Mesenchymal Stem Cell-Derived Exosomes and Other Extracellular Vesicles as New Remedies in the Therapy of Inflammatory Diseases

There is growing evidence that mesenchymal stem cell (MSC)-based immunosuppression was mainly attributed to the effects of MSC-derived extracellular vesicles (MSC-EVs). MSC-EVs are enriched with MSC-sourced bioactive molecules (messenger RNA (mRNA), microRNAs (miRNAs), cytokines, chemokines, immunomodulatory factors) that regulate phenotype, function and homing of immune cells. In this review article we emphasized current knowledge regarding molecular mechanisms responsible for the therapeutic effects of MSC-EVs in attenuation of autoimmune and inflammatory diseases. We described the disease-specific cellular targets of MSC-EVs and defined MSC-sourced molecules, which were responsible for MSC-EV-based immunosuppression. Results obtained in a large number of experimental studies revealed that both local and systemic administration of MSC-EVs efficiently suppressed detrimental immune response in inflamed tissues and promoted survival and regeneration of injured parenchymal cells. MSC-EVs-based anti-inflammatory effects were relied on the delivery of immunoregulatory miRNAs and immunomodulatory proteins in inflammatory immune cells (M1 macrophages, dendritic cells (DCs), CD4+Th1 and Th17 cells), enabling their phenotypic conversion into immunosuppressive M2 macrophages, tolerogenic DCs and T regulatory cells. Additionally, through the delivery of mRNAs and miRNAs, MSC-EVs activated autophagy and/or inhibited apoptosis, necrosis and oxidative stress in injured hepatocytes, neurons, retinal cells, lung, gut and renal epithelial cells, promoting their survival and regeneration.


Introduction
Epidemiological studies revealed a significant increase in the incidence of autoimmune and inflammatory diseases during the last two decades [1]. Accordingly, the total number of patients taking immunosuppressive drugs has been continuously increasing [2]. Long-term administration of immunosuppressive medications is inevitably associated with increased risk of infection and malignancy due to the sustained suppression of anti-microbial and anti-tumor immunity [3]. Therefore, new therapeutic agents, which could suppress detrimental immune response without causing life-threatening immunosuppression are urgently needed for the treatment of autoimmune and inflammatory diseases.

Macrophages: The Main Cellular Targets of MSC-Derived EVs in Alleviation of Colon Inflammation
Macrophages have been identified as the most important cells for the induction of colon inflammation [22,23]. Massive release of damage-associated molecular patterns (DAMPs) from injured epithelial cells activates NF-κB signaling pathway in colon macrophages, resulting in increased expression of inducible nitric oxide synthase (iNOS) and enhanced secretion of inflammatory cytokines (tumor necrosis factor alpha (TNF-α), IL-1β), nitric oxide (NO) and lymphocyte and monocyte-recruiting chemokines (CCL-17 and CCL-24) [24]. Macrophage-derived TNF-α and IL-1β induce enhanced expression of E and P selectins on endothelial cells enabling massive influx of circulating monocytes and lymphocytes in the injured gut [22]. Macrophage-sourced CCL-17 and CCL-24 attract inflammatory M1 macrophages and IFN-γ producing CD4+Th1 cells, which either directly damage epithelial cells (NO-producing M1 macrophages) or activate macrophages in IFN-γ-dependent manner (Th1 cells) and indirectly promote colon injury and inflammation by enabling creation of "positive inflammatory loop" in the gut [22].
Several recently published studies indicated that MSC-based alleviation of colitis was mainly relied on MSC-EV-induced suppression of colon macrophages [25][26][27][28] (Figure 1). Cao and colleagues showed that MSC-EVs significantly alleviated dextran sulphate sodium (DSS)-induced colitis in mice by inducing polarization of colon macrophages in immunosuppressive, M2 phenotype [25]. Higher number of IL-10-producing M2 macrophages, observed in MSC-EVs-treated mice, correlated with reduced weight loss, alleviated injury of gut epithelial cells and increased colon length [25]. Concentration of macrophage-sourced inflammatory cytokines and chemokines (TNF-α, CCL-17 and CCL-24) and Th1-derived IFN-γ were significantly attenuated in the gut of DSS-treated mice that received MSC-EVs. Additionally, MSC-EVs managed to increase colon concentration of immunosuppressive cytokines (IL-10 and transforming growth factor beta (TGF-β)), enabling enhanced repair and regeneration of DSS-injured epithelial cells [25]. Importantly, in vitro obtained results confirmed that MSC-EVs entered in lipopolysaccharides (LPS)-activated colon macrophages, suppressed production of inflammatory cytokines and induced generation of immunosuppressive M2 phenotype [25].
received MSC-EVs. Additionally, MSC-EVs managed to increase colon concentration of immunosuppressive cytokines (IL-10 and transforming growth factor beta (TGF-β)), enabling enhanced repair and regeneration of DSS-injured epithelial cells [25]. Importantly, in vitro obtained results confirmed that MSC-EVs entered in lipopolysaccharides (LPS)-activated colon macrophages, suppressed production of inflammatory cytokines and induced generation of immunosuppressive M2 phenotype [25].

Figure 1.
Modulation of phenotype and function of colonic macrophages as the main mechanism for mesenchymal stem cell-derived extracellular vesicle (MSC-EV)-based attenuation of ulcerative colitis: MSC-EVs reduced cleavage of caspase-3, -8 and -9 and alleviated release of damage-associated molecular patterns (DAMPs) from injured gut epithelial cells, resulting in attenuated activation of NF-κB signaling pathway in colon macrophages. Through the delivery of miR-146a, MSC-EVs inhibited TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1) expression, down-regulated phosphorylation of NF-κB p65 and inhibited generation of inflammatory M1 phenotype in macrophages, which was manifested by down-regulated expression of inducible nitric oxide synthase (iNOS), significantly reduced production of nitric oxide (NO), inflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokines (CCL-17 and CCL-24) and resulted in reduced influx of circulating neutrophils, monocytes and lymphocytes in the inflamed gut. Additionally, MSC-EVs induced polarization of colon macrophages in anti-inflammatory M2 phenotype, manifested by increased secretion of immunosuppressive cytokines TGF-β and IL-10 and alleviation of colitis.
Yang and colleagues suggested that modulation of anti-oxidant/oxidant balance in the injured gut was responsible for MSC-EVs-induced effects on macrophage phenotype and function [26]. MSC-EV-mediated suppression of NO-driven injury in the gut was accompanied by decreased activity of myeloperoxidase and malondialdehyde and increased superoxide dismutase and glutathione activity. Furthermore, MSC-EVs reduced cleavage of caspase-3, -8 and -9 and alleviated release of Figure 1. Modulation of phenotype and function of colonic macrophages as the main mechanism for mesenchymal stem cell-derived extracellular vesicle (MSC-EV)-based attenuation of ulcerative colitis: MSC-EVs reduced cleavage of caspase-3, -8 and -9 and alleviated release of damage-associated molecular patterns (DAMPs) from injured gut epithelial cells, resulting in attenuated activation of NF-κB signaling pathway in colon macrophages. Through the delivery of miR-146a, MSC-EVs inhibited TNF receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1) expression, down-regulated phosphorylation of NF-κB p65 and inhibited generation of inflammatory M1 phenotype in macrophages, which was manifested by down-regulated expression of inducible nitric oxide synthase (iNOS), significantly reduced production of nitric oxide (NO), inflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokines (CCL-17 and CCL-24) and resulted in reduced influx of circulating neutrophils, monocytes and lymphocytes in the inflamed gut. Additionally, MSC-EVs induced polarization of colon macrophages in anti-inflammatory M2 phenotype, manifested by increased secretion of immunosuppressive cytokines TGF-β and IL-10 and alleviation of colitis.
Yang and colleagues suggested that modulation of anti-oxidant/oxidant balance in the injured gut was responsible for MSC-EVs-induced effects on macrophage phenotype and function [26]. MSC-EV-mediated suppression of NO-driven injury in the gut was accompanied by decreased activity of myeloperoxidase and malondialdehyde and increased superoxide dismutase and glutathione activity. Furthermore, MSC-EVs reduced cleavage of caspase-3, -8 and -9 and alleviated release of DAMPs from injured gut epithelial cells, resulting in attenuated activation of NF-κB signaling pathway in colon macrophages, which consequently led to the generation of immunosuppressive M2 phenotype [26].
Mao and coworkers suggested that, in addition to the inhibition of NF-κB and iNOS, MSC-EVs exert their beneficial effects in colitis through the inhibition of IL-7 signaling in colon macrophages, as well [28]. IL-7 displays strong chemotactic property for circulating monocytes enabling their massive accumulation in inflamed tissues [29]. Additionally, IL-7 induces increased production of NO, TNF-α and IL-1β in macrophages, enhancing their inflammatory properties [29]. MSC-EVs contain miR17, which impairs IL-7:IL-7 receptor signaling by preventing synthesis and transactivation of Janus kinase 1 [30,31]. In line with these findings, MSC-EVs treatment significantly reduced activation of IL-7 and iNOS-signaling pathways in colon macrophages, resulting in attenuated production of TNF-α, IL-1β, IL-6 and increased secretion of IL-10, which led to the alleviation of colitis [28].

Molecular Mechanisms Responsible for MSC-EVs-Based Protection of Hepatocytes in Acute Liver Injury and Fibrosis
As recently evidenced by us and others, MSC-derived secretome efficiently attenuated acute liver failure and liver fibrosis in mice by suppressing major effector cells: natural killer T cells (NKT) cells in fulminant hepatitis and CD4+ T helper lymphocytes and hepatic stellate cells (HSCs) in fibrosis [32][33][34][35][36].
MSC-sourced secretome contains high concentration of NO and reactive nitrogen species, which decrease proliferation of liver NKT cells [37]. Accordingly, administration of MSC-derived secretome significantly reduced total number of inflammatory NKT cells in the injured livers of mice with fulminant hepatitis [32,34]. Additionally, MSC-derived secretome contains Kynurenine, which maintains immunosuppressive phenotype of FoxP3-expressing NKT cells in the inflamed livers and suppress their transdifferentiation in inflammatory, IL-17-producing NKT17 cells [34]. Liver NKT cells cultured in the presence of MSC-sourced secretome have reduced capacity for production of hepatotoxic (TNF-α) and inflammatory cytokines (IFN-γ, IL-17) [32,34]. Moreover, reduced expression of molecules, which are responsible for NKT cell-dependent apoptosis of hepatocytes (Fas ligand, CD107a and NKG2D) was observed in liver NKT cells cultured in the presence of MSC-derived secretome [34].
In addition to immunosuppressive effects against NKT cells, MSC-sourced secretome may directly protect hepatocytes from cell death [38,39]. Injection of human menstrual blood-derived MSC-Exos significantly attenuated d-galactosamine/lipopolysaccharide (d-GalN/LPS)-induced acute liver injury and increased survival rate of experimental mice by suppressing caspase-3-driven apoptosis of hepatocytes [38]. In line with these findings are results obtained by Chen and colleagues who provided additional evidence of anti-apoptotic capacity of MSC-EVs in a murine model of autoimmune hepatitis (AIH) [39]. Hepatoprotective effects of MSC-Exos were relied on suppression of NLRP3-dependent activation of caspase-1 and on inhibition of caspase-1-driven pyroptosis, characterized by plasma membrane rupture, cytoplasmic swelling, osmotic lysis, DNA cleavage and massive release of pro-inflammatory cytokines (IL-1β and IL-18) [40]. Accordingly, by suppressing pyroptosis, MSC-Exos inhibited cell death of hepatocytes and attenuated IL-1β and IL-18-driven inflammation. MSC-derived miR-233 was crucially important for these hepatoprotective effects of MSC-Exos since administration of Exos derived from miR-233 deficient MSCs did not attenuate AIH [39]. The analysis of NLRP3-signaling pathway revealed that exosomal miR-233 suppressed NLRP3:caspase-1-induced pyroptosis by inducing degradation of NLRP3 mRNA in hepatocytes [39]. MSC-Exos attenuate oxidative stress in inflamed livers, as well [41]. MSC-Exo-derived glutathione peroxidase 1 (GPX1) was mainly responsible for MSC-Exo-dependent suppression of reactive oxygen species (ROS) formation in injured hepatocytes [41].
In addition to their hepatoprotective effects, MSC-Exos may induce proliferation of hepatocytes. As recently evidenced by Du and colleagues intravenous injection of Exos, obtained from human-induced pluripotent stem cell-derived MSCs (hiPSC-MSCs-Exos) attenuated hepatic ischemia-reperfusion (I/R) injury by suppressing necrosis of hepatocytes and by promoting their proliferation [42]. The serum levels of hepatocyte injury markers (aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly lower and the expression levels of proliferation markers (proliferation cell nuclear antigen (PCNA) and phosphohistone-H3 (PHH3)) were greatly increased in the livers of I/R-injured mice that received hiPSC-MSCs-Exos [42]. Significantly increased proliferation of hiPSC-MSCs-Exos-treated primary hepatocytes and HL7702 human hepatocytes was confirmed in vitro. Mechanistically, hiPSC-MSCs-Exos directly fused with target hepatocytes or HL7702 cells and increased the activity of sphingosine kinase (SK1) resulting in synthesis of sphingosine-1-phosphate (S1P), which promoted hepatocyte growth, survival and proliferation [42,43]. This phenomenon was completely abrogated after inhibition of either SK1 or S1P receptor, confirming crucial importance of SK1/S1P signaling for hiPSC-MSCs-Exos-induced enhanced proliferation of hepatocytes [42].
Several lines of evidence demonstrated that MSC-EVs protected hepatocytes during chronic liver inflammation and fibrosis, as well [44]. Results obtained by Li and colleagues showed that human umbilical cord-MSCs-derived Exos attenuated carbon tetrachloride (CCl4)-induced liver fibrosis in mice, as evidenced by recovered serum AST levels and reduced deposition of collagen type I and III in the liver [44]. Significantly decreased expression of TGF-β1 and phosphorylated Smad2 was observed in the CCl4-injured livers of MSC-Exo-treated mice, indicated that MSC-Exo-dependent inhibition of TGF-β1 signaling pathway in hepatocytes was crucially important for anti-fibrotic effects of MSC-Exos. Upon phosphorylation, Smad2 formed complexes with phosphorylated Smad3 and Smad4 and, subsequently, translocated into the nucleus to regulate the transcription of genes responsible for epithelial-to-mesenchymal transition (EMT) of hepatocytes [45]. Significant increase in E-cadherin-positive cells and decrease in N-cadherin-and vimentin-positive cells in MSC-Exo-treated fibrotic livers, suggested that MSC-Exos prevented TGF-β1/Smad2-induced EMT of hepatocytes [44]. This hypothesis was confirmed in vitro. MSC-Exos completely reversed spindle-shaped morphology and abrogated expression of EMT-associated markers in HL7702 human hepatocytes that underwent EMT after treatment with recombinant TGF-β1 [44].

MSC-EVs as Next-Generation Therapeutics for the Treatment of Lung Inflammatory Diseases
There is growing evidence that MSC-EVs protect lung epithelial cells from reactive oxidative species and proteolytic enzymes released by lung-infiltrating neutrophils and monocytes [49][50][51][52].
Li and colleagues demonstrated that MSC-EV-based protection of lung epithelial cells against oxidative stress-induced cell death is dependent on anti-apoptotic properties of miR-21-5p [50]. Intratracheal administration of MSC-Exos inhibited both intrinsic and extrinsic apoptotic pathways in lung epithelial cells. However, pre-treatment of MSCs with miR-21-5p antagomir completely abrogated MSC-Exos-mediated suppression of caspase-3, -8 and -9 and diminished MSC-Exo-based protective effects [50]. Western blot analysis revealed that pro-apoptotic phosphatase and tensin homolog (PTEN) and programmed cell death protein 4 (PDCD4) were the main targets of MSC-derived miR-21-5p since their expression was significantly decreased in lung epithelial cells of MSC-Exo-treated mice. When I/R-injured mice received Exos derived from miR-21-5p-antagomir-treated MSCs, expression of PTEC and PDCD4 and apoptosis of lung epithelial cells were not reduced, indicating crucial importance of miR-21-5p-dependent suppression of PTEN and PDCD4 for anti-apoptotic effects of MSC-Exos in I/R lung injury [50].
In addition to their anti-oxidative effects, MSC-EVs may protect lung epithelial cells by regulating protease/antiprotease balance in the inflamed lungs [51]. Alpha-1-antitrypsin (AAT) is a potent inhibitor of neutrophil-derived proteolytic enzymes, which protects lung epithelial cells and exerts important anti-inflammatory and immunomodulatory effects in the lungs [52]. Most recently, Bari and colleagues revealed that AAT was aggregated and/or adsorbed on the surface of adipose-tissue derived MSC-EVs that served as natural carriers of AAT, promoting its stability and activity in vivo [51]. Importantly, MSC-EVs derived from IL-β-primed MSCs showed significantly higher expression of AAT gene and had increased anti-elastase activity compared to MSC-EVs obtained from IL-β-non-primed MSCs [51].
Importantly, MSC-EVs, in addition to AAT, contained 46 proteins involved in the response to Gram-negative bacteria, implying potent anti-microbial activity of MSC-EVs [51]. In line with these findings are results obtained by Hao and colleagues who demonstrated that administration of MSC-EVs remarkably reduced severity of bacterial pneumonia in mice [53]. MSC-EVs increased phagocytic and anti-microbial activity of lung-infiltrating neutrophils and monocytes by promoting synthesis of leukotriene B4 (LTB4) [53]. LTB4 is well-known activator of leucocytes, which augments phagocytosis and promotes release of anti-microbial agents, contributing to the bacterial clearance [54]. Hao and colleagues demonstrated that miR-145, contained within MSC-EVs, reduced expression of multidrug resistance-associated protein 1 (MRP1) in lung macrophages [53]. MRP1 is ATP-binding cassette transporter, which inhibits synthesis and release of LTB4 [53]. Accordingly, MSC-EV-induced suppression of MRP1 resulted in enhanced release of LTB4 by alveolar macrophages that, due to its anti-microbial activity, increased bacterial clearance and reduced severity of bacterial pneumonia in mice [53].
It is important to highlight that capacity of MSC-EVs to modulate phenotype and function of alveolar macrophages depends on the phase of anti-microbial inflammatory response [55]. During the onset of inflammation, MSC-EVs, in a miR-145/LTB4-dependent manner, promote phagocytic activity of alveolar macrophages contributing to the elimination of bacterial pathogens from the lungs. However, during the resolution of inflammation, MSC-EVs promote expansion of alternatively activated M2 macrophages that are involved in tissue repair and regeneration [55]. It is well known that alveolar macrophages, through the production of inflammatory cytokines and chemokines, orchestrate influx of circulating monocytes and lymphocytes in inflamed lungs, promoting chronic inflammation [55]. Therefore, MSC-EV-based suppression of chronic, macrophage-driven inflammatory lung diseases was mainly relied on MSC-EV-dependent polarization of alveolar macrophages. MSC-Exos significantly decreased iNOS mRNA expression and remarkable increased expression of Arginase-1 mRNA in alveolar macrophages, inducing their polarization from inflammatory M1 towards immunosuppressive M2 phenotype [50,55]. Accordingly, concentration of M1-related inflammatory cytokines (IL-8, IL-1β, IL-6 and TNF-α) was significantly reduced and concentration of M2 macrophage-derived immunosuppressive cytokines (IL-10 and TGF-β) was increased in the lungs of I/R-injured mice that received MSC-Exos [50].
Interestingly, as recently revealed by Huang and colleagues, aging MSC-EVs did not manage to induce generation of M2 macrophages in the inflamed lungs [56]. Although aging and young MSC-EVs had similar phenotypic characteristics (expression of CD63, CD81, CD105 and CD44), their capacity to alter the phenotype of alveolar macrophages was different. Internalization of aging MSC-EVs by alveolar macrophages was significantly lower compared to the young MSC-EVs. Furthermore, aging MSC-EVs had reduced capacity to inhibit production of inflammatory, M1-related cytokines (IL-6, IL-1β and TNF-α) and to induce expression of M2-related Arginase-1 in alveolar macrophages [56]. Most importantly, aging and young MSC-EVs differed in levels of miRNAs (miR-223-5p, miR-127-3p and miR-125b-5p) that regulate macrophage polarization. Compared with aging MSC-EVs, young MSC-EVs showed higher expression of miR-223-5p (which is responsible for induction of M2 phenotype in alveolar macrophages) and lower expression of miR-127-3p and miR-125b-5p (which promote generation of M1 phenotype in macrophages) [56]. Since aging MSC-Exos had significantly reduced capacity to attenuate M1 macrophage driven inflammation in the lungs, MSC-Exos used for the therapy of inflammatory lung diseases should be obtained only from young donors.
Mansouri and colleagues recently revealed that single intravenous administration of Exos, obtained from human bone marrow-derived MSC, managed to significantly attenuate bleomycin-induced lung fibrosis in mice through the modulation of phenotype and function of alveolar macrophages [57]. An improved Ashcroft score and reduced deposition of collagen were observed in bleomycin-injured lungs of MSC-Exo-treated animals. MSC-Exo-based alleviation of fibrosis was followed by significantly reduced number of TGF-β1-producing, Arginase-1 and CD206-expressing alveolar macrophages, indicating that macrophages were the main cellular targets of MSC-Exos in alleviation of pulmonary fibrosis. Importantly, anti-fibrotic effects were not observed in bleomycin-injured mice that received fibroblasts-derived Exos or Exos free iodixanol, suggesting that immunomodulatory properties of MSCs were responsible for beneficial effects of MSC-Exos [57].
In addition to alveolar macrophages, MSC-EVs may also modulate phenotype and function of lung-infiltrating dendritic cells (DCs) [58]. As recently evidenced by Cho and colleagues, MSC-EV-based alleviation of Th2 cell-driven immune response against Aspergillus protease antigen was dependent on suppression of antigen-presenting properties of DCs [45]. MSC-Exos induced increased expression of immunosuppressive IL-10 and TGF-β that suppressed maturation of lung DCs [58]. Immature DCs of MSC-Exos-treated mice had reduced expression of co-stimulatory molecules (CD40, CD80 and CD86) and were not capable to optimally activate CD4+Th2 cells, resulting in alleviation of Th2 cell-driven lung inflammation [58].
The lung is a portal of entry for numerous microbial pathogens, which are, immediately after invasion, captured and efficiently eliminated by alveolar macrophages and lung DCs, resulting in the activation of antigen specific, T cell-driven immune response [59,60]. Upon activation, alveolar macrophages and lung DCs produce large amount of inflammatory chemokines and cytokines and orchestrate both local and systemic immune response [59]. Accordingly, lung macrophages and DCs have been considered as the cells that are crucially important for the generation and development of chronic inflammatory diseases [59]. Since most of intratracheally and intravenously administered MSC-EVs accumulate in the lungs where, in similar manner as microbial pathogens, become phagocyted by lung-infiltrated macrophages and DCs, capacity of MSC-EVs to modulate phenotype and function of these professional antigen-presenting cells could be used not only for alleviation of inflammatory lung diseases but also for modulation of detrimental macrophage and DC-driven systemic immune response.

Modulation of Microglial Activity: The Main Mechanism Responsible for MSC-EVs-Dependent Attenuation of Neuroinflammatory Diseases
Microglia, the resident immune cells of the central nervous system (CNS), maintain tissue homeostasis under physiological conditions [61]. However, after neuronal injury, microglia secrete pro-inflammatory cytokines that either have direct neurotoxic effects or, in combination with inflammatory chemokines, promote influx of circulating neutrophils in inflamed tissue [61]. An excessive microglial activation damages the surrounding healthy neural tissue and induces the release of alarmins and DAMPs from dead or dying neurons, which in turn, activates microglia enabling creation of "positive inflammatory loop" in CNS, that results in a massive and progressive loss of neurons [61]. In line with these findings, Ding and colleagues recently revealed that modulation of microglial activity was the main mechanism responsible for beneficial effects of MSC-EVs in alleviation of Alzheimer's disease (AD) [62]. Excessive accumulation of the amyloid-β peptide (Aβ) in the brain is considered as the most common pathological characteristic of AD, which triggers dysfunction of cognitive behavior [63]. Intravenously injected Exos, obtained from human umbilical cord-derived MSCs, managed to reduce Aβ deposition and increased spatial learning and memory function in AβPP/PS1 transgenic mice, used as murine model of AD [62]. Additionally, Bodart-Santos and colleagues recently revealed that MSC-EVs prevented neuronal damage in AD by suppressing oxidative stress-induced injury of hippocampal neurons [64]. Catalase was mainly responsible for MSC-EV-based protection against ROS-induced injury since MSC-EVs with inactivated catalase were unable to prevent ROS formation in hippocampal neurons [64]. MSC-Exos induced polarization of microglia towards immunosuppressive M2 phenotype. Significantly higher number chitinase 3-like 3, arginase-1 and mannose receptor C type 1 (MRC1)-expressing M2 microglia cells were found in the brains of MSC-Exos-treated AβPP/PS1 mice [62]. M2 cells produce Aβ-degrading enzymes (neprilysin (NEP) and insulin-degrading enzyme (IDE)) and anti-inflammatory cytokines (IL-10 and TGF-β), contributing to the reduced Aβ deposition and alleviated inflammation [61]. Significantly increased levels of NEP, IDE, IL-10 and TGF-β, and greatly reduced concentration of inflammatory cytokines (TNF-α and IL-1β) were noticed in the brains of MSC-Exos-treated AβPP/PS1 mice, indicating that MSC-Exos induce conversion of microglia from inflammatory M1 towards immunosuppressive M2 phenotype [62]. MSC-Exo-induced alternative microglial activation was confirmed in vitro, since significantly higher concentration of IL-10 and TGF-β and lower concentration of TNF-α and IL-1β were measured in supernatants of MSC-Exo-treated BV2 murine microglia cells [62].
As recently revealed by Shiue and colleagues [66], continuous intrathecal injection of MSC-Exos enabled functional recovery from nerve ligation-induced injury [66]. MSC-Exos suppressed production of inflammatory cytokines (TNF-α and IL-1β) and promoted synthesis of anti-inflammatory cytokines (IL-10 and TGF-β) in microglia, resulting in the alleviation of inflammation within the site of neural injury [66]. The analgesic effects of MSC-Exos involved their actions on neurons, as well. MSC-Exos delivered brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor in the ipsilateral L5/6 dorsal root ganglion of nerve-ligated rats, enabling better recovery from nerve ligation-induced injury [66]. Protein analysis demonstrated that vascular endothelial growth factor C, angiopoietin-2 and fibroblast growth factor-2 were also present in the MSC-Exos, indicating that induction of neo-angiogenesis may be, at least partially responsible for beneficial effects of MSC-Exos. Importantly, immunofluorescence staining showed that MSC-Exos were presented in the ipsilateral L5 spinal dorsal horn, dorsal root ganglion and peripheral axons, suggesting a high homing ability of MSC-Exos [66].
Huang and colleagues provided evidence that MSC-Exos ameliorated cerebral I/R injury by preventing neural cell death through the inhibition of caspase-9 and caspase-3 [67]. MSC-sourced pigment epithelium-derived factor (PEDF), which exhibits anti-inflammatory, antioxidative and neuroprotective properties, was mainly responsible for beneficial effects of MSC-Exos [68]. Through the delivery of PEDF, MSC-Exos increased expression of autophagy-associated protein LC3 and suppressed caspase-3-driven apoptosis in neurons, significantly reducing I/R-induced injury [67]. Exos, obtained from PEDF-overexpressing MSCs showed better therapeutic effects, while inhibition of autophagy significantly reduced neuroprotection elicited by PEDF-containing MSC-Exos, indicating crucial importance of PEDF-induced autophagy for MSC-Exo-based attenuation of cerebral I/R injury [67].

Molecular Mechanisms Responsible for MSC-EVs-Based Renal Protection
MSC-EVs-dependent renal protection is relied on the inhibition of apoptosis, necrosis and oxidative stress in renal tubular epithelial cells as well as suppression of detrimental immune response in the kidneys (Figure 2) [69]. MSC-sourced mRNAs, miRNAs and immunosuppressive factors were mainly responsible for beneficial effects of MSC-EVs in alleviation of acute and chronic renal inflammation [69][70][71][72][73][74][75][76][77][78][79][80]. . MSC-EVs activated autophagy in PTEC and protected against cisplatin-induced AKI by delivering trophic factor 14-3-3ζ, which interacted with ATG-16L, a protein essential for autophagy induction. MSC-EVs enhanced activation of NF-E2-related factor 2/antioxidant responsive element, decreased expression of NADPH oxidase and reduced production of reactive oxygen species in ischemic kidneys and promoted their regeneration. Additionally, through the delivery of miR-21, MSC-EVs significantly attenuated capacity for antigen-presentation of renal dendritic cells, which resulted in reduced activation of Th1 and Th17 cells and alleviation of Th1 and Th17 cell-driven inflammation in the kidneys. Through the delivery of microRNAs (miRNAs), particularly let-7b, MSC-EVs induced conversion of inflammatory M1 macrophages into immunosuppressive M2 cells, which produced lower amount of inflammatory cytokines (TNF-α and IL-1β) and chemokine CXCL1, resulting in alleviated acute and chronic renal inflammation. MSC-sourced miRNA, particularly let-7c, targeted pro-fibrotic genes (collagen IVα1, TGF-β1 and TGFβR1) in inflamed kidneys, crucially contributing to the therapeutic effects of MSC-EVs in renal fibrosis. Additionally, neo-angiogenesis, induced by MSC-derived vascular endothelial growth factor (VEGF) was also responsible for beneficial effects of MSC-EVs in alleviation of renal fibrosis.
Several lines of evidence demonstrated that MSC-derived mRNAs were involved in MSC-EVsbased attenuation of acute kidney injury (AKI) [70][71][72][73]. Bruno and colleagues noticed significantly improved renal function in glycerol and cisplatin-injured kidneys of experimental animals [70][71][72]. They revealed that mRNAs, which regulate transcription (e.g., CLOCK, IRF6 and LHX6), cell cycle regulation (e.g., SENP2, RBL1 and CDC14B) and DNA/RNA repair (e.g., HMGN4, TOPORS and . MSC-EVs activated autophagy in PTEC and protected against cisplatin-induced AKI by delivering trophic factor 14-3-3ζ, which interacted with ATG-16L, a protein essential for autophagy induction. MSC-EVs enhanced activation of NF-E2-related factor 2/antioxidant responsive element, decreased expression of NADPH oxidase and reduced production of reactive oxygen species in ischemic kidneys and promoted their regeneration. Additionally, through the delivery of miR-21, MSC-EVs significantly attenuated capacity for antigen-presentation of renal dendritic cells, which resulted in reduced activation of Th1 and Th17 cells and alleviation of Th1 and Th17 cell-driven inflammation in the kidneys. Through the delivery of microRNAs (miRNAs), particularly let-7b, MSC-EVs induced conversion of inflammatory M1 macrophages into immunosuppressive M2 cells, which produced lower amount of inflammatory cytokines (TNF-α and IL-1β) and chemokine CXCL1, resulting in alleviated acute and chronic renal inflammation. MSC-sourced miRNA, particularly let-7c, targeted pro-fibrotic genes (collagen IVα1, TGF-β1 and TGFβR1) in inflamed kidneys, crucially contributing to the therapeutic effects of MSC-EVs in renal fibrosis. Additionally, neo-angiogenesis, induced by MSC-derived vascular endothelial growth factor (VEGF) was also responsible for beneficial effects of MSC-EVs in alleviation of renal fibrosis.
In line with these findings are results reported by Gatti and colleagues who demonstrated that MSC-EVs alleviated I/R-induced AKI by reducing apoptosis and by increasing proliferation of renal tubular cells [74]. Similarly as it was observed by Bruno et al. [70] and Ju and et al [73], MSC-EV-based renoprotection was diminished by RNase pretreatment [74], confirming the hypothesis that beneficial effects of MSCs-EVs were mainly mediated by MSC-sourced mRNA.
Wang and colleagues indicated that activation of autophagy in proximal tubular epithelial cells (PTEC) was responsible for greatly improved renal function of cisplatin + MSC-EVs-treated mice [75]. They showed that beneficial effects of MSC-EVs were completely abrogated by autophagy inhibitor, 3-methyladenine. Similarly, Jia and coworkers demonstrated that MSC-EVs activated autophagy in cisplatin-injured PTEC and protected against AKI by delivering trophic factor 14-3-3ζ, which interacted with ATG-16L, a protein essential for autophagy induction [76].
By using the I/R model of AKI, Zhou and colleagues indicated that attenuation of oxidative stress was mainly responsible for MSC-EVs-based renoprotection in AKI [77,78]. This hypothesis was based on enhanced activation of NF-E2-related factor 2/antioxidant responsive element, decreased expression of NADPH oxidase and reduced production of ROS, which were observed in the I/R-injured kidneys of MSC-EV-treated mice [77,78]. In line with these findings, Gu and coworkers observed preserved mitochondrial morphology in renal tubular cells of MSC-EV-treated mice [79]. They showed that miR-30 antagomirs remarkably reduced renoprotective effects of MSC-EVs, implying critical role of miR-30 in MSC-EV-based attenuation of AKI [79]. Song and colleagues further emphasized importance of MSC-derived miRNAs in renoprotection by demonstrating anti-inflammatory properties of miR-21 in alleviation of I/R-induced AKI [80]. MSC-sourced miR-21 reduced NF-κB activity in renal infiltrating DCs and suppressed their maturation [80]. Accordingly, administration of miR-21-containing MSC-EVs significantly attenuated capacity of renal DCs for production of inflammatory cytokines and reduced activation of Th1 and Th17 cell-driven inflammation in I/R-injured kidneys leading to the attenuation of AKI [80].
In addition to miR-21 and miR-30, members of the let-7 miR family, contained within MSC-EVs, have been shown to regulate multiple genes involved in apoptosis and proliferation of renal tubular epithelial cells, including CCNA2, CDC34, AURA/STK6, AURKB/STK12, E2F5, and CDK8 [81]. Moreover, MSC-derived let-7b was responsible for MSC-EV-induced generation of immunosuppressive M2 phenotype in renal macrophages [82]. Accordingly, significantly lower concentration of M1-derived inflammatory cytokines TNF-α and IL-1β were measured in I/R-injured kidneys of mice that received let-7b-containing MSC-EVs.
MSC-sourced miRNAs, particularly let-7c, targeted pro-fibrotic genes (collagen IVα1, TGF-β1 and TGFβR1) in inflamed kidneys, crucially contributing to the therapeutic effects of MSC-EVs in renal fibrosis and diabetic nephropathy [83][84][85]. In line with these findings were results obtained by Zou and colleagues who indicated that MSC-EV-dependent down-regulation of CXCL1 production was responsible for significantly decreased number of CD68+ macrophages in fibrotic kidneys of MSC-EVs-treated mice [84]. Since remarkably increased expression of MSC-sourced vascular endothelial growth factor (VEGF) was observed in the MSC-EV-treated kidneys, Zou and coworkers suggested that MSC-induced neo-angiogenesis was, in addition to MSC-EV-based immunosuppression, also responsible for beneficial effects of MSC-EVs in alleviation of renal fibrosis [86]. Since activation of MSCs with inflammatory cytokines (TNF-α and IFN-γ) significantly enhanced production of immunosuppressive and pro-angiogenic factors in MSCs-Exos [87], TNF-α and IFN-γ-priming of MSCs should be further explored as a new approach for the generation of MSC-EVs with optimal renoprotective characteristics.

MSC-EV-Based Attenuation of Autoimmune and Inflammatory Eye Disease
A large number of experimental and clinical studies demonstrated beneficial effects of MSC-Exos in the suppression of autoimmune and chronic inflammatory eye diseases [88][89][90][91][92][93][94][95]. Intravenous as well as periocular administration of MSC-Exos efficiently attenuated experimental autoimmune uveitis (EAU) [89,90]. MSC-Exos suppressed production of CCL2 and CCL21, which resulted in significantly reduced presence of Gr-1-expressing granulocytes, CD68-expressing macrophages and CD4+T cells in injured retinas [89]. While massive infiltration of inflammatory cells resulted in severe disruption of the retinal photoreceptor layers in vehicle-treated EAU mice, only little structural damage of retinal cells and few inflammatory infiltrates were observed in the eyes of MSC-Exo-treated EAU mice [90]. In addition to their effect on chemokine production, MSC-Exos inhibited antigen-presenting function of retinal-infiltrating DCs, as well. MSC-Exos significantly reduced expression of costimulatory molecules (CD40, CD80 and CD86) and MHC class II proteins on DCs, attenuating their capacity for activation of naive CD4+ T cells [90]. The transcript levels of DC-derived Th1 and Th17-related cytokines (IL-1β, IL-6 and IL-12) were significantly lower in MSC-Exos-treated animals [90]. Accordingly, remarkably reduced number of IFN-γ-producing Th1 and IL-17-producing Th17 cells, that play crucially important pathogenic role in progression of EAU, were noticed in the eyes of MSC-Exo-treated EAU mice, implying that therapeutic effects of MSC-Exos in alleviation of EAU were relied on suppression of Th1 and Th17 cell-driven inflammation [90].
Th17 cells are the main inflammatory, effector cells in dry eye disease (DED), chronic inflammatory disease of the tears and ocular surface that is manifested by symptoms of discomfort, visual disturbance, and tear film instability [91]. MSC-Exos contain a growth related oncogene (GRO), which suppresses production of Th17-inducing cytokines (IL-1β, IL-6 and IL-23) in DCs and prevent Th17 cell-driven inflammation [91,92]. In addition to GRO, MSC-sourced Indoleamine 2-3 dioxygenase (IDO) was responsible for MSC-Exo-based suppression of DC-dependent generation of Th17 cells [93,94]. Exos obtained from IDO-overexpressing MSCs down-regulated expression of co-stimulatory molecules and suppressed production of Th17-inducing cytokines in DCs, attenuating their capacity for activation of naïve T cells and generation of inflammatory Th17 cells [93,94]. Additionally, MSC-derived IDO acts as a critical molecular switch that maintains immunosuppressive phenotype of FoxP3-exspressing Tregs in inflamed tissues and prevents their re-programming into inflammatory Th17 cells [14]. Furthermore, MSC-derived IDO promotes expansion of TGFβ and IL-10-producing-immunosuppressive Tregs, contributing to the creation of immunosuppressive microenvironment in the inflamed eyes [88]. Accordingly, IDO-dependent regulation of Th17:T regulatory cells (Tregs) ratio, is also responsible for MSC-Exo-based suppression of Th17 cell driven inflammation in the eyes [88]. In line with these findings, we recently designed an ophthalmic solution (Exo-d-MAPPS), which activity was based on therapeutic effects of GRO and IDO-containing MSC-Exos [94]. Exo-d-MAPPS treatment significantly attenuated production of inflammatory cytokines in T cells and managed to alleviate dryness, grittiness, scratchiness, irritation, burning and eye fatigue in DED patients [94].
In addition to their anti-inflammatory effects, MSC-Exos promoted repair and regeneration of injured neurons in the eye [88]. Exos, obtained from bone marrow-derived MSCs, increased survival and neuritogenesis of retinal ganglion cells (RGCs) [95]. By using nerve crush model, Mead and Tomarev showed that intravitreal administration of MSC-Exos significantly reduced loss of RGCs and improved their function [95]. Therapeutic effects of MSC-Exos were relied on the delivery of miR-17-92, miR21 and miR-146 into the injured RGCs. MSC-sourced miR-17-92 and miR21 down-regulated expression of PTEN (an important suppressor of RGC axonal growth), while MSC-derived miR-146a reduced expression of epidermal growth factor receptor (involved in inhibition of axon regeneration) [95]. Importantly, beneficial effects of MSC-Exos in protection, repair and regeneration of RGCs were observed only in animals that received MSC-Exos and were not noticed after injection of fibroblasts-derived Exos [95], implying specific therapeutic potential of MSCs-Exos in regeneration of injured RGCs. Since gradual loss of RGCs is the hallmark of glaucoma, MSC-Exos represent potentially new therapeutic agents for glaucoma treatment, which efficacy should be explored in up-coming clinical trials.

Delivery of MSC-Sourced mRNAs into the Injured Cardiomyocytes Was Mainly Responsible for MSC-EVs-Based Cardioprotection
Several lines of evidence demonstrated that injection of MSC-EVs efficiently protected cardiomyocytes from ischemic injury [96,97]. By using animal model of I/R-induced myocardial injury, Lai and colleagues showed that Exos, isolated form human embryonic stem cells derived MSCs, significantly reduced infarct size and remarkably improved cardiac function in experimental animals [96]. MSC-Exos attenuated oxidative stress in I/R-injured hearts, as evidenced by greatly increased tissue levels of ATP and nicotine adenine dinucleotide and significantly decreased levels of reactive oxygen species [97]. MSC-Exos contain Parkinson protein 7/DJ-1 (DJ-1), which binds to the PARKIN protein in oxidative stress conditions, protecting the mitochondria from oxidative stress [98,99]. SinceDJ-1 protects murine heart from oxidative damage [100], MSC-sourced DJ-1 may be responsible for MSC-Exo-based modulation of oxidative balance in ischemic hearts [96]. Accordingly, Exos obtained from DJ-1-overexpressing MSCs should be explored in up-coming preclinical studies as new agents that could promote cardiac regeneration after ischemic injury. Cardioprotective effects of MSC-Exos were also relied on increased phosphorylation and activation of kinases that prevented apoptosis of injured cardiomyocytes (Akt and Glycogen synthase kinase 3 (GSK3)) and on suppression of c-Jun-N-terminal kinase, which promoted apoptosis in ischemic hearts [96].
Results obtained by Yu and coworkers supported the hypothesis that Akt kinase was the main intracellular target for MSC-EV-based cardioprotection [97]. They showed that Exos, obtained from Gata-4-overexpressing bone marrow derived MSCs, significantly reduced the size of ischemic lesion and restored cardiac function in the rat model of acute myocardial infarction (AMI) by activating Akt-dependent signaling pathway in injured cardiomyocytes [97]. Yu and colleagues revealed that among several MSC-Exo-containing miRNAs that regulate survival and proliferation of cardiomyocytes, miR-19a was mainly responsible for MSC-Exos-induced anti-apoptotic effects in ischemic hearts. MSC-sourced miR-19a down-regulated activation of PTEN and promoted phosphorylation and activation of Akt resulting in the up-regulation of anti-apoptotic Bcl-2 protein, resulting in reduced apoptotic loss of cardiomyocytes [97]. In line with these findings are results obtained by Wang and colleagues who demonstrated that Exos, obtained from endometrium-derived MSCs, significantly improved recovery of cardiac function after AMI by promoting Akt-dependent up-regulation of Bcl-2 activity in injured cardiomyocytes [101]. Wang et al. suggested that MSC-derived miR-21 was mainly responsible for cardioprotective effects of MSC-EVs. They demonstrated that, in addition to anti-apoptotic effects, miR21-containing MSC-Exos induced enhanced expression of vascular endothelial growth factor (VEGF) and promoted neovascularization in ischemic hearts, significantly improving cardiac function after AMI [101].
A crucially important role of MSC-sourced miRNAs for MSC-EV-based cardioprotection was confirmed by Feng and colleagues [102]. They suggested that MSC-Exo-mediated delivery of miR-22 in ischemic cardiomyocytes was mainly responsible for improved cardiac function that was noticed in MSC-Exo-treated mice with AMI [81]. Significantly reduced infarct size and cardiac fibrosis was a consequence of miR-22-dependent down-regulation of methyl-CpG-binding protein 2, epigenetic regulator, which was up-regulated in ischemic hearts [102].
It should be emphasized that, in addition to their anti-apoptotic effects, MSC-EVs also suppressed the influx of circulating leucocytes in injured hearts, contributing to the attenuation of on-going inflammation [97]. Significantly reduced release of alarmins and DAMPs from MSC-EV-treated cardiomyocytes resulted in decreased secretion of leucocyte-attracting chemokines by resident macrophages. Accordingly, after reperfusion, a significantly lower number of neutrophils, monocytes and lymphocytes infiltrated myocardium of MSC-Exo-treated animals, indicating that MSC-Exos-based suppression of inflammatory response also contributed to the enhanced repair and regeneration of injured cardiomyocytes [97].

Conclusions and Future Directions
MSC-EVs represent new, cell-free agents that could be used for efficient attenuation of organ-specific and systemic inflammation. Both local and systemic administration of MSC-EVs efficiently suppressed detrimental immune response in inflamed tissues and promoted survival and regeneration of injured parenchymal cells.
It should be noted that although experimental findings strongly suggested therapeutic potential of MSC-EVs, there is still a lot of experimental work to be done before MSC-EVs could be offered as universal human remedy for the therapy of inflammatory diseases.
MSC-EVs exhibit most of the properties of MSCs and fundamental challenges relating to MSC heterogeneity affect biological properties and therapeutic potential of MSC-EVs, as well [103]. Differences in the proliferation rate, potential for multi-lineage differentiation and immunosuppressive properties of MSCs from different sources are well-documented [104]. Furthermore, even when MSCs were obtained from the same tissue of origin, they could have prodigious donor-to-donor variation in expression of membrane markers, transcriptional and proteomic profile [104]. Aging also has a negative influence on self-renewal capacity, differentiation and immunosuppressive characteristics of MSCs, attenuating their therapeutic potential [105]. In line with these findings, several recently published studies indicated that MSC-EVs have significant tissue source and age-dependent differences in their capacity for immunosuppression and tissue regeneration [106][107][108]. Additionally, culture conditions in which MSCs were exposed may also influence the concentration of immunomodulatory factors within MSC-EVs. Significantly higher concentration of immunosuppressive cytokines were observed in EVs that were obtained from the MSCs, which were primed with inflammatory cytokines (TNF-α and IFN-γ) than in EVs that were derived from MSCs, which were grown under standard culture conditions [103].
Since large number of different mRNAs, miRNAs, anti-apoptotic and immunosuppressive proteins have been proposed as crucially important for beneficial effects of MSC-EVs, further experimental studies should identify the exact disease-specific MSC-sourced molecule(s) responsible for long-term protection of injured cells and/or sustained immunosuppression. Additionally, the precise dose and route of administration of MSC-EVs should be defined for each organ-specific and systemic inflammatory disease in order to prevent the development of uncontrolled immunosuppression in MSC-EVs recipients.
It should be noted that different laboratories use diverse methods to isolate and purify MSC-EVs and, accordingly, it is critical to define and standardize highly effective method for MSC-EV yields [31]. Additionally, clinical applications of MSC-EVs require their long-term use and considerable thought must be given to the preservation of their immunosuppressive potential [21]. A large number of studies demonstrated that the most convenient mode of storage for MSC-EVs remains −80 • C [109]. Nevertheless, due to the complex cold chain logistics, alternatives such as lyophilization and the incorporation of additives might be necessary to improve MSC-EV storage stability during transportation [21,109].
In summing up, due to their unique biological and immunosuppressive properties, MSC-EVs represents potentially new therapeutic agents in regenerative medicine. Once the critical questions around isolation, long-term preservation, donor and tissue source of MSC-EVs are answered, MSC-EVs will meet their full versatile potential as a new remedies in the therapy of inflammatory diseases.

Conflicts of Interest:
The authors declare no conflict of interest.