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Human MSCs shed from their surface a diverse subpopulation of vesicles. To characterize these vesicles, we performed electron microscopy on those recovered from MSC-conditioned medium by differential ultracentrifugation (100,000 g for 18 h). This analysis demonstrated the presence of 50–100-nm vesicles that are morphologically consistent with exosomes (Fig. 1a, left). Flotation of the 100,000 g pellets on sucrose gradients followed by western blot and fluorescent activated cell sorting (FACS) further demonstrated that these vesicles expressed the exosomal markers’ milk fat globule factor 8 (Mfge8) and the tetraspanins CD9 and CD63, respectively (Supplementary Fig. 1A). Centrifugation of conditioned medium at low speeds (10,000 g) revealed the presence of larger vesicles (>100 nm) that contain subcellular mitochondrial structures including outer and inner membranes and cristae, and expressed the mitochondria-specific protein ATP synthase as evidenced by immuno-gold labelling (Fig. 1a, centre and Supplementary Fig. 1B). MSCs also release larger multivesicular bodies containing lysosome-like vesicles and entire mitochondria, suggesting that these organelles were selected for mitophagy by targeting to autophagosomes (Fig. 1a, right).

To examine mitophagy in MSCs in more detail, MSCs were infected with baculoviruses encoding green fluorescent protein (GFP) fused to the E1alpha pyruvate dehydrogenase leader peptide, which drives transport to the mitochondria, and LC3 fused to a red fluorescent protein (RFP) to allow tracking to the phagophore^{39, 40}. Fluorescent microscopy confirmed that the GFP-labelled mitochondrial network is in close proximity to RFP-LC3-labelled phagosomes (Fig. 2a). Live cell imaging further revealed that mitochondria are loaded in the cytoplasm into LC3-containing vesicles, which migrate towards the cell periphery and are incorporated into outward budding blebs in the plasma membrane (Fig. 2b–d and Supplementary Movie 1). Western blot analysis further revealed that RFP-LC3-MVs also expressed the endosomal sorting complex required for transport (ESCRT)-associated proteins’ tumour suppressor gene 101 (TSG101) and arrestin domain-containing protein 1 (ARRDC1)^{11, 41} (Supplementary Fig. 1D). Collectively, these results indicate that MSCs employ the release of ARMMs to extrude mitochondria at their cell surface. Moreover, MSCs exhibited marked increases in apoptosis when treated with Bafilomycin A1 or low concentrations (3–5 μM) of chloroquine, which block the mitophagy flux, indicating that this process is critical for MSC survival (Supplementary Movie 2).

Next, we co-cultured GFP-labelled human MSCs from above with primary human or mouse macrophages. Live cell imaging revealed that macrophages nibble the plasma membrane of MSCs, establishing cell contact at areas where membrane blebs are enriched in RFP-labelled vesicles, which are subsequently stripped by the macrophage (Fig. 2e–h and Supplementary Movie 3). This activity was also observed between mouse macrophages and primary human MSCs (Fig. 3a and Supplementary Movies 4 and 5) but was not evident when macrophages were co-cultured with mouse or human fibroblasts (Supplementary Fig. 2). In a subsequent experiment, we co-cultured the macrophage cell line RAW 264.7 with human MSCs containing RFP-labelled mitochondria (10:1 ratio) for 4 h and recovered macrophages using FACS after staining with antibodies that recognize macrophage epitopes (that is, F4/80) not expressed by MSCs. Sorted macrophages were cultured for up to 2 weeks in RPMI media, which do not support MSC expansion and survival. Fluorescent microscopy of these macrophages revealed clear evidence of cell-associated RFP derived from human MSCs (Fig. 3b). To confirm these findings, we demonstrated using PCR amplification that these macrophages expressed the mitochondrial specific transcript human cytochrome c oxidase I (MT-COX I), which was confirmed on the basis of the restriction fragment pattern obtained after digestion of the PCR product with Bfa1 (Fig. 3b). This PCR product was not detected in mouse macrophages because of limited sequence homology between the two genes³⁰ but was detected in human MSC-derived MVs as expected (Fig. 3c and Supplementary Fig. 3A). Lastly, we co-cultured Cy5-labelled human MSCs with macrophages that were pre-incubated with or without dextran sulfate (100 μg ml⁻¹), a nonspecific inhibitor of phagocytosis. Live cell imaging showed phagocytosis of MVs by macrophages over a period of 18 min, and confocal microscopy confirmed that the engulfed Cy5-labelled vesicles resided within the cell body of the macrophage (Supplementary Movie 6). However, MV uptake was blocked in macrophages pre-treated with dextran sulfate as evidenced by the accumulation of Cy5-labelled MVs on the macrophage surface (Supplementary Movie 7).

To track the in vivo transfer of mitochondria, we systemically administered RFP-labelled human MSCs into C57BL/6 mice expressing a GFP reporter under control of the endothelial specific Tie2 promoter. At 24 h post injection, GFP-labelled endothelial cells, epithelial cells and macrophages that contained RFP-labelled mitochondria were visible (Supplementary Fig. 3B). Bfa1 digestion of mouse lung DNA following intravenous administration of human MSCs, exosomes or MVs yielded a pattern of restriction similar to those observed in RAW 264.7 macrophages (Fig. 3d). To follow the fate of viable human MSCs in the mouse lung, we measured the abundance of human-specific GAPDH transcripts via reverse transcriptase–PCR (RT–PCR)⁴². Human GAPDH mRNA was not detected in the lung tissue of untreated mice but was detected at 3 days post injection of human MSCs or human fibroblasts (Fig. 3d). However, expression rapidly declined and was no longer evident by 14 or 28 days post transplant, consistent with the clearance rate of cells from lung tissue. Expression of human COXI mRNA in mouse lung mirrored that of human GAPDH following injection of human fibroblasts and was detected at 3 days but not 14 or 28 days post transplant. In contrast, human COXI transcripts were detected up to 28 days post injection of human MSCs, indicating that mouse lung tissue retained mtDNA long after the disappearance of viable human MSCs (Fig. 3d). Thus, MSC-derived vesicles constitute an effective mechanism to transfer mtDNA into the mouse lung.

MSC extracellular vesicles enhance macrophage energetics

To study the effect of MVs on macrophage bioenergetics, we analysed oxygen consumption rates (OCRs) using the SeaHorse technology. Human macrophages exhibit higher basal OCR than human MSCs or human fibroblasts (Fig. 4a). Co-culture of macrophages with human MSCs (Mac+hMSC) or MSC-derived exosomes (Mac+Exo) but not human fibroblasts (Mac+Fibro) significantly (analysis of variance (ANOVA) followed by Student–Neuman Keuls (SNK) post-hocpairwise comparisons) increased their OCR, suggesting that MSCs or MSC-derived exosomes alter macrophage bioenergetics (Fig. 4a). Next, we repeated these measurements after treatment of cells with oligomycin A, an inhibitor of ATP synthase, which is required for the oxidative phosphorylation of ADP to ATP. These conditions differentiate ATP-linked respiration from the proton leak. Macrophages exhibited a higher level of proton leak as compared with human MSCs and fibroblasts, and proton leak was significantly (ANOVA followed by SNK post hoc pairwise comparisons) reduced following co-culture with human Mac+Exo but not Mac+Fibro (Fig. 4a). Co-culture with human MSCs (Mac+hMSC) also significantly (ANOVA followed by SNK post hocpairwise comparisons) reduced proton leak in macrophages. We also repeated the OCR measurements following treatment of cells with the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy) phenylhydraone (FCCP) to determine how cells respond to an increase in ATP demand. All three cell types responded to FCCP treatment with increased OCR, and the magnitude of the response was greater in macrophages as compared with human MSCs and fibroblasts. Moreover, OCR was significantly increased in FCCP-treated macrophages following co-culture with human MSCs (Mac+hMSC) or human Mac+Exo but not Mac+Fibro (Fig. 4a).

To examine the effect of MSC or exosomes on macrophage bioenergetics under conditions of altered homeostasis, we exposed macrophages to silica particles. Silica exposure results in a burst of mtROS production as evidenced by changes in MitoSOX Red fluorescence intensity; however, this effect is largely mitigated in macrophages incubated with human MSC-derived exosomes (Fig. 4b,c). Silica exposure also decreased macrophage OCR, but this decrease was reversed by co-culture with human MSCs or human MSC-derived exosomes but not with human fibroblasts (Fig. 4d). The fact that transfer of partially depolarized mitochondria from MSCs to macrophages enhances that macrophage bioenergetics appears paradoxical. However, loss of mitochondrial membrane potential as a result of MSC expansion is not absolute as mitochondria exhibit residual membrane potential as evidenced by the concentration of JC-1 aggregates (Fig. 1c). This indicates that the mitochondrial membrane is not collapsed and the mitochondria are still capable of undergoing fusion. To determine whether these mitochondria are recycled in macrophages by fusion, we co-cultured human MSCs with macrophages after labelling cells with two different MitoTracker dyes (Red and Green)⁴³. Live cell imaging clearly demonstrated the transfer and subsequent fusion (yellow colour in merged images) of RFP-labelled, human MSC-derived mitochondria with GFP-labelled mitochondria within human macrophages (Fig. 5). These data indicate that under oxidative stress MSCs outsource mitophagy to macrophages to unload partially depolarized mitochondria, which are recycled via fusion by macrophages thereby enhancing their bioenergetics.

Exosomes transfer RNAs between cells⁸. We hypothesized that this process may be exploited by MSCs to tolerize macrophages against mitochondrial transfer. To explore this possibility, we analysed the RNA content of human MSC-derived exosomes. Using microRNA microarray analysis, we identified 156 (45 increased; 111 decreased) microRNAs that differed (log2>1.0,P<0.05 (ANOVA followed by Holm–Sidak post hoc pairwise comparisons) in abundance between exosomes compared with their parent MSCs. The 10 microRNAs that exhibited the greatest increase included miR451a (316-fold), miR1202 (45-fold), miR630 (40-fold) and miR638 (28-fold), while microRNAs that exhibited the greatest decrease in exosomes and were enriched in MSCs included miR125b (148-fold) and miR21 (91-fold; Fig. 6a,b). This pattern of microRNA expression was conserved in MSC-derived exosomes obtained from five human donors (Fig. 6c,d).

Mitochondrial uptake can induce inflammation via activation of pattern recognition receptors³⁴. Therefore, given the presence of mtDNA and microRNAs in MSC-derived MVs and exosomes, respectively, we hypothesized that exposure to these vesicles would tolerize macrophages to mitochondrial transfer by inducing changes in TLR expression. Subsequently, we profiled the expression of 84 TLR-associated transcripts in mouse macrophages. We contrasted these results with those observed in macrophages that were co-cultured with mouse or human MSCs, human MSC-derived exosomes or silica particles, which when phagocytized induce macrophage activation⁴⁴. Co-culture of macrophages with MSC-derived exosomes induced nuclear translocation of the transcription factor NF-κB (Fig. 7a) resulting in significant changes (>2.5-fold increase or decrease) in expression of 50 of the 84 TLR-associated transcripts (Fig. 7b). For example, compared with silica-exposed macrophages those treated with exosomes exhibited significant (>2.5-fold) increases in transcripts associated with cytokine signalling including interleukin (IL)-1β, prostaglandin endoperoxide synthase 2 (PTGS2, aka COX2), granulocyte colony-stimulating factor 3 (CSF3), IL-10, chemokine (C–C motif) ligand 2 (CCL2, aka MCP-1), NF-κB-chemokine (C–X–C motif) ligand 10 (CXCL10), tumour necrosis factor (TNF) and reticuloendotheliosis oncogene (Rel; Fig. 7b). In contrast, transcripts encoding proteins involved in MyD88-dependent signalling (MyD88, TLR 1,4,5,7,8 and 9, IRAK1 and TRAF6), TRIF-dependent signalling (TLR adaptor molecule 1 (TICAM1) and TICAM2) and TLR-related signalling (CD80, CD86, IL-2, IL-12, Interferon gamma, PGLYRP1 and CSF2) were downregulated.

MSCs secrete PGE2 that acts on prostaglandin receptors of LPS-stimulated macrophages to enhance their production of the anti-inflammatory cytokine IL-10 (ref. 45). However, this effect of MSCs was abrogated in macrophages from TLR4, MyD88, TNFR1 or COX2-deficient mice⁴⁵. Consistent with these results, exosome treatment of non-stimulated macrophages augmented secretion of PGE2, TNF, IL-10 and IL-1-receptor antagonist (Fig. 7c), which may reprogramme macrophages³. These responses recapitulate those observed when macrophages are exposed to intact human or mouse MSCs, except that IL-6, CSF2 and IL-1 receptor 1 were increased more following exposure to mouse MSCs (Fig. 7b).

Subsequently, we treated TLR-signalling-deficient macrophages (TLR4^−/−, TLR9^−/−, MyD88^−/−) or scavenger receptor-deficient macrophages (MARCO^−/−) with MSC-derived exosomes. As shown inFig. 7c, PGE2 production was similar following exosome treatment in all signalling-deficient macrophages as compared with wild-type cells from strain-matched C57BL/6J or BALB/CJ mice. In contrast, secretion of TNF and IL-10 was significantly (ANOVA followed by SNK post hoc pairwise comparisons) reduced in TLR4^−/− and MYD88^−/− macrophages as compared with wild-type cells following exosome treatment, and IL-10 secretion was also significantly reduced in macrophages from TLR9^−/− mice (Fig. 7c). These data confirm the importance of TLRs and in particular MyD88-dependent pathways in mediating exosome-induced effects on macrophage function. Lastly, we showed that pre-incubation with dextran sulfate significantly (ANOVA followed by SNK post hocpairwise comparisons) reduced the release of PGE2, TNF and IL-10 by exosome-treated macrophages, confirming the need for phagocytosis of MSC-derived vesicles in this process (Supplementary Fig. 4A).

To examine the role of microRNAs in macrophage tolerization, we treated RAW 264.7 cells, which use TLRs to recruit autophagy proteins in phagosomes to degrade its cargo⁴⁶, with exosomes derived from human MSCs transfected with or without an short-hairpin RNA (shRNA) designed to inhibit DICER expression in the presence or absence of indomethacin, a cyclooxygenase inhibitor (Supplementary Fig. 4B). Treatment of naive RAW 264.7 macrophages with native exosomes enhanced TNF and reduced TLRs and MyD88 mRNA expression over 24 h (Fig. 7c), while treatment of silica-exposed macrophages with exosomes ameliorated TLR7 induction following silica exposure (Fig. 7c). Pre-incubation of RAW 264.7 macrophages with indomethacin before treatment with native exosomes, or treatment with exosomes from DICER knockout MSCs significantly (ANOVA followed by SNK post hoc pairwise comparisons) reduced the observed effects on TLR mRNA expression (Fig. 7c) and reduced secretion of proteins such as TNF, MIP, MCP1, KC and IP-10 associated with macrophage activation (Supplementary Fig. 4B,C). The inhibitory effects of indomethacin were restricted to TLR4 and MyD88 mRNA, while the effects of DICER-deficient exosomes were of greater magnitude and also involved negative regulation of TLR 7 and 9 (Fig. 7c). Concomitant treatment with indomethacin and exosomes from DICER-deficient exosomes demonstrated additive effects (Fig. 7c).

Importantly, miR-451 is one of the most abundantly expressed microRNAs in MSC-derived exosomes, but its maturation occurs independent of DICER⁴⁷. Therefore, its expression is not altered in exosomes from DICER knockdown MSCs. MiR-451 negatively regulates cytokine production in dendritic cells infected with influenza virus⁴⁸. Consistent with these results, transfection of RAW 264.7 macrophages with a miR-451 mimic significantly (Student’s t-test) decreased TNF mRNA expression in non-stimulated macrophages, and inhibited mRNA expression and protein release in silica-exposed macrophages (Supplementary Fig. 4D). In contrast, treatment of cells with a miR-451 antagomir yielded the opposite result. These data confirm a role of exosome-derived microRNAs in regulating cytokine expression in macrophages.

MSC exosomes attenuate monocyte activation and silicosis

Circulating MVs enter the bone marrow and reprogramme cells to express proteins of the tissue of vesicle origin⁴⁹. Ly6C^hi monocytes are recruited from the bone marrow into the lung in response to injury and play an important role in the pathogenesis of lung fibrosis^{50, 51}. Therefore, we investigated whether MSC or their exosomes are capable of altering the lung recruitment and cytokine production of Ly6C^hi monocytes in mice following silica exposure. As shown in Fig. 8a, FACS identified a limited number of Ly6C^hi monocytes in the normal mouse lung, which was significantly (ANOVA followed by SNK post hoc pairwise comparisons) increased by 72 h post exposure to silica. Moreover, high expression of CCR2 and release of inflammatory (TNF, CCL2 and CXCL1) and fibrotic (transforming growth factor β (TGFβ) and IL-10) mediators indicate that these monocytes are activated (Fig. 8b). In contrast, intravenous administration of human MSCs (500,000 cells) or freshly isolated human MSC-derived exosomes (~3 × 10¹¹ exosomes containing 40 μg protein) at 24 h post-silica exposure (0.2 g·kg⁻¹) significantly reduced the extent of Ly6C^himonocyte infiltration into the lung and secreted levels of inflammatory mediators (Fig. 8a,b).

Silica induces inflammation and collagen deposition in peri-bronchiolar, silicotic nodule and peri-vascular regions of the lung (Fig. 9a). These lesions are associated with enhanced numbers of inflammatory cells (although the percentage of macrophages decreases, there is an increase in neutrophils and lymphocytes) in bronchoalveolar lavage fluid (BALF, Fig. 9b), significant deposition of lung collagen as measured by hydroxyproline (Fig. 9c) and enhanced expression of pro-inflammatory cytokines (TNF and IL-6) and fibrotic mediators (IL-10 and α(I) collagen) by 14 and 28 days after silica exposure (Fig. 9d). Intravenous administration of human MSCs or exosomes 3 days after silica exposure reduced the size of the silicotic nodules (Fig. 9a), the total number of white blood cells in BALF (Fig. 9b) and expression of inflammatory and pro-fibrotic genes in the lung (Fig. 9d). Administration of exosomes significantly (ANOVA followed by SNK post hocpairwise comparisons) reduced the accumulation of neutrophils and lymphocytes in BALF, while MSCs only reduced the accumulation of neutrophils and induced a slight (<1%) increase in eosinophil count (Fig. 9b). In contrast, the intravenous administration of human fibroblasts significantly exacerbated the inflammatory and fibrotic responses to silica (Fig. 9a–c). Exosomes, but not MSC or fibroblast administration, reduced the accumulation of hydroxyproline in lung tissue 28 days after silica (Fig. 9c).

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MSCs modulate macrophage function by a variety of mechanisms, and this crosstalk contributes to their anti-inflammatory activity but the physiological relevance of this crosstalk remains obscure particularly as it relates to the survival and function of MSCs. In this study, we report that during their ex vivo culture MSCs transfer partially depolarized mitochondria to macrophages as a pro-survival mechanism in response to oxidative stress and that these mitochondria are repurposed via a process involving fusion to increase macrophage bioenergetics. Moreover, we show that MSCs also desensitize macrophages to mitochondrial transfers by repressing TLR-signalling. Our data indicate that MSCs employ two different types of MVs to achieve these goals. MSCs load mitochondria in the cytoplasm into LC3 containing MVs that are recovered from cell culture media with low-speed centrifugation. These MVs express the ESCRT-I-associated proteins TSG101 and ARRDC1 and are extruded from cells in ARMMs¹¹, which bud outwards directly from the plasma membrane where they are identified by macrophages. MSCs also shed exosomes that modulate TLR signalling and cytokine secretion in macrophages, in part, by transfer of regulatory microRNAs.

Previous reports indicate that mitochondria transferred by MSCs improve the energetic activity of the alveolar epithelium of LPS-treated mice^{30, 31, 32, 33} and animal models of rotenone-induced airway injury⁵². However, the beneficial effects of mitochondria were limited to acceptor cells almost completely deficient of mitochondrial function^{30, 53}. Therefore, it is unclear whether rescue is because of the transfer of mitochondria, mtDNA or release of other mediators by MSCs³³. Importantly, the bone marrow niche contains few, if any, epithelium, so the physiological relevance of this is unclear.

Our data suggest that mitochondrial transfer by MSCs is not altruistic but rather may serve to enhance MSCs’ cell survival by unloading partially depolarized mitochondria. Elimination of depolarized mitochondria is a priority for MSCs that experience high mtROS generation when cultured under atmospheric oxygen tension²⁸ since inhibitors of the mitophagy flux induce MSC apoptosis. Unexpectedly, MV-mediated mitochondrial transfer augments macrophage function by improving mitochondrial bioenergetics. As reported for the alveolar epithelial cells, recovery of the energetic function of macrophages is characterized by an increased ability to generate ATP under conditions in which the cells exhibit mitochondrial uncoupling or an enhanced proton leak, and involves protection of the macrophage by reducing mtROS generation. This outcome is consistent with data indicating that transfer of mitochondria, even if partially depolarized, is followed by fusion inside the acceptor macrophage. Notably, several studies have reported that transfer of only a few mitochondria is sufficient to rescue cells depleted of mtDNA by culture in ethidium bromide^{30, 54,55}. Furthermore, the current study confirms evidence that exosomes, which do not carry mitochondria, contain nucleic acids⁵⁶, including mtDNA that can be transferred, long term, in vivoto the lung. Presence of mtDNA inside exosomes is not surprising as mtDNAs are dispersed throughout the mitochondrial network as histone-free nucleoids with an average size in mammals under 100 nm, and contain a single copy of mtDNA per nucleoid⁵⁷. However, we cannot completely exclude the possibility that the exosome preparations could be contaminated by apoptotic bodies.

Accumulation of mtDNA that escapes mitophagy induces TLR 9-mediated inflammation that in the case of cardiac muscle is associated with heart failure³⁵ and mice transplanted with cells harbouring allogeneic mtDNA trigger MyD88 responses to reject these cells³⁶. Therefore, silencing TLR responses in macrophages is likely necessary to induce tolerance to transferred mitochondria. Consistent with this hypothesis, we demonstrate that uptake of MSC-derived exosomes represses TLR signalling in macrophages and the production of inflammatory mediators by targeting pathways (TLRs and NF-κB) central to inflammation.

Interestingly, microRNAs present in MSC-derived exosomes are highly conserved between human MSC donors. One such microRNA, miR-451, is highly abundant in exosomes but is expressed at low levels in macrophages and dendritic cells where it regulates cytokine production^{58, 59, 60}. Mir-451 is known to suppress TNF, and macrophage migration inhibitory factor, which inhibits the anti-inflammatory effects of glucocorticoids and negatively regulates p38 MAPK signalling to protect from diabetic nephropathy^{48, 58, 59, 60}. Indeed, ectopic expression of a mir-451 mimic in macrophages inhibits TNF secretion in response to silica. Consistent with these findings, MSC-derived exosomes prevent the recruitment of Ly6C^hi monocytes and reduces secretion of pro-fibrotic IL-10 and TGFβ by these cells in the lung of silica-exposed mice. Therefore, these data suggest that, as tested in vitro, immunomodulatory activities may have evolved, in part, as a mechanism by which MSCs survive oxidative stress and serendipitously confers on cells the ability to suppress inflammation, in lung injury models. Indeed, our data illustrate a physiological role for the innate immune regulatory activity of MSCs, and in doing so further highlights the important association between MSCs and macrophages in vivo.