P032 IFN-γ controls the immunomodulatory properties of bone marrow mesenchymal stem cells via a microRNA29a/STAT-3 dependent mechanism
A. Oikonomopoulos*1, P. Lacey1, T. Tomakili1, D. Iliopoulos2, D. W. Hommes1
1UCLA Centre for Inflammatory Bowel Diseases, Division of Digestive Diseases, Los Angeles, California, United States, 2UCLA Centre for Systems Biomedicine, Division of Digestive Diseases, Los Angeles, California, United States
Mesenchymal stem cells (MSC) offer new therapeutic options for fistulising refractory Crohn’s disease (CD). Priming of MSC with pro-inflammatory cytokines, such as IFN-γ further enhances their immunomodulatory properties. However, the exact molecular mechanisms controlling the MSC immunomodulatory properties remain elusive. We hypothesised that manipulation of microRNA levels might potentiate the salutary properties of bone marrow mesenchymal stem cells (BMMSC).
MicroRNA profiling was performed using the Nanostring nCounter platform following IFN-γ stimulation (priming, 50 ng/mL, 24 h) of human donor-derived BMMSC. Gene expression was determined by qRT-polymerase chain reaction (PCR). Cytokine expression in cell supernatants was measured by multiplex ELISA immunoassays. MSC immunomodulatory properties were accessed by co-culture assays of non-primed or primed BMMSC with human Carboxyfluorescein diacetate succinimidyl ester (CFSE)-preloaded stimulated human T-cells, for a period of 96 hours. Transfection of BMMSC with microRNA-29a mimics or inhibitors was performed by lipofectamine assays.
Molecular profiling revealed that microRNA-29a was the most highly up regulated in BMMSC, following IFN-γ priming (1.7-fold, n = 3). Independent assessment of microRNA29-a expression by qRT-PCR verified the above results (2-fold, n = 3). Time course analysis (6h, 12, 24h, and 48h) revealed that IFN-γ up-regulated microRNA-29a in healthy donor-derived BMMSC (n = 3) but not in CD patient-derived cells (n = 6). Non-primed healthy donor-derived cells (n = 3) exhibited higher levels of microRNA-29a compared with CD patient-derived cells (n = 6). Inhibition of microRNA-29a in IFN-γ-primed BMMSC (n = 3) enhanced their immunosuppressive properties, whereas over-expression of microRNA-29a (n = 3) had the opposite effect, as shown by CFSE assays. Moreover, microRNA-29a inhibition (n = 3) in IFN-γ-primed BMMSC increased the levels of IDO-1, IL-10, CCL-1, CCL-21, CCL-25, CCL-26, CXCL-1, CXCL-6, CXCL-8, CXCL-10, and CXCL-11. Over-expression of microRNA-29a had the opposite effect on the expression of all cytokines except for IDO-1. Finally, microRNA-29a inhibition increased the expression of STAT-3 in non-primed and IFN-γ-primed BMMSC. Accordingly, overexpression of microRNA-29a resulted in decreased expression of STAT-3.
Our data show that miRNA-29a is differentially regulated by IFN-γ in CD patients and healthy donors. Further, we provide strong evidence suggesting that microRNA-29a is a novel regulator of the immunosuppressive properties of human BMMSC, controlling the expression of numerous immunomodulators through a STAT-3 mediated mechanism. Elucidation of the molecular circuits regulating the MSC immunomodulatory properties will greatly benefit the design of future clinical trials in CD and other autoimmune diseases.