. Kiyosawa, K. Yoneda, M. Manabe, Activation of mast cells within a tumor of angiosarcoma: ultrastructural study of five cases, J Dermatol 29(5) (2002) 280-9. [62] S. Iwamoto, Y. Asada, N. Ebihara, K. Hori, Y. Okayama, J. Kashiwakura, Y. Watanabe, S. Kawasaki, N. Yokoi, T. Inatomi, K. Shinomiya, A. Murakami, A. Matsuda, Interaction between conjunctival epithelial cells and mast cells induces CCL2 expression and piecemeal degranulation in mast cells, Invest Ophthalmol Vis Sci 54(4) (2013) 2465-73. [63] B. Frossi, F. D'Inca, E. Crivellato, R. Sibilano, G. Gri, M. Mongillo, L. Danelli, L. Maggi, C.E. Pucillo, Single-cell dynamics of mast cell-CD4+ CD25+ regulatory T cell interactions, Eur J Immunol 41(7) (2011) 1872-82. [64] R.A. Caruso, F. Fedele, L. Rigoli, C. Inferrera, Mast cell interaction with tumor cells in small early gastric cancer: ultrastructural observations, Ultrastruct Pathol 21(2) (1997) 173-81. [65] G. Greenberg, G. Burnstock, A novel cell-to-cell interaction between mast cells and other cell types, Exp Cell Res 147(1) (1983) 1-13. [66] H.N. Claman, Mast cell changes in a case of rapidly progressive scleroderma-ultrastructural analysis, J Invest Dermatol 92(2) (1989) 290-5. [67] R. Giorno, J. Lieber, H.N. Claman, Ultrastructural evidence for mast cell activation in a case of neurofibromatosis, Neurofibromatosis 2(1) (1989) 35-41.

[68] K.C. Dines, H.C. Powell, Mast cell interactions with the nervous system: relationship to mechanisms of disease, J Neuropathol Exp Neurol 56(6) (1997) 627-40. [69] M. Wilhelm, R. Silver, A.J. Silverman, Central nervous system neurons acquire mast cell products via transgranulation, Eur J Neurosci 22(9) (2005) 2238-48. [70] A.M. Dvorak, H.F. Dvorak, S.P. Peters, E.S. Shulman, D.W. MacGlashan, Jr., K. Pyne, V.S. Harvey, S.J. Galli, L.M. Lichtenstein, Lipid bodies: cytoplasmic organelles important to arachidonate metabolism in macrophages and mast cells, J Immunol 131(6) (1983) 2965-76. [71] A.M. Dvorak, Ultrastructure of human mast cells, Int Arch Allergy Immunol 127(2) (2002) 100-5. [72] W.E. Greineisen, M. Speck, L.M. Shimoda, C. Sung, N. Phan, K. Maaetoft-Udsen, A.J. Stokes, H. Turner, Lipid body accumulation alters calcium signaling dynamics in immune cells, Cell Calcium 56(3) (2014) 169-80. [73] A. Dichlberger, S. Schlager, J. Lappalainen, R. Kakela, K. Hattula, S.J. Butcher, W.J. Schneider, P.T. Kovanen, Lipid body formation during maturation of human mast cells, J Lipid Res 52(12) (2011) 2198-208. [74] W.E. Greineisen, L.M. Shimoda, K. Maaetoft-Udsen, H. Turner, Insulin-containing lipogenic stimuli suppress mast cell degranulation potential and up-regulate lipid body biogenesis and eicosanoid secretion in a PPARgamma-independent manner, J Leukoc Biol 92(3) (2012) 653-65. [75] A.M. Dvorak, E.S. Morgan, P.F. Weller, RNA is closely associated with human mast cell lipid bodies, Histol Histopathol 18(3) (2003) 943-68. [76] W.J. Beil, P.F. Weller, M.A. Peppercorn, S.J. Galli, A.M. Dvorak, Ultrastructural immunogold localization of subcellular sites of TNF-alpha in colonic Crohn's disease, J Leukoc Biol 58(3) (1995) 284-98. [77] A.M. Dvorak, E.S. Morgan, P.F. Weller, Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells, Histochem J 33(7) (2001) 397-402. [78] D.J. Murphy, The biogenesis and functions of lipid bodies in animals, plants and microorganisms, Prog Lipid Res 40(5) (2001) 325-438. [79] R. Aoki, T. Kawamura, F. Goshima, Y. Ogawa, S. Nakae, A. Nakao, K. Moriishi, Y. Nishiyama, S. Shimada, Mast cells play a key role in host defense against herpes simplex virus infection through TNF-alpha and IL-6 production, J Invest Dermatol 133(9) (2013) 2170-9. [80] H. Matsushima, N. Yamada, H. Matsue, S. Shimada, TLR3-, TLR7-, and TLR9-mediated production of proinflammatory cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells, J Immunol 173(1) (2004) 531-41. [81] K.V. Vukman, T. Visnovitz, P.N. Adams, M. Metz, M. Maurer, S.M. O'Neill, Mast cells cultured from IL-3-treated mice show impaired responses to bacterial antigen stimulation, Inflamm Res 61(1) (2012) 79-85. [82] H. Sandig, S. Bulfone-Paus, TLR signaling in mast cells: common and unique features, Front Immunol 3 (2012) 185. [83] A.A. Gupta, I. Leal-Berumen, K. Croitoru, J.S. Marshall, Rat peritoneal mast cells produce IFNgamma following IL-12 treatment but not in response to IgE-mediated activation, J Immunol 157(5) (1996) 2123-8. [84] C. Song, Q. Zhang, X. Liu, Y. Shan, IL-12 and IL-10 production are differentially regulated by phosphatidylinositol 3-kinase in mast cells, Scand J Immunol 75(3) (2012) 266-72. [85] E. Mortaz, M.E. Givi, C.A. Da Silva, G. Folkerts, F.A. Redegeld, A relation between TGF-beta and mast cell tryptase in experimental emphysema models, Biochim Biophys Acta 1822(7) (2012) 115460. [86] K. Feuser, K.P. Thon, S.C. Bischoff, A. Lorentz, Human intestinal mast cells are a potent source of multiple chemokines, Cytokine 58(2) (2012) 178-85. [87] T.J. Olynych, D.L. Jakeman, J.S. Marshall, Fungal zymosan induces leukotriene production by human mast cells through a dectin-1-dependent mechanism, J Allergy Clin Immunol 118(4) (2006) 837-43.

[88] K. Palgan, Z. Bartuzi, Platelet activating factor in allergies, Int J Immunopathol Pharmacol 28(4) (2015) 584-9. [89] G. Gri, B. Frossi, F. D'Inca, L. Danelli, E. Betto, F. Mion, R. Sibilano, C. Pucillo, Mast cell: an emerging partner in immune interaction, Front Immunol 3 (2012) 120. [90] S. Bulfone-Paus, R. Bahri, Mast Cells as Regulators of T Cell Responses, Front Immunol 6 (2015) 394. [91] M.A. Brown, B.A. Sayed, A. Christy, Mast cells and the adaptive immune response, J Clin Immunol 28(6) (2008) 671-6. [92] S. Nakae, H. Suto, M. Iikura, M. Kakurai, J.D. Sedgwick, M. Tsai, S.J. Galli, Mast cells enhance T cell activation: importance of mast cell costimulatory molecules and secreted TNF, J Immunol 176(4) (2006) 2238-48. [93] G.D. Gregory, A. Bickford, M. Robbie-Ryan, M. Tanzola, M.A. Brown, MASTering the immune response: mast cells in autoimmunity, Novartis Found Symp 271 (2005) 215-25; discussion 225-31. [94] S.J. Galli, S. Nakae, M. Tsai, Mast cells in the development of adaptive immune responses, Nat Immunol 6(2) (2005) 135-42. [95] P.J. Bryce, M.L. Miller, I. Miyajima, M. Tsai, S.J. Galli, H.C. Oettgen, Immune sensitization in the skin is enhanced by antigen-independent effects of IgE, Immunity 20(4) (2004) 381-92. [96] A. Mazzoni, R.P. Siraganian, C.A. Leifer, D.M. Segal, Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T cells, J Immunol 177(6) (2006) 3577-81. [97] B. Gyorgy, T.G. Szabo, M. Pasztoi, Z. Pal, P. Misjak, B. Aradi, V. Laszlo, E. Pallinger, E. Pap, A. Kittel, G. Nagy, A. Falus, E.I. Buzas, Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles, Cell Mol Life Sci 68(16) (2011) 2667-88. [98] J. Kowal, G. Arras, M. Colombo, M. Jouve, J.P. Morath, B. Primdal-Bengtson, F. Dingli, D. Loew, M. Tkach, C. Thery, Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes, Proc Natl Acad Sci U S A 113(8) (2016) E968-77. [99] V.R. Minciacchi, M.R. Freeman, D. Di Vizio, Extracellular vesicles in cancer: exosomes, microvesicles and the emerging role of large oncosomes, Semin Cell Dev Biol 40 (2015) 41-51. [100] X. Osteikoetxea, A. Balogh, K. Szabo-Taylor, A. Nemeth, T.G. Szabo, K. Paloczi, B. Sodar, A. Kittel, B. Gyorgy, E. Pallinger, J. Matko, E.I. Buzas, Improved characterization of EV preparations based on protein to lipid ratio and lipid properties, PLoS One 10(3) (2015) e0121184. [101] X. Osteikoetxea, A. Nemeth, B.W. Sodar, K.V. Vukman, E.I. Buzas, Extracellular vesicles in cardiovascular disease: are they Jedi or Sith?, J Physiol 594(11) (2016) 2881-94. [102] J.C. Akers, D. Gonda, R. Kim, B.S. Carter, C.C. Chen, Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies, J Neurooncol 113(1) (2013) 111. [103] C. Lasser, G.V. Shelke, A. Yeri, D.K. Kim, R. Crescitelli, S. Raimondo, M. Sjostrand, Y.S. Gho, K. Van Keuren Jensen, J. Lotvall, Two distinct extracellular RNA signatures released by a single cell type identified by microarray and next-generation sequencing, RNA Biol (2016) 1-15. [104] J.R. Chevillet, Q. Kang, I.K. Ruf, H.A. Briggs, L.N. Vojtech, S.M. Hughes, H.H. Cheng, J.D. Arroyo, E.K. Meredith, E.N. Gallichotte, E.L. Pogosova-Agadjanyan, C. Morrissey, D.L. Stirewalt, F. Hladik, E.Y. Yu, C.S. Higano, M. Tewari, Quantitative and stoichiometric analysis of the microRNA content of exosomes, Proc Natl Acad Sci U S A 111(41) (2014) 14888-93. [105] R. Crescitelli, C. Lasser, T.G. Szabo, A. Kittel, M. Eldh, I. Dianzani, E.I. Buzas, J. Lotvall, Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes, J Extracell Vesicles 2 (2013). [106] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J.J. Lee, J.O. Lotvall, Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells, Nat Cell Biol 9(6) (2007) 654-9. [107] K. Ekstrom, H. Valadi, M. Sjostrand, C. Malmhall, A. Bossios, M. Eldh, J. Lotvall, Characterization of mRNA and microRNA in human mast cell-derived exosomes and their transfer to other mast cells and blood CD34 progenitor cells, J Extracell Vesicles 1 (2012).

[108] M. Eldh, K. Ekstrom, H. Valadi, M. Sjostrand, B. Olsson, M. Jernas, J. Lotvall, Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA, PLoS One 5(12) (2010) e15353. [109] J. Lotvall, H. Valadi, Cell to cell signalling via exosomes through esRNA, Cell Adh Migr 1(3) (2007) 156-8. [110] D. Skokos, S. Le Panse, I. Villa, J.C. Rousselle, R. Peronet, A. Namane, B. David, S. Mecheri, Nonspecific B and T cell-stimulatory activity mediated by mast cells is associated with exosomes, Int Arch Allergy Immunol 124(1-3) (2001) 133-6. [111] G. Raposo, D. Tenza, S. Mecheri, R. Peronet, C. Bonnerot, C. Desaymard, Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation, Mol Biol Cell 8(12) (1997) 2631-45. [112] H. Vincent-Schneider, C. Thery, D. Mazzeo, D. Tenza, G. Raposo, C. Bonnerot, Secretory granules of mast cells accumulate mature and immature MHC class II molecules, J Cell Sci 114(Pt 2) (2001) 323-34. [113] H. Xiao, C. Lasser, G.V. Shelke, J. Wang, M. Radinger, T.R. Lunavat, C. Malmhall, L.H. Lin, J. Li, L. Li, J. Lotvall, Mast cell exosomes promote lung adenocarcinoma cell proliferation - role of KIT-stem cell factor signaling, Cell Commun Signal 12 (2014) 64. [114] F. Mion, F. D'Inca, L. Danelli, B. Toffoletto, C. Guarnotta, B. Frossi, A. Burocchi, A. Rigoni, N. Gerdes, E. Lutgens, C. Tripodo, M.P. Colombo, J. Rivera, G. Vitale, C.E. Pucillo, Mast cells control the expansion and differentiation of IL-10-competent B cells, J Immunol 193(9) (2014) 4568-79. [115] D. Skokos, S. Le Panse, I. Villa, J.C. Rousselle, R. Peronet, B. David, A. Namane, S. Mecheri, Mast cell-dependent B and T lymphocyte activation is mediated by the secretion of immunologically active exosomes, J Immunol 166(2) (2001) 868-76. [116] K. Laulagnier, C. Motta, S. Hamdi, S. Roy, F. Fauvelle, J.F. Pageaux, T. Kobayashi, J.P. Salles, B. Perret, C. Bonnerot, M. Record, Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization, Biochem J 380(Pt 1) (2004) 161-71. [117] K.L. Cheung, R. Jarrett, S. Subramaniam, M. Salimi, D. Gutowska-Owsiak, Y.L. Chen, C. Hardman, L. Xue, V. Cerundolo, G. Ogg, Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a, J Exp Med (2016). [118] T. Hugle, V. Hogan, K.E. White, J.M. van Laar, Mast cells are a source of transforming growth factor beta in systemic sclerosis, Arthritis Rheum 63(3) (2011) 795-9. [119] D. Skokos, H.G. Botros, C. Demeure, J. Morin, R. Peronet, G. Birkenmeier, S. Boudaly, S. Mecheri, Mast cell-derived exosomes induce phenotypic and functional maturation of dendritic cells and elicit specific immune responses in vivo, J Immunol 170(6) (2003) 3037-45. [120] K. Al-Nedawi, J. Szemraj, C.S. Cierniewski, Mast cell-derived exosomes activate endothelial cells to secrete plasminogen activator inhibitor type 1, Arterioscler Thromb Vasc Biol 25(8) (2005) 1744-9. [121] D. Skokos, H. Goubran-Botros, M. Roa, S. Mecheri, Immunoregulatory properties of mast cellderived exosomes, Mol Immunol 38(16-18) (2002) 1359-62. [122] C. Subra, D. Grand, K. Laulagnier, A. Stella, G. Lambeau, M. Paillasse, P. De Medina, B. Monsarrat, B. Perret, S. Silvente-Poirot, M. Poirot, M. Record, Exosomes account for vesiclemediated transcellular transport of activatable phospholipases and prostaglandins, J Lipid Res 51(8) (2010) 2105-20. [123] C. Tkaczyk, P. Frandji, H.G. Botros, P. Poncet, J. Lapeyre, R. Peronet, B. David, S. Mecheri, Mouse bone marrow-derived mast cells and mast cell lines constitutively produce B cell growth and differentiation activities, J Immunol 157(4) (1996) 1720-8. [124] S. Merluzzi, E. Betto, A.A. Ceccaroni, R. Magris, M. Giunta, F. Mion, Mast cells, basophils and B cell connection network, Mol Immunol 63(1) (2015) 94-103. [125] H. Vincent-Schneider, P. Stumptner-Cuvelette, D. Lankar, S. Pain, G. Raposo, P. Benaroch, C. Bonnerot, Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells, Int Immunol 14(7) (2002) 713-22. [126] F. Li, Y. Wang, L. Lin, J. Wang, H. Xiao, J. Li, X. Peng, H. Dai, L. Li, Mast Cell-Derived Exosomes Promote Th2 Cell Differentiation via OX40L-OX40 Ligation, J Immunol Res 2016 (2016) 3623898.

[127] A. Veerappan, M. Thompson, A.R. Savage, M.L. Silverman, W.S. Chan, B. Sung, B. Summers, K.C. Montelione, P. Benedict, B. Groh, A.G. Vicencio, H. Peinado, S. Worgall, R.B. Silver, Mast cells and exosomes in hyperoxia-induced neonatal lung disease, Am J Physiol Lung Cell Mol Physiol 310(11) (2016) L1218-32. [128] B. Chen, M.Y. Li, Y. Guo, X. Zhao, H.C. Lim, Mast cell-derived exosomes at the stimulated acupoints activating the neuro-immuno regulation, Chin J Integr Med (2016). [129] N.R. Smalheiser, Exosomal transfer of proteins and RNAs at synapses in the nervous system, Biol Direct 2 (2007) 35. [130] K. Bechter, B. Schmitz, Cerebrospinal fluid outflow along lumbar nerves and possible relevance for pain research: case report and review, Croat Med J 55(4) (2014) 399-404. [131] T. Groot Kormelink, G.J. Arkesteijn, C.H. van de Lest, W.J. Geerts, S.S. Goerdayal, M.A. Altelaar, F.A. Redegeld, E.N. Nolte-'t Hoen, M.H. Wauben, Mast Cell Degranulation Is Accompanied by the Release of a Selective Subset of Extracellular Vesicles That Contain Mast Cell-Specific Proteases, J Immunol 197(8) (2016) 3382-3392. [132] A. Biswas, J.E. Richards, J. Massaro, M. Mahalingam, Mast cells in cutaneous tumors: innocent bystander or maestro conductor?, Int J Dermatol 53(7) (2014) 806-11. [133] D.E. Woolley, The mast cell in inflammatory arthritis, N Engl J Med 348(17) (2003) 1709-11. [134] O. Bakharevski, P.F. Ryan, Mast cells as a target in the treatment of rheumatoid arthritis, Inflammopharmacology 7(4) (1999) 351-62. [135] B.Y. De Winter, R.M. van den Wijngaard, W.J. de Jonge, Intestinal mast cells in gut inflammation and motility disturbances, Biochim Biophys Acta 1822(1) (2012) 66-73. [136] T. Gulen, H. Hagglund, B. Dahlen, G. Nilsson, Mastocytosis: the puzzling clinical spectrum and challenging diagnostic aspects of an enigmatic disease, J Intern Med 279(3) (2016) 211-28.

Legends for figures Figure 1: The mast cell secretome Mast cells can be activated by various stimuli such as allergens, pathogens or Ca2+-channel ligands through crosslinking IgE bound to FcεRI receptors, pathogen recognition by PRRs and ligand binding to Ca2+-channels, respectively. Crosslinking of suface FcεRI-bound IgE molecules induces formation, fusion and secretion of granules. Mast cells can secrete preformed mediators either by conventional degranulation or piecemeal degranulation (PMD). They also secrete de novo synthetized molecules through exocytosis of secretory vesicles. These processes require the increase of Ca2+ in the cytoplasm. The source of this Ca2+ can be either extracellular (through Ca2+-channels), or intracellular (from the ER). Finally, mast cells release microvesicles (MVs) by shedding from their plasma membrane or exosomes (EXOs) by exocytosis of the multivesicular bodies (MVBs). PM: plasma membrane, PRR: pattern recognition receptor, ER: endoplasmic reticulum.

Figure 2: Mast cell-derived exosomes play numerous roles in cell to cell communication. Mast cells-derived exosomes carry and transfer functional RNAs (both mRNA and miRNA) to other mast cells, induce maturation of dendritic cells and proliferation of B-cells. Mast cellderived exosomes are also able to stimulate cytotoxic T-cells (however, the presence of dendritic cells is also required), and induce IL-2 secretion by CD4+ T lymphocytes. They induce plasminogen activator inhibitor type 1 (PAI1) expression of endothelial cells. They play role in tumour growth by affecting angiogenesis and the recruitment of macrophages. They participate in pain signalling and modulate synaptic functions in the nervous system.

Table 1 Possible roles of molecules associated with mast cell-derived EVs in cell-to cell communication Molecules target cells effects reference mRNA, miRNA, sRNA MHC II

CD117 TGF-β hsp60, hsp70 Factor V precursor, prothrombin, angiotensinogen, TNF precursors CD13, ribosomal protein S6 kinase, annexin V, CDC25, γ-actin bioactive lipids like phospholipid scramblase, fatty acid binding protein phospholipases GTPase, PAP1, PGE2 CD40,

CD80, CD86 OX40L

CD63+ cells, mast cells

migration, development, mediator secretion

[104, 106109]

CD4+, CD8+ Tcell, dendritic cells, B-cells adenocarcinoma cells fibroblasts

stimulation, affect antigen presentation, proliferation

[111, 112, 125]

PI3K/AKT signalling, migration and cell proliferation stimulation

[113]

splenocytes, Tcells, dendritic cells

immunoregulation, functional maturation

[106, 114, 119]

thrombin and PAI-1 generation under inflammatory conditions

[120]

splenocytes, Tcells

stimulation

[121]

T-cells

activation during psoriasis

[117]

dendritic cells

functional maturation

[106, 112]

dendritic cells, Bcells

proliferation, maturation, cytokine secretion

[110, 114, 115, 119]

CD4+ T-cells

proliferation, differentiation to Th2

[126]

[118]