Exosomes derived from mesenchymal stem cells inhibit catabolism in human chondrocytes by activating autophagy via inhibition of the NF-κB pathway

Objective: We aimed to determine the signicance of MSC-derived exosomes (MSC-Exos) in chondrocyte autophagy under normal and inammatory conditions. Design: Human umbilical cord-derived MSCs (hMSCs) were cultured in vitro. hMSC-Exos( EX (cid:0) were isolated by an ultracentrifugation method. Transmission electron microscopy and western analysis were used to identify exosomes. Human chondrocytes were extracted from ve adult males with OA undergoing total knee arthroplasty. Primary cultures of chondrocytes from OA patients were stimulated with 50 ng/ml tumor necrosis factor-α (TNF-α) in the presence or absence of hMSC-Exos. Autophagy levels were determined based on expression of autophagic marker LC3, StubRFP-SensGFP-LC3 analysis, and electron microscopy. Catabolic gene and chemokine expression were evaluated using quantitative PCR. The NF-κB inhibitor NS398 was used to analyze the role of the NF-κB pathway in autophagic activation. Results: hMSC-Exos increased LC3-II levels as well as autophagosome number in chondrocytes. hMSC-Exos inhibited TNF-α–induced expression of MMP-3, -9, and -13; ADAMTS5; CCL-2 and -5; and CXCL1. NF-κB inhibition activated autophagy in TNF-α–treated chondrocytes. These results indicate that hMSC-Exos might suppress the levels of catabolic and inammatory factors in chondrocytes by promoting autophagy via NF-κB pathway inhibition. Conclusions: Our data support the interest in hMSC-Exos to develop new therapeutic approaches for joint conditions.


Introduction
Osteoarthritis (OA) is the most prevalent musculoskeletal disorder and a major cause of joint pain and disability. Damage to joints as a result of trauma, obesity, aging, and genetic background are the major risk factors for OA 1 . Chondrocytes are the only resident cells in cartilage and are responsible for both synthesis and turnover of the abundant extracellular matrix (ECM). Therefore, maintenance of healthy chondrocytes appears to be an important factor in maintaining cartilage and preventing degeneration of cartilage 2 . Mesenchymal stem cells (MSCs)-multipotent precursors of connective tissue cells that can be isolated from many adult tissues, including those of the diarthrodial joint-have emerged as a potential therapy 3 . MSC therapies have demonstrated e cacy in cartilage repair in animal and clinical studies. The e cacy of MSC-based therapies, which were previously predicated on the chondrogenic potential of MSCs, is increasingly attributed to paracrine secretion, particularly of exosomes 4 . The crucial role of MSC-derived exosomes for the regulation of cell migration, proliferation, differentiation, and ECM synthesis has been increasingly supported by recent ndings 5 . A report in 2016 showed that adiposederived stem cells (ADSCs) are able to activate autophagy and inhibit catabolism in chondrocytes during in ammation, and the mTOR pathway might be involved in activation of autophagy 6 .
Autophagy, a mechanism of organelle recycling that promotes cell survival, has been previously implicated in osteoarthritis (OA) 7 . Currently, researchers suggest that autophagy may increase as an adaptive response to protect chondrocytes from various environmental changes, whereas failure of the adaptation may lead to progression of cartilage degradation 8 .Djavaheri-Mergny M demonstrated that the decline in autophagy during aging creates problems in cellular housekeeping functions that stimulate NF-κB signaling in order to, directly or via in ammasomes, trigger an age-related proin ammatory phenotype. Moreover, there are indications that in ammatory signaling can repress autophagy and thus induce this destructive interplay between autophagy and in ammasomes. For instance, tumor necrosis factor-α (TNF-α), an in ammatory cytokine, can induce or repress autophagy in an NF-κB-dependent manner 9 . Yi et al suggested that inhibiting the NF-κB pathway can promote autophagy and decrease apoptosis and the in ammatory response in lipopolysaccharide (LPS)-induced nucleus pulposus cells. Meanwhile, autophagy triggered by NF-κB inhibition plays a protective role against apoptosis and in ammation 10 .

Materials And Methods
Human umbilical-cord-derived MSCs (hMSCs) hMSCs were isolated from umbilical cords of ve healthy donors. The experimental design was approved by the Institutional Ethical Committee (Zhengzhou Central Hospital, Henan, China). Samples were obtained from donors after they provided informed consent according to the Helsinki Declaration of 1975, as revised in 2013.
Samples were washed with phosphate-buffered saline (PBS) and minced and digested at 37 °C for 1 h with 2% type I collagenase (Gibco, Life Technologies, Madrid, Spain). Digested tissue was ltered through a 100-µm cell strainer (BD Biosciences, Durham, NC, USA). Cells were then washed with Dulbecco's Modi ed Eagle Medium (DMEM)/Ham's F12 (Sigma-Aldrich, St. Louis, MO, USA) containing penicillin and streptomycin (1%), seeded into tissue culture asks (1-2 × 10 6 cells/mL) in DMEM/Ham's F12 medium with penicillin and streptomycin (1%), supplemented with 15% extracellular vesicle-free human serum, and incubated at 37 °C in a humidi ed atmosphere of 5% CO 2 . Human serum was obtained from wholeblood donations of AB blood-group-typed donors according to the criteria of Valencia Transfusion Centre. To eliminate the extracellular vesicle fraction, serum was centrifuged for 18 h at 120,000 × g and 4 °C using a SW-28 swinging-bucket rotor (Beckman Coulter, Brea, CA, USA). When cells reached semicon uence, culture plates were washed and the MSC phenotype con rmed by ow cytometry (Cyto ex, Beckman Coulter) using the speci c antibodies anti-CD73-PE, anti-CD90-APC, anti-CD105-APC-A750, anti-CD34,anti-CD45, anti-CD11b, anti-CD19, and anti-HLA-DR-FITC (Biolegend, San Diego, CA USA), and measuring cell viability with propidium iodide staining. Finally, conditioned medium (CM) was collected from cultured cells at passage 0 every 48 h of culture. CM was pooled, centrifuged, and stored under sterile conditions at -80 °C prior to further use. Isolation Of Exosomes Exosomes were obtained from hMSC CM using a ltration/centrifugation-based protocol 34 . Cellular debris was eliminated by centrifugation at 300 × g for 10 min. Vesicles were then collected from the supernatant through differential centrifugation steps. CM was ltered through an 800-nm lter (Merck, Darmstadt, Germany) and centrifuged at 12,200 × g for 20 min at 4 °C to pellet microvesicles. Then, supernatants were ltered through a 200-nm lter (Merck) and centrifuged at 100,000 × g for 90 min at 4 °C. Pellets were washed once with sterile PBS, resuspended in 15 µL PBS, and stored at -80 °C until further use.

Human Chondrocyte Cultures
The knee specimens were obtained from three females and two males, 69.4 ± 7.2 years of age (mean ± standard error of the mean [SEM]), with diagnosis of advanced OA undergoing total knee arthroplasty. Diagnosis was based on clinical, laboratory, and radiological evaluation. The study design was approved by the Institutional Ethical Committee (Zhengzhou Central Hospital, Henan, China). Samples were obtained with patient's consent according to the Declaration of Helsinki. Knee articular cartilages samples were obtained and cut into about 1mm 3 pieces. Tissues were digested with 0.25% trypsin-EDTA for 30 min and collagenase II for 4 h and then ltered. After rinsing, the chondrocytes were cultured in DMEM with high-dose (4.5 g/L) glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin at 37 °C with 5% CO 2 . Second-passage chondrocytes were used in the experiments to eliminate the in uence of dedifferentiation on experimental results.

Toluidine Blue Staining For Morphological Identi cation Of Chondrocytes
The chondrocytes were inoculated into a six-well plate and, when the cells reached 50-60% con uence, the culture medium was discarded. The chondrocytes were then xed in 4% paraformaldehyde for 30 min, stained with 1% toluidine blue at room temperature for 10-30 min, washed with absolute ethyl alcohol until the cells were colorless, and observed under an inverted microscope (Olympus, Tokyo, Japan). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI).

Experimental Design
The H + -ATPase inhibitor ba lomycin A1 (0.1 µM) was used to measure the role of hMSC-Exos or hMSC-Exos combined with 50 ng/mL TNF-α in autophagic ux. Then, the NF-κB pathway involved in autophagic activation was investigated. When cells were treated with 50 ng/mL TNF-α and hMSC-Exos or speci c COX-2 inhibitor NS398 (10 µM), hMSC-Exos and NS398 were always added to the medium 1 h prior to TNF-α addition. In all experiments, chondrocytes were treated with TNF-α for 24 h. During the autophagic ux assay, 0.1 µM ba lomycin A1 was added to the medium 1 h prior to addition of hMSC-Exos or hMSC-Exos combined with 50 ng/mL TNF-α, followed by co-incubation for 24 hours.

Transmission Electron Microscopy (TEM)
Chondrocytes cultured in 10-cm dishes were treated with TNF-α and/or hMSC-Exo for 24 h and then harvested by manual scraping. After centrifugation, chondrocytes were xed with 2.5% glutaraldehyde overnight and post-xed with 1% osmium tetroxide for 2 h at 4 °C. After staining with 2% uranyl acetate, chondrocyte pellets were dehydrated through an acetone series and then embedded in Epon 812. After semi-thin sectioning, chondrocytes were stained with toluidine blue and observed under a light microscope. Finally, ultrathin sections were prepared based on light microscopic observations. Cellular ultra-structures were visualized using a transmission electron microscope (Hitachi, Japan)

Statistical analysis
Statistical assay was performed using GraphPad Prism 7.0 (GraphPad, San Diego, CA USA). All data in this study are shown as the mean ± standard error of the mean (SEM) of three independent experiments. The signi cance of the differences in mean values between and within multiple groups was examined by one-way ANOVA followed by Duncan's multiple range test. P value < 0.05 was considered statistically signi cant.

Results
Characterization of exosomes derived from hMSCs Exosome fractions were isolated as indicated in Materials and Methods. NTA2.3(Nanosight LM10,UK) analysis indicated a mean diameter of 30-150 nm and a concentration of 0.97 × 10 10 particles/mL. Figure 1a shows a representative NTA analysis of exosome fractions. Exosome morphology was studied using TEM (Fig. 1b). Expression of proteins in exosomes, including CD9, CD81, and transforming growth factor β1 (TGF-β1) was assessed by western blotting (Fig. 1c).

Successful Isolation And Identi cation Of Chondrocytes
Chondrocytes were successfully isolated from OA cartilage tissue and stained with toluidine blue. As shown in Fig. 2, toluidine blue staining illustrated that the chondrocytes were long-fusiform or irregular in shape. Overall, the above-mentioned ndings demonstrate the successful isolation of chondrocytes from normal and OA cartilage tissue.

MSC-derived exosomes activate autophagy in TNF-α-treated chondrocytes
LC3 is a classic marker of autophagy. Western blotting of LC3 and stubRFP-sensGFP-LC3, therefore, were used to demonstrate the stimulatory effects of exosomes on autophagy. Exosomes signi cantly enhanced LC3-II expression in chondrocytes treated with TNF-α for 24 h (Fig. 3a, b). Interestingly, we found that LC3-II expression was increased in chondrocytes treated with exosomes alone and was lower than that in cells co-treated with both exosomes and TNF-α, indicating that in ammation enhances the stimulatory effect of exosomes on autophagy. stubRFP-sensGFP-LC3 is also widely used to monitor accumulation of the cytoplasmic LC3-positive puncta and autophagosomes. We established chondrocytes stably expressing stubRFP-sensGFP-LC3 using a lentiviral vector and assessed the autophagic ux. We used stubRFP-sensGFP-LC3-mut lentiviral vector as negative control. sensGFP is sensitive to pH change owing to the fusion of autophagosomes and lysosomes, whereas stubRFP is stable. Consistent with the results described above, we observed enhanced autophagosome-lysosome fusion in chondrocytes using a laser scanning confocal microscope. The number of yellow dots(LC3positive puncta) in exosome-treated chondrocytes under in ammatory conditions induced by TNF-α was higher than that in chondrocytes treated with TNF-α alone (Fig. 3c), suggesting accumulation of autophagosomes in the chondrocytes. TEM is the gold standard by which activation of autophagy is con rmed. Autophagosomes and autolysosomes with double membranes appeared in the chondrocytes treated with TNF-α and hMSC-Exos for 24 h (Fig. 3d). hMSC-Exos signi cantly enhanced the number of autophagosomes in TNF-α-treated chondrocytes compared with hMSC-Exos-treated cells. We further investigated autophagic ux by treatment with ba lomycin A1 to inhibit the fusion of lysosomes with autophagosomes during the late phase of autophagy, leading to LC3-II accumulation in the cytoplasm. Ba lomycin A1 signi cantly elevated the LC3-II/ β-actin level in hMSC-Exos-treated chondrocytes, suggesting that hMSC-Exos increased autophagic ux (Fig. 3e, f). Furthermore, the stimulatory effect of hMSC-Exos combined with TNF-α on autophagic ux was also demonstrated by the increase in LC3-II/β-actin ratio following the addition of ba lomycin A1 (Fig. 3g, h).

Involvement of NF-κB pathway in hMSC-Exos-induced autophagic activation
Inhibition of the NF-κB pathway involved in autophagic activation was investigated using NS398, an NF-κB inhibitor. NF-κB expression was enhanced after NS398 treatment in chondrocytes treated with TNF-α for 24 hours, suggesting inhibition of the NF-κB pathway. Importantly, LC3-II levels were increased by NS398 in TNF-α-induced chondrocytes (Fig. 5a, b). stubRFP-sensGFP-LC3 analysis showed that the number of yellow puncta in the cytoplasm was also increased after treatment with NS398 (Fig. 5c), indicating activation of autophagy.

Discussion
The crucial role of MSC-derived exosomes for the regulation of cell migration, proliferation, differentiation, and ECM synthesis has been increasingly supported by recent ndings [11][12][13] . A report in 2010 showed that exosomes are secreted as active factors by MSCs responding to damage caused by myocardial ischemia reperfusion (I/R) 14 . Moreover, reports have demonstrated that MSC exosomes contribute to the repair and regeneration of cartilage via regulating immune reactivity, diminishing apoptosis, and increasing proliferation [15][16][17] . Exosomes, which function as intercellular communication vehicles, are small lipid-bilayer membrane-bound vesicles between 50 and 150 nm in diameter.
Exosomes are able to transfer cargos of nucleic acids (mRNAs and microRNAs), proteins, and bioactive lipids 18 . Exosomes can produce biological responses in recipient cells 28 . Some ndings have shown ambiguous effects of exosomes on the immune response or possible tumorigenicity, which may be considered unfavorable properties of exosomes 19,20 . Nevertheless, there have been few studies conducted to investigate the precise molecular mechanisms by which MSC exosomes can promote chondrogenesis 16,[20][21][22][23] . In the present study, we demonstrated that MSC-derived exosomes activate autophagy and enhance autophagic ux in TNF-α-treated chondrocytes, as demonstrated by western and mRFP-GFP-LC3 analysis. The NF-κB pathway was inhibited in chondrocytes treated with exosomes, suggesting that the NF-κB pathway might be involved in autophagic activation. Finally, the anti-catabolic effect of exosomes on chondrocytes was shown by real-time PCR.
Autophagy is a self-degradative process that is important for balancing sources of energy at critical times during development and in response to cell stress. Recent data support the idea that autophagy can occur in combination with apoptosis in OA 24 . Indeed, Almonte-Becerril and collaborators demonstrated that, in early stages of OA, chondrocytes from the super cial zone showed increased expression of both apoptotic cell death and autophagic markers, even though the authors suggested that autophagy is activated as an adaptive response to sublethal conditions, with the aim of avoiding cell death 25 .
Researchers have demonstrated that autophagy is a constitutively active and apparently protective process for the maintenance of homeostasis in normal cartilage. In contrast, human OA and agingrelated and surgically-induced OA in mice are associated with reduction and loss of ULK1, Beclin1, and LC3 expression in articular cartilage, suggesting that autophagy was decreased in the surgical OA mouse model. Increased expression of proin ammatory cytokines in cartilage, synovial membrane, and subchondral bone are believed to be linked to the development and progression of structural changes in the OA joint 26 . In this study, the mechanisms involved in the effects of MSC-derived exosomes on catabolism and in ammation in chondrocytes were investigated. Autophagy was found to mediate the effects of exosomes in chondrocytes. The stimulatory role of exosomes in autophagy was demonstrated by western analysis of LC3-II and mRFP-GFP-LC3 assay, TEM, and autophagic ux assay using ba lomycin A1.
OA is a joint disorder identi ed by ECM degradation initiated by abnormal joint tissue metabolism followed by anatomic, and/or physiologic derangements. It is the end result of a combination of genetic, metabolic, biochemical, in ammatory, and mechanical predispositions and insults, but the loss of articular cartilage is certainly a consequence of this 27 . The loss of articular cartilage can, in turn, aggravate in ammation, joint misalignment, and bony remodeling (subchondral bone destruction and osteophyte development), loss of muscular and ligamentous joint support, and ultimately the de ning clinical symptoms of joint pain, instability and stiffness 28 . Chondrocytes are sensitive to physical injury, and extreme mechanical forces alter the chondrocyte balance of anabolic and catabolic factors, compounding injury and inducing in ammation 29 . Catabolic enzymes, such as matrix metalloproteinase-13 (MMP-13), the dominant factor in collagen type II degradation, and disintegrin and metalloproteinases with the thrombospondin motifs (ADAMTS4 and 5) that degrade the predominant proteoglycan, aggrecan, are upregulated in chondrocytes and synovial cells during OA. In our study, catabolic genes, including MMP-3, -9, and-13 and ADAMTS5 and the proin ammatory cytokines, including CCL-2 and − 5 and CXCL1, were also inhibited by hMSC-Exos treatment, suggesting the potential role of hMSC-Exos in maintaining cellular homeostasis and treating OA. Therefore, the effect of hMSC-Exos on catabolism requires further investigation.
The NF-κB transcription factor family is ubiquitously expressed in all cell types and regulates essential cellular responses including survival, differentiation, apoptosis, and autophagy 30 . It is clear that chronic and low-grade in ammation is involved in the progression of OA that leads to catabolic responses in chondrocytes via upregulation of factors such as nuclear NF-κB 31 . Li-Bo J 32 suggested that ADSCs activate autophagy and enhance autophagic ux in chondrocytes treated with IL-1ß or LPS, and the mTOR pathway might be involved in the activation. Activation of NF-κB and autophagy are two processes involved in the regulation of cell death, but whether there is crosstalk between these two signaling pathways is largely unknown. Rapamycin can inhibit the overexpression of in ammatory catabolic genes by activating autophagy, and can suppress the NF-κB signaling pathway in chondrocytes to break the positive feedback loop with in ammatory factors and reduce the rate and level of in ammation progression 33 . Although the inhibitory role of MSCs on NF-κB has been extensively studied, the association between NF-κB and autophagy in chondrocytes is unclear. It has been reported that LPS can induce in ammation by promoting secretion of various in ammatory factors, including IL-1ß and activating the COX-2/PGE2 pathway. In our study, the NF-κB inhibitor NS398(COX-2 inhibitor) was shown to increase LC3-II levels and autophagosome number in TNF-α-treated chondrocytes. In porcine primary granulosa cells, Gao et al. found that NF-κB activated autophagy via activation of the JNK pathway. The JNK pathway might also be associated with upregulation of autophagy in cancer cells. However, the detailed mechanisms underlying NF-κB inhibitory effects on autophagic activation require further investigation.

Conclusions
In conclusion, the present study demonstrates that MSC-derived exosomes induce autophagic activation and inhibit the expression of TNF-α-induced catabolic genes and chemokines. hMSC-Exos suppresses the activation of the NF-κB pathway stimulated by TNF-α. Finally, the NF-κB pathway inhibitor NS398 activates autophagy in TNF-α-treated chondrocytes. Our ndings further our understanding of hMSC-Exos-induced autophagic activation via the NF-κB pathway. Declarations (1) The conception and design of the study, or acquisition of data, or analysis and interpretation of data.
(2) Drafting the article or revising it critically for important intellectual content.
(3) Final approval of the version to be submitted.

Founding
The research was supported by the National Natural Science Foundation of China (81970312), the research was also supported by henan province medical science and technology project LHGJ20191045 Availability of data and materials All data generated or analyzed during this study are included in this article.