Mesenchymal stem cell enhances the function of MDSCs in experimental Sjögren syndrome mice

Primary Sjögren’s syndrome (pSS) is a chronic systemic autoimmune disease characterized by lymphocytic inltrates in exocrine glands. Mesenchymal stem cells (MSCs) have been proved to be effective in the treatment of different autoimmune diseases. Although MSC transplantation has been demonstrated to be an effective therapeutic approach to treat SS, the underlying mechanisms are still elusive. Our previous study has identied the reduced suppressive capacity of MDSCs advanced the progression of experimental Sjögren’s syndrome (ESS). The ESS mouse model was induced with murine salivary glands proteins emulsied in an equal volume of Freund’s adjuvant. Both frequency and phenotype of MDSCs during ESS development in mice were analyzed by ow cytometry. The suppressive capacity of MDSCs was examined by CD4 + T cell proliferation test, CFSE-labelled CD4 + T cell was analyzed by ow cytometry. Both phenotype and function of MDSCs upon MSCs treatment were analyzed in vitro and in vivo. The function of MSCs in modulating the suppressive function of MDSCs was further evaluated by silencing TGF-β in MSCs.


Background
Primary Sjögren's syndrome (pSS) is a chronic, systemic autoimmune disease characterized by lymphocytic in ltrates in salivary and lacrimal glands, leading to the destruction of these exocrine glands.
The common symptoms are xerostomia and xerophthalmia (1). Besides the characteristic glandular symptoms, other systemic extraglandular manifestations, including synovitis, interstitial lung disease, vasculitis and renal diseases (2). Moreover, approximately 5% of patients with pSS may develop lymphoma, mainly the mucosa-associated lymphoid tissue non-Hodgkin lymphoma, which is the most severe complication of the disease (3). pSS is considered to be essentially driven by a complex interaction between epithelial barrier and adaptive and innate immunity. Macrophages, dendritic cells, NK cells, T cells (Th1, Th2, Th17, Tfh, Tfr, Treg) and B cells have been reported to be involved in the pathogenesis of the disease (3)(4)(5)(6). Additionally, our previous work has clari ed the essential role of MDSCs in the progression of pSS (7). Currently, treatment of Sjögren's syndrome patients is still challenging due to the complex pathogenesis of the disease, approaches such as biologic agents and traditional disease-modifying antirheumatic drugs can't cure this disease and have some side effects (8). Therefore, exploring novel therapeutic approaches is critically necessary for the treatment of pSS.
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which has emerged as a universal regulator of immune function under many pathologic conditions (9).
MDSCs can be subdivided into two major subpopulations: polymorphonuclear MDSCs (PMN-MDSCs) with a CD11b + Ly-6G + Ly-6C lo phenotype and monocytic MDSCs (M-MDSCs) with a CD11b + Ly-6G − Ly-6C hi phenotype, both are characterized by the expression of CD11b + Gr-1 + . PMN-MDSCs exert their suppressive effect mainly by high levels of arginase 1 and reactive oxygen species (ROS) whereas M-MDSCs produce NO. In healthy individuals, MDSCs are generated in bone marrow and quickly differentiate into mature dendritic cells, macrophages, or granulocytes. However, under pathological conditions, the differentiation of MDSCs will be blocked and cause the expansion of this population in vivo (9). Recently, MDSCs have been demonstrated to be involved in the pathogenesis of various autoimmune diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 1 diabetes and multiple sclerosis (MS) (10)(11)(12)(13)(14)(15). Our previous studies have characterized a pivotal role of MDSCs in the development of SS.
We found that MDSCs were signi cantly increased in mice with experimental Sjögren's syndrome (ESS), but their suppressive function of MDSCs was gradually decreased with the progression of the disease, and eventually leading to the uncontrollable in ammatory responses and irreversible tissue injury. Therefore, restoring or enhancing the suppressive capacity of MDSCs are supposed to be a promising therapeutic strategy for pSS.
Mesenchymal stem cells (MSCs) is a group of mesodermal and ectodermal origin multipotent stromal cells with the capacity of self-renewal and differentiation into osteoblasts, adipocytes, and chondrocytes (16). The properties of rapid proliferation and powerful immunomodulation have entitled their potential application in the treatment of various debilitating diseases (17). Indeed, MSCs have been reported to exert immunomodulatory effects on T cells, B cells, dendritic cells and natural killer cells (18), which makes them a promising therapy for various autoimmune diseases, including systemic lupus erythematosus (19,20), rheumatoid arthritis (21), in ammatory bowel disease (22) and systemic sclerosis (23). However, much less is known about the effects of MSCs in treating Sjögren's syndrome, and the underlying mechanism still remains to be elucidated.
In this study, we characterized the effect of BM-MSCs on the suppressive capacity of MDSCs in ESS mice, and clari ed the regulation was predominantly mediated by the TGF-β/Smad pathway. Our study offers new insights into the mechanisms of the application of MSCs as a therapy for pSS.

Mice
Female C57BL/6 mice at 8-week-old were purchased from Experimental Animal Center of Yangzhou University. Mice were housed in a speci c pathogen-free animal facility and all the experiments were approved by the Jiangsu University Animal Ethics and Experimentation Committee.

Induction of ESS model
The ESS mouse model was induced as previously described (7). Brie y, bilateral salivary glands were isolated from female C57BL/6 mice for homogenization in PBS to prepare SG proteins. Naïve mice were immunized with SG proteins emulsi ed in an equal volume of CFA (Sigma-Aldrich) to a concentration of 2 mg/mL (100µl /mouse) s.c. on the neck on days 0 and 7. On day 14, the booster injection was performed with a dose of 1 mg/mL SG proteins emulsi ed in Freund's incomplete adjuvant (Sigma-Aldrich).

Detection of saliva ow rate
Saliva ow rates were measured as previously described (7). Brie y, mice were anesthetized and injected intraperitoneally with pilocarpine (Sigma-Aldrich) at a dose of 5 mg/kg body weight. Saliva was then collected using a 20-μl pipet tip from the oral cavity for 15min.

Autoantibody and cytokine detection
Autoantibodies against SG proteins and anti-M3 muscarinic receptor (M3R) antibodies were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) as previously described (7). Mouse serum levels of IL-17 and IFN-γ were measured with ELISA Kits (eBioscience) following the manufacturer's protocol.

Isolation and Culture of BM-MSCs
For the culture of BM-MSCs, bone marrow (BM) cells were cultured in the medium (DMEM supplemented with 15% fetal calf serum) (Gibco) for 3 days. Non-adherent cells were then removed and when the remaining cells reached 80% con uence in the dish, the adherent cells were expanded for three passages and used for the subsequent experiments.

T cell suppression assay
Mouse CD4 + T cells were sorted from wild-type mice using CD4 + T cell microbeads (Miltenyi Biotec). CD4 + T cells were labeled with carboxy uorescein succinimidyl ester (CFSE, 5 mM; Invitrogen), and then cocultured with MDSCs at a ratio of 1:1 in 96-well plates (Costar) in the presence of anti-CD3 and anti-CD28 mAbs (eBioscience) for 3 days. CFSE uorescence intensity was analyzed to determine the proliferation of CD4 + T cells by ow cytometry.

Western blot
Proteins extracted from cells were prepared as previously described. Equal amounts of proteins were separated by 12% SDS-PAGE, then transferred onto Immobilon polyvinylidene di uoride membranes (Bio-Rad). Antibodies against Smad2/3 and p-Smad2/3 were purchased from Cell Signaling Technology.

Histologic analysis
After mice were euthanatized, submandibular glands were collected and immediately xed in 4% paraformaldehyde. Paraformaldehyde-xed tissues were embedded in para n. Serial 4-μm sections were cut and stained with hematoxylin and eosin (H&E) for morphologic examination.
Detection of arginase activity and NO production The activity of arginase and NO concentration were measured as previously described (24).
Transfection TGF-β siRNA and the negative control were synthesized by RiboBio. Oligonucleotide transfection was performed with Entranster-R (Engreen Biosystem) according to the manufacturer's instructions.

Statistical analysis
The statistical signi cance was determined by the Student's t test or one-way ANOVA. All analyses were performed using SPSS 16.0 software. p Values less than 0.05 were considered statistically signi cant.

Results
Adoptive transfer of BM-MSCs effectively alleviates the progression of ESS BM-MSCs were adoptively transferred into ESS mice on days 18 and 25, and then the therapeutic effect of the cells in SS was evaluated ( Figure 1A). Remarkably, BM-MSCs treatment effectively ameliorated the saliva ow rate and reduced the serum autoantibodies against total SG antigens, ANA, and anti-M3R Abs ( Figure 1B-E). Notably, the BM-MSCs treated group displayed smaller cervical lymph nodes (CLNs) and salivary glands (SG) while compared to the control group. In addition, histological analysis showed only a small amount of lymphocytic in ltration in local SG from ESS mice treated with BM-MSCs ( Figure 1G).

BM-MSCs expand MDSCs with strong suppressive function in ESS
Our previous ndings have shown that MDSCs in ESS mice gradually lost their suppressive effect during the progression of the disease, which has been determined to be a critical element in the pathogenesis of pSS (7). Therefore, restoring the suppressive capacity of MDSCs in ESS mice might be an e cient strategy for the immunotherapy in pSS. As shown in Figure 2A (Figure 3 A-D). Additionally, the expression of CD40, CD80, CD86 and MHCII was also down-regulated when compared to the group without BM-MSCs treatment ( Figure 3E). Together, the in vitro experiment further con rmed that BM-MSCs could reverse MDSCs to the immature state with strong suppressive function directly.

Enhanced suppressive function of MDSCs is mediated by BM-MSC-secreted TGF-β
It has been reported that TGF-β plays an important role in the inhibitory effect of MSCs (25,26). As shown in Figure 4A and 4B, a high level of TGF-β was measured in BM-MSCs when co-cultured with MDSCs. Moreover, the phosphorylation of Smad2/3 in MDSCs was signi cantly increased after BM-MSCs treatment ( Figure 4C). To further clarify the critical role of TGF-β in regulating the suppressive effect of MDSCs, TGF-β was neutralized by anti-TGF-β antibody. After the blockade of TGF-β pathway, the effect of BM-MSCs on the regulation of MDSCs was almost disappeared, MDSCs still exhibited low immunosuppressive function on T cell proliferation ( Figure 4D), and the production of arginase and NO were also at low levels ( Figure 4E, F). These in vitro data suggest that the enhanced suppressive effect of MDSCs was mainly mediated by TGF-β released by BM-MSCs.

Silencing TGF-β in BM-MSCs attenuates their capacity in alleviating ESS progression
To further determine the role of TGF-β from BM-MSCs in regulating the function of MDSCs in vivo, we adoptively transferred BM-MSCs with silenced TGF-β expression into ESS mice ( Figure 5A). As expected, the therapeutic effect of BM-MSCs in inhibiting ESS development was almost abolished after knocking down the TGF-β. Notably, the saliva ow rate was decreased, and the serum autoantibodies against total SG antigens, ANA, and anti-M3R Abs were remarkably increased in siTGFβ-MSCs-treated ESS mice ( Figure 5B-E). In addition, the histological analysis showed serious lymphocytic in ltration in SG when compared to the Ctrl-MSCs-treated group ( Figure 5F). Moreover, MDSCs in siTGFβ-MSCs-treated ESS mice displayed week suppressive capacity while the control group possessed strong suppression on T cells. Taken together, these data suggest that BM-MSCs modulated the function of MDSCs is predominantly mediated by TGF-β.

Discussion
Extensive studies have described the role of MDSCs in the progression of various autoimmune diseases, including MS, RA, SLE and type I diabetes (10-12, 14, 15, 27). Although there have been a large number of studies on MDSCs in autoimmune diseases, the exact effect of MDSCs in these diseases is still controversial. It has been found that adoptive transfer of MDSCs from EAE mice could obviously inhibit the in ammatory immune responses and suppress the progression of EAE (14). However, some other studies have shown that MDSCs in EAE could promote the differentiation of Th17 cells, and the severity of disease can be alleviated after depletion of MDSCs in vivo (15). The similar con ict results were also observed in RA. Some data showed the protective role of MDSCs during the development of collageninduced arthritis (CIA) while some others found MDSCs signi cantly exacerbated the disease (10, 11). The controversial results on the role of MDSCs in autoimmune disorders are mainly due to the high heterogeneity and plasticity of MDSCs whose phenotypes and functions are largely dependent on the local microenvironment (9,28,29). MDSCs at different stages of disease may play a different role in either exacerbating or alleviating the disease. In our previous studies, we have investigated the role of MDSCs and their subsets in pSS. We found that MDSCs gradually lost their suppressive capacity during the development of ESS, thus leading to the progression of the disease. Early-stage MDSCs in ESS showed strong suppressive capacity while the late-stage MDSCs exhibited weak inhibitory effects on T cells. Therefore, reversing or enhancing the suppressive capacity of MDSCs in vivo might be a promising strategy for the treatment of SS. As expected, in this study, we found that BM-MSCs with immunomodulatory capacity could e ciently enhance the suppressive function of MDSCs, reserving the phenotype of MDSCs to an immature status with low levels of CD40, CD80, CD86 and MHC-II, and eventually alleviating the development of the disease.
Due to the strong immune regulatory property, MSCs have been applied in a number of autoimmune diseases. Indeed, the mechanisms for the immunomodulatory effect of MSCs has been extensively investigated. Abundant evidence has shown that MSCs exert modulatory effects on both innate and adaptive immune cells. MSCs can inhibit the activation, proliferation and differentiation of T cells (30)(31)(32). Krampera et al demonstrated that murine BMSCs inhibited naive and memory T-cell responses to their cognate antigens (33). MSCs can also regulate the balance of Th1/Th2 cells, MSCs inhibit the production of IFN-γ by Th1 cells and increase the production of IL-4 by Th2 cells (34). Rafei et al reported that MSCs could suppress Th17 cell activation in a CC chemokine ligand 2-dependent manner (32).
Recently, MSCs are found to suppress Tfh cell differentiation in RA partially through the production of indoleamine 2,3-dioxygenase (IDO) (35), and the similar results are also observed in pSS patients and lupus-prone mice (36,37). Besides, BM-MSCs can induce the differentiation of CD4 + CD25 hi Foxp3 + regulatory T cells and maintain their suppressive function (38). In addition to the regulation on T cells, Corcione et al found that BM-MSC could inhibit the function, differentiation and the chemotactic properties of B cells (39). In relation to innate immune cells, MSCs have been demonstrated to inhibit the differentiation and function of DCs (40,41). In pSS, it has been reported that MSCs can alleviate the disease by inhibiting Th1, Th17 and Tfh cell responses (36,42). Moreover, MSCs can also ameliorate SS via suppressing IL-12 production in DCs (43). A recent study by Yao et al has found that MSC-secreted interferon-β (IFN-β) promoted DCs to produce IL-27 and then suppressing the SS-like syndrome (44). In this study, we were the rst to observe the regulation of MSCs on MDSCs. Our previous study has clari ed the critical role of MDSCs during the progression of ESS, and the suppressive function of MDSCs was decreased during the disease development. We then found that BM-MSCs could directly modulate the suppressive function of MDSCs both in vitro and vivo. After BM-MSCs treatment, the suppressive function of MDSCs was signi cantly enhanced with high levels of arginase and NO. Furthermore, the expression of CD40, CD80, CD86 and MHCII was also down-regulated. These data suggest that BM-MSCs can e ciently restore the strong suppressive function and the immature status of MDSCs and then alleviate the progression of ESS.
MSCs exhibited a range of immunomodulatory effect through releasing soluble factors and cell-cell contact. It has been reported that soluble factors, including TGF-β1, prostaglandin E2, indoleamine-pyrrole 2,3-dioxygenase, have been proposed to mediate the immunosuppressive function of MSCs (45). In this study, we observed high concentration of TGF-β in MSCs when co-cultured with MDSCs. TGF-β1 is a pleiotropic cytokine which has broad effects on the differentiation and function of various cell (46). Lee et al have found Treg-derived TGF-β could e ciently promote MDSC proliferation and function in murine colitis (47). A recent study also reported that TGF-β could increase the expansion of MDSCs and enhance the suppressive capacity of MDSCs in vitro (48). Similarly, in our experiment, the canonical signaling of TGF-β was activated, the phosphorylation of Smad2 and Smad3 was strikingly enhanced in MDSCs cocultured with BM-MSCs. Furthermore, while the TGF-β pathway in MDSCs was blocked by anti-TGF-β neutralizing antibody, the effect of BM-MSCs induced was almost inhibited, the suppressive function of MDSCs was reversed to the primary weak status. Concurrently, the in vivo experiment also showed TGF-βsilenced MSCs displayed a worse therapeutic effect in treating mice with ESS when compared with the control group. All these data indicate the regulation of BM-MSCs on the function of MDSCs was predominantly mediated by TGF-β.

Conclusions
In conclusion, our ndings suggest that BM-MSCs are capable of enhancing the suppressive capacity of MDSCs, thus alleviating the progression of ESS. Further exploration revealed the regulation of BM-MSCs on MDSCs was mainly through TGF-β/Smad pathway. Our study further enriches the mechanism of MSCs in the cell-based immunotherapy for autoimmune diseases.

Declarations
Ethics approval and consent to participate This study was approved by the Jiangsu University Animal Ethics and Experimentation Committee.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in the published article. The datasets used and/or analyzed in this study are available on request from the corresponding author.

Competing interests
The authors declare that they have no competing interests.

Authors' Contributions
JT and YH performed the experiments, analyzed the data, and wrote the paper; QZ, HZ and HG performed the experiments; YZ, ZS and Yue Z analyzed the data; SW, KR and HX designed the study and wrote the paper. All authors read and approved the nal manuscript.    groups were isolated, and then co-cultured with CD4+T cells in the presence of anti-CD3 and anti-CD28 mAbs for 72 h (MDSC:T cell ratio 1:1). CD4+ T cell proliferation was evaluated by staining with CFSE.