Mesenchymal stem cell–secreted extracellular vesicles carrying TGF‐β1 up‐regulate miR‐132 and promote mouse M2 macrophage polarization

Abstract The effects of mesenchymal stem cells (MSCs) on different types of diseases are controversial, and the inner mechanisms remain unknown, which retards the utilization of MSCs in disease therapy. In this study, we aimed to elucidate the mechanisms of MSCs‐extracellular vesicles (EVs) carrying transforming growth factor‐beta 1 (TGF‐β1) in M2 polarization in mouse macrophages via the microRNA‐132 (miR‐132)/E3 ubiquitin ligase myc binding protein 2 (Mycbp2)/tuberous sclerosis complex 2 (TSC2) axis. Mouse MSCs were isolated for adipogenic and osteogenic induction, followed by co‐culture with mouse macrophages RAW264.7. Besides, mouse macrophages RAW264.7 were co‐cultured with MSCs‐EVs in vitro, where the proportion of macrophages and inflammation were detected by flow cytometry and ELISA. The experimental data revealed that MSCs‐EVs promoted M2 polarization of macrophages, and elevated interleukin (IL)‐10 expression and inhibited levels of IL‐1β, tumour necrosis factor (TNF)‐α and IL‐6. MSC‐EV‐treated macrophages RAW264.7 increased TGF‐β1 expression, thus elevating miR‐132 expression. MiR‐132 directly bound to Mycbp2, as confirmed by luciferase activity assay. Meanwhile, E3 ubiquitin ligase Mycbp2 could ubiquitinate TSC2 protein. Furthermore, silencing TGF‐β1 inhibited M2 polarization of MSC‐EV‐treated macrophages. Taken conjointly, this study provides evidence reporting that MSC‐secreted EVs carry TGF‐β1 to promote M2 polarization of macrophages via modulation of the miR‐132/Mycbp2/TSC2 axis.


| INTRODUC TI ON
Macrophages are known as tissue-resident or recruited cells, and they have essential functions in pathogen recognition, initiation of host defence through the protective inflammation. 1 Two differentiation patterns, namely M1 and M2, have been characterized. M1 macrophages function as modulators of the host defence system, which are able to protect from infection because of protozoa, bacteria and viruses. 2 M1 macrophages have a pro-inflammatory effect and can produce inflammatory factors such as interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6. 3 M2 macrophages are responsible for tissue repair and reconstruction by secreting anti-inflammatory cytokines that modulate cell replacement, angiogenesis and matrix remodelling. 4 Macrophage polarization is emerged as a vital pathogenetic factor in neoplastic and inflammatory diseases. 5 Polarization from M1 macrophages to M2 macrophages can reduce inflammatory responses and promote tissue repair and regeneration. 1 Therefore, controlling excessive inflammatory response and maintaining a balance between pro-inflammatory and anti-inflammatory responses play a key role in promoting M2 polarization of macrophages.
Mesenchymal stem cells (MSCs) have become a hot issue as potent therapeutic tools for cell-based therapy due to their characteristics of self-renewal and differentiation into various tissues. 6 According to previous literature, 7,8 MSCs can affect the polarization of macrophages in different diseases. MSCs-extracellular vesicles (EVs) are secreted in large numbers, including both large micro-vesicles and smaller exosomes with microRNAs (miRNAs), proteins as well as DNA that could modulate the gene expression along with functionality of recipient cells. 9 Evidence has shown that MSCs-EVs stimulated by lipopolysaccharide (LPS) can differentiate macrophages into a protective phenotype, thereby impacting cytokine secretion and enhancing phagocytic activity as well as inducing haematopoiesis and tissue repair. 10 Besides being secreted by tumour educated-stromal cells and carcinoma cells, transforming growth factor-beta 1 (TGF-β1) can also be produced by MSCs in the tumour environment. 11 Meanwhile, a study has demonstrated that 12 TGF-β1 can promote M2 polarization of macrophages. Li et al 13 have found that miR-132 expression could be up-regulated by TGF-β1, and miR-132 induces M2 polarization in macrophages through regulating multiple transcription factors and adaptor proteins. 13 E3 ubiquitin ligase myc binding protein 2 (Mycbp2) is a highly conserved protein that directly interacts with the transcriptional activation domain of myc. 14 Mycbp2 has been proposed to possess the ability to ubiquitinate and degrade tuberous sclerosis complex 2 (TSC2) protein, 15 and TSC2 protein can promote M2 polarization of macrophages. 16 Since great achievements have been achieved in regulating the polarized activation of macrophages, the mechanisms remain to be further understood.
Therefore, we conducted this study to investigate the mechanisms of MSCs-EVs carrying TGF-β1 in regulating M2 polarization of mouse macrophages via the miR-132/Mycbp2/TSC2 axis.

| Cell culture and treatment
Mouse macrophages RAW264.7 (American Type Culture Collection) were thawed in a 37°C water bath box and then transferred to a 15-mL centrifuge tube to remove the supernatant. The suspended cell suspension was moved to a 25 cm 2 culture bottle and then incubated at 37°C with 5% CO 2 . The liquid was renewed every 2 days. Upon reaching 80%-90% confluence (approximately 1 × 10 7 cells), RAW264.7 cells were treated with 500 ng/ mL LPS (strain O55:B5; Sigma-Aldrich) for 24 hours. Afterwards, the supernatant of each group was taken, and the expression of inflammatory factors IL-1β, TNF-α, IL-6 and IL-10 was detected by enzyme-linked immunosorbent assay (ELISA). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was employed to detect the viability of cells following treatment. The cells in each group were inoculated into 96-well plates at a density of 1 × 10 4 cells/well, with eight parallel wells in each group.
Additionally, a blank control well with only culture medium and no cells was set, and three time-points: 24, 48 and 72 hours, respectively, were set, followed by subsequent experiments. When cells grew to 70% confluence, 5 mg/mL MTT solution (10 μL; ST316; Beyotime Institute of Biotechnology) was added to each well, and incubated in a 37°C incubator for 4 hours, followed by supernatant removal. After PBS washing, each well was added with 100 μL DMSO (D5879; Sigma) and incubated by shaking for 10 minutes.
Thereafter, a microplate reader (MK3; Thermo) was applied to measure the optical density (OD) value of each well at 490 nm.
Cell viability = (OD value of the experimental well − OD value of the blank well)/OD value of the blank well. The experiment was repeated three times, and the average value was taken. RAW264.7 cells at the logarithmic growth phase were seeded into a 6-well cell culture plate at a density of 4 × 10 5 cells/well. When cell confluence reached 80%-90%, the plasmids of different groups were connected to pLV-Neo (Inovogen Tech. Co.). Each sequence was provided by Sigma-Aldrich. After sequencing, the plasmid and pLV-Neo were cotransfected into HEK293T cells, and the supernatant of the culture medium containing lentiviral particles was collected to infect RAW264.7 cells, and stably transfected cell lines were selected. The cells were treated with miR-132 mimic, overexpression (oe)-Mycbp2, oe-TSC2 or their corresponding negative controls (NCs).

| Culture and identification of MSCs
The well-grown C57BL/6 mice were killed and soaked in alcohol for 10 minutes. The femur and tibia of the mice were taken in a sterile environment and then placed in Dulbecco's modified Eagle's medium (DMEM; Gibco by Life Technologies) after the leg meat was removed with instruments. Both ends of the femur and tibia were removed with clean and sterile scissors, and a syringe was utilized to flush bone marrow cells into a 15-mL centrifuge tube with DMEM to discard the supernatant. Cells were resuspended in DMEM containing 10% foetal bovine serum (FBS; Biowest) and 100 U/mL penicillinstreptomycin (Gibco by Life Technologies).
Mesenchymal stem cells at passage 3 were detached with trypsin (Gibco by Life Technologies) and suspended with phosphate buffered saline (PBS) to adjust the cell concentration to 1 × 10 6 cells/mL. Cell suspension (200 μL) was sub-packaged into Eppendorf (EP) tubes, added with 5 μL of different fluorescence-labelled monoclonal antibodies (LA, CD11b, Sca-1, CD105, CD34, CD45, CD31 and CD29) and incubated at 4°C for 15 minutes. After that, each tube was supplemented with 2 mL PBS and centrifuged at 1000 r/min for 5 minutes to discard the supernatant and unbound antibody. Subsequently, each tube was suspended with 400 μL of 0.01 mol/L PBS containing 0.5% paraformaldehyde and mixed well. The isotype control group was set with the fluorescence-labelled IgG antibodies in the same colour, and the cells in each tube were detected by flow cytometer.
Mesenchymal stem cells at passage 3 were detached with trypsin to prepare single cell suspension. The cell concentration was altered to 1 × 10 5 cell/mL and spread on a six-well plate. When the cells reached 60%-70% confluence, the supernatant was discarded. The experimental group was added with 2 mL osteogenic differentiation induction complete medium (MUBMX-90021; Cyagen Biosciences) for induction.
The control group was added with an equal amount of DMEM. The fresh medium was renewed in the experimental and the control groups every 3 days, for 2-3 weeks. Next, each well was fixed with 2 mL of 4% neutral formaldehyde for 30 minutes and dyed with 1.5 mL alkaline phosphatase dye for 5 minutes, and observed under a microscope.
Mesenchymal stem cells at passage 3 were detached with trypsin to prepare for single cell suspension. The cell concentration was altered to 3 × 10 5 cell/mL and spread on a six-well plate. When the cells reached 80%-90% confluence, the supernatant was discarded.
The experimental group was added with 2 mL adipogenic differentiation medium A (MUBMX-90031; Cyagen Biosciences) and then 2 mL adipogenic differentiation medium B. The induction was performed with alternate induction by medium A and B. Next, each well was fixed with 2 mL of 4% neutral formaldehyde for 30 minutes and dyed with 1.5 mL oil red O dye for 5 minutes, and observed under a microscope.

| EVs extraction and identification
The well-grown bone marrow MSCs were cultured overnight in serum-free DMEM. When the cell confluence reached 80%-90%, the supernatant was collected. The cells were centrifuged at 2000 g at 4°C for 20 minutes to remove the cell debris, and the obtained supernatant was centrifuged at 10 000 g at 4°C for 1 hour at high speed. After that, the precipitate was suspended and precipitated in serum-free DMEM containing 25 mmol/L hydroxyethyl piperazine ethanesulfonic acid (pH = 7.4), and the high-speed centrifugation was repeated again. The supernatant was discarded, and the precipitate was stored at −80°C for use. 17 Identification of EVs by a transmission electron microscopy: 30 μL EVs were added dropwise on a copper net. One minute later, the liquid was dried from the side with filter paper. Next, the EVs were supplemented with 30 μL of phosphotungstic acid solution (pH = 6.8), counterstained at room temperature for 5 minutes and photographed under a transmission electron microscope.
Extracellular vesicles particles were dissolved in radioimmunoprecipitation assay (RIPA) buffer and quantitatively identified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific).

| RT-qPCR
TRIzol reagent (Invitrogen) was used to extract the total RNA from the tissues or cells according to the instructions, and the RNA concentration was then determined. The primers used in this study were synthesized by Takara (Table 1). For miRNAs, polyAtailed detection kit (B532451; Sangon Biotech) was used to obtain cDNA of the polyA-tailed miRNA (containing universal PCR primers reverse (R) and U6 universal PCR primers R). Non-miRNA reverse transcription was performed in the light of the instructions of cDNA reverse transcription kit (K1622; Beijing Reanta Biotechnology Co., Ltd.). Detection was performed in a real-time PCR instrument (ViiA 7; Da'an Gene Co., Ltd.). GAPDH was used as an internal reference primer to calculate the relative transcription level of the target gene using a relative quantitative method (2 −ΔΔC t method). 20

| Western blot analysis
Total protein was extracted from tissues or cells using high-efficiency RIPA lysis buffer (C0481; Sigma-Aldrich) following the instructions.
The supernatant was extracted, and the protein concentration of each sample was determined using a BCA kit (23227; Thermo). The protein was quantified according to different concentrations. After separation by polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane, which was then blocked with 5% bovine serum albumin at room temperature for 1 hour. Thereafter, the membrane was probed with primary antibodies against inducible nitric oxide synthase (iNOS; 1:100, ab15323), software (Bio-Rad) was used for quantitative protein analysis. The protein expression was analysed by the ratio of grey values of target band to that of the internal reference.

| Flow cytometry
The cells were made into single cell suspension and resuspended in staining buffer (BD Biosciences). The cells were stained with

| Co-immunoprecipitation (Co-IP) assay
Transfected cells were lysed in lysis buffer (50 mmol/L Tris-HCl, pH = 7.4; 150 mmol/L NaCl, 10% glycerol, 1 mmol/L ethylenediaminetetraacetic acid, 0.5% NP-40 and a protease inhibitor mixture), and the cell debris was removed by centrifugation. The cleared cell lysate was incubated with anti-HA or anti-FLAG antibody (Sigma-Aldrich Chemical Company) and 15Si protein A/G beads (Santa Cruz Biotechnology) for 2 hours. The lysate was then centrifuged at 3000 rpm and 4°C for 3 minutes to enable agarose beads to the bottom of the tube, with the supernatant removed. The complex of HES5 and antigen antibody at the bottom of the tube was subsequently collected and quantified.
The agarose beads were washed with 1 mL of lysis buffer for three to four times and added with 15 μL of 2 × SDS sample buffer. After extensive washing, the beads were boiled at 100°C for 5 minutes. After denaturation, proteins were separated by sodium lauryl sulphate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (Millipore) and then immunoblotted. 22

| Statistical analysis
The data were processed using SPSS 19.0 statistical software (IBM Corp.). Measurement data were expressed as mean ± SD. Comparison between two groups was conducted using unpaired t test, while comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) with Tukey's post hoc test. The data at different time-points were compared by two-factor ANOVA followed by Dunnett's correction. A value of P < .05 indicated significant difference.

| Isolation and identification of MSCs
The isolated and cultured MSCs were uniformly fibrous, long spindle-shaped and vortex-shaped ( Figure 1A). Flow cytometric analysis showed that the cells overexpressed Sca-1, CD29 and CD105, but they did not express or poorly expressed CD31, CD34, CD45, LA and CD11b ( Figure 1B). Oil red O staining results suggested that a large number of red lipid droplets appeared after isolation of the cultured MSCs ( Figure 1C). After osteogenesis induction, alkaline phosphatase activity was increased ( Figure 1D). These results indicate the successful isolation of MSCs.

| MSCs promote M2 polarization of LPS-treated macrophages through EVs in vitro
With the aim to probe into the effect of MSCs and EVs on mouse macrophage polarization, we co-cultured RAW264.7 cells with MSCs or GW4869-treated MSCs. Flow cytometry was used to detect the

| TGF-β1 in MSC-derived EVs promotes miR-132 expression in macrophages and thus promotes M2 polarization
Next, we used ultra-high-speed centrifugation to extract EVs from MSCs. EVs observed under a transmission electron microscopy ( Figure 3A) presented round or oval membrane vesicles with basically the same shape. EVs range in size from 30 to 120 nm by dynamic light scattering ( Figure 3B). As shown in Figure 3C, EVs highly expressed positive markers CD63, CD81 and TSG101, but did not express negative marker calnexin, confirming the successful extrac-  The next step was to investigate whether MSCs-EVs could carry TGF-β1 to promote the expression of miR-132 in macrophage RAW264.7. The expression of miR-132 determined using RT-qPCR ( Figure 3O) was found to be enhanced in RAW264.7 cells co-cultured with MSCs-EVs, which was decreased in RAW264.7 cells co-cultured with MSCs-EVs treated with si-TGF-β1. This result indicates that MSCs-EVs carry TGF-β1 to promote miR-132 expression in RAW264.7 cells.

| miR-132 targets Mycbp2 in macrophages
In order to further elucidate the downstream mechanism of miR-132 in regulating macrophage differentiation, we predicted through an online prediction software starbase that miR-132 could target Mycbp2 ( Figure 4A). Meanwhile, the results of the dual-luciferase reporter gene assay ( Figure 4B) revealed that the luciferase activ-

| MSC-derived EVs carrying TGF-β1 elevate miR-132 expression and down-regulate MYCBP2 expression, polarizing macrophages towards M2 phenotype in vitro
The aforementioned results had revealed that MSCs-EVs could carry TGF-β1 to promote the expression of miR-132 and thus promote

| Mycbp2 enhances ubiquitination and degradation of TSC2 in macrophages
Mycbp2 has been reported to be able to ubiquitinate and degrade TSC2 protein, 15 and TSC2 protein can promote M2 polarization of macrophages. 16 In order to further study whether the stability of   Additionally, the increased expression of miR-132 has been found in murine macrophages cultured with ginseng stem-leaf saponins (GSLS) and/or thimerosal (TS). 33 miR-132 expresses highly during the inflammatory phase of wound repair, and concurrently, TGF-β1

| D ISCUSS I ON
can promote miR-132 expression in keratinocytes. 13 miR-132 in human macrophages has been described as a regulator of the interferon-γ-induced macrophage activation pathway. 34 Additionally, miR-132 has been elucidated to exert anti-inflammatory functions in alveolar macrophages, and LPS-induced rat alveolar macrophages contributed to an enhancement in miR-132 expression and high TNF-α, IL-1β and IL-6 levels. 35 Nevertheless, the presence of other mRNAs or miRNAs in EVs cannot be ruled out, which may play a role in cell crosstalk. This needs to be further investigated in future studies.
In order to further elucidate the downstream mechanism of miR-132 in regulating macrophage differentiation, we found that miR-132 could target Mycbp2. Mycbp2 is an inhibitor of the M2 macrophage polarization, which executes its suppressive effect via various signalling pathway. 36 In addition to that, we also found that overexpression of Mycbp2 reversed the effects of overexpressed miR-132 on M2 polarization of macrophages, indicating that miR-132 can target Mycbp2 in macrophages to promote M2 polarization of macrophages.
Mycbp2 can act as an E3 ubiquitin ligase towards tuberin and modulate mTOR signalling, implying that Mycbp2 in turn controls cell growth and neuronal function via the TSC/mTOR pathway in mammalian cells. 15 Translation processes are modulated by Mycbp2 mainly through the mTOR signalling where Mycbp2 has a double function by ubiquitylating the Rheb inhibitor TSC2. 37 As reported, TSC1/2 complex inhibits the Ras GTPase pathway to restrict M1 response, and its vital role in M2 activation is basically regulated by suppressing the mTOR pathway. 38 Finally, we found that MSC-secreted EVs carried TGF-β1 to promote M2 polarization of macrophages via the miR-132/Mycbp2/TSC2 axis.
Similar to our study, a recent article has revealed that MSCs-EVs treatment decreased the atherosclerotic plaque and greatly attenuating the infiltration of macrophages, thereby inducing M2 polarization of macrophages through the miR-let7/IGF2BP1/PTEN pathway. 39 In conclusion, the current study highlights that EVs derived from MSCs carrying TGF-β1 promote M2 polarization of mouse macrophages through regulating the miR-132/Mycbp2/TSC2 axis ( Figure 9). These findings may provide a molecular basis for the application of MSCs-EVs as an agent for macrophage M2 polarization.
Additionally, the identification of the regulatory role of the miR-132/ Mycbp2/TSC2 axis in macrophage activation provides potent targets for macrophage-associated diagnostic and therapeutic strategies.

ACK N OWLED G EM ENTS
This study was supported by Natural Science Foundation of Science and Technology Department of Gansu Province (No. 17JR5RA264).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest associated with the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.