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Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis
Biologicals xxx (2017) 1e7
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Stem cells from human-exfoliated deciduous teeth reduce tissueinfiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis ~o a, Sandra B.R. Castro b, Danilo C. de Almeida a, Cristiano Rossato a, Wesley N. Branda b Carlos M.C. Maranduba , Niels O.S. Camara a, Jean P.S. Peron a, 1, Fernando S. Silva b, *, 1 a b
~o Paulo, Sa ~o Paulo, Brazil Universidade de Sa Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2017 Received in revised form 8 June 2017 Accepted 21 June 2017 Available online xxx
Stem cells from human exfoliated deciduous teeth (SHED) have great therapeutic potential and here, by the first time, we evaluated their immunomodulatory effect on experimental model of autoimmune encephalomyelitis (EAE). Specifically, we investigated the effect of SHED administration on clinical signs and cellular patterns in EAE model using Foxp3 GFP þ transgenic mice (C57Bl/6-Foxp3GFP). The results showed that SHED infusion ameliorated EAE clinical score with reduced number of infiltrating IFNgþCD8þ, IL-4þCD8þ, IFN-gþCD4þ and IL-4þCD4þ T cells into the central nervous system (CNS). In addition, we observed that SHED promoted a significant increase in CD4þFOXP3þ T cells population in the spleen of EAE-affected animals. Taken together, our results provide strong evidence that SHED can modulate peripherally the CD4þ T cell responses suggesting that SHED would be explored as part of cellular therapy in autoimmune diseases associated with CNS. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Dental pulp stem cells SHED Multiple sclerosis Experimental autoimmune encephalomyelitis EAE Immunomodulation
1. Introduction Experimental Autoimmune encephalomyelitis (EAE) is characterized by an autoimmune response against central nervous system (CNS) resident proteins such as the presence of infiltrating inflammatory cell, myelin defragmentation and neuronal degeneration [1]. Considering its similar clinical and cellular manifestation with multiple sclerosis (MS), EAE model can be useful for study and interpretation of molecular pattern in MS. In MS patients, accumulative lesions can be found in the spinal cord, optic nerve, or brainstem/cerebellum [2]; [3]. In addition, distinct clinical courses are also observed in MS patients: i) secondary-progressive, ii) primary-progressive, iii) progressive-relapsing and iv) relapsingeremitting (RRMS) type, which corresponds to 80% of the diagnosed MS patients. Sequelae and residual deficit upon recovery
* Corresponding author. Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, Minas Gerais, Brazil. E-mail address: [email protected] (F.S. Silva). 1 J.P.S and F.S.S share last authorship.
are usually seen, while relapses followed by complete recovery are rare [3e6]. Then, it is speculated that effective therapeutic strategies could improve these variable clinical conditions of MS [5,7,8]. The use of stem cells (SC) for the treatment of MS patients has been currently proposed such as an innovative therapeutic strategy [9e12]. In fact, it was observed that distinct SC subtypes can ameliorate clinical signs of EAE, for instance: i) human embryonic stem cell-derived mesenchymal stroma cells can reduce the clinical score and prevent demyelination in EAE [11]; ii) human bone marrow-derived mesenchymal stem cells induced Th2 cell polarization and improved EAE [13] (Bai et al., 2009); iii) human endometrialderived mesenchymal stem cells reduced Th17 cells in CNS [14]; iv) human adipose-derived stem cells also improved EAE clinical signs [15]. However, studies using stromal stem cells from human exfoliated deciduous teeth (SHED) for treatment and improvement of EAE have not been reported. SHED expresses Nestin, the marker for ectodermal and neuronal lineages, which allows them to differentiate into mesenchymal lineages (i.e. bone, adipose, muscle and cartilage) and nervous-like cells [16]. Additionally, we have previously demonstrated that
http://dx.doi.org/10.1016/j.biologicals.2017.06.007 1045-1056/© 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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SHED can induce a shift in the immune profile of monocyte-derived dendritic cells, which resulted in an increased frequency of regulatory T cells in vitro [17], suggesting that SHED could be applied specifically for treatment of autoimmune associated diseases. In this regard, we aimed to investigate the therapeutic effect of SHED in EAE model using Foxp3 GFP þ transgenic mice (C57Bl/6Foxp3GFP). The results showed that SHED infusion ameliorates EAE clinical signs and cellular pattern with reduced frequency of infiltrating inflammatory cells into the central nervous system (CNS), beyond a significant increase in CD4þFOXP3þ T cells in the spleen.
cage, with light/dark cycle of 12:12-hs and water and chow ad libitum. All animals were bred and experiments were performed at the Animal Care Facility of the Institute of Biomedical Sciences, ~o Paulo, Brazil. All experiments University of Sao Paulo (USP), Sa were performed in accordance with the guidelines of the Committee on Animal Research of USP; with approval of the Committee Animal Research of IPEN/USP (number 81/11). 2.4. EAE induction and SHED treatment
SHED were injected intraperitoneally (i.p.) one day before immunization (EAE induction) and two days post-immunization (p.i) in C57Bl/6-Foxp3þ Green fluorescent protein (GFP) mice (EAESHED group; n ¼ 4). Additionally, a group of animals were maintained without treatment (EAE group; n ¼ 4). On day 14 (peak of disease), the all animals were euthanized. Total splenocytes were evaluated by flow cytometry for FOXP3 expression (mean fluorescence intensity) and CD4 and CD8 cells markers. IFN-g and IL-4producing T cells were also evaluated in suspension cells of CNS.
For EAE induction, mice were subcutaneously injected with 200 ml (on the tail base) of the Freund's Adjuvant, Complete (CFA; v/ v) (Sigma, USA) emulsion containing 1 mg/ml of M. tuberculosis H37RA (Difco, USA) and 200 mg of myelin oligodendrocyte glycoprotein (MOG)35-55 (Proteimax, BRA). Mice also received two intraperitoneal doses of 200 ng of pertussis toxin (Sigma), before immunization and 48 hs later. All animals were scored daily, according to: 0, no disease; 1, limp tail; 2, weak/partially paralyzed hind legs; 3, completely paralyzed hind legs; 4, complete hind and partial front leg paralysis; and 5, total paralysis (moribund) [14]. Animals were divided into two groups: i) the treated group (EAESHED) was intraperitoneally treated with 1 106 SHED in 200 ml (PBS) one day before and two days post EAE induction; and ii) the control group (EAE) received PBS one day before and two days post EAE induction. All animals were euthanized on day 14 post EAE.
2.2. Stem cells isolation, culture and characterization
2.5. Isolation of CNS mononuclear cells
Cellular isolation, culture and characterization were performed as previously described in Silva et al. (2014) [17]. SHED were obtained from healthy volunteers after written consent and approval by the Institutional Review Board at Dentistry School/University of ~o Paulo (USP) (number 129/10). The pulp extraction process was Sa performed by tearing and culturing until adhesion and release of the cells to the culture dish. The cells (passage P7-P10) were cultivated with basal medium, consisting of F12 medium (Gibco, USA) supplemented with 15% serum Hyclone (Thermo, USA), antibiotic-antimycotic solution (100 U/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml amphotericin; Gibco), 2.5 mM Lglutamine and nonessential amino acids (Gibco). After culture, the cells were harvested by treatment with trypsin (Gibco), washed and suspended in phosphate-buffered saline (PBS); approximately 1 105 cells were incubated on ice for 20 min with conjugated monoclonal antibodies (1:100) against CD73, CD90, CD105, CD45, CD34, (BD Biosciences, USA). The acquisition was done in a FACSCanto II flow cytometer (BD Biosciences) and the analysis performed using the FlowJo software, Ver.7.2.4 (Tree Star, Ashland, OR, USA). SHED adipogenic and osteogenic differentiation were evaluated, as described by Pittenger et al. (1999) [18]. SHED were cultivated until total confluence and, then, induced to differentiation by the Mesenchymal Stem Cell Adipogenesis Kit and Mesenchymal Stem Cell Osteogenesis Kit respectively (Chemicon, USA), according to the manufacturer instructions. The medium was replaced every three to four days over a period of 21 days. Adipogenesis was determined by staining with oil red O, to verify neutral lipids accumulation in fat vacuoles, and osteogenesis was detected by accumulation of calcium compounds (by Alizarin Red staining). SHED were positive for CD73, CD90 and CD105 and negative for CD45 and CD34 markers; and were capable of differentiating into mesodermal lineages (Fig. 1).
Brain and spinal cords were harvested, macerated and maintained in 4 ml of DMEM (Gibco, USA) supplemented with collagenase D (250 mg/ml; Roche, USA) and incubated at 37 C with CO2 5%. 45 min later, the reaction was stopped with EDTA (2 mM) and suspensions were centrifuged (5 min, 450g, 4 C; Eppendorf, GER). Cells were then suspended in percoll 37% (GE Healthcare, UK) and gently laid over percoll 70% in 15 ml tubes. The tubes were centrifuged (20 min, 950g, 24 C) without brakes. Then, the interface containing mononuclear cells was collected, washed and centrifuged (5 min, 450g). Cellular suspensions were then suspended in PBS with bovine serum albumins (BSA) 0,2% for cell staining (Gibco).
2. Materials and methods 2.1. Experimental design
2.3. Animals Around 6-8-week-old C57Bl/6-Foxp3GFP mice (20-25 g) were used in this study. Mice were housed at four mice (per group) per
2.6. In vitro stimulation of CNS-Infiltrating cells Infiltrating mononuclear cells were seeded (5 105 cells/well) in 96 flat-bottom plates and stimulated with MOG35-55 peptide (50 mg/ml) with Brefeldin A (1:1000; Biolegend, USA) overnight, followed by PMA (50 ng/ml) and ionomycin (1 mg/ml) for 4 hs. Cells were then stained with anti-mouse CD4-APC or CD8-PerCP (Biolegend) for 30 min at 4 C. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences, USA) according to manufacturer protocol and incubated with antiIFN-g-FITC and anti-IL-4-PE (Biolegend). Cells were washed, and acquired by BD Accuri 6 (BD Biosciences, USA) equipment, and analyzed using C6 Software (BD Biosciences, USA). 2.7. Isolation and characterization of splenocytes On day 14 post EAE, the spleen was mechanically dissociated and homogenized in 5 ml of sterile DMEM (Gibco) to form a cell suspension. This suspension was centrifuged (Eppendorf) at 450 g for 5 min, resuspended in 9 mL of sterile ammonium chloride to lyse erythrocytes and centrifuged again. Total splenocytes were stained with CD4-APC and CD8-PE antibodies (Biolegend), as previously described. Cells were washed, fixed in paraformaldehyde 1%, acquired, counted and analyzed using flow cytometry (BD
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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Fig. 1. SHED express characteristic of MSC as predicted markers and differentiation potential into mesodermal lineages. (A) SHED was characterized by negative expression CD34 and CD45, and positive expression of mesenchymal markers CD90, CD73 and CD105. For prove stem cell plasticity of SHED, this cell was differentiated to: (B) osteogenic lineage and Alizarin Red was used for stained mineral deposition; (C) adipogenic lineage analyzed by neutral lipids accumulation in fat vacuoles stained with oil red O.
Accuri 6 and C6 Software; BD Biosciences).
SHED also demonstrated mesodermal differentiation potential for osteogenic (Fig. 1A) and adipogenic lineages (Fig. 1C).
2.8. Statistics 3.2. Clinical score GraphPad Prism 5.0 software was used for statistical analysis. Data represent two independent experiments: i) two-way ANOVA with Bonferroni post-tests that was run for score progression, and ii) Mann-Whitney test was used for flow cytometric analysis. Values of P < 0.05 were considered statistically significant. 3. Results
The clinical course of EAE was investigated during 14 days after immunization. EAE severity was recorded daily in both groups (treated and untreated with SHED). On day 14 post-immunization, the EAE group (only immunized mice) presented higher score than EAE-SHED group (immunized and SHED-treated mice) (i.e. 3.5 ± 0.5 vs 2.0 ± 1.2, respectively), suggesting that SHED can had conferred a protection against EAE (Fig. 2).
3.1. Morphological characterization of SHED SHED are known to share many features with bone marrowderived MSC, in this study SHED presented classical surface MSCs profile such as positive cells for CD73, CD90 and CD105 and negative cells for hematopoietic markers: CD34 and CD45 (Fig. 1A).
3.3. SHED treatment diminished the number of IL-4 and IFN-gexpressing CD4þ and CD8þ T cells in the CNS After verify an evident clinical protection in EAE after SHED infusion, we investigated whether SHED treatment could alter
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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number of IFN-gþCD4þ and IL-4þCD4þ T cells in the EAE-SHED group (Fig. 3B and C). Conversely, although a higher frequency of IL-4 þ CD4 þ T cells was observed, the frequency of IFN-gþ- and IL4þ-expressing CD4þ cells was not significantly altered among treated and untreated groups (Fig. 3E and F). After, we pointed up at CD8þ cells population and found similar findings at those observed in CD4þ cells (Fig. 4). The absolute numbers of total CD8þ cells, IFNgþCD8þ and IL-4þCD8þ T cells in the CNS were all lower at EAESHED group than EAE control group (Fig. 4B and C). Again, no difference was observed in frequencies of CD8þ, IFN-gþCD8þ and IL4þCD8þ T cells between EAE-SHED and EAE groups (Fig. 4E and F). 3.4. SHED treatment increased FOXP3 expression in CD4þ cells in the spleen
Fig. 2. Clinical score of C57Bl/6 mice immunized with MOG35-55 peptide. The clinical signs were registered from day 0 to day 14 post-immunization, when the mice were euthanized (arrow). EAE-SHED group was treated with 1 106 cells in 200 mL (PBS) intraperitoneally injected one day before immunization and two days p.i; the control group received PBS injection (EAE). Bars represent mean ± SEM of five mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
inflammatory cellular infiltration during EAE progression. The total number of CNS mononuclear cells showed significant reduction in EAE-SHED group in comparison with EAE group (6,1 ± 1,4 105 cells vs 13,3 ± 0,6 105 cells, respectively; p 0,05). Similarly, flow cytometry analysis of infiltrating mononuclear cells detected a decreasing in CNS CD4þ cells (frequency and absolute number) in EAE-SHED group when compared to EAE group (Fig. 3A,D). Moreover, we also observed a reduction in absolute
In order to understand the possible mechanism of immunomodulation of SHED in EAE progression, we searched for peripheral immunomodulation in the spleen of these EAE-affected animals. Thus, the splenocytes analysis showed that the absolute number for CD4þ or Foxp3þCD4þ cells were unaltered (Fig. 5A and B), however, the FOXP3 expression in regulatory T cells (Foxp3þCD4þ) was highly increased in SHED-treated EAE mice when compared to EAE control group (Fig. 5C). These findings indicate, in part, an immunoregulatory role of SHED in the EAE attenuation via peripheral immunomodulation. 4. Discussion Stem cell-based therapies have been considered an innovative and alternative treatment for autoimmune diseases. Specially in this study, we demonstrated, for the first time, that SHED can be a promisor treatment to improve EAE functional and clinical
Fig. 3. Numbers and proportion of infiltrating CD4þ cells in CNS. CD4þ (A,D), IFN-gþCD4þ (B,E) and IL-4þCD4þ (C,F) in mononuclear cells isolated from the CNS stimulated in vitro. The mononuclear cells were isolated from C57Bl/6Foxp3-GFP mice immunized with MOG35-55 peptide treated with SHED (EAE-SHED group) or not (EAE group). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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Fig. 4. Numbers and proportion of infiltrating CD8þ cells in CNS. CD8þ (A,D), IFN-gþCD8þ (B,E) and IL-4þCD8þ (C,F) in mononuclear cells isolated from the CNS stimulated in vitro. Mononuclear cells were isolated from C57Bl/6Foxp3-GFP mice immunized with MOG35-55 peptide treated with SHED (EAE-SHED group) or not (EAE group). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
Fig. 5. Numbers and proportion of cells expressing FOXP3 in the spleen. CD4þ (A), Foxp3GFPþCD4þ (B) numbers and Mean Fluorescence Intensity of Foxp3GFPþCD4þ in total splenocytes (C). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
parameters. We observed that SHED infusion decreased CD4þ and CD8þ T cell absolute number in CNS of EAE-affected mice. Further, we also documented that SHED promoted the reduction of total inflammatory cell numbers in the spleen, with a correspondent increasing of FOXP3 expression in CD4þ T cells. In this context, we can speculate that SHED would be able to suppresses immune response in EAE model, corroborating with previous reports involving stem cell treatments [16,19,20]. In EAE and MS, CD4þ T cell infiltration has been documented as the main factor for damage progression in the CNS [21]. Between these inflammatory cells, the Th1 cells (IFN-gþCD4þ) have been considered to play an important role in EAE development, principally by producing IFN-g [22]. In this sense, infiltrating T cells secrete large amounts of IFN-g which will activate resident glial cells during EAE onset [22,23]. IFN-g can enhances proinflammatory cytokines (IL-1b, IL-12) and MHC class II molecules via monocytes and macrophages activation [24]. On the other hand, it was shown that mesenchymal stem cells over-expressing IL-4
improve EAE score reducing demyelination in the CNS. This finding suggests that a shift from a pro-inflammatory (Th1) to an antiinflammatory (Th2) profile is important for an EAE improvement [25,26]. In our study, we found a slight increase in IL-4þCD4þ T cell frequency in SHED-treated animals, considering that IFN-gþCD4þ and IL-4þCD4þ T cells had a prominent reduction in the CNS. All together, these data corroborate the results of Wang et al. (2014) which associated the reduction in inflammatory cells with the decreasing of EAE severity [11]. In the last two decades, CD8þ T cells have gained special attention in EAE when researchers have isolated Myelin Basic Protein (MBP)-specific CD8þ T cells from MS patients [27,28] and adoptively transferred these cells into wild-type mice. In this cellular transplant, it was demonstrated that mice developed similar disease pattern as seen in MS patients [29,30]. Therefore, activated CD8þ T cells, are likely to be expanded in response to myelin antigen which is cross-presented by dendritic cells, macrophages, microglial or others antigen-presenting cells (APCs). The
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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authors also have demonstrated that CD8þ T cells can directly attack oligodendrocytes expressing MHC class I and myelin epitopes [26,31]. In MS patients, the information is limited due the accessibility of living cells in CNS, however, MBP-specific CD8þ T cells isolated from the peripheral blood of MS patients showed higher expression of pro-inflammatory cytokines (e.g. IFN-g and TNF-a), thereby determined as Tc1 phenotype [29,32]. In our set of experiments, CD8þ and CD4þ T cell was reduced in the CNS after SHED treatment. Concomitantly, total IFN-gþCD8þ and IL-4þCD8þ T cell numbers were also diminished in SHED-injected group. Similarly, one report showed that human embryonic stem cell-derived mesenchymal stromal cells reduced clinical signs and prevented demyelination in EAE with less CD4þ and CD8þ T cell infiltrate in the CNS [11]. In our study, SHED also suppressed CD8þ and CD4þ T cell subtypes infiltration in CNS highlighting that SHED is capable of modulating T cells function. Peripheral activation of myelin-specific T cells has also been evaluated. Studies evidenced that activation of APCs can be triggered by soluble myelin antigens drained from CNS to peripheral lymph nodes [26]. Peripherally, regulatory T cells (Tregs) suppress immune responses considering that FOXP3 expression is essential to establish functional Tregs [33e36]. FOXP3 can be expressed after TCR stimulation, which has been correlated with hyporesponsiveness of activated T cells [37e40]. In our findings, although the frequency of Foxp3þCD4þ T cells did not change in spleen after SHED treatment, the FOXP3 expression (intensity) in splenocytes CD4þ was significantly improved in SHED-treated animals when compared to EAE control group. In line with these findings, it was found that peripheral blood from MS patients had a reduced frequency of FOXP3-positive T cells and reduced expression of FOXP3 at the single-cell level [39]. Moreover, it was also detected elevated FOXP3 expression in cells derived from spinal cord and in the spleen of ethyl-eicosapentaenoic acid-treated EAE animals in comparison to the control group [41,42]. In the light of this evidence, we have previously demonstrated that SHED could generates Tregs by modulating dendritic cells in vitro [17]. Thus, with this perspective, our data suggests that SHED is capable of modulating the immune system also in vivo through an immune cross-talk among stem cells, APCs and T cells in the periphery systems. Hence, we believe that SHED could release a myriad of immunosuppressive molecules (i.e. TGF-b, NO, IDO, TSG6, prostaglandin E2, IL-1 receptor antagonist, IL-10 and an antagonistic variant of the chemokine CCL2) which will suppress immune response in EAE. Consequently, a change from pro-inflammatory to an antiinflammatory profile can improves not only regulatory T cells, but also Th2 cells, regulatory dendritic cells and M2 macrophages, suggesting that SHED regulates the overall immune response (see to review: [12,43,44]). 5. Conclusions Thus, our study showed that SHED were able to suppress EAE clinical score. In addition, this improvement occurred by decreasing CD4þ and CD8þ T cell infiltrates in CNS. Furthermore, our results provide strong evidence that SHED can modulates peripherally the CD4þ T cell responses suggesting that these cells could be explored as part of cellular therapy in autoimmune diseases associated with CNS, which will elucidate possible and intriguing mechanisms of immune cells regulation in central and peripheral encephalomyelitis. Funding This work was supported by Fapesp (grant number 2011/187032); Fapemig (grant number APQ-00496-14).
Ethical approval Stem cells from human exfoliated deciduous teeth (SHED) were obtained after approval by the Institutional Review Board at ~o Paulo (USP) (number 129/10). Dentistry School/University of Sa Animals were used after approval by Committee Animal Research of IPEN/USP (number 81/11). Informed consent Stem cells from human exfoliated deciduous teeth (SHED) were obtained from healthy volunteers after written consent. Authors' contributions CR, WB and DA participated in the immunological experiments (in vivo and in vitro). CR, SC and FS performed the statistical analysis and drafted and corrected the manuscript. DA, CM and NC isolated and characterized stem cells. JP and FS share last authorship, conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Conflict of interest All authors declare that there is no conflict of interest. Appendix A. Supplementary data Supplementary data related to this chapter can be found at http://dx.doi.org/10.1016/j.biologicals.2017.06.007. References [1] Rodriguez M. Effectors of demyelination and remyelination in the CNS: implications for multiple sclerosis. Brain Pathol 2007;17(2):219e29. [2] Compston A, Coles A. Multiple sclerosis. Lancet 2002;359(9313):1221e31. [3] Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 1996;46(6):907e11. [4] Simmons SB, Pierson ER, Lee SY, Goverman JM. Modeling the heterogeneity of multiple sclerosis in animals. Trends Immunol 2013;34(8):410e22. [5] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005;23:683e747. [6] Weinshenker BG, Bass B, Rice GP, Noseworthy J, Carriere W, Baskerville J, et al. The natural history of multiple sclerosis: a geographically based study 2. Predictive value of the early clinical course. Brain 1989;112(6):1419e28. [7] Milo R, Kahana E. Multiple sclerosis: geoepidemiology, genetics and the environment. Autoimmun Rev 2010;9(5):387e94. [8] Steinman L. Multiple sclerosis: a two-stage disease. Nat Immunol 2001;2(9): 762e4. [9] Karussis D, Karageorgiou C, Vaknin-Dembinsky A, Gowda-Kurkalli B, Gomori JM, Kassis I, et al. Safety and immunological effects of mesenchymal stem cell transplantation in patients with multiple sclerosis and amyotrophic lateral sclerosis. Arch Neurol 2010;67(10):1187e94. [10] Matysiak M, Orlowski W, Fortak-Michalska M, Jurewicz A, Selmaj K. Immunoregulatory function of bone marrow mesenchymal stem cells in EAE depends on their differentiation state and secretion of PGE2. J Neuroimmunol 2011;233(1e2):106e11. [11] Wang X, Kimbrel EA, Ijichi K. Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Rep 2014;3(1):115e30. [12] Wang Y, Chen X, Cao W, Shi Y. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol 2014;15(11):1009e16. [13] Bai L, Lennon DP, Eaton V, Maier K, Caplan AI, Miller SD, et al. Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia 2009;57(11):1192e203. [14] Peron JP, Jazedje T, Brand~ ao WN, Perin PM, Maluf M, Evangelista LP, et al. Human endometrial-derived mesenchymal stem cells suppress inflammation in the central nervous system of EAE mice. Stem Cell Rev 2012;8(3):940e52. [15] Scruggs BA, Semon JA, Zhang X, Zhang S, Bowles AC, Pandey AC, et al. Age of the donor reduces the ability of human adipose-derived stem cells to alleviate symptoms in the experimental autoimmune encephalomyelitis mouse model.
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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Stem cells from human-exfoliated deciduous teeth reduce tissueinfiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis ~o a, Sandra B.R. Castro b, Danilo C. de Almeida a, Cristiano Rossato a, Wesley N. Branda b Carlos M.C. Maranduba , Niels O.S. Camara a, Jean P.S. Peron a, 1, Fernando S. Silva b, *, 1 a b
~o Paulo, Sa ~o Paulo, Brazil Universidade de Sa Universidade Federal de Juiz de Fora, Juiz de Fora, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2017 Received in revised form 8 June 2017 Accepted 21 June 2017 Available online xxx
Stem cells from human exfoliated deciduous teeth (SHED) have great therapeutic potential and here, by the first time, we evaluated their immunomodulatory effect on experimental model of autoimmune encephalomyelitis (EAE). Specifically, we investigated the effect of SHED administration on clinical signs and cellular patterns in EAE model using Foxp3 GFP þ transgenic mice (C57Bl/6-Foxp3GFP). The results showed that SHED infusion ameliorated EAE clinical score with reduced number of infiltrating IFNgþCD8þ, IL-4þCD8þ, IFN-gþCD4þ and IL-4þCD4þ T cells into the central nervous system (CNS). In addition, we observed that SHED promoted a significant increase in CD4þFOXP3þ T cells population in the spleen of EAE-affected animals. Taken together, our results provide strong evidence that SHED can modulate peripherally the CD4þ T cell responses suggesting that SHED would be explored as part of cellular therapy in autoimmune diseases associated with CNS. © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Keywords: Dental pulp stem cells SHED Multiple sclerosis Experimental autoimmune encephalomyelitis EAE Immunomodulation
1. Introduction Experimental Autoimmune encephalomyelitis (EAE) is characterized by an autoimmune response against central nervous system (CNS) resident proteins such as the presence of infiltrating inflammatory cell, myelin defragmentation and neuronal degeneration [1]. Considering its similar clinical and cellular manifestation with multiple sclerosis (MS), EAE model can be useful for study and interpretation of molecular pattern in MS. In MS patients, accumulative lesions can be found in the spinal cord, optic nerve, or brainstem/cerebellum [2]; [3]. In addition, distinct clinical courses are also observed in MS patients: i) secondary-progressive, ii) primary-progressive, iii) progressive-relapsing and iv) relapsingeremitting (RRMS) type, which corresponds to 80% of the diagnosed MS patients. Sequelae and residual deficit upon recovery
* Corresponding author. Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, Minas Gerais, Brazil. E-mail address: [email protected] (F.S. Silva). 1 J.P.S and F.S.S share last authorship.
are usually seen, while relapses followed by complete recovery are rare [3e6]. Then, it is speculated that effective therapeutic strategies could improve these variable clinical conditions of MS [5,7,8]. The use of stem cells (SC) for the treatment of MS patients has been currently proposed such as an innovative therapeutic strategy [9e12]. In fact, it was observed that distinct SC subtypes can ameliorate clinical signs of EAE, for instance: i) human embryonic stem cell-derived mesenchymal stroma cells can reduce the clinical score and prevent demyelination in EAE [11]; ii) human bone marrow-derived mesenchymal stem cells induced Th2 cell polarization and improved EAE [13] (Bai et al., 2009); iii) human endometrialderived mesenchymal stem cells reduced Th17 cells in CNS [14]; iv) human adipose-derived stem cells also improved EAE clinical signs [15]. However, studies using stromal stem cells from human exfoliated deciduous teeth (SHED) for treatment and improvement of EAE have not been reported. SHED expresses Nestin, the marker for ectodermal and neuronal lineages, which allows them to differentiate into mesenchymal lineages (i.e. bone, adipose, muscle and cartilage) and nervous-like cells [16]. Additionally, we have previously demonstrated that
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Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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SHED can induce a shift in the immune profile of monocyte-derived dendritic cells, which resulted in an increased frequency of regulatory T cells in vitro [17], suggesting that SHED could be applied specifically for treatment of autoimmune associated diseases. In this regard, we aimed to investigate the therapeutic effect of SHED in EAE model using Foxp3 GFP þ transgenic mice (C57Bl/6Foxp3GFP). The results showed that SHED infusion ameliorates EAE clinical signs and cellular pattern with reduced frequency of infiltrating inflammatory cells into the central nervous system (CNS), beyond a significant increase in CD4þFOXP3þ T cells in the spleen.
cage, with light/dark cycle of 12:12-hs and water and chow ad libitum. All animals were bred and experiments were performed at the Animal Care Facility of the Institute of Biomedical Sciences, ~o Paulo, Brazil. All experiments University of Sao Paulo (USP), Sa were performed in accordance with the guidelines of the Committee on Animal Research of USP; with approval of the Committee Animal Research of IPEN/USP (number 81/11). 2.4. EAE induction and SHED treatment
SHED were injected intraperitoneally (i.p.) one day before immunization (EAE induction) and two days post-immunization (p.i) in C57Bl/6-Foxp3þ Green fluorescent protein (GFP) mice (EAESHED group; n ¼ 4). Additionally, a group of animals were maintained without treatment (EAE group; n ¼ 4). On day 14 (peak of disease), the all animals were euthanized. Total splenocytes were evaluated by flow cytometry for FOXP3 expression (mean fluorescence intensity) and CD4 and CD8 cells markers. IFN-g and IL-4producing T cells were also evaluated in suspension cells of CNS.
For EAE induction, mice were subcutaneously injected with 200 ml (on the tail base) of the Freund's Adjuvant, Complete (CFA; v/ v) (Sigma, USA) emulsion containing 1 mg/ml of M. tuberculosis H37RA (Difco, USA) and 200 mg of myelin oligodendrocyte glycoprotein (MOG)35-55 (Proteimax, BRA). Mice also received two intraperitoneal doses of 200 ng of pertussis toxin (Sigma), before immunization and 48 hs later. All animals were scored daily, according to: 0, no disease; 1, limp tail; 2, weak/partially paralyzed hind legs; 3, completely paralyzed hind legs; 4, complete hind and partial front leg paralysis; and 5, total paralysis (moribund) [14]. Animals were divided into two groups: i) the treated group (EAESHED) was intraperitoneally treated with 1 106 SHED in 200 ml (PBS) one day before and two days post EAE induction; and ii) the control group (EAE) received PBS one day before and two days post EAE induction. All animals were euthanized on day 14 post EAE.
2.2. Stem cells isolation, culture and characterization
2.5. Isolation of CNS mononuclear cells
Cellular isolation, culture and characterization were performed as previously described in Silva et al. (2014) [17]. SHED were obtained from healthy volunteers after written consent and approval by the Institutional Review Board at Dentistry School/University of ~o Paulo (USP) (number 129/10). The pulp extraction process was Sa performed by tearing and culturing until adhesion and release of the cells to the culture dish. The cells (passage P7-P10) were cultivated with basal medium, consisting of F12 medium (Gibco, USA) supplemented with 15% serum Hyclone (Thermo, USA), antibiotic-antimycotic solution (100 U/ml penicillin, 100 mg/ml streptomycin, and 25 mg/ml amphotericin; Gibco), 2.5 mM Lglutamine and nonessential amino acids (Gibco). After culture, the cells were harvested by treatment with trypsin (Gibco), washed and suspended in phosphate-buffered saline (PBS); approximately 1 105 cells were incubated on ice for 20 min with conjugated monoclonal antibodies (1:100) against CD73, CD90, CD105, CD45, CD34, (BD Biosciences, USA). The acquisition was done in a FACSCanto II flow cytometer (BD Biosciences) and the analysis performed using the FlowJo software, Ver.7.2.4 (Tree Star, Ashland, OR, USA). SHED adipogenic and osteogenic differentiation were evaluated, as described by Pittenger et al. (1999) [18]. SHED were cultivated until total confluence and, then, induced to differentiation by the Mesenchymal Stem Cell Adipogenesis Kit and Mesenchymal Stem Cell Osteogenesis Kit respectively (Chemicon, USA), according to the manufacturer instructions. The medium was replaced every three to four days over a period of 21 days. Adipogenesis was determined by staining with oil red O, to verify neutral lipids accumulation in fat vacuoles, and osteogenesis was detected by accumulation of calcium compounds (by Alizarin Red staining). SHED were positive for CD73, CD90 and CD105 and negative for CD45 and CD34 markers; and were capable of differentiating into mesodermal lineages (Fig. 1).
Brain and spinal cords were harvested, macerated and maintained in 4 ml of DMEM (Gibco, USA) supplemented with collagenase D (250 mg/ml; Roche, USA) and incubated at 37 C with CO2 5%. 45 min later, the reaction was stopped with EDTA (2 mM) and suspensions were centrifuged (5 min, 450g, 4 C; Eppendorf, GER). Cells were then suspended in percoll 37% (GE Healthcare, UK) and gently laid over percoll 70% in 15 ml tubes. The tubes were centrifuged (20 min, 950g, 24 C) without brakes. Then, the interface containing mononuclear cells was collected, washed and centrifuged (5 min, 450g). Cellular suspensions were then suspended in PBS with bovine serum albumins (BSA) 0,2% for cell staining (Gibco).
2. Materials and methods 2.1. Experimental design
2.3. Animals Around 6-8-week-old C57Bl/6-Foxp3GFP mice (20-25 g) were used in this study. Mice were housed at four mice (per group) per
2.6. In vitro stimulation of CNS-Infiltrating cells Infiltrating mononuclear cells were seeded (5 105 cells/well) in 96 flat-bottom plates and stimulated with MOG35-55 peptide (50 mg/ml) with Brefeldin A (1:1000; Biolegend, USA) overnight, followed by PMA (50 ng/ml) and ionomycin (1 mg/ml) for 4 hs. Cells were then stained with anti-mouse CD4-APC or CD8-PerCP (Biolegend) for 30 min at 4 C. For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm kit (BD Biosciences, USA) according to manufacturer protocol and incubated with antiIFN-g-FITC and anti-IL-4-PE (Biolegend). Cells were washed, and acquired by BD Accuri 6 (BD Biosciences, USA) equipment, and analyzed using C6 Software (BD Biosciences, USA). 2.7. Isolation and characterization of splenocytes On day 14 post EAE, the spleen was mechanically dissociated and homogenized in 5 ml of sterile DMEM (Gibco) to form a cell suspension. This suspension was centrifuged (Eppendorf) at 450 g for 5 min, resuspended in 9 mL of sterile ammonium chloride to lyse erythrocytes and centrifuged again. Total splenocytes were stained with CD4-APC and CD8-PE antibodies (Biolegend), as previously described. Cells were washed, fixed in paraformaldehyde 1%, acquired, counted and analyzed using flow cytometry (BD
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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Fig. 1. SHED express characteristic of MSC as predicted markers and differentiation potential into mesodermal lineages. (A) SHED was characterized by negative expression CD34 and CD45, and positive expression of mesenchymal markers CD90, CD73 and CD105. For prove stem cell plasticity of SHED, this cell was differentiated to: (B) osteogenic lineage and Alizarin Red was used for stained mineral deposition; (C) adipogenic lineage analyzed by neutral lipids accumulation in fat vacuoles stained with oil red O.
Accuri 6 and C6 Software; BD Biosciences).
SHED also demonstrated mesodermal differentiation potential for osteogenic (Fig. 1A) and adipogenic lineages (Fig. 1C).
2.8. Statistics 3.2. Clinical score GraphPad Prism 5.0 software was used for statistical analysis. Data represent two independent experiments: i) two-way ANOVA with Bonferroni post-tests that was run for score progression, and ii) Mann-Whitney test was used for flow cytometric analysis. Values of P < 0.05 were considered statistically significant. 3. Results
The clinical course of EAE was investigated during 14 days after immunization. EAE severity was recorded daily in both groups (treated and untreated with SHED). On day 14 post-immunization, the EAE group (only immunized mice) presented higher score than EAE-SHED group (immunized and SHED-treated mice) (i.e. 3.5 ± 0.5 vs 2.0 ± 1.2, respectively), suggesting that SHED can had conferred a protection against EAE (Fig. 2).
3.1. Morphological characterization of SHED SHED are known to share many features with bone marrowderived MSC, in this study SHED presented classical surface MSCs profile such as positive cells for CD73, CD90 and CD105 and negative cells for hematopoietic markers: CD34 and CD45 (Fig. 1A).
3.3. SHED treatment diminished the number of IL-4 and IFN-gexpressing CD4þ and CD8þ T cells in the CNS After verify an evident clinical protection in EAE after SHED infusion, we investigated whether SHED treatment could alter
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number of IFN-gþCD4þ and IL-4þCD4þ T cells in the EAE-SHED group (Fig. 3B and C). Conversely, although a higher frequency of IL-4 þ CD4 þ T cells was observed, the frequency of IFN-gþ- and IL4þ-expressing CD4þ cells was not significantly altered among treated and untreated groups (Fig. 3E and F). After, we pointed up at CD8þ cells population and found similar findings at those observed in CD4þ cells (Fig. 4). The absolute numbers of total CD8þ cells, IFNgþCD8þ and IL-4þCD8þ T cells in the CNS were all lower at EAESHED group than EAE control group (Fig. 4B and C). Again, no difference was observed in frequencies of CD8þ, IFN-gþCD8þ and IL4þCD8þ T cells between EAE-SHED and EAE groups (Fig. 4E and F). 3.4. SHED treatment increased FOXP3 expression in CD4þ cells in the spleen
Fig. 2. Clinical score of C57Bl/6 mice immunized with MOG35-55 peptide. The clinical signs were registered from day 0 to day 14 post-immunization, when the mice were euthanized (arrow). EAE-SHED group was treated with 1 106 cells in 200 mL (PBS) intraperitoneally injected one day before immunization and two days p.i; the control group received PBS injection (EAE). Bars represent mean ± SEM of five mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
inflammatory cellular infiltration during EAE progression. The total number of CNS mononuclear cells showed significant reduction in EAE-SHED group in comparison with EAE group (6,1 ± 1,4 105 cells vs 13,3 ± 0,6 105 cells, respectively; p 0,05). Similarly, flow cytometry analysis of infiltrating mononuclear cells detected a decreasing in CNS CD4þ cells (frequency and absolute number) in EAE-SHED group when compared to EAE group (Fig. 3A,D). Moreover, we also observed a reduction in absolute
In order to understand the possible mechanism of immunomodulation of SHED in EAE progression, we searched for peripheral immunomodulation in the spleen of these EAE-affected animals. Thus, the splenocytes analysis showed that the absolute number for CD4þ or Foxp3þCD4þ cells were unaltered (Fig. 5A and B), however, the FOXP3 expression in regulatory T cells (Foxp3þCD4þ) was highly increased in SHED-treated EAE mice when compared to EAE control group (Fig. 5C). These findings indicate, in part, an immunoregulatory role of SHED in the EAE attenuation via peripheral immunomodulation. 4. Discussion Stem cell-based therapies have been considered an innovative and alternative treatment for autoimmune diseases. Specially in this study, we demonstrated, for the first time, that SHED can be a promisor treatment to improve EAE functional and clinical
Fig. 3. Numbers and proportion of infiltrating CD4þ cells in CNS. CD4þ (A,D), IFN-gþCD4þ (B,E) and IL-4þCD4þ (C,F) in mononuclear cells isolated from the CNS stimulated in vitro. The mononuclear cells were isolated from C57Bl/6Foxp3-GFP mice immunized with MOG35-55 peptide treated with SHED (EAE-SHED group) or not (EAE group). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
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Fig. 4. Numbers and proportion of infiltrating CD8þ cells in CNS. CD8þ (A,D), IFN-gþCD8þ (B,E) and IL-4þCD8þ (C,F) in mononuclear cells isolated from the CNS stimulated in vitro. Mononuclear cells were isolated from C57Bl/6Foxp3-GFP mice immunized with MOG35-55 peptide treated with SHED (EAE-SHED group) or not (EAE group). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
Fig. 5. Numbers and proportion of cells expressing FOXP3 in the spleen. CD4þ (A), Foxp3GFPþCD4þ (B) numbers and Mean Fluorescence Intensity of Foxp3GFPþCD4þ in total splenocytes (C). Bars represent mean ± SEM of four mice/group; * ¼ p < 0.05 EAE versus EAE-SHED.
parameters. We observed that SHED infusion decreased CD4þ and CD8þ T cell absolute number in CNS of EAE-affected mice. Further, we also documented that SHED promoted the reduction of total inflammatory cell numbers in the spleen, with a correspondent increasing of FOXP3 expression in CD4þ T cells. In this context, we can speculate that SHED would be able to suppresses immune response in EAE model, corroborating with previous reports involving stem cell treatments [16,19,20]. In EAE and MS, CD4þ T cell infiltration has been documented as the main factor for damage progression in the CNS [21]. Between these inflammatory cells, the Th1 cells (IFN-gþCD4þ) have been considered to play an important role in EAE development, principally by producing IFN-g [22]. In this sense, infiltrating T cells secrete large amounts of IFN-g which will activate resident glial cells during EAE onset [22,23]. IFN-g can enhances proinflammatory cytokines (IL-1b, IL-12) and MHC class II molecules via monocytes and macrophages activation [24]. On the other hand, it was shown that mesenchymal stem cells over-expressing IL-4
improve EAE score reducing demyelination in the CNS. This finding suggests that a shift from a pro-inflammatory (Th1) to an antiinflammatory (Th2) profile is important for an EAE improvement [25,26]. In our study, we found a slight increase in IL-4þCD4þ T cell frequency in SHED-treated animals, considering that IFN-gþCD4þ and IL-4þCD4þ T cells had a prominent reduction in the CNS. All together, these data corroborate the results of Wang et al. (2014) which associated the reduction in inflammatory cells with the decreasing of EAE severity [11]. In the last two decades, CD8þ T cells have gained special attention in EAE when researchers have isolated Myelin Basic Protein (MBP)-specific CD8þ T cells from MS patients [27,28] and adoptively transferred these cells into wild-type mice. In this cellular transplant, it was demonstrated that mice developed similar disease pattern as seen in MS patients [29,30]. Therefore, activated CD8þ T cells, are likely to be expanded in response to myelin antigen which is cross-presented by dendritic cells, macrophages, microglial or others antigen-presenting cells (APCs). The
Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007
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authors also have demonstrated that CD8þ T cells can directly attack oligodendrocytes expressing MHC class I and myelin epitopes [26,31]. In MS patients, the information is limited due the accessibility of living cells in CNS, however, MBP-specific CD8þ T cells isolated from the peripheral blood of MS patients showed higher expression of pro-inflammatory cytokines (e.g. IFN-g and TNF-a), thereby determined as Tc1 phenotype [29,32]. In our set of experiments, CD8þ and CD4þ T cell was reduced in the CNS after SHED treatment. Concomitantly, total IFN-gþCD8þ and IL-4þCD8þ T cell numbers were also diminished in SHED-injected group. Similarly, one report showed that human embryonic stem cell-derived mesenchymal stromal cells reduced clinical signs and prevented demyelination in EAE with less CD4þ and CD8þ T cell infiltrate in the CNS [11]. In our study, SHED also suppressed CD8þ and CD4þ T cell subtypes infiltration in CNS highlighting that SHED is capable of modulating T cells function. Peripheral activation of myelin-specific T cells has also been evaluated. Studies evidenced that activation of APCs can be triggered by soluble myelin antigens drained from CNS to peripheral lymph nodes [26]. Peripherally, regulatory T cells (Tregs) suppress immune responses considering that FOXP3 expression is essential to establish functional Tregs [33e36]. FOXP3 can be expressed after TCR stimulation, which has been correlated with hyporesponsiveness of activated T cells [37e40]. In our findings, although the frequency of Foxp3þCD4þ T cells did not change in spleen after SHED treatment, the FOXP3 expression (intensity) in splenocytes CD4þ was significantly improved in SHED-treated animals when compared to EAE control group. In line with these findings, it was found that peripheral blood from MS patients had a reduced frequency of FOXP3-positive T cells and reduced expression of FOXP3 at the single-cell level [39]. Moreover, it was also detected elevated FOXP3 expression in cells derived from spinal cord and in the spleen of ethyl-eicosapentaenoic acid-treated EAE animals in comparison to the control group [41,42]. In the light of this evidence, we have previously demonstrated that SHED could generates Tregs by modulating dendritic cells in vitro [17]. Thus, with this perspective, our data suggests that SHED is capable of modulating the immune system also in vivo through an immune cross-talk among stem cells, APCs and T cells in the periphery systems. Hence, we believe that SHED could release a myriad of immunosuppressive molecules (i.e. TGF-b, NO, IDO, TSG6, prostaglandin E2, IL-1 receptor antagonist, IL-10 and an antagonistic variant of the chemokine CCL2) which will suppress immune response in EAE. Consequently, a change from pro-inflammatory to an antiinflammatory profile can improves not only regulatory T cells, but also Th2 cells, regulatory dendritic cells and M2 macrophages, suggesting that SHED regulates the overall immune response (see to review: [12,43,44]). 5. Conclusions Thus, our study showed that SHED were able to suppress EAE clinical score. In addition, this improvement occurred by decreasing CD4þ and CD8þ T cell infiltrates in CNS. Furthermore, our results provide strong evidence that SHED can modulates peripherally the CD4þ T cell responses suggesting that these cells could be explored as part of cellular therapy in autoimmune diseases associated with CNS, which will elucidate possible and intriguing mechanisms of immune cells regulation in central and peripheral encephalomyelitis. Funding This work was supported by Fapesp (grant number 2011/187032); Fapemig (grant number APQ-00496-14).
Ethical approval Stem cells from human exfoliated deciduous teeth (SHED) were obtained after approval by the Institutional Review Board at ~o Paulo (USP) (number 129/10). Dentistry School/University of Sa Animals were used after approval by Committee Animal Research of IPEN/USP (number 81/11). Informed consent Stem cells from human exfoliated deciduous teeth (SHED) were obtained from healthy volunteers after written consent. Authors' contributions CR, WB and DA participated in the immunological experiments (in vivo and in vitro). CR, SC and FS performed the statistical analysis and drafted and corrected the manuscript. DA, CM and NC isolated and characterized stem cells. JP and FS share last authorship, conceived of the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Conflict of interest All authors declare that there is no conflict of interest. Appendix A. Supplementary data Supplementary data related to this chapter can be found at http://dx.doi.org/10.1016/j.biologicals.2017.06.007. References [1] Rodriguez M. Effectors of demyelination and remyelination in the CNS: implications for multiple sclerosis. Brain Pathol 2007;17(2):219e29. [2] Compston A, Coles A. Multiple sclerosis. Lancet 2002;359(9313):1221e31. [3] Lublin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of an international survey. Neurology 1996;46(6):907e11. [4] Simmons SB, Pierson ER, Lee SY, Goverman JM. Modeling the heterogeneity of multiple sclerosis in animals. Trends Immunol 2013;34(8):410e22. [5] Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005;23:683e747. [6] Weinshenker BG, Bass B, Rice GP, Noseworthy J, Carriere W, Baskerville J, et al. The natural history of multiple sclerosis: a geographically based study 2. Predictive value of the early clinical course. Brain 1989;112(6):1419e28. [7] Milo R, Kahana E. Multiple sclerosis: geoepidemiology, genetics and the environment. Autoimmun Rev 2010;9(5):387e94. [8] Steinman L. Multiple sclerosis: a two-stage disease. Nat Immunol 2001;2(9): 762e4. [9] Karussis D, Karageorgiou C, Vaknin-Dembinsky A, Gowda-Kurkalli B, Gomori JM, Kassis I, et al. Safety and immunological effects of mesenchymal stem cell transplantation in patients with multiple sclerosis and amyotrophic lateral sclerosis. Arch Neurol 2010;67(10):1187e94. [10] Matysiak M, Orlowski W, Fortak-Michalska M, Jurewicz A, Selmaj K. Immunoregulatory function of bone marrow mesenchymal stem cells in EAE depends on their differentiation state and secretion of PGE2. J Neuroimmunol 2011;233(1e2):106e11. [11] Wang X, Kimbrel EA, Ijichi K. Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis. Stem Cell Rep 2014;3(1):115e30. [12] Wang Y, Chen X, Cao W, Shi Y. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat Immunol 2014;15(11):1009e16. [13] Bai L, Lennon DP, Eaton V, Maier K, Caplan AI, Miller SD, et al. Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia 2009;57(11):1192e203. [14] Peron JP, Jazedje T, Brand~ ao WN, Perin PM, Maluf M, Evangelista LP, et al. Human endometrial-derived mesenchymal stem cells suppress inflammation in the central nervous system of EAE mice. Stem Cell Rev 2012;8(3):940e52. [15] Scruggs BA, Semon JA, Zhang X, Zhang S, Bowles AC, Pandey AC, et al. Age of the donor reduces the ability of human adipose-derived stem cells to alleviate symptoms in the experimental autoimmune encephalomyelitis mouse model.
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Please cite this article in press as: Rossato C, et al., Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis, Biologicals (2017), http://dx.doi.org/10.1016/ j.biologicals.2017.06.007