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small extracellular vesicles in tissue regeneration
ARTICLE IN PRESS Cytotherapy, 2018; 0001 8
Immune regulatory targets of mesenchymal stromal cell exosomes/ small extracellular vesicles in tissue regeneration
D1X XWEI SEONG TOHD2X1,2, X *, D3X XBIN ZHANGD4X3,X *, D5X XRUENN CHAI LAID6X3X & D7X XSAI KIANG LIMD3,4 8X X 1
Faculty of Dentistry, National University of Singapore, Singapore,2Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore,3Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, and 4Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Abstract Mesenchymal stromal cell (MSC) therapies have demonstrated therapeutic efficacy in a wide-ranging array of tissue injury and disease indications. An important aspect of MSC-mediated therapeutic activities is immune modulation. Consistent with the concentration of MSC therapeutic potency in its secretion, a significant proportion of MSC immune potency resides in the small extracellular vesicles (sEVs) secreted by MSCs. These sEVs, which also include exosomes, carry a large cargo enriched in proteins with potent immunomodulatory activities. They have been reported to exert potent effects on humoral and cellular components of the immune system in vitro and in vivo, and may have the potential to support the diametrically opposite pro- and antiinflammatory functions necessary for tissue repair and regeneration following injury. Following injury, pro-inflammatory activities are necessary to neutralize injury and remove dead or injured tissue, while anti-inflammatory activities to facilitate migration and proliferation of reparative cell types and to increase vascularization and nutrient supply are necessary to repair and regenerate new tissue. Therefore, a critical immunomodulatory requisite of MSC sEVs in tissue regeneration is the capacity to support the appropriate immune activities at the appropriate time. Here, we review how some of the immune regulatory targets of MSC sEVs could support the dynamic immunomodulatory activities during tissue repair and regeneration.
Key Words: exosome, extracellular vesicles, immunomodulation, mesenchymal stromal cell, proteome, tissue regeneration
Introduction Arnold Caplan who coined the original term mesenchymal stem cells (MSCs) has recently proposed that MSCs should be re-named as “Medicinal Signaling Cell” to better reflect the in vivo function of MSCs as secretory cells rather than stem cells with differentiation potential [1]. This proposal essentially captures the evolving paradigm shift from a cell- to a secretion-based mechanism of action for the therapeutic use of MSCs. It is long recognized that MSCs secrete a myriad of factors that could modulate the tissue microenvironment and response to facilitate tissue repair and recovery [2]. To identify the active agent that underpins the MSC therapeutic potency, many secreted factors have been proposed and these include small molecules, such as the growth factors and cytokines, and relatively large complexes, such
as the extracellular vesicles (EVs). The most promising active agents proposed to date are the EVs, including the 50 1000 nm microvesicles that protect against acute kidney injury (AKI) in a mouse model of glycerol-induced AKI [3] and the 100 200 nm exosomes that protect against myocardial ischemiareperfusion injury in mice [4]. EVs are particularly attractive candidates because their cargos are sufficiently complex and diverse to rationalize the reported potencies of MSC secretion. Consistent with EVs being the active agents in mediating MSC therapeutic activity, immunomodulation, which has long been recognized as a cornerstone in MSCmediated tissue repair [5], is also a widely observed phenomenon in MSC-mediated tissue repair and regeneration. This review will focus on the family of small MSC EVs to which exosomes belong, and their
Correspondence: Sai Kiang Lim, PhD, Institute of Medical Biology, 8A Biomedical Grove, #05-505 Immunos, Singapore 138648. E-mail: [email protected] * These authors contributed equally to this work. (Received 21 August 2018; accepted 19 September 2018) ISSN 1465-3249 Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2018.09.008
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MSCs cultured in chemically defined serum-free media may be more suitable for manufacture of MSC EVs.
Therapeutic MSC exosomes Exosomes were first described in 1983 [6,7]. They are presently classified as a member of the small EV family comprising EVs of 50 200 nm. Exosomes are unique from all other known EVs in having an endosomal biogenesis. In the first report of MSC exosomes [4], the EV preparation was characterized as exosomes based on several parameters, namely, a hydrodynamic radius of 55 65 nm, a flotation density of 1.10 1.18 g mL¡1, enrichment of tetraspannin proteins CD9 and CD81 and endosomal proteins such as ALIX and TSG101 and presence of RNA and membrane lipids. This preparation was effective in reducing the infarct size in a mouse model of myocardial ischemia-reperfusion injury. It was also found to be effective against drug-induced liver injury [8] in attenuating immune rejection in graft-versus-host disease (GVHD) [9] and in promoting repair in osteochondral injuries [10,11]. It is notable that MSC EVs have successfully treated acute GVHD in patients [12]. Others in the field have also reported that similar EV preparations were effective against other indications, such as hypoxic pulmonary hypertension [13], lupus [14], burns [15], cutaneous wounds [16], autoimmune uveitis [17], liver failure or injury [18,19] and arthritic diseases including osteoarthritis [20,21] and rheumatoid arthritis [22] in animals. However, this wide spectrum of therapeutic efficacies will have to be carefully examined to determine if such wide-ranging efficacies are typical of MSC exosome preparation per se or if each efficacy is specific to a type of MSC exosome preparation. MSC exosomes generally refer to exosomes prepared from a MSC culture. The tissue source, culture conditions and exosome purification method differ significantly among different research groups. Although the impact of such differences on the therapeutic efficacy of the exosome preparations has not yet been systematically studied, it is likely to be significant. For example, some groups used chemically defined medium for exosome isolation, whereas others used EV-depleted serum or platelet lysate. It has been reported that, in commercial EV-depleted serum or ultracentrifuged EV-depleted serum, the residual particle concentration in a 25% EV-depleted serum was »5 £ 109 particles/mL, representing a 50% reduction in particle concentration [23]. Therefore, for studies using MSC exosomes prepared in EVdepleted serum or platelet lysate, the residual serum EV will have to be considered when evaluating the purity and function of MSC exosomes. To this end,
“MSC exosome” versus “MSC sEV” The term “exosome” specifically defines a small EV of 50 200 nm that is formed when the membrane of endosome invaginates to produce a multivesicular body (MVB) that upon fusion with cell membrane releases the vesicles within as exosomes. Consequently, the key markers of exosomes are proteins associated with endocytosis and endosomal trafficking such as caveolins, clathrin, transferrin receptors, tetraspanins (CD81, CD63, CD9), ALIX, and TSG101 (reviewed [24]). We had shown through pulse chase studies that MSC exosomes bearing these markers are derived from endosomes [25]. In these studies, it was observed that when MSC endocytosed biotin-conjugated transferrin or biotin-conjugated cholera toxin B chain (CTB) a fraction of endocytosed extracellular ligands was secreted in sEVs bearing exosome markers. CTB has a high-binding affinity for GM1 ganglioside, which is highly enriched in lipid rafts where most endocytosis occurs. The receptor for transferrin, commonly known as the transferrin receptor, is the first exosome marker to be identified [6,7] and transferrin receptor 2, which is enriched in lipid rafts, has been reported to be released into exosomes with CD81 [26]. It was further demonstrated that the exosome markers co-localized with transferrin receptors in CTB-binding EVs, which are about 100 200 nm. Hence, we established that MSC exosomes are CTB-binding sEVs marked by the presence of endosome-associated proteins such as caveolins, clathrin, transferrin receptors, tetraspanins (CD81, CD63, CD9), ALIX and TSG101. In addition to exosomes, MSCs were found to secrete similarly sized non-exosomal EVs of 100 200 nm [27]. Unlike exosomes, these EVs do not bind CTB or carry endosome-associated proteins. Instead, they bind Annexin V (AV) or Shiga toxin B chain (ST), which in turn has high-binding affinity for membrane lipids, phosphatidylserine and globotriaosylceramide. Because all these EV types are similar in size and density to exosomes, the present methods of preparing exosomes by size and density will not differentiate these non-exosomal EV types from exosomes. Such exosome preparations will include non-exosomal EVs and the exosome preparations should be more appropriately described as small EV (sEV) preparation unless it is determined that exosomes constitute a significant proportion of the EV types. However, the term “exosomes” is widely used in the literature to describe sEVs that had not been characterized as such.
ARTICLE IN PRESS Immunoregulation of MSC-EVs in tissue regeneration The MSC sEVs are generally prepared from MSCconditioned medium by separating the relatively larger vesicular components from the soluble non-vesicular components using size-based separation methodologies such as ultrafiltration, ultracentrifugation, size exclusion chromatography, tangential flow filtration and precipitation. As previously highlighted by members of the International Society for Extracellular Vesicles (ISEV) and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore (SOCRATES), there is presently no accepted metrics to assess the purity or degree of purity in a sEV preparation [28]. Furthermore, having harsh purification procedures to obtain a pure product could result in loss of function through damage to EV-intrinsic effectors or even to loss of extrinsic, loosely associated factors that act with EVs to exert function. As such, it is presently not possible to exclude a complementary or synergistic contribution of nonvesicular MSC-secreted components to the functions of sEVs. MSC immune activity is conveyed by sEVs A key feature of MSC therapeutic potency is its modulatory effects on the activity of both autologous and allogeneic adaptive and innate immune cells [29], such as inhibition of mitogen-activated T-cell proliferation [30 34], induction of an anti-inflammatory tolerant dendritic cells (DCs) [35], naive and effector T cells and natural killer (NK) cells [36], inhibition of complement activation [37] and inhibition of B-cell proliferation [38]. Consistent with these immune activities, MSCs have been approved for the treatment of GVHD in Canada, New Zealand and Japan, and they are undergoing testing for efficacy against immune diseases such as Crohn’s disease [39] and type 1 diabetes [40]. There is substantial evidence that the immunomodulatory activity of MSCs is modulated at least in part by soluble mediators secreted by the cell [36]. Over the years, numerous small molecules with potent immunomodulatory activity were found to be secreted by MSCs and they include prostaglandin E2 (PGE2), transforming growth factor (TGF)-b, interferon-g (IFNg), human leukocyte antigen (HLA)-G5, interleukin (IL)-10 and indoleamine 2,3-dioxygenase (IDO) (reviewed [41]). However, Ghannam et al. [42], in a review of the literature on these factors, argued that many of these secreted factors by themselves cannot account for the immunomodulatory activity of MSCs. For example, the absence of IDO production caused by either defective IFNg receptor 1 or IDO inhibitors [43], which would result in loss of IFNg [44 46] and IDO [47], did not compromise MSC immune activity. Presently, the most promising candidate for the paracrine
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mediator of MSC immunomodulatory activity is MSC sEVs because MSC sEVs alone could recapitulate the potency of MSCs in treating immune diseases such as GVHD [12].
Immune modulation and tissue regeneration Immunomodulation is a critical aspect of tissue repair and regeneration. However, unlike immune disorders such as GVHD or Crohn’s disease where attenuation of a reactive immune response is the primary therapeutic endpoint, tissue repair and regeneration after injury requires a dynamic flux in the immune microenvironment during which it transits from a pro-inflammatory microenvironment to neutralize injury and remove dead or injured tissue to an anti-inflammatory microenvironment to facilitate migration and proliferation of reparative cell types and to increase vascularization and nutrient supply to repair and regenerate new tissue. Therefore, both pro- and anti-inflammation are critical to the process of tissue repair and regeneration following injury or disease. A critical immunomodulatory requisite of MSC sEVs is the capacity to support the diametrically opposite pro- and anti-inflammatory functions at the appropriate time. The importance of MSC sEVs in mediating the immune reactivity of MSC secretion during tissue regeneration is reflected in the number of scientific publications. However, of the 104 articles found in a literature search (Pubmed, accessed on August 20, 2018) using the keywords ‘mesenchymal stem cell, extracellular vesicles, immune”, 54 are reviews. Although the preponderance of reviews over scientific reports highlights the scientific excitement in the immune regulatory role of MSC EVs, it also reflects the nascent state of our understanding in this field. In this review, we will focus on how MSC sEVs could potentially exert pro- and anti-inflammatory functions for tissue repair and regeneration.
Diverse immune potencies in MSC sEVs Immunomodulation to attenuate injury-induced inflammation and promote a regenerative microenvironment for tissue repair and regeneration could potentially be effected through the humoral and cellular components in either innate or adaptive immune systems (Figure 1). Proteomic profiling of MSC sEVs has revealed an enrichment of proteins involved in inflammation or complement activation [48 52]. This suggests that MSC sEVs have the proteomic potency to exert diverse effects on both humoral and cellular components of the immune system.
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Figure 1. MSC sEVs can modulate humoral and cellular components of the immune system to promote tissue regeneration following injury. They can attenuate injury-induced inflammation by dampening complement activation through CD59, enhancing polarization of Tregs in the presence of activated antigen-presenting cells and suppressing Th1 and Th17. They can also promote a regenerative microenvironment by suppressing M1 macrophages and promoting regenerative M2 macrophages and production of associated anti-inflammatory cytokines such as IL-10.
Humoral immunity The complement system is a key player in innate immunity and is activated through one of three pathways, namely, classical, lectin or alternative pathways. These pathways converged at the cleavage of C3 to activate a proteolytic cascade to form several potent complement peptides and complexes. The peptides include the following: C3b, an opsonin to enhance phagocytosis; C3a, an anaphylatoxin to activate inflammation; and C5a, a chemoattractant to recruit or activate macrophages, neutrophils and mast cells. The different complement peptides could also assemble to form membrane attack complexes (MAC) that insert into membranes of bacteria or cells and cause lysis. These complement-mediated activities are not only important in destroying foreign or damaged cells, they are also critical to the process of tissue repair and regeneration (reviewed [53]). For example, complement-activated inflammation is known to increase vascular permeability to facilitate entry of reparative and restorative cells. However, this may potentially cause injurious edema in the tissue. Similarly, complement-induced opsonization and phagocytosis not only kill foreign or damaged cells, they clear cellular debris for new tissue formation and could also potentially damage newly regenerated tissues. Therefore, the complement system is a ‘double-edged sword’ that requires careful management to ensure sufficient but not excessive tissue destruction or remodeling for optimal destruction of damaged tissue without compromising newly regenerated tissue.
MSC-conditioned medium has been shown to suppress complement-mediated sheep erythrocyte hemolysis through the secretion of Factor H, an inhibitor of complement activation [37]. In addition, Factor H production was also found to be suppressed by two known suppressors of immunosuppressive activity of MSC, namely, indomethacin or 1-methyl-d-tryptophan (1-MT), suggesting the role of Factor H in the immunosuppressive activity of MSC. However, we and others have not detected Factor H in MSC sEVs based on the proteomic databases at ExoCarta and Vesiclepedia (accessed June 13, 2018). Nevertheless, MSC sEVs could suppress complement-mediated hemolysis of sheep erythrocytes. This suppressive activity was attributed to the presence of CD59 because it was abrogated in the presence of CD59 neutralizing antibodies [52]. Unlike Factor H, which inhibits C3bBb, a C3convertase, and, therefore, the initiation of the alternative pathway of complement activation, CD59 acts on MAC formation, which represents the endproduct of the complement activation cascade. This is a critical difference because the use of CD59 to attenuate complement activation will not inhibit complement activation per se, whereas Factor H will abrogate the initiation of complement activation. Therefore, CD59 in MSC sEVs could attenuate inflammation to facilitate tissue regeneration by dampening but not abrogating complement activation. This provides for a more calibrated management of the complement system for optimal tissue destruction/remodeling in tissue repair and regeneration.
ARTICLE IN PRESS Immunoregulation of MSC-EVs in tissue regeneration Cellular immunity MSC sEVs have been reported to modulate immune cell response to promote tissue repair. In a mouse model of myocardial ischemia-reperfusion injury, MSC exosomes attenuated ischemic injury induced inflammatory response, as evidenced by reduced white blood cell count and tissue infiltration of neutrophils [54]. More recently, MSC exosomes were found to enhance infiltration of M2 over M1 macrophages, together with suppression of synovial pro-inflammatory IL-1b and tumor necrosis factor (TNF)-a to promote osteochondral repair in an immunocompetent rat model [11]. Similarly, in a mouse model of hyperoxia-induced lung injury, MSC exosomes promoted the influx of M2 macrophages into the tissues and attenuated the level of the M1 macrophages and pro-inflammatory cytokines such as TNF-a. Together, these led to reduced fibrosis and microvasculature loss with improved overall lung function [55]. MSC sEVs also reportedly enhanced M2 macrophage polarization and reduced pro-inflammatory cytokine expression to improve survival and tissue repair in sepsis [56], peritonitis [57], incisional hernia [58] and diabetic wound [59]. One of the key immunomodulatory properties of MSC sEVs involves the promotion of antiinflammatoryand pro-regenerative (M2) macrophages over the pro-inflammatory M1 macrophages and concomitantly enhances the expression of antiinflammatory cytokines such as IL-10 instead of proinflammatory cytokines (e.g., IL-1b and TNF-a) to promote tissue repair. Although the exact mechanism remains to be clarified, the macrophage polarization observed in MSC sEV mediated tissue repair could be attributed to the ability of MSC sEVs to interact directly with the monocytes and modulate the cytokine production. Despite having the potency to activate toll-like receptor (TLR)-4, MSC exosomes unlike lipopolysaccharide (LPS) enhanced the expression of anti-inflammatory IL-10 and TGF-b1 instead of pro-inflammatory IL-1b, IL-6, TNF-a and IL-12P40 to induce a M2-like phenotype in THP-1 cell, monocytes [9]. However, in another study by Li et al., MSC sEVs were found to downregulate TLR-4 signaling pathway to counteract LPS-induced inflammation in macrophages by enhancing IL-10 production and inhibiting levels of TNF-a and IL-1b [60]. More recently, Zhao et al. showed that MSC exosomes induced M2 macrophage polarization through transactivation of arginase-1 by active exosomal STAT3 [61]. Collectively, these findings suggest that MSC sEV/exosomes are indeed immunomodulatory with the potential to act through multiple pathways to promote
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anti-inflammation and enhance M2 macrophage polarization. However, it is not clear at this point in time if the promotion of anti-inflammatory activities by MSC sEVs is concomitant with or independent of an inhibition of pro-inflammatory activities. Beside macrophages, regulatory T cells (Tregs) are increasingly associated with MSC EV mediated attenuation of an activated immune system. In mice, MSC sEVs delayed skin graft rejection with a concomitant induction of Tregs in mice grafted with allogenic skin but not in un-grafted mice, implying that the Treg induction by MSC sEVs is dependent on an activated immune system and MSC sEVs are not immunosuppressive per se [9]. Similarly, MSC exosomes have been found to enhance production of anti-inflammatory IL-10 and TGF-b1 in peripheral blood mononuclear cells (PBMCs) from patients with asthma and alleviate airway inflammation in mice with asthma through increased proliferation and immunosuppressive capacity of activated Tregs [62]. This induction of Tregs by MSC EVs was also similarly observed in a concanavalin A induced liver injury model, an acute inflammatory liver disease model [63] and in a mouse model of acute GVHD [9]. However, not all MSC EVs exert their immunosuppressive effects through Tregs. Although Shigemoto-Kuroda et al. observed increased expression of IL-10 in two mouse models of autoimmune diseases (type 1 diabetes and uveoretinitis), they did not observe an increase in Tregs [64]. Instead, they observed inhibition of antigen-presenting cell (APC) activation and suppression of T helper type 1 (Th1) and IL17 producing effector T (Th17) cells. Similarly, Chen et al. observed in vitro that MSC exosomes induced conversion of Th1 into Th2 cells, while inhibiting the differentiation of T cells into Th17 cells [65]. These studies suggest that increased Treg production in MSC EV mediated immune regulation cannot be attributed to IL-10 alone and may involve more molecules or cell types, such as DCs. MSC sEVs could induce IL-10 expressing regulatory DCs to suppress Th1 and Th17 cell development without inducing Tregs. Additionally, it was observed that MSC sEVs polarized CD4+ T cells to Tregs when the CD4+ T cells were activated by allogenic CD11C+ APCs but not by CD3/CD28 co-stimulation [66]. Based on these studies, it is evident that MSC sEVs induced polarization of the immune cells toward an immunosuppressive phenotype only in the presence of an activated immune system such that the immune system is attenuated but still retains the capacity to mount an immune response. Hence, MSC sEVs are immunosuppressive only in the presence of an immune-reactive environment. This unique MSC sEV
ARTICLE IN PRESS 6 W.S. TOH et al. immunomodulatory property may attenuate the immune system without causing adverse side effects, such as increased risk of infection or cancer observed with the use of immunosuppressive drugs. Clinical translation of MSC sEVs The therapeutic potential of MSCs has been extensively studied, and is currently being evaluated in >850 clinical trials (https://clinicaltrials.gov/; accessed on September 11, 2018). Although cell-based MSC therapies have demonstrated efficacy and safety in several clinical trials [40,67,68], there exists inherent risks associated with cell transplantation and significant challenges related to the maintenance of viability and vitality of cells at different stages from manufacture and storage to delivery to patients. The use of MSC sEVs as therapeutic offers several advantages over that of MSCs. MSC sEVs are potentially safer therapeutic agents than MSCs (reviewed [69,70]). Unlike cells, sEVs are small non-living agents that could be sterile filtered and frozen without cryopreservatives. The need to preserve cell viability and function from manufacture to storage and delivery to patient is also eliminated. The use of sEVs also reduces the safety risks associated with the transplantation of viable replicating cells. Cell transplantation is considered a permanent treatment and could not be reversed. The transplanted cells may continue to persist or amplify even after the disease has resolved. In event of an adverse effect, the permanence of cell transplantation may have a devastating effect on patients. MSCs are relatively large cells and have been previously reported to cause occlusions in distal microvasculature [71]. The differentiation potential of MSCs into osteocytes and chondrocytes has also reportedly resulted in a high frequency of ossifications and/or calcifications in tissues in animal studies [72]. These therapeutic advantages of MSC sEVs provide a compelling rationale to develop a MSC sEV based therapy. Conclusion MSC sEVs have the immunomodulatory potential to promote tissue regeneration after injury. They can attenuate injury-induced inflammation by dampening but not inhibiting complement activation through CD59, and enhancing polarization of immune regulatory cell types, such as M2 macrophages, Tregs or regulatory DCs, to attenuate the immune response. They can also promote a regenerative microenvironment by suppressing M1 macrophages and associated proinflammatory cytokines, including IL-1b and TNF-a, while promoting regenerative M2 macrophages. Together, MSC sEVs have the potential to modulate immune activities to facilitate tissue regeneration.
Acknowledgments B.Z., R.C.L. and S.K.L. are funded by the Agency for Science, Technology and Research (A*STAR), Singapore. W.S.T. was partially supported by grants from the National University of Singapore (R221000090112 and R221000114114) and the National Medical Research Council Singapore (R175000144213). Disclosure of interests:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S.K. L. is a founder of Paracrine Therapeutics.
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Immune regulatory targets of mesenchymal stromal cell exosomes/ small extracellular vesicles in tissue regeneration
D1X XWEI SEONG TOHD2X1,2, X *, D3X XBIN ZHANGD4X3,X *, D5X XRUENN CHAI LAID6X3X & D7X XSAI KIANG LIMD3,4 8X X 1
Faculty of Dentistry, National University of Singapore, Singapore,2Tissue Engineering Program, Life Sciences Institute, National University of Singapore, Singapore,3Institute of Medical Biology, Agency for Science, Technology and Research, Singapore, and 4Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
Abstract Mesenchymal stromal cell (MSC) therapies have demonstrated therapeutic efficacy in a wide-ranging array of tissue injury and disease indications. An important aspect of MSC-mediated therapeutic activities is immune modulation. Consistent with the concentration of MSC therapeutic potency in its secretion, a significant proportion of MSC immune potency resides in the small extracellular vesicles (sEVs) secreted by MSCs. These sEVs, which also include exosomes, carry a large cargo enriched in proteins with potent immunomodulatory activities. They have been reported to exert potent effects on humoral and cellular components of the immune system in vitro and in vivo, and may have the potential to support the diametrically opposite pro- and antiinflammatory functions necessary for tissue repair and regeneration following injury. Following injury, pro-inflammatory activities are necessary to neutralize injury and remove dead or injured tissue, while anti-inflammatory activities to facilitate migration and proliferation of reparative cell types and to increase vascularization and nutrient supply are necessary to repair and regenerate new tissue. Therefore, a critical immunomodulatory requisite of MSC sEVs in tissue regeneration is the capacity to support the appropriate immune activities at the appropriate time. Here, we review how some of the immune regulatory targets of MSC sEVs could support the dynamic immunomodulatory activities during tissue repair and regeneration.
Key Words: exosome, extracellular vesicles, immunomodulation, mesenchymal stromal cell, proteome, tissue regeneration
Introduction Arnold Caplan who coined the original term mesenchymal stem cells (MSCs) has recently proposed that MSCs should be re-named as “Medicinal Signaling Cell” to better reflect the in vivo function of MSCs as secretory cells rather than stem cells with differentiation potential [1]. This proposal essentially captures the evolving paradigm shift from a cell- to a secretion-based mechanism of action for the therapeutic use of MSCs. It is long recognized that MSCs secrete a myriad of factors that could modulate the tissue microenvironment and response to facilitate tissue repair and recovery [2]. To identify the active agent that underpins the MSC therapeutic potency, many secreted factors have been proposed and these include small molecules, such as the growth factors and cytokines, and relatively large complexes, such
as the extracellular vesicles (EVs). The most promising active agents proposed to date are the EVs, including the 50 1000 nm microvesicles that protect against acute kidney injury (AKI) in a mouse model of glycerol-induced AKI [3] and the 100 200 nm exosomes that protect against myocardial ischemiareperfusion injury in mice [4]. EVs are particularly attractive candidates because their cargos are sufficiently complex and diverse to rationalize the reported potencies of MSC secretion. Consistent with EVs being the active agents in mediating MSC therapeutic activity, immunomodulation, which has long been recognized as a cornerstone in MSCmediated tissue repair [5], is also a widely observed phenomenon in MSC-mediated tissue repair and regeneration. This review will focus on the family of small MSC EVs to which exosomes belong, and their
Correspondence: Sai Kiang Lim, PhD, Institute of Medical Biology, 8A Biomedical Grove, #05-505 Immunos, Singapore 138648. E-mail: [email protected] * These authors contributed equally to this work. (Received 21 August 2018; accepted 19 September 2018) ISSN 1465-3249 Copyright © 2018 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2018.09.008
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MSCs cultured in chemically defined serum-free media may be more suitable for manufacture of MSC EVs.
Therapeutic MSC exosomes Exosomes were first described in 1983 [6,7]. They are presently classified as a member of the small EV family comprising EVs of 50 200 nm. Exosomes are unique from all other known EVs in having an endosomal biogenesis. In the first report of MSC exosomes [4], the EV preparation was characterized as exosomes based on several parameters, namely, a hydrodynamic radius of 55 65 nm, a flotation density of 1.10 1.18 g mL¡1, enrichment of tetraspannin proteins CD9 and CD81 and endosomal proteins such as ALIX and TSG101 and presence of RNA and membrane lipids. This preparation was effective in reducing the infarct size in a mouse model of myocardial ischemia-reperfusion injury. It was also found to be effective against drug-induced liver injury [8] in attenuating immune rejection in graft-versus-host disease (GVHD) [9] and in promoting repair in osteochondral injuries [10,11]. It is notable that MSC EVs have successfully treated acute GVHD in patients [12]. Others in the field have also reported that similar EV preparations were effective against other indications, such as hypoxic pulmonary hypertension [13], lupus [14], burns [15], cutaneous wounds [16], autoimmune uveitis [17], liver failure or injury [18,19] and arthritic diseases including osteoarthritis [20,21] and rheumatoid arthritis [22] in animals. However, this wide spectrum of therapeutic efficacies will have to be carefully examined to determine if such wide-ranging efficacies are typical of MSC exosome preparation per se or if each efficacy is specific to a type of MSC exosome preparation. MSC exosomes generally refer to exosomes prepared from a MSC culture. The tissue source, culture conditions and exosome purification method differ significantly among different research groups. Although the impact of such differences on the therapeutic efficacy of the exosome preparations has not yet been systematically studied, it is likely to be significant. For example, some groups used chemically defined medium for exosome isolation, whereas others used EV-depleted serum or platelet lysate. It has been reported that, in commercial EV-depleted serum or ultracentrifuged EV-depleted serum, the residual particle concentration in a 25% EV-depleted serum was »5 £ 109 particles/mL, representing a 50% reduction in particle concentration [23]. Therefore, for studies using MSC exosomes prepared in EVdepleted serum or platelet lysate, the residual serum EV will have to be considered when evaluating the purity and function of MSC exosomes. To this end,
“MSC exosome” versus “MSC sEV” The term “exosome” specifically defines a small EV of 50 200 nm that is formed when the membrane of endosome invaginates to produce a multivesicular body (MVB) that upon fusion with cell membrane releases the vesicles within as exosomes. Consequently, the key markers of exosomes are proteins associated with endocytosis and endosomal trafficking such as caveolins, clathrin, transferrin receptors, tetraspanins (CD81, CD63, CD9), ALIX, and TSG101 (reviewed [24]). We had shown through pulse chase studies that MSC exosomes bearing these markers are derived from endosomes [25]. In these studies, it was observed that when MSC endocytosed biotin-conjugated transferrin or biotin-conjugated cholera toxin B chain (CTB) a fraction of endocytosed extracellular ligands was secreted in sEVs bearing exosome markers. CTB has a high-binding affinity for GM1 ganglioside, which is highly enriched in lipid rafts where most endocytosis occurs. The receptor for transferrin, commonly known as the transferrin receptor, is the first exosome marker to be identified [6,7] and transferrin receptor 2, which is enriched in lipid rafts, has been reported to be released into exosomes with CD81 [26]. It was further demonstrated that the exosome markers co-localized with transferrin receptors in CTB-binding EVs, which are about 100 200 nm. Hence, we established that MSC exosomes are CTB-binding sEVs marked by the presence of endosome-associated proteins such as caveolins, clathrin, transferrin receptors, tetraspanins (CD81, CD63, CD9), ALIX and TSG101. In addition to exosomes, MSCs were found to secrete similarly sized non-exosomal EVs of 100 200 nm [27]. Unlike exosomes, these EVs do not bind CTB or carry endosome-associated proteins. Instead, they bind Annexin V (AV) or Shiga toxin B chain (ST), which in turn has high-binding affinity for membrane lipids, phosphatidylserine and globotriaosylceramide. Because all these EV types are similar in size and density to exosomes, the present methods of preparing exosomes by size and density will not differentiate these non-exosomal EV types from exosomes. Such exosome preparations will include non-exosomal EVs and the exosome preparations should be more appropriately described as small EV (sEV) preparation unless it is determined that exosomes constitute a significant proportion of the EV types. However, the term “exosomes” is widely used in the literature to describe sEVs that had not been characterized as such.
ARTICLE IN PRESS Immunoregulation of MSC-EVs in tissue regeneration The MSC sEVs are generally prepared from MSCconditioned medium by separating the relatively larger vesicular components from the soluble non-vesicular components using size-based separation methodologies such as ultrafiltration, ultracentrifugation, size exclusion chromatography, tangential flow filtration and precipitation. As previously highlighted by members of the International Society for Extracellular Vesicles (ISEV) and the Society for Clinical Research and Translation of Extracellular Vesicles Singapore (SOCRATES), there is presently no accepted metrics to assess the purity or degree of purity in a sEV preparation [28]. Furthermore, having harsh purification procedures to obtain a pure product could result in loss of function through damage to EV-intrinsic effectors or even to loss of extrinsic, loosely associated factors that act with EVs to exert function. As such, it is presently not possible to exclude a complementary or synergistic contribution of nonvesicular MSC-secreted components to the functions of sEVs. MSC immune activity is conveyed by sEVs A key feature of MSC therapeutic potency is its modulatory effects on the activity of both autologous and allogeneic adaptive and innate immune cells [29], such as inhibition of mitogen-activated T-cell proliferation [30 34], induction of an anti-inflammatory tolerant dendritic cells (DCs) [35], naive and effector T cells and natural killer (NK) cells [36], inhibition of complement activation [37] and inhibition of B-cell proliferation [38]. Consistent with these immune activities, MSCs have been approved for the treatment of GVHD in Canada, New Zealand and Japan, and they are undergoing testing for efficacy against immune diseases such as Crohn’s disease [39] and type 1 diabetes [40]. There is substantial evidence that the immunomodulatory activity of MSCs is modulated at least in part by soluble mediators secreted by the cell [36]. Over the years, numerous small molecules with potent immunomodulatory activity were found to be secreted by MSCs and they include prostaglandin E2 (PGE2), transforming growth factor (TGF)-b, interferon-g (IFNg), human leukocyte antigen (HLA)-G5, interleukin (IL)-10 and indoleamine 2,3-dioxygenase (IDO) (reviewed [41]). However, Ghannam et al. [42], in a review of the literature on these factors, argued that many of these secreted factors by themselves cannot account for the immunomodulatory activity of MSCs. For example, the absence of IDO production caused by either defective IFNg receptor 1 or IDO inhibitors [43], which would result in loss of IFNg [44 46] and IDO [47], did not compromise MSC immune activity. Presently, the most promising candidate for the paracrine
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mediator of MSC immunomodulatory activity is MSC sEVs because MSC sEVs alone could recapitulate the potency of MSCs in treating immune diseases such as GVHD [12].
Immune modulation and tissue regeneration Immunomodulation is a critical aspect of tissue repair and regeneration. However, unlike immune disorders such as GVHD or Crohn’s disease where attenuation of a reactive immune response is the primary therapeutic endpoint, tissue repair and regeneration after injury requires a dynamic flux in the immune microenvironment during which it transits from a pro-inflammatory microenvironment to neutralize injury and remove dead or injured tissue to an anti-inflammatory microenvironment to facilitate migration and proliferation of reparative cell types and to increase vascularization and nutrient supply to repair and regenerate new tissue. Therefore, both pro- and anti-inflammation are critical to the process of tissue repair and regeneration following injury or disease. A critical immunomodulatory requisite of MSC sEVs is the capacity to support the diametrically opposite pro- and anti-inflammatory functions at the appropriate time. The importance of MSC sEVs in mediating the immune reactivity of MSC secretion during tissue regeneration is reflected in the number of scientific publications. However, of the 104 articles found in a literature search (Pubmed, accessed on August 20, 2018) using the keywords ‘mesenchymal stem cell, extracellular vesicles, immune”, 54 are reviews. Although the preponderance of reviews over scientific reports highlights the scientific excitement in the immune regulatory role of MSC EVs, it also reflects the nascent state of our understanding in this field. In this review, we will focus on how MSC sEVs could potentially exert pro- and anti-inflammatory functions for tissue repair and regeneration.
Diverse immune potencies in MSC sEVs Immunomodulation to attenuate injury-induced inflammation and promote a regenerative microenvironment for tissue repair and regeneration could potentially be effected through the humoral and cellular components in either innate or adaptive immune systems (Figure 1). Proteomic profiling of MSC sEVs has revealed an enrichment of proteins involved in inflammation or complement activation [48 52]. This suggests that MSC sEVs have the proteomic potency to exert diverse effects on both humoral and cellular components of the immune system.
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Figure 1. MSC sEVs can modulate humoral and cellular components of the immune system to promote tissue regeneration following injury. They can attenuate injury-induced inflammation by dampening complement activation through CD59, enhancing polarization of Tregs in the presence of activated antigen-presenting cells and suppressing Th1 and Th17. They can also promote a regenerative microenvironment by suppressing M1 macrophages and promoting regenerative M2 macrophages and production of associated anti-inflammatory cytokines such as IL-10.
Humoral immunity The complement system is a key player in innate immunity and is activated through one of three pathways, namely, classical, lectin or alternative pathways. These pathways converged at the cleavage of C3 to activate a proteolytic cascade to form several potent complement peptides and complexes. The peptides include the following: C3b, an opsonin to enhance phagocytosis; C3a, an anaphylatoxin to activate inflammation; and C5a, a chemoattractant to recruit or activate macrophages, neutrophils and mast cells. The different complement peptides could also assemble to form membrane attack complexes (MAC) that insert into membranes of bacteria or cells and cause lysis. These complement-mediated activities are not only important in destroying foreign or damaged cells, they are also critical to the process of tissue repair and regeneration (reviewed [53]). For example, complement-activated inflammation is known to increase vascular permeability to facilitate entry of reparative and restorative cells. However, this may potentially cause injurious edema in the tissue. Similarly, complement-induced opsonization and phagocytosis not only kill foreign or damaged cells, they clear cellular debris for new tissue formation and could also potentially damage newly regenerated tissues. Therefore, the complement system is a ‘double-edged sword’ that requires careful management to ensure sufficient but not excessive tissue destruction or remodeling for optimal destruction of damaged tissue without compromising newly regenerated tissue.
MSC-conditioned medium has been shown to suppress complement-mediated sheep erythrocyte hemolysis through the secretion of Factor H, an inhibitor of complement activation [37]. In addition, Factor H production was also found to be suppressed by two known suppressors of immunosuppressive activity of MSC, namely, indomethacin or 1-methyl-d-tryptophan (1-MT), suggesting the role of Factor H in the immunosuppressive activity of MSC. However, we and others have not detected Factor H in MSC sEVs based on the proteomic databases at ExoCarta and Vesiclepedia (accessed June 13, 2018). Nevertheless, MSC sEVs could suppress complement-mediated hemolysis of sheep erythrocytes. This suppressive activity was attributed to the presence of CD59 because it was abrogated in the presence of CD59 neutralizing antibodies [52]. Unlike Factor H, which inhibits C3bBb, a C3convertase, and, therefore, the initiation of the alternative pathway of complement activation, CD59 acts on MAC formation, which represents the endproduct of the complement activation cascade. This is a critical difference because the use of CD59 to attenuate complement activation will not inhibit complement activation per se, whereas Factor H will abrogate the initiation of complement activation. Therefore, CD59 in MSC sEVs could attenuate inflammation to facilitate tissue regeneration by dampening but not abrogating complement activation. This provides for a more calibrated management of the complement system for optimal tissue destruction/remodeling in tissue repair and regeneration.
ARTICLE IN PRESS Immunoregulation of MSC-EVs in tissue regeneration Cellular immunity MSC sEVs have been reported to modulate immune cell response to promote tissue repair. In a mouse model of myocardial ischemia-reperfusion injury, MSC exosomes attenuated ischemic injury induced inflammatory response, as evidenced by reduced white blood cell count and tissue infiltration of neutrophils [54]. More recently, MSC exosomes were found to enhance infiltration of M2 over M1 macrophages, together with suppression of synovial pro-inflammatory IL-1b and tumor necrosis factor (TNF)-a to promote osteochondral repair in an immunocompetent rat model [11]. Similarly, in a mouse model of hyperoxia-induced lung injury, MSC exosomes promoted the influx of M2 macrophages into the tissues and attenuated the level of the M1 macrophages and pro-inflammatory cytokines such as TNF-a. Together, these led to reduced fibrosis and microvasculature loss with improved overall lung function [55]. MSC sEVs also reportedly enhanced M2 macrophage polarization and reduced pro-inflammatory cytokine expression to improve survival and tissue repair in sepsis [56], peritonitis [57], incisional hernia [58] and diabetic wound [59]. One of the key immunomodulatory properties of MSC sEVs involves the promotion of antiinflammatoryand pro-regenerative (M2) macrophages over the pro-inflammatory M1 macrophages and concomitantly enhances the expression of antiinflammatory cytokines such as IL-10 instead of proinflammatory cytokines (e.g., IL-1b and TNF-a) to promote tissue repair. Although the exact mechanism remains to be clarified, the macrophage polarization observed in MSC sEV mediated tissue repair could be attributed to the ability of MSC sEVs to interact directly with the monocytes and modulate the cytokine production. Despite having the potency to activate toll-like receptor (TLR)-4, MSC exosomes unlike lipopolysaccharide (LPS) enhanced the expression of anti-inflammatory IL-10 and TGF-b1 instead of pro-inflammatory IL-1b, IL-6, TNF-a and IL-12P40 to induce a M2-like phenotype in THP-1 cell, monocytes [9]. However, in another study by Li et al., MSC sEVs were found to downregulate TLR-4 signaling pathway to counteract LPS-induced inflammation in macrophages by enhancing IL-10 production and inhibiting levels of TNF-a and IL-1b [60]. More recently, Zhao et al. showed that MSC exosomes induced M2 macrophage polarization through transactivation of arginase-1 by active exosomal STAT3 [61]. Collectively, these findings suggest that MSC sEV/exosomes are indeed immunomodulatory with the potential to act through multiple pathways to promote
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anti-inflammation and enhance M2 macrophage polarization. However, it is not clear at this point in time if the promotion of anti-inflammatory activities by MSC sEVs is concomitant with or independent of an inhibition of pro-inflammatory activities. Beside macrophages, regulatory T cells (Tregs) are increasingly associated with MSC EV mediated attenuation of an activated immune system. In mice, MSC sEVs delayed skin graft rejection with a concomitant induction of Tregs in mice grafted with allogenic skin but not in un-grafted mice, implying that the Treg induction by MSC sEVs is dependent on an activated immune system and MSC sEVs are not immunosuppressive per se [9]. Similarly, MSC exosomes have been found to enhance production of anti-inflammatory IL-10 and TGF-b1 in peripheral blood mononuclear cells (PBMCs) from patients with asthma and alleviate airway inflammation in mice with asthma through increased proliferation and immunosuppressive capacity of activated Tregs [62]. This induction of Tregs by MSC EVs was also similarly observed in a concanavalin A induced liver injury model, an acute inflammatory liver disease model [63] and in a mouse model of acute GVHD [9]. However, not all MSC EVs exert their immunosuppressive effects through Tregs. Although Shigemoto-Kuroda et al. observed increased expression of IL-10 in two mouse models of autoimmune diseases (type 1 diabetes and uveoretinitis), they did not observe an increase in Tregs [64]. Instead, they observed inhibition of antigen-presenting cell (APC) activation and suppression of T helper type 1 (Th1) and IL17 producing effector T (Th17) cells. Similarly, Chen et al. observed in vitro that MSC exosomes induced conversion of Th1 into Th2 cells, while inhibiting the differentiation of T cells into Th17 cells [65]. These studies suggest that increased Treg production in MSC EV mediated immune regulation cannot be attributed to IL-10 alone and may involve more molecules or cell types, such as DCs. MSC sEVs could induce IL-10 expressing regulatory DCs to suppress Th1 and Th17 cell development without inducing Tregs. Additionally, it was observed that MSC sEVs polarized CD4+ T cells to Tregs when the CD4+ T cells were activated by allogenic CD11C+ APCs but not by CD3/CD28 co-stimulation [66]. Based on these studies, it is evident that MSC sEVs induced polarization of the immune cells toward an immunosuppressive phenotype only in the presence of an activated immune system such that the immune system is attenuated but still retains the capacity to mount an immune response. Hence, MSC sEVs are immunosuppressive only in the presence of an immune-reactive environment. This unique MSC sEV
ARTICLE IN PRESS 6 W.S. TOH et al. immunomodulatory property may attenuate the immune system without causing adverse side effects, such as increased risk of infection or cancer observed with the use of immunosuppressive drugs. Clinical translation of MSC sEVs The therapeutic potential of MSCs has been extensively studied, and is currently being evaluated in >850 clinical trials (https://clinicaltrials.gov/; accessed on September 11, 2018). Although cell-based MSC therapies have demonstrated efficacy and safety in several clinical trials [40,67,68], there exists inherent risks associated with cell transplantation and significant challenges related to the maintenance of viability and vitality of cells at different stages from manufacture and storage to delivery to patients. The use of MSC sEVs as therapeutic offers several advantages over that of MSCs. MSC sEVs are potentially safer therapeutic agents than MSCs (reviewed [69,70]). Unlike cells, sEVs are small non-living agents that could be sterile filtered and frozen without cryopreservatives. The need to preserve cell viability and function from manufacture to storage and delivery to patient is also eliminated. The use of sEVs also reduces the safety risks associated with the transplantation of viable replicating cells. Cell transplantation is considered a permanent treatment and could not be reversed. The transplanted cells may continue to persist or amplify even after the disease has resolved. In event of an adverse effect, the permanence of cell transplantation may have a devastating effect on patients. MSCs are relatively large cells and have been previously reported to cause occlusions in distal microvasculature [71]. The differentiation potential of MSCs into osteocytes and chondrocytes has also reportedly resulted in a high frequency of ossifications and/or calcifications in tissues in animal studies [72]. These therapeutic advantages of MSC sEVs provide a compelling rationale to develop a MSC sEV based therapy. Conclusion MSC sEVs have the immunomodulatory potential to promote tissue regeneration after injury. They can attenuate injury-induced inflammation by dampening but not inhibiting complement activation through CD59, and enhancing polarization of immune regulatory cell types, such as M2 macrophages, Tregs or regulatory DCs, to attenuate the immune response. They can also promote a regenerative microenvironment by suppressing M1 macrophages and associated proinflammatory cytokines, including IL-1b and TNF-a, while promoting regenerative M2 macrophages. Together, MSC sEVs have the potential to modulate immune activities to facilitate tissue regeneration.
Acknowledgments B.Z., R.C.L. and S.K.L. are funded by the Agency for Science, Technology and Research (A*STAR), Singapore. W.S.T. was partially supported by grants from the National University of Singapore (R221000090112 and R221000114114) and the National Medical Research Council Singapore (R175000144213). Disclosure of interests:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. S.K. L. is a founder of Paracrine Therapeutics.
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