Mysm1 epigenetically regulates the immunomodulatory function of adipose‐derived stem cells in part by targeting miR‐150

Abstract Adipose‐derived stem cells (ASCs) are highly attractive for cell‐based therapies in tissue repair and regeneration because they have multilineage differentiation capacity and are immunosuppressive. However, the detailed epigenetic mechanisms of their immunoregulatory capacity are not fully defined. In this study, we found that Mysm1 was induced in ASCs treated with inflammatory cytokines. Adipose‐derived stem cells with Mysm1 knockdown exhibited attenuated immunosuppressive capacity, evidenced by less inhibition of T cell proliferation, more pro‐inflammatory factor secretion and less nitric oxide (NO) production in vitro. Mysm1‐deficient ASCs exacerbated inflammatory bowel diseases but inhibited tumour growth in vivo. Mysm1‐deficient ASCs also showed depressed miR‐150 expression. When transduced with Mysm1 overexpression lentivirus, ASCs exhibited enhanced miR‐150 expression. Furthermore, Mysm1‐deficient cells transduced with lentivirus containing miR‐150 mimics produced less pro‐inflammatory factors and more NO. Our study reveals a new role of Mysm1 in regulating the immunomodulatory activities of ASCs by targeting miR‐150. These novel insights into the mechanisms through which ASCs regulate immune reactions may lead to better clinical utility of these cells.


| INTRODUC TI ON
Adipose-derived stem cells (ASCs) are similar to bone marrow-derived stem cells (BMSCs) in their ability to differentiate into multiple cell types, including bone, cartilage, adipocytes and neurons. 1,2 Additionally, ASCs are immunosuppressive and express similar surface markers to BMSCs. 6 Unlike BMSCs, clinically applicable numbers of ASCs can easily be obtained from adipose tissue collected through minimally invasive procedures such as lipoplasty. 7 Due to these properties, ASCs are appealing for use in cell-based therapies for tissue repair and regeneration.
Allogeneic ASCs are immune privileged and have immunomodulatory capabilities. In vitro, ASCs inhibit the proliferation and function of activated immune cells through cell-cell binding and paracrine signalling. 8 In vivo, ASCs have demonstrated therapeutic potential in numerous immune-mediated conditions in both pre-clinical and clinical studies, including graft-vs-host disease (GvHD) and chronic inflammatory autoimmune diseases. 9,10 There are several possible mechanisms through which ASCs function to suppress immunity. A series of factors and molecules produced by ASCs, such as prostaglandin (PG) E2, 13 transforming growth factor-β (TGF-β) 14 and interleukin (IL)-10, 14,15 have been shown to be critical for their immunoregulatory functions. While many of these factors have been well characterized, much remains unknown about the immunomodulatory function and therapeutic efficacy of ASCs.
Mysm1, a histone deubiquitinase, mediates the deubiquitination of lysine 119 (K119) of histone H2A, 16 and removes K63 polyubiquitins attached to TRAF3 and TRAF6. 17 We and several other groups have previously demonstrated that Mysm1 plays a crucial role in stem cell maintenance and immune cell development and function. 18,19 Mysm1 can control essential lineage-specific developmental regulators and miRNA expression at a transcriptional level, and it regulates the p53 stress response pathway in a cell-specific manner. 25,26 Despite these observations, knowledge of the biological functions of Mysm1 remains incomplete and its role in ASC immunoregulatory function has not been investigated.
In the present study, we demonstrate that Mysm1-deficient ASCs showed attenuated inhibition of T cell proliferation in vitro, while exacerbating inflammatory bowel diseases and inhibiting tumour growth in vivo. Further mechanistic studies revealed that Mysm1 regulates the immunomodulatory capacity of ASCs by targeting miR-150 expression. Taken together, our data reveal a novel role of Mysm1 in regulating the immunomodulatory activities of ASCs.

| Animals
Groups of 3-4-week-old and 8-12-week-old C57BL/6 mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences of China (Beijing). Mysm1-deficient (KO) mice were generated as described previously. 18 In all experiments, age-and sex-matched wild type (WT) littermates were used for controls. Mice were maintained in a pathogen-free barrier facility.

| ASC isolation and culture
Wild type and Mysm1deficient mice (3-4 weeks old) were euthanized by cervical dislocation and sterilized in 75% ethanol for 5 minutes.
Adipose-derived stem cells were obtained from inguinal subcutaneous adipose tissue with abdomens facing up. The collected tissue was rinsed with phosphate buffered solution (PBS) and minced, followed by digestion with 1 mg/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 1 mg/mL dispase (Sigma-Aldrich) for 35 minutes at 37°C with agitation. Cells were added to α-MEM (Gibco, Carlsbad, CA) containing 10% fetal bovine serum (FBS) (Gibco) to stop digestion and filtered through a 40 μm cell strainer (Biologix, Lenexa, KS) to generate single-cell suspensions. After centrifugation at 400 g for 5 minutes, cells were resuspended in α-minimum essential medium (α-MEM) containing 10% FBS and cultured at 37°C in 5% CO 2 . Upon 80%-90% confluence, cells were subcultured in a ratio of 1:3.

| Immunofluorescence staining
For immunofluorescence, cells were fixed in 4% formaldehyde for 20 minutes at room temperature, permeabilized with 0.3% Triton X-100 in PBS for 10 minutes and then blocked with 2% bovine serum albumin and 0.05% sodium azide in PBS for 1 hour at room temperature to block non-specific antibody binding. Subsequently, cells were incubated with the primary antibodies overnight at 4°C. Cells were then washed three times for 10 minutes with PBS and incubated for 1 hour at room temperature with Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA). Then the cells were counterstained with 4′,6-diamidino-2-phenylindole in D-PBS (Sigma-Aldrich) and analyzed under a Zeiss confocal microscope.

| Quantitative RT-PCR
Total RNA was extracted with TRIzol (Sigma-Aldrich) and reverse transcribed into cDNA with a reverse transcriptase kit (Toyobo, Osaka, Japan). cDNA was used as a template in quantitative PCR with Synergy Brands Synergy Brands (SYBR) Green (Toyobo) to determine specific gene expression. Total mRNA was normalized to endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA.

| Western blot
Cells were lysed with lysis buffer and protein samples were separated on 12% SDS-polyacrylamide gel, and then the proteins were transferred to 0.45 μm polyvinylidene fluoride blotting membranes.
The membrane was blocked in 5% non-fat dry milk for 1 hour, were then probed with primary antibodies against the proteins of interest in blocking solution overnight at 4°C, washed and then incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Finally, enhanced chemiluminescence substrate (Thermo Fisher, Waltham, MA) was added to the membranes and the proteins were assayed according to manufacturer instructions. Antibodies against GAPDH, Mysm1 were purchased from Cell Signaling Technology, Inc.

| Induction of acute colitis
Acute colitis was induced in C57BL/6 mice by administering 3% dextran

| Statistical Analysis
All data were analyzed with Prism 5.0 software (GraphPad Software, San Diego, CA) and are presented as the means ± SDs. Statistical significance was assessed by unpaired two-tailed Student's t tests (*P < 0.05; **P < 0.01).  Figure 1C). These data indicate that Mysm1 may be involved in the immunomodulatory activity of ASCs.

| Mysm1 knockdown attenuates the immunosuppressive capacity of ASCs
To test the involvement of Mysm1 in ASC immunomodulatory activity, ASCs from Mysm1 KO and WT mice were isolated. The deficient expression of Mysm1 in KO ASCs was confirmed by quantitative RT-PCR and Western blot analysis (Figure 2A). Surface markers and cell cycle were examined by flow cytometry and no significant differences were observed between cultures of KO ASCs and their WT counterparts (data not shown). Next, T cell proliferation was used as an immune response model, in which a reduction of CFSE intensity was measured to determine T cell proliferation ( Figure 2B).
As shown in Figure 2C,  weighed after 13 days. In contrast to WT ASCs that promoted tumour growth, infusion of KO ASCs was found to significantly inhibit tumour growth ( Figure 4A,B). Therefore, modulation of ASCs could provide a novel strategy for tumour immunotherapy. ASCs, but not in KO ASCs ( Figure 5C), which might account for the lower expression of pri-miR-150 in KO ASCs ( Figure 5D).

| D ISCUSS I ON
In addition to their use in tissue repair and regenerative medicine, ASCs have been utilized in treating immune disorders due to their immunomodulatory properties. 9

| CON CLUS IONS
This study reveals that Mysm1 regulates the immunosuppressive capacity of ASCs by targeting miR-150, and thus uncovers a previously undescribed role of Mysm1 in regulating the immunomodulatory activities of ASCs. These novel insights into the mechanisms through which ASCs regulate immune reactions may help to improve the clinical utility of these cells in many inflammation related diseases.

E THI C S APPROVAL AND CON S ENT TO PARTI CIPATE
All experimental animal protocols for this study are in accordance with the national guidelines for the use of animals in scientific research. Additional approval was granted by the Animal Care and Use Committee of the Academy of Military Medical Sciences.
F I G U R E 7 A proposed model of the mechanism by which Mysm1 regulates the immunomodulatory effect of adiposederived stem cells (ASCs). Mysm1 is induced by tumour necrosis factor-α (TNF-α) and IFNγ in ASCs. Mysm1 then promotes miR-150 transcription, which enhances inducible nitric oxide synthases (iNOS) production. Nitric oxide (NO) is catalyzed by iNOS that is essential for the immunosuppressive capacity of ASCs