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      Ex vivo immunosuppressive effects of mesenchymal stem cells on Crohn’s disease mucosal T cells are largely dependent on indoleamine 2,3-dioxygenase activity and cell-cell contact

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          Abstract

          Introduction

          Crohn’s disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application.

          Methods

          T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors’ MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes.

          Results

          A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4 +CD25 + subset and increase of the CD3 +CD69 + population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-β and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used.

          Conclusion

          MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13287-015-0122-1) contains supplementary material, which is available to authorized users.

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          Most cited references 69

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          Interleukin-10-deficient mice develop chronic enterocolitis.

          Interleukin-10 (IL-10) affects the growth and differentiation of many hemopoietic cells in vitro; in particular, it is a potent suppressor of macrophage and T cell functions. In IL-10-deficient mice, generated by gene targeting, lymphocyte development and antibody responses are normal, but most animals are growth retarded and anemic and suffer from chronic enterocolitis. Alterations in intestine include extensive mucosal hyperplasia, inflammatory reactions, and aberrant expression of major histocompatibility complex class II molecules on epithelia. In contrast, mutants kept under specific pathogen-free conditions develop only a local inflammation limited to the proximal colon. These results indicate that the bowel inflammation in the mutants originates from uncontrolled immune responses stimulated by enteric antigens and that IL-10 is an essential immunoregulator in the intestinal tract.
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            Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli.

            CD2(+) T lymphocytes obtained from either the donor of bone marrow stromal cells (BMSCs) or a third party were cultured in mixed lymphocyte reactions (MLRs) with either allogeneic dendritic cells (DCs) or peripheral blood lymphocytes (PBLs). When autologous or allogeneic BMSCs were added back to T cells stimulated by DCs or PBLs, a significant and dose-dependent reduction of T-cell proliferation, ranging from 60% +/- 5% to 98% +/- 1%, was evident. Similarly, addition of BMSCs to T cells stimulated by polyclonal activators resulted in a 65% +/- 5% (P =.0001) suppression of proliferation. BMSC- induced T-cell suppression was still evident when BMSCs were added in culture as late as 5 days after starting of MLRs. BMSC-inhibited T lymphocytes were not apoptotic and efficiently proliferated on restimulation. BMSCs significantly suppressed both CD4(+) and CD8(+) T cells (65% +/- 5%, [P =.0005] and 75% +/- 15% [P =.0005], respectively). Transwell experiments, in which cell-cell contact between BMSCs and effector cells was prevented, resulted in a significant inhibition of T-lymphocyte proliferation, suggesting that soluble factors were involved in this phenomenon. By using neutralizing monoclonal antibodies, transforming growth factor beta1 and hepatocyte growth factor were identified as the mediators of BMSC effects. In conclusion, our data demonstrate that (1) autologous or allogeneic BMSCs strongly suppress T-lymphocyte proliferation, (2) this phenomenon that is triggered by both cellular as well as nonspecific mitogenic stimuli has no immunologic restriction, and (3) T-cell inhibition is not due to induction of apoptosis and is likely due to the production of soluble factors.
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              Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.

              Mesenchymal stem cells (MSCs) can become potently immunosuppressive through unknown mechanisms. We found that the immunosuppressive function of MSCs is elicited by IFNgamma and the concomitant presence of any of three other proinflammatory cytokines, TNFalpha, IL-1alpha, or IL-1beta. These cytokine combinations provoke the expression of high levels of several chemokines and inducible nitric oxide synthase (iNOS) by MSCs. Chemokines drive T cell migration into proximity with MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). This cytokine-induced immunosuppression was absent in MSCs derived from iNOS(-/-) or IFNgammaR1(-/-) mice. Blockade of chemokine receptors also abolished the immunosuppression. Administration of wild-type MSCs, but not IFNgammaR1(-/-) or iNOS(-/-) MSCs, prevented graft-versus-host disease in mice, an effect reversed by anti-IFNgamma or iNOS inhibitors. Wild-type MSCs also inhibited delayed-type hypersensitivity, while iNOS(-/-) MSCs aggravated it. Therefore, proinflammatory cytokines are required to induce immunosuppression by MSCs through the concerted action of chemokines and NO.
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                Author and article information

                Contributors
                +39 (0)382 502786 , rachele.ciccocioppo@unipv.it
                cangemi.giusy@gmail.com
                +420 608 352 569 , peter.kruzliak@savba.sk
                ale_gallia@hotmail.com
                elena.betti19@gmail.com
                c.badulli@smatteo.pv.it
                m.martinetti@smatteo.pv.it
                marila.cervio@unipv.it
                alessandro.pecci@unipv.it
                bozzivaleria@libero.it
                paolo.dionigi@unipv.it
                livia.visai@unipv.it
                ti.ri@libero.it
                c.alvisi@smatteo.pv.it
                c.picone@smatteo.pv.it
                m.monti@smatteo.pv.it
                mebernardo@gmail.com
                gobbipg@smatteo.pv.it
                gr.corazza@smatteo.pv.it
                Journal
                Stem Cell Res Ther
                Stem Cell Res Ther
                Stem Cell Research & Therapy
                BioMed Central (London )
                1757-6512
                24 July 2015
                24 July 2015
                2015
                : 6
                : 1
                Affiliations
                [ ]Clinica Medica I, Dipartimento di Medicina Interna, Fondazione IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Centre for the Study and Cure of Inflammatory Bowel Disease, Clinica Medica I, IRCCS San Matteo Hospital Foundation, University of Pavia, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]International Clinical Research Center, St. Anne’s University Hospital and Masaryk University, Pekarska 53, Brno, 656 91 Czech Republic
                [ ]Servizio di Immunogenetica, Immunoematologia e Medicina Trasfusionale, Fondazione IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Clinica Medica III, Dipartimento di Medicina Interna, Fondazione IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Chirurgia Generale I, Fondazione IRCCS Policlinico San Matteo, Università di Pavia, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Dipartimento di Medicina Occupazionale, Ergonomia e Disabilità, Laboratorio di Nanotecnologia, Fondazione IRCCS Salvatore Maugeri, Università di Pavia, Via Maugeri 8-10, Pavia, 27100 Italy
                [ ]Dipartimento di Medicina Molecolare, Centro di Ingegneria Tissutale, INSTM UdR Pavia, Università di Pavia, Pavia, 27100 Italy
                [ ]Laboratori di Oncoematologia Pediatrica, Fondazione IRCCS Policlinico San Matteo, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Laboratorio di Ematologia, Fondazione IRCCS Policlinico San Matteo, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Centro di Ricerca di Medicina Rigenerativa, Fondazione IRCCS Policlinico San Matteo, Piazzale Golgi 19, Pavia, 27100 Italy
                [ ]Dipartimento di Onco-Ematologia Pediatrica e Medicina Trasfusionale, Ospedale Pediatrico Bambino Gesù, Via Sant’Onofrio 4, Rome, 00165 Italy
                Article
                122
                10.1186/s13287-015-0122-1
                4529692
                26206376
                © Ciccocioppo et al. 2015

                Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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                © The Author(s) 2015

                Molecular medicine

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