Human Gingiva-Derived Mesenchymal Stem Cells Ameliorate Streptozoticin-induced T1DM in mice via Suppression of T effector cells and Up-regulating Treg Subsets

There is yet no cure for type 1 diabetes (T1DM) so far. A significant body of evidence has demonstrated that bone marrow-derived mesenchymal stem cells (BMSCs) showed great potential in controlling T1DM. But there exists much difficulty in using BMSCs as a clinical therapy. We here test whether a new population of mesenchymal stem cells from human gingiva (GMSCs), which has many advantages over BMSCs, can delay or prevent progress of T1DM. GMSCs were adoptively transferred to multiple low-dose streptozotocin (STZ)-induced T1DM. Blood glucose levels and disease severities were analyzed. T cells subsets in blood, spleen and lymph nodes were detected dynamically by flow cytometry. GMSC distribution was dynamically analyzed. We found that infusion of GMSCs but not fibroblast cells significantly controlled blood glucose levels, delayed diabetes onset, ameliorated pathology scores in pancreas, and down-regulated production of IL-17 and IFN-γ in CD4+ and CD8+ T cells in spleens, pancreatic lymph nodes (pLN) and other lymph nodes. GMSCs also up-regulated the levels of CD4+ Treg induced in the periphery. Mechanismly, GMSCs could migrate to pancreas and local lymph node and function through CD39/CD73 pathway to regulate effector T cells. Thus, GMSCs show a potential promise in treating T1DM in the clinic.


GMSCs ameliorated the development of T1DM in mice.
To investigate the effect of GMSCs in experimental diabetes, we used STZ-induced T1DM model in male C57BL/6-FoxP3 gfp mice. We chose male mice because rodents were reported to show a substantial gender difference in STZ sensitivity with male mice and rats tend to be more susceptible to STZ-induced diabetes compared to little or no response in female mice and severe hyperglycemia was present in male mice receiving identical doses 32 . We wanted to use flow cytometry to dynamically test Treg cell numbers and phenotype in blood by tail vein bleeding, so we used a Foxp3-GFP-reporter mice, which required quite little lymphocytes than other methods. Diabetes onset was markedly delayed following GMSCs treatment, although GMSCs treatment did not completely prevent the onset of diabetes eventually (Fig. 2a). GMSCs significantly diminished blood glucose levels compared with the model and fibroblast treatment groups (P < 0.001) (Fig. 2b). The body weight of the three groups were monitored, and showed that GMSCs could inhibit weight loss, but there was no significant difference among three groups (P > 0.05) (Fig. 2c). Thus, we show that GMSCs transfer could delay T1DM onset and ameliorate disease severity.
Decreased severity of insulitis following treatment with GMSCs. Histological examination revealed that islets from GMSCs-treated mice exhibited bigger size than other two groups (Fig. 3a). GMSCs treated mice also showed higher proportions of insulin-positive beta cells (Fig. 3c) and lower proportions of glucogon-positive alpha cells (Fig. 3d) by immunohistochemistry using the anti-insulin and anti-glucagon antibodies on day 10. Insulitis score was significantly decreased in GMSCs treated mice compared with the model and fibroblast groups (Fig. 3b). We noted that GMSCs treatment could not prevent diabetes onset, insulitis and alpha/beta proportion has also no difference among three groups on day 30 ( Supplementary Fig. S1).
GMSCs down-regulated IL-17 and IFN-γ expression on both CD4 + and CD8 + T cells in STZ-induced T1DM model. We next investigated the mechanisms underlying the decreased severity of diabetes following administration of GMSCs. Interleukin-17 (IL-17) has been associated with the pathogenesis of T1DM and our in vitro study found that GMSCs had the ability to inhibit Th17/Tc17 cells differentiation 16 , so we wondered whether  GMSCs ameliorated the development and severity of T1DM in mice. T1DM was induced in male C57BL/6-FoxP3 gfp mice using multiple low doses of STZ injection, 1 × 10 6 GMSCs or human skin fibroblast cells were injected into mice via intraperitoneal route on days 0, 7, 14, 21, 28. (a) Incidence of diabetes. Blood glucose concentration exceeding 300 mg/dL in two consecutive daily measurements was considered diabetes. (b) Nonfasting blood glucose of mice in three groups. (c) Body weight in different groups. Data are presented as the mean ± SEM from two independent experiments (n = 5). ***P < 0.001, versus the fibroblast or model groups.
GMSCs could suppress IL-17 expression in T1DM. Consistently with our in vitro study, we found that GMSCs significantly reduced the percentages of Th17 cells and Tc17 cells in pLN of mice with T1DM on day 10 after STZ induction compared with model, while fibroblast conversely had the tendency to increase Th17/Tc17 cells ( Fig. 4a and c). In addition to IL-17 production, we also analyzed IFN-γ expression on T cells, since T1DM is considered a Th1 cells-mediated disease 33 . We also observed a suppressive function of GMSCs on IFN-γ expression on both CD4 + T cells and CD8 + T cells ( Fig. 4b and d). IL-17 and IFN-γ expression on T cells in spleens and other lymph nodes were also decreased in GMSC treated group. These effects were consistent in spleens and other lymph nodes on day 30 after STZ induction ( Supplementary Fig. S2). Thus, GMSCs delayed and treated diabetes at least partly through suppressing proliferation and activation of autoreactive T subsets, like Th17, Tc17, Th1, and Tc1 cells.
GMSCs also promoted Treg production in STZ-induced T1DM mice model. Evidence has showed that Tregs are defective in numbers and/or function in animal model of T1DM and T1DM patients, so we tried to study the effects of GMSCs on Tregs in vivo. GMSCs treated group had a significantly larger number of CD4 + Treg cells in spleens and LNs than model and fibroblast groups on day 30 (Fig. 5a,b and c). According to previous studies, Nrp-1 and Helios could be used as specific cell markers to distinguish natural Tregs (nTregs) from induce Tregs (iTregs) 34,35 , we then tried to figure out whether these up-regulated Tregs originated from nTregs subsets or were induced by GMSCs in vivo. As shown in Fig. 5d, the majority of Tregs in GMSCs-treated mice were Helios negative and had a decrease of Nrp-1 expression compared with control groups, suggesting that GMSCs may induce the generation of iTregs in vivo rather than expand endogenous nTregs. CD4 + Tregs circulating in the peripheral blood and those residing in pLN of T1DM mice among the three groups had no statistical differences ( Supplementary Fig. S3). As to the newly identified Treg cell population 36 , we found that GMSCs did not affect CD8 + CD103 + CD122 + CD28 − GFP -Treg cells in the diabetes model (data not shown).
Injected GMSCs migrated to pancreas and local lymph nodes. Previous studies suggested that therapeutic efficacy of MSCs was greatly dependent on their ability to produce juxtacrine or paracrine factors and migration of MSCs to the diseased organs/tissues was required for the juxtacrine effects 37 . We therefore also investigated the ultimate fate of the GMSCs following intraperitoneal injection. The intravital fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), which does not interfere with cytotoxic function, has been used extensively for studying lymphocyte migration and study reported that adoptively transferred lymphocytes labeled with CFSE could be indentified in vivo at least 2-3 months after injection 38 . To this end, we labeled GMSCs with CFSE before i.p. injection. GMSCs organ distribution and engraftment was then examined at different time points post-injection by flow cytometry. The results showed that i.p. injected GMSCs mainly migrated to lymph nodes ( Fig. 6a and b) but barely exsisted in brain, lung, kidney or liver (data not shown) on day 3, 7, 14 and 28. Interestingly, GMSCs homed to pancreas lymph nodes in a large percentage and the target organ-pancreas regardless of a lower percentage at different time points ( Fig. 6a and b).

GMSCs functioned through CD39/CD73 pathway to regulate T effector cells. We used an
in vitro naïve CD4 + T cells differentiation experiment to explore the possible mechanisms responsible for GMSCs-mediated suppression of T effector cells in STZ-induced T1DM mice model. Same as previous reports, GMSCs significantly inhibited murine CD4 + T cells differentiation into Th1 or Th17 cells in a dose-dependent manner ( Fig. 7a and b). To exclude the possible effect of different solube factors on T cells, we pre-treated GMSCs with CD39, CD73 and IDO inhibitor, respectively and then co-cultured GMSCs with T cells in an inhibitor-free system for 3 days. Results showed that after CD39 or CD73 inhibitor but not 1-MT treatment, GMSCs almost completely lost their immunosuppression on T effector cell differentiation, if any, this even lead a tendency to up-regulate IL-17 or IFN-γ expression ( Fig. 7c and d), suggesting a vital role for CD39/CD73 signals in mediating the immunoregulatory function of GMSCs.

Discussion
MSCs are multipotent progenitor cells, which can be found in bone marrow, adipose tissue, umbilical cord blood, the placenta of humans and human gingival tissues 39 . A decade of experiments has established that MSCs can inhibit the proliferation and function of T and B cells, NK cells, as well as promote the expansion of CD4 + Tregs 4-6 . MSCs-mediated immunosuppression ability is also associated with inhibition of pro-inflammatory cytokine induction 40 . Results of in vivo administration of MSCs in autoimmune diseases is consistent with in vitro studies 41,42 . MSCs as a therapy for T1DM has been studied before 42,43 . BMSC is the most intensively studied type of MSCs, however, there are substantial weaknesses of BMSC clinical applications: 1) an invasive process is needed to obtain BMSC; 2) BMSCs are not stable after multi cycle proliferation; 3) BMSCs are dysfunctional in patients with autoimmune diseases 44,45 , use of autologous BMSC is unlikely to be feasible; 4) BMSCs have a tumorigenesis risk [46][47][48] .
GMSCs have many advantages over BMSCs as this population is abundant and more stable than BMSC as well as not having tumorigenic potential 11 . GMSCs have been showed to have significant therapeutic effect in experimental colitis 15 and rheumatoid arthritis 16 . Our recent study demonstrated that GMSC can suppress human cell-mediated xeno-GVHD 17 , implying a potential for clinical translation. In the current study, we have provided a line of new evidence that GMSCs administration can be a potential therapy for autoimmune diabetes since they could delay STZ-induced diabetes onset, control blood glucose level, prevent weight loss, decrease insulitis score and preserve beta cells. Moreover, as far as we know, our group is the first to show that GMSCs have such function on diabetes.
T1DM, which was previously thought to be a Th1-mediated autoimmune disease, is now considered also associated with Th17 cells 49 . IL-17 expression in the islet correlates with insulitis in spontaneous autoimmune diabetes model in nonobese diabetic (NOD) mice 50 . Inhibition of Th17 cells significantly suppressed development of diabetes 51 . IL-17 deficiency delayed onset of diabetes, with reduced insulitis in NOD mice, and ameliorates STZ-induced diabetes 52,53 . Adoptive transfer of in vitro differentiated islet antigen specific Th17 cells to immunodeficient mice can lead to rapid onset of diabetes with extensive insulitic lesions 50 . In addition to Th17 cells, CD8 + T cells that are characterized by the expression of the transcription factor retinoic acid-related orphan receptor (RORγt) and the production of IL-17, termed Tc17 cells, have recently been identified 54 . These cells have a role in diabetes pathogenesis, since percentages of circulating Tc17 cells in children with new-onset T1DM was increased 55 and a role in diabetes was also demonstrated in a murine model 56 . All these findings clearly highlight the major role of IL-17 and IFN-γ in the development of diabetes. In accordance with these findings, our results showed that GMSCs can suppress mouse naïve CD4 + T cells differentiate into Th17 and Th1 cells in vitro. In the STZ-induced T1DM mice model, GMSCs could significantly inhibit IL-17 expression on CD4 + T cells and CD8 + T cells compared with model and fibroblasts groups. We also observed an gorgeous effect of GMSCs on IFN-γ expression, which was consistent with other reports 16 . So, it can be illustrated that effects of GMSCs on the diabetes model may be partly mediated by suppressing IL-17 and IFN-γ expression on autoreactive T cells.
MSCs are reported to increase the percentages of regulatory T and B cells 16,57,58 . In the present study, although we did not see any significant difference in CD4 + Tregs number in blood or pLN, we did find an increased number of CD4 + Foxp3 GFP+ Tregs in GMSCs treatment group in spleen and MLN. Through analysis of the expression of nTregs cell markers, Nrp-1 and Helios, we found that GMSCs mainly induced iTregs rather than expanded nTregs thus resulting in up-regulation of Tregs subsets. Similar results have been reported in other models 16 . We recently identified a new CD8 + Treg cell population that requires CD103 for their development and function but are independent of the cytotoxic effect 59 . In this study we also analyzed CD8 + CD103 + CD122 + CD28 − GFP -Treg cells in peripheral blood, spleen, MLN and pLN of three groups. We did not observe significant augmentation of CD8 + Tregs. Thus, GMSCs mainly function through inducing CD4 + Treg subsets, but not by affecting CD8 + Tregs, in delaying T1DM.
Number of therapeutic cells that can migrate and colonize at the injury site is a decisive prerequisite for the success of cell-based therapy. In line with other reports, which showed that i.p. injected MSCs engrafted more to inflamed colon in a DSS-induced colitis mouse model but showed fewer trapped cells in lung, liver and spleen compared to i.v. injection, thus resulting in better experimental colitis recovery 60 , our results showed that i.p. injected GMSCs barely existed in lung, liver, brain or kidney but mostly homed to lymph nodes, including pancreas lymph nodes and somehow to pancreas at different time points, which could attribute to the immunosuppressive role of GMSCs in T1DM model. The immunoregulatory function of murine multipotent mesenchymal stromal cells, human BMSCs and regulatory T cells all have been reported to be mediated at least partially through CD39/CD73 signal pathways 25,26,61,62 . Studies from our own group also reported a CD39/CD73 dependent immunomodulatory function of human GMSCs 16,17 . In accordance with these reports, we found that GMSCs, which highly expressed CD39 and CD73, could inhibit T effector cells differentiation in vitro and this function was significantly abrogated by CD39 or CD73 inhibitor treatment, which could also be a mechanism of the in vivo suppression of GMSCs on T1DM. However, IDO was not significantly involved in this suppression. It is likely because immunosuppressive properties of IDO by MSCs could be affected by specific microenvironments 62 , expression of IDO is not constitutive but could be induced by IFN-γ and induction of functional IDO activity required MSC activation 27 .
In this study we only observe a delay but not complete prevention of diabetes onset after GMSCs treatment. It can not be explaned by a too short life span of GMSCs in vivo. Using tracking experiments, we can clearly observe the distribution of GMSCs even at day 28. It is possible that GMSCs might need a sufficient local concentration to exert their effect in vivo. Although GMSCs could migrate to target organs, the percentages of GMSCs in pancreas could be too low to exert an efficient immunosuppressive effect. Additionally, the in vivo inhibitory effect of GMSCs could be overcome by a high level of inflammation in T1DM model. As GMSCs were hardly detected in blood, lung and liver, and diabetes was a multiorgan-associated disease, the proinflammatory cytokines production in these organs might not be suppressed by GMSCs, and if these proinflammatory cytokines accumulated to a certain extent, it could offset or even overcome the immunosuppressive effect of GMSCs.
Based on evidence presented above,we conclude that GMSCs have the ability to migrate to pancreas and local lymph nodes and alter the imbalance between Tregs and T effector cells thus ameliorate STZ-induced T1DM in mice model. Mechanismly,GMSCs could function through CD39/CD73 pathway to regulate effector T cells. Our findings serve as evidence supporting that GMSCs, a unique population of MSCs, which has similar function with but much advantage over BMSCs, are a promising cell source for stem cell-based therapies of autoimmune diabetes and other autoimmune and inflammatory diseases.

Materials and Methods
Mice. Wild-type C57BL/6 (male, 6-8 week old, 20-26 g body weight) mice were obtained from Jackson Laboratory. C57BL/6-FoxP3 gfp mice were generously provided by Dr. Talil Chatilla (University of Southern California, Los Angeles). All animals were housed in SPF system in a temperature (72 ± 3 °F) and air (50 ± 20% relative humidity)-controlled room with a 12 h light-dark cycle and were given standard diet and tap water. All experiments using mice were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Third Affiliated Hospital of Sun Yat-sen University.
GMSCs isolation, culture and identification. Ten samples of healthy human gingival tissues (from healthy Asian male or female who experienced an operation of wisdom tooth extraction surgery, aged between 20 and 30 years old, without any autoimmune or inflammatory diseases, all from Guangdong Province of China) were obtained as remnant or discarded tissues following routine dental procedures at the Third Affiliated Hospital of Sun-Yat-sen University, in accordance with the ethical standards and with the approval of Institutional Review Board protocol of Sun Yat-sen University. Informed consent was obtained from all participating human subjects for the collection of fresh tissue. GMSCs isolation and culture and all reagents used in this study were as previously described 16 . Cells from third to sixth passages were used in experiments. GMSC and human dermal fibroblasts (a cell line from American Type Culture Collection, Manassas, VA) was stained for CD14, CD26, CD29, CD34, CD39, CD44, CD45, CD73, CD90 and CD105 for phenotypes study. For each experiment, cells from one single donor were used.
In vitro suppression assay of GMSC. To validate that GMSCs but not fibroblast cells have immunomodulatory function, as a big difference between these two cell types, in vitro suppressive assay on mouse splenic T cells was performed. Mouse splenic CD25 − T cells were isolated using magnetic isolation from C57BL/6 mice and labeled with CFSE using the CellTrace ™ CFSE cell proliferation kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions and then stimulated with anti-CD3 (0.025 μg/ml) and irradiated (30 cGy) syngeneic non-T cells. GMSCs or fibroblasts were plated in triplicate in 96-well plates and allowed to adhere to the plate overnight. The ratio of GMSCs or fibroblast cells to mouse CD25 − T cells ranged from 1:1 to 1:200. Cells were cultured for 3 days and CFSE dilution of CD8 + T cells was tested by Flow Cytometry.
Induction of diabetes. T1DM was induced in C57BL/6-FoxP3 gfp mice (male, 6-8 week old, 20-26 g body weight) using multiple low doses of STZ (Sigma-Aldrich, St Louis, MO, USA) injection. STZ was administered via intraperitoneal route at a dose of 40 mg/kg, dissolved in freshly made and cold 0.01 M citrate buffer (pH 4.5) within 20 minutes for 5 consecutive days. Mice were fasted for 4-6 hours prior to STZ injection and 10% sucrose water were supplied overnight after the first injection to avoid sudden hypoglycemia. To determine intervention effects, 1.0 × 10 6 GMSCs suspended in 200ul phosphate-buffered saline (PBS) were given through i.p. injection on days 0, 7, 14, 21 and 28 after STZ administration. A similar dose of human dermal fibroblasts was given as a control. Meanwhile, STZ-induced model receiving 200ul PBS was considered an additional control. Non-fasting blood glucose level was monitored twice a week over the following 30 days using an Embrace one touch blood glucometer (OMNIS Health, USA). Blood glucose concentration exceeding 300 mg/dL in two consecutive daily measurements was considered diabetes. Blood samples were collected every 3 days from tail vessels and assayed for CD4 + and CD8 + Treg. Body weight was recorded every 3 days. Cages were changed every 3-4 days since mice would develop symptom of polyuria.
Histology and immunohistochemistry. Pancreas glands were harvested on days 10 and 30. Pancreas samples were fixed overnight in 10% formalin, and embedded in paraffin. All the embedding, slicing, and H&E staining were done by the Penn State Pathology and Laboratory Medicine facility. For assessment of insulin and glucogon production in pancreas, sections were stained with anti-insulin (1:1600, AbCam63820) and anti-glucagon (1:8000, AbCam ab92517) and were revealed using anti-rabbit DAB-HRP (OMap) as secondary antibodies for visualizing, and then dying the nucleus with hematoxylin and bluing reagent. Six sections per pancreas, evenly sectioned and separated by 200μm, were stained with H&E, anti-insulin and anti-glucogon antibodies. Images were captured by Olympus microscope (Tokyo, Japan). For morphometric analysis, images of islets were traced and analyzed with the use of Image-pro-plus 6.0 software manually. Islet sizes were examined as the total islets area divided by the total number of islets. Sections were measured from at least three individual pancreases from each group by at least two different people to avoid subject bias. All islets were evaluated and the insulitis scores were determined as follows: 0, no intraislet mononuclear cell infiltration; 1, mild peri-islet mononuclear cell infiltration (granulation of <30%); 2, intraislet moderate mononuclear cell infiltration (granulation of <50%); 3, severe to massive cell infiltration (granulation of >50%) 63 .
Tracking intraperitoneal injected GMSCs. GMSCs were labeled with CFSE using the CellTrace ™ CFSE cell proliferation kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. In brief, cells were pelleted and resuspended in PBS containing 1 μM CFSE at a concentration of 1 × 10 7 / ml and incubated for 10 min at 37 °C in a water bath. After staining, cells were washed twice in Roswell Park Memorial Institute medium (RPMI-1640) using centrifugation at 400 g for 5 min. After the final wash, a sigle dose of 2.0 × 10 6 cells, resuspended in 200uL PBS were then injected into C57BL/6 mice. GMSCs migration and homing to different organs was examined by flow cytometry at day 3, 7, 14, and 28.
Murine naive CD4 + T cell differentiation in vitro. Naive CD4 + CD62L + T cells were purified from the spleens or lymph nodes of C57BL/6 mice via magnetic isolation (Miltenyi Biotec). GMSCs were cocultured with naive CD4 + CD62L + T cells (1:10 or 1:20) during their in vitro differentiation into T helper cells. GMSCs were allowed to adhere to the plate overnight before coculture. Naive CD4 + T cells were stimulated with anti-CD3 (1ug/ml) and anti-CD28 (1ug/ml) antibodies (both from Biolegend) in the presence of irradiated (30 cGy) syngeneic non-T cells (1:1), along with cytokines for Th1 or Th17 cell polarization differentiation, as previously described 16 . After 3 days in culture, differentiated cells were restimulated with PMA and ionomycin for 5 hours and brefeldin A for 4 hours. The expression of IFN-γ and IL-17 was then measured by flow cytometry.
Flow cytometry analysis. GMSCs were digested using 0.05% Trypsin-EDTA(Gibco by Life Technologies) and suspended in PBS. GMSCs and fibroblast cells phenotype identification are as previously described 16 . Mouse blood samples were collected using 0.1%EDTA(Gibco by Life Technologies) and lysis of red blood cells was processed using Red Blood Cell Lysing Buffer (Sigma-Aldrich). If lysis is incomplete, steps are repeated. Antibodies against CD4(GK1.5, PerCP/Cy5.5), CD8(53-6.7, PerCP/Cy5. Statistical analyses. All data were expressed as mean ± SEM from at least three independent experiments. For comparison of more than two groups, one-way ANOVA for normally distributed data or Kruskal-Wallis for skewed data was performed. All statistical analyses were carried out using SPSS software (version 17.0). P < 0.05 was considered statistically significant.
Data availability statements. The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.