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.

Keywords: Mysm1; adipose-derived stem cells; miR-150; nitric oxide.

Figure 1

Inflammatory cytokines induce Mysm1 expression. A, Immunofluorescence analysis of Mysm1 expression in murine‐derived adipose‐derived stem cells (ASCs) and bone cells. Scale bars: 50 µm. B, Adipose‐derived stem cells were treated with tumour necrosis factor‐α (TNF‐α) plus IFNγ for 12 h at different concentrations and then collected in TRIzol. Mysm1 mRNA levels were determined with quantitative RT‐PCR. C, Adipose‐derived stem cells were treated with 10 ng/mL TNF‐α and 10 ng/mL IFNγ for 30 min, 60 min and 24 h, then Mysm1 protein levels were determined by Western blot. **P < 0.01

Figure 2

Properties of adipose‐derived stem cells (ASCs) with Mysm1 knockdown. A, Adipose‐derived stem cells were isolated from wild type (WT) and Mysm1‐deficient (KO) mice. Mysm1 mRNA levels (top) were determined by quantitative RT‐PCR, and protein expression (bottom) was examined by Western blot. B, Flow cytometry analysis of CD3⁺ T cell proliferation as indicated by reduced carboxy fluorescein diacetate succinimidyl ester (CFSE) intensity. CD3⁺ T cells were isolated from murine spleens with CD3e MicroBead Kits and labelled with or without CFSE. CD3⁺ T cells were stimulated with or without PMA (50 ng/mL) plus ionomycin (1 µg/mL) for 72 h, and then cells were collected for flow cytometry analysis. C, Attenuated inhibition of T cell proliferation by KO ASCs. CD3⁺ T cells were labelled with CFSE and stimulated with PMA (50 ng/mL) plus ionomycin (1 µg/mL) for 24 h, and then cultured alone (left), with WT ASCs, or with KO ASCs at different ratios (ASCs:T cells) (right). After 48 h, cells were analyzed by flow cytometry for T cell proliferation as indicated by reduced CFSE intensity. Data are representative of two independent experiments. D, mRNA levels of inflammatory cytokines in WT and KO ASCs were determined by quantitative RT‐PCR. E, After treating WT and KO ASCs with tumour necrosis factor‐α (TNF‐α) plus IFNγ for 24 h, the amount of nitrate in the supernatant was determined by a Griess test. *P < 0.05 and **P < 0.01

Figure 3

Mysm1‐deficient adipose‐derived stem cells (ASCs) exacerbated dextran sodium sulphate (DSS)‐induced colitis. A, Schematic representation of mouse colitis experiments. Mice received 3% DSS in drinking water from day 0 to day 8. Adipose‐derived stem cells (1 × 10⁶/mouse) were infused intraperitoneally on day 1. Weight loss (B) and disease activity scores (C) were observed daily. Colon weight (D) and colon length (E) were measured on day 8. Control mice received no DSS in drinking water. n = 6 mice/group. *DSS + WT ASCs vs DSS + KO ASCs, *P < 0.05 and **P < 0.01

Figure 4

Mysm1‐deficient adipose‐derived stem cells (ASCs) suppress tumour growth in vivo. On day 1, 5 × 10⁵ B16‐F0 cells were injected subcutaneously to the back of C57BL/6 mice, with or without co‐injection of WT or KO ASCs (1 × 10⁶ cells per mouse). Seven days later, tumour size was measured daily (A). On day 13, mice were euthanized and the tumours were weighed (B, top), and pictures of the representative tumour of each group were taken under a microscope (B, bottom). Each treatment group included five mice, and data are representative of three independent experiments. Wild type (WT) ASCs vs KO ASCs, *P < 0.05 and **P < 0.01

Figure 5

Mysm1 epigenetically regulates miR‐150 expression. A, Wild type (WT) and KO adipose‐derived stem cells (ASCs) were treated with tumour necrosis factor‐α (TNF‐α) plus IFNγ for 24 h and cells were collected in TRIzol. Levels of miR‐150, miR‐155‐5p, and miR‐155‐3p were determined with quantitative RT‐PCR. B, Ectopic expression of Mysm1 in KO ASCs. Cells were transduced with lentivirus containing GFP (LV‐GFP) or Mysm1 (LV‐Mysm1) (left). Quantitative RT‐PCR results show increased expression of Mysm1, miR‐150 and inducible nitric oxide synthases and Griess test show the increase amount of nitrate in KO ASCs transduced with LV‐Mysm1. Data are representative of three independent experiments and shown as the mean ± SD. C, Chromatin immunoprecipitation assays of WT ASCs and KO ASCs via an Mysm1 antibody probing for the pri‐miR‐150 promoter sequence or an IgG nonspecific antibody. D, Quantitative RT‐PCR result shows decreased expression of pri‐miR‐150 in KO ASCs. *P < 0.05 and **P < 0.01

Figure 6

miR‐150 regulates inducible nitric oxide synthases (iNOS) expression. A, KO adipose‐derived stem cells (ASCs) were transduced with lentivirus containing GFP (LV‐GFP) or LV‐miR‐150, and miR‐150 expression was detected by quantitative RT‐PCR. B, mRNA levels of inflammatory cytokines in KO ASCs transduced with LV‐GFP or LV‐miR‐150 was determined by quantitative RT‐PCR. C, KO ASCs transduced with LV‐GFP or LV‐miR‐150 were treated with tumour necrosis factor‐α (TNF‐α) plus IFNγ at different concentrations for 24 h, then levels of nitrate in the supernatant was measured by a Griess test. D, After C3H/10T1/2 cells were transduced with LV‐GFP or LV‐miR‐150, miR‐150 expression was detected by quantitative RT‐PCR. E, mRNA levels of inflammatory cytokines in C3H/10T1/2 cells transduced with LV‐GFP or LV‐miR‐150 was determined by quantitative RT‐PCR. F, C3H/10T1/2 cells transduced with LV‐GFP or LV‐miR‐150 were treated with TNF‐α plus IFNγ at different concentrations for 24 h, then levels of nitrate in the supernatant was measured by a Griess test. **P < 0.01

Figure 7

A proposed model of the mechanism by which Mysm1 regulates the immunomodulatory effect of adipose‐derived 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