Event Abstract

Back to Event

IL17RA knockout mesenchymal stem cells lose their immunosuppressive capacity and exerts deleterious effects on EAE mice

Mónica Kurte1, Patricia Luz-Crawford2, Ana Maria Vega-Letter1, Catalina Fernández1, Melanie Gauthier1, Ivón Moya1, Christian Díaz1, Daniela Ruiz-Higgs1, Farida Djouad2 and Flavio Carrión1*
  • 1 Laboratorio de Inmunología/Universidad de los Andes, Centro de Investigaciones Biomedicas, Chile
  • 2 INSERM, U844, France

INTRODUCTION Mesenchymal Stem Cells (MSCs) are adherent, undifferentiated, pluripotent and non-hematopoietic progenitor cells. MSCs have the potential to differentiate into mesodermal lineages including osteoblasts, chondrocytes, and adipocytes among others, which is why MSCs have great therapeutic value for regenerative medicine (1-2). Currently, MSCs are also known for their ability to regulate the immune system. MSCs have potent immunosuppressive properties and have been proposed as a new therapy for autoimmune diseases (3-4). However, MSCs are not spontaneously immunosuppressive; they require a “licensing” step provided by inflammatory molecules, such as IFN-γ, TNF-α and IL-1β (5-6). We propose that IL-17, also has a role in this “licensing” step, therefore the absence on MSCs of IL-17 receptor type A (IL17RA) on MSCs (the most important subunit of IL17 receptor) will decrease their immunosuppressive capacity. Results of our group showed that MSCs stimulation with recombinant IL-17 as well as supernatants from differentiated Th17 cells induce the expression of immunosuppressive molecules such as Nitric Oxide, TGF-b and COX-2 (7). Moreover, we observed that IL17RA expression is involved in the suppressive effect of MSCs on Th17 differentiation, because the co-culture of CD4+T cells induced to differentiate to Th17 with MSC- IL17RA siRNA lost the ability to inhibit Th17 differentiation, compare to control MSC. The aim of this work was to evaluate the role of IL-17/IL17RA on the immunosuppressive capacity of MSCs, in vitro and in vivo. METHODS Animals: 10-14 week old Female C57BL/6mice purchased from the Central Animal Facility, Instituto de Salud Pública (ISP), Chile, were housed under standard laboratory conditions and maintained with food and water ad libitum. Experimental procedures and protocols were performed according to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of the Universidad de los Andes, and the Fondecyt Bioethics Advisory Committee in Chile. Cells: MSC knockout for IL17RA (MSC IL17RA KO) and MSC wild type (MSC wt) were kindly provided by Farida Djouad, Inserm U488, Montpellier, France. MSC IL17RA KO were isolated from bone marrow of genetically deficient in IL-17RA (IL-17R KO) mice, generated by homologous recombination in ES cells using a targeting vector that replaces exons 4–11 with a PGK-neo cassette and then were backcrossed to C57BL/6 background for 10 generations. T cell proliferation assay: Splenocytes isolated from C57BL/6 mice were stained with CellTraceTM Violet probe (CTV) following the manufacturer's instructions and stimulated with Concanavalin A (1 ug/ml) for 4 days. CTV-splenocytes were cultured in presence or absence of MSCs (1:10 ratio, MSCs:splenocytes) in RPMI complete medium, at 37°C in a 5% CO2. CD3+ lymphocytes were analyzed on FACS Canto II flow cytometer and FacsExpress 4 Plus Research Edition software. EAE Induction and MSCs Administration: For EAE induction, 10- to 14-week-old female C57BL/6 mice were injected subcutaneously with 50ug of MOG35–55 peptide emulsified in complete Freund’s adjuvant supplemented with heat-inactivated Mycobacterium tuberculosis H37RA. After 2 and 48 hrs mice also received 350 ng of Pertussis toxin via intraperitoneal injection. MSCs (1x106) diluted in PBS were administrated after 5 days of EAE induction. The clinical score and mice weights were recorded daily for 22 days. Lymphocytes from EAE mice were stimulated ex vivo with PMA/Ionomicin (50 ng/ml, 1 μg/ml) for 4 hours in the presence of brefeldin A. Th1 and Th17 analysis were performed using commercial Kit CytoFix/CytoPerm BD according to the manufacturer's instructions. Treg analysis were done using Treg Detection Kit (CD4/CD25/FoxP3, Miltenyi). The samples were analyzed on FACS Canto II flow cytometer and FacsExpress 4 Plus Research Edition software. ELISA: Plasma cytokine concentrations were measured by a Milliplex mouse Th17 magnetic beads array Kit (MTH17MAG-47K, Millipore). Statistical Analysis: Statistical analyses were performed using the software GraphPad Prism 5.0. Unpaired Mann-Whitney or Wilcoxon tests were used to compare between the different experimental groups. Data were expressed as mean ± SEM. All p values <0.05 were considered statistically significant. RESULTS MSC IL17RA KO has decreased immunosuppressive capacity. To evaluate the effect of MSC wt and MSC IL17RA KO on T cell proliferation we stimulated CTV stained-splenocytes with Concanavalin A (ConA, 1ug/ml) for 4 days and then analyzed CD3+ T cell proliferation by flow cytometry. Both, MSC wt and MSC IL17RA KO reduced the percentage of CD3+ T cell proliferation. However, MSC wt was by far more effective to inhibit lymphocyte proliferation. MSC wt inhibited the proliferation of T cells by 80%, whereas the MSC IL17RA KO only did by 30% (p<0.005 and p< 0.05, respectively). Cytokines profiles of MSC wt and MSC IL17RA. In order to evaluate the cytokine profile of both MSCs, wt and IL17RA KO, we stimulated the MSCs for 48h. in presence or absence of IL-17 (50ng/ml) or IFN-g/LPS (20ng/ml / 0.5ug/ml). The supernatants were recovered and stored at -80°C until their measuring. Ten different cytokines were evaluated using Milliplex mouse magnetic bead panel Kit. First, we analyzed Th1 associated cytokines, IFN-g, IL12p40, TNF-a and TNF-b. We observed after IFN-g/LPS stimulation, that MSC wt, but not MSC IL17RA KO, was able to increase TNF-a and TNF-b(p<0,05) Interestingly, MSC IL17RA KO expressed lower levels of TNF-B in presence or absence of pro-inflammatory stimulus (p<0,05). No differences were found on IFN-g and IL12p40 levels. Second, we evaluated cytokines associated to the IL-17 family, including IL-17A (commonly referred to as IL-17), IL-17E (also known as IL-25) and IL-17F (8). We also included the analysis for IL-6, a pleiotropic cytokine involved in Th17 differentiation. We observed an important increased of IL-17 and Il-6 after MSCs were stimulated with IFN-g/LPS (p<0.05). Both cell types expressed similar levels of IL-6, but curiously MSC IL17RA KO expressed higher levels of IL-17 (p<0.05). On the other hand, MSC IL17RA KO expressed less IL-17E than MSC wt. This cytokine is associated to Th2 phenotype, unlike IL-17A and IL-17F, which are associated directly to Th17 phenotype. Lastly, we evaluated anti-inflammatory cytokines, IL-10 and IL-27. It is well known that both cytokines are involved in the induction of Treg lymphocytes. IL-27 is also associated to inhibition of Th17 response (9-10). Surprisingly, we observed that both cytokines are secreted mainly by the MSC wt, compared to MSC IL17RA KO, especially when stimulated with IFN-g/LPS (p<0.05). MSC IL17RA KO increased the clinical symptoms in EAE animals. To elucidate the therapeutic effect of MSC IL17RA KO we induced EAE on C57BL/6 using MOG35-55 peptide. Four experimental groups were evaluated: EAE (without MSCs), EAE+MSC wild type, EAE+MSC IL17RA KO and healthy mice. MSCs were injected intraperitoneally (1x106 cells) at day 5 after EAE induction. Consistent with previous results, MSC wt induced a significant improvement of clinical score (p<0.0005). For the contrary, MSC IL17RA KO administration induced an increase of the clinical signs (p<0.0005). MSC IL17RA KO did not induce Treg in vivo and neither anti-inflammatory cytokines in EAE mice. It is well known that EAE model are associated to a T helper response, therefore we analyzed Th1, Th17 and Treg in lymph node samples from EAE mice. We observed that MSC wt is able to reduce the percentage of Th1, Th17 and the double positive cells CD4+IFN-g+IL-17+ cells (p<0.05) and significantly increased the percentage of Treg (p<0.005). Contrary, MSC IL17RA KO decrease Th1 subsets, inducing an increase on Th17/Th1 ratio and more importantly, MSC IL17RA KO was not able to induce Treg. Finally, we evaluated the cytokine profiles in plasma samples of EAE mice. We observe two major differences. First, the MSC IL17RA KO was unable to reduce IL-6 plasma levels. It has been reported that IL-6 alters the permeability of the blood brain barrier (BBB), which facilitates the entry into the CNS of pro-inflammatory cells, therefore inducing higher demyelination (11-12). On the other hand, we found that the MSC wt is capable to induce an increase of IL-27 in plasma samples of EAE mice, which is not observed in mice injected with MSC IL17RA KO. The dendritic cell-derived cytokine IL-27 is a potent inhibitor of Th17 cell differentiation. Finally, we did not observe significant changes on IFN-g, TNF-a, TNF-b, IL-12p70, IL-10, IL-17A, IL-17F and IL-17E levels. CONCLUSIONS In this study, the immunomodulatory capacity and the therapeutic effects of MSC IL17RA KO on EAE progression were evaluated. These results demonstrate that the expression of IL17RA is important for MSCs to exert their immunosuppressive effects, as well as to improve EAE symptoms. These data also suggest that IL-17/IL17RA pathway participate in the MSCs licensing process, which increased the immunosuppressive capacity of MSCs. The absence of IL17RA on MSCs prevent the downregulation of pro-inflammatory T cells, especially Th17 lymphocytes and do not increase Tregs in vivo. These changes are probably associated with a shift in the cytokine profile in periphery. Undoubtedly, the increase of IL-27 would favor an increase of Treg and the inhibition of Th17, according also to our ex vivo results. Once again, IL-6 appears to be associated to the worsening of clinical signs of EAE (13), the interaction of IL-6 and blood-brain barrier permeability would have a major role, which should be studied in more detail.

Acknowledgements

Research was supported by FONDECYT 1130444 project. We thank to Biomedical Sciences PhD program of Faculty of Medicine, Universidad de los Andes for travel award for congress assistance.

References

[1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak., “Multilineage potential of adult human mesenchymal stem cells,” Science, Vol. 284, no. 5411, pp. 143-147, 1999.
[2] A. Caplan, S. Bruder, “Mesenchymal stem cells: building blocks for molecular medicine in the 21st century.” Trends Mol Med. Vol. 7 no. 6, pp. 259-264, 2001.
[3] A. Bartholomew, C. Sturgeon, M. Siatskas, K. Ferrer, K. McIntosh, S. Patil, W. Hardy, S. Devine, D. Ucker, R. Deans, A. Moseley, R. Hoffman, “Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo,” Experimental Hematology, Vol. 30 no. 1, pp. 42-48, 2002.
[4] FE. Figueroa, F. Carrión, S. Villanueva, M. Khoury, “Mesenchymal stem cell treatment for autoimmune diseases: a critical review.” Biological Research, Vol. 45, no. 3, pp. 269-77, 2012.
[5] M. Krampera, “Mesenchymal stromal cell 'licensing': a multistep process.” Leukemia, vol. 25, no. 9, pp. 1408-1414, 2011.
[6] G. Ren, L. Zhang, X. Zhao, et al., “Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide”. Cell Stem Cell, vol 7; no.2, pp. 141-150, 2008.
[7] Luz-Crawford P, Noël D, Fernandez X, Khoury M, Figueroa F, Carrión F, Jorgensen C, Djouad F. “Mesenchymal stem cells repress Th17 molecular program through the PD-1 pathway” PLoS One;7(9), 2012.
[8] Wei Jin1 and Chen Dong. “IL-17 cytokines in immunity and inflammation” Emerging Microbes and Infections 2, e60; doi:10.1038/emi.2013.58, 2013.
[9] C. Hunter C and R. Kastelein, “Interleukin-27: balancing protective and pathological immunity,” Immunity, Vol. 14, no. 6, pp. 960-969, 2012.
[10] Stumhofer JS, Laurence A, Wilson EH, Huang E, Tato CM, Johnson LM, Villarino AV, Huang Q, Yoshimura A, Sehy D, Saris CJ, O’Shea JJ, Hennighausen L, Ernst M, Hunter CA Interleukin 27 negatively regulates the development of interleukin 17-producing T helper cells during chronic inflammation of the central nervous system. Nat Immunol 7:937–945, 2006.
[11] K.D. Rochfort, L.E. Collins, R.P. Murphy, P.M. Cummins, “Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions”. PLoS One, Vol. 9, no. 7, e101815, 2014
[12] K.D. Rochfort, L.E. Collins, R.P. Murphy, P.M. Cummins, “Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions”. PLoS One, Vol. 9, no. 7, e101815, 2014
[13] Mónica Kurte, Javiera Bravo-Alegría, Alexander Torres, Vania Carrasco, Cristina Ibañez, Ana María Vega-Letter, Catalina Fernández-O´Ryan, Carlos E. Irarrázabal, Fernando Figueroa, Rodrigo A. Fuentealba, Claudia Riedel, and Flavio Carrión. “Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cells Induce a Switch from Classical to Atypical Symptoms in Experimental Autoimmune Encephalomyelitis (EAE)”. Stem cell International. Received 13 November 2014; Revised 23 January 2015; Accepted 23 January 2015.

Keywords: MSc, IL17, EAE, Immunotherapy, Autoimmunity

Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.

Presentation Type: Oral Presentation

Topic: Immunotherapy

Citation: Kurte M, Luz-Crawford P, Vega-Letter A, Fernández C, Gauthier M, Moya I, Díaz C, Ruiz-Higgs D, Djouad F and Carrión F (2015). IL17RA knockout mesenchymal stem cells lose their immunosuppressive capacity and exerts deleterious effects on EAE mice. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00363

Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.

The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.

Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.

For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.

Received: 15 Apr 2015; Published Online: 15 Sep 2015.

* Correspondence: Dr. Flavio Carrión, Laboratorio de Inmunología/Universidad de los Andes, Centro de Investigaciones Biomedicas, Santiago, Las Condes, Chile, fcarrion@udd.cl