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Dental pulp-derived stem cell-conditioned media attenuates secondary Sjögren's syndrome via suppression of inflammatory cytokines in the submandibular glands

Introduction Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease, which affects the exocrine glands. Its primary symptoms are decreased moisture in the mouth and eyes. Therapies are limited to treatment with steroids, which has unpleasant side effects, so new treatments would be beneficial. One possibility might be stem cells, such as bone marrow mesenchymal stem cells (BMMSCs) or dental pulp-derived stem cells (DPSCs); these have been reported to exert immunomodulatory effects on activated lymphoid cells. This study aimed to evaluate the effects of conditioned media from DPSCs (DPSC-CM) or BMMSCs (BMMSC-CM) on salivary functions in SS. Methods Cytokine array analysis was performed to assess the types of cytokines present in the media. DPSC-CM or BMMSC-CM was administered in an SS mouse model. Histological analysis of the salivary glands was performed, and gene expression levels of inflammatory and anti-inflammatory cytokines in the submandibular glands (SMGs) were evaluated. Results DPSC-CM contained more anti-inflammatory factors than BMMSC-CM. The mice that were given DPSC-CM had a lower number of inflammation sites in the SMGs than those in the other experimental groups, and their salivary flow rate increased. The expression levels of interleukin (IL)-10 and transforming growth factor-β1 increased in the DPSC-CM group, while those of Il-4, Il-6, and Il-17a decreased. The mice that received DPSC-CM showed a significantly increased percentage of regulatory T cells and a significantly decreased percentage of type T helper 17 cells compared to other groups. Conclusions These results indicate that DPSC-CM could be an effective therapy for SS-induced hyposalivation, since it decreases the number of inflammatory cytokines and regulates the local inflammatory microenvironment in the SMGs.


Introduction
Sj€ ogren's syndrome (SS) is a chronic inflammatory autoimmune disease that interferes with the functions of the exocrine glands and is marked by decreased moisture secreted from the mucous glands of the mouth and eyes [1]. This disease is more common in middle-aged women; it can occur alone (primary SS) or in association with other rheumatic diseases (secondary SS), such as rheumatoid arthritis, systemic sclerosis, polymyositis, mixed cryoglobulinemia, and systemic lupus erythematosus (SLE) [2].
SS is most often associated with SLE and occurs in 9%e33% of SLE patients [3e5]. The most prominent feature of SLE is the production of multiple circulating autoantibodies; some reactivities, such as the anti-double-stranded DNA (dsDNA), anti-Sj€ ogren's syndrome A (SSA), and anti-Sj€ ogren's syndrome B (SSB) antibody, are specific serologic markers of SLE [6]. The diagnostic boundaries between primary SS and SLE can be difficult to discern because they share clinical and laboratory features.
Three drugs are currently licensed for the treatment of lupus: corticosteroids, hydroxychloroquine, and belimumab [7]. Immunosuppressants, such as azathioprine, methotrexate, and mycophenolate, are also used; despite these treatments, however, lupus patients face serious morbidity. Steroids are not likely to cause serious side effects if they are taken for a short time or at a low dose [8], but there are unpleasant minor unpleasant side effects, such as an increased appetite, mood changes, and insomnia. Therefore, new treatments are needed for the management of the secondary SS consequences of active lupus.
Extensive clinical trials have recently been conducted using mesenchymal stem cell (MSC)-based therapies for treating a range of diseases [9e11]. Studies have shown that transplanted cells can play multiple and important roles; they can not only migrate in their host tissues and participate directly in the regeneration of tissue but also display paracrine effects [12e15]. MSCs produce cytokines that can decrease inflammation, enhance progenitor cell proliferation, improve tissue repair, and decrease infection.
Studies have shown that dental pulp-derived stem cellconditioned media (DPSC-CM) has immunoregulatory properties that contribute to tissue repair and anti-inflammatory effects comparable to bone marrow-derived mesenchymal stem cellconditioned media (BMMSC-CM) [16]. In this study, we will evaluate the therapeutic effects of DPSC-CM and BMMSC-CM in mouse models of secondary SS.

Ethics statement
The study design and methods were approved by and carried out according to the Institutional Review Board of the Center for Clinical and Translational Research of Kyushu University Hospital (IRB serial numbers 25e287 and 26e86) and the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients or their relatives prior to inclusion in the study.
All animal protocols were approved by the Animal Experiments Care and Use Committee of Kyushu University (approval nos. A19-060-0 and 29e104). Maximum efforts were made to minimize animal suffering; all measurements under catheter insertion were performed with the animals under deep anesthesia.

Cell preparation
Human DPSCs and BMMSCs were purchased from Lonza, Inc. (Walkersville, MD, USA). DPSCs were cultured in DPSC basal medium (Lonza, Inc.) containing DPSC SingleQuots (Lonza, Inc.) at 37 C in 5% CO 2 and 95% air. The BMMSCs were cultured in MSC basal medium (Lonza, Inc.), containing MSC-GM SingleQuots (Lonza, Inc.), at 37 C in 5% CO 2 and 95% air. After primary culture, the cells were subcultured at a density of approximately 1 Â 10 4 cells/cm 2 . Cells from the third to sixth passages were used for the experiments.

Preparation of CM
After achieving 80% confluence, the DPSCs or BMMSCs were replenished with serum-free Dulbecco's Modified Eagle's Medium (DMEM (À); Gibco, Rockville, MD, USA) containing an antibioticeantimycotic solution. The cell-cultured CM was collected after 48 h of incubation and centrifuged at 440 Â g for 5 min at 4 C. The supernatant was collected, centrifuged at 17,400 Â g for 3 min at 4 C, and filtered using 0.22 mm pore filters (Millex-GP; Merck Millipore Ltd., Billerica, MA, USA). The DPSC-CM and BMMSC-CM were stored at À80 C before use in the experiments.

Cytokine antibody array
Cytokine array analysis was performed via laser scanning using 174 human-cytokine array plates to assess the cytokines present in the DPSC-CM and BMMSC-CM (Quantibody® Human Cytokine Array 6000; RayBiotech, Inc., Norcross, GA, USA). Each scan was performed in duplicate, and data were calculated as the ratio of the cytokine levels in DPSC-CM to that in BMMSC-CM.

Measurement of stimulated saliva flow
The MRL/lpr mice were anesthetized with chloral hydrate (0.4 g/ kg body weight), and the stimulated saliva flow was measured as previously described [18,19]. At 3 min after intraperitoneal injection of pilocarpine (0.05 mg/100 g body weight), a micropipette was used to collect whole saliva from the oral cavity for 10 min, and the amount of saliva collected was then calculated.

Histological analysis
Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed as previously described [12]: dissected submandibular glands (SMGs) were fixed in 4% paraformaldehyde, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin. The samples were cut to create 5-mm-thick histological sections, which were stained with H&E and analyzed under a light microscope.
To clearly assess the inflammation, focus scores were analyzed. The ratio of the foci area to the total area of SMGs was calculated and presented (six independent experiments for each group).

Terminal deoxynucleotidyl transferase-mediated dUTP nickend labeling) staining
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed (Click-iT Plus TUNEL Assay with Alexa Fluor 647; Thermo Fisher Scientific, Inc., Waltham, MA, USA) to detect apoptotic cells. Images of the sections were taken with a fluorescence microscope (BZ-X810; Keyence, Osaka, Japan; n ¼ 10 per group). We calculated the percentage of TUNEL-positive cells per total number of cells in the SMGs of each group.

Enzyme-linked immunosorbent assay (ELISA) analysis
The concentrations of anti-dsDNA and anti-SSA in the mice were measured using mouse anti-dsDNA and mouse anti-Ro52/SSA ELISA kits (Signosis, Inc., Santa Clara, CA, USA). The serum samples were diluted at a ratio of 1:50.
The sections were rehydrated, subjected to antigen retrieval using Dako Target Retrieval Solution (pH 9.0; Dako North America, Inc., Carpinteria, CA, USA) for 10 min at 121 C, blocked for endogenous peroxidase with 0.3% H 2 O 2 in methanol, and incubated for 30 min. After washing with phosphate-buffered saline, the sections were blocked for nonspecific binding using Blocking One Histo (Nacalai Tesque, Inc., Kyoto, Japan) for 15 min at room temperature and then incubated with the primary antibody overnight at 4 C. The sections were reacted using peroxidase stain 3,3diaminobenzidine (DAB) kit (Nacalai Tesque, Inc.) for 1 h and developed with DAB solution. Hematoxylin counterstaining was performed following the DAB reaction.

Statistical analysis
All experiments were conducted in triplicate and repeated at least twice. Group means and standard deviations were calculated for each measured parameter. Statistical differences were evaluated using the Student's t-test, ManneWhitney U test, and Tukey's honest significant difference test. A p-value < 0.05 was considered to be statistically significant, and a p-value of <0.01 was considered to be highly significant.

DPSC-CM prevents a decrease in saliva and inhibits inflammation in SMGs
Our protocols for the administration of DMEM (À), BMMSC-CM, and DPSC-CM are shown in Fig. 2a. As shown in Fig. 2b, DPSC-CM alleviated inflammation in the SMGs of the mice, and the focus scores in the DPSC-CM group were lower than those in the nontreatment, DMEM (À), and BMMSC-CM groups (Fig. 2c).
The stimulated saliva flow rate increased in the 12-week-old mice injected with DPSC-CM than that in the other groups (Fig. 2d). Interestingly, anti-dsDNA and anti-Ro52/SSA were highly detected in the serum collected from the non-treated, DMEM (À), or BMMSC-CM-administered mice than those in the DPSC-CM group (Fig. 2e).

Relative mRNA expression levels of inflammatory or antiinflammatory cytokines in the SMGs
The SMGs of mice who received DPSC-CM had significantly decreased relative mRNA expression levels of inflammatory cytokines, such as Il-2, Ifn-g, Il-4, Il-6, and Il-17a (Fig. 3a). On the other hand, levels of Il-10 and Tgf-b1, both anti-inflammatory cytokines, were significantly increased in these mice (Fig. 3a).

DPSC-CM administration decreases the number of apoptotic cells in the SMGs
We then investigated apoptotic cells and found that they increased in the SMGs of the non-treatment and DMEM (À) groups than the BMMSC-CM and DPSC-CM groups (Fig. 3b). The number of apoptotic cells significantly decreased in the group receiving DPSC-CM relative to the group receiving BMMSC-CM (Fig. 3b).

DPSC-CM induces FOXP3-expressing cells and inhibits RORgexpressing cells in the mouse spleen
We investigated the T helper subset in the mouse spleen tissue to confirm the mechanism by which DPSC-CM influences the whole ***p < 0.00, **p < 0.01, *p < 0.05). (b) TUNEL assays of the SMGs of the MRL/lpr mice reveal that the nuclei were stained with DAPI (blue) (Bars ¼ 50 mm). Percentage of TUNELpositive cells per total number of cells in the SMGs (right side). Data are representative of the mean ± standard deviation (n ¼ 6, **p < 0.01, *p < 0.05).
body. As shown in Fig. 4, FOXP3-expressing cells, which were used as the marker of Treg cells, increased in the DPSC-CM-treated group. However, RORg-expressing and GATA3-expressing cells which were used as markers of the type T helper 17 [Th17] and Th2 cells, respectively, decreased in the DPSC-CM-treated group than in the other groups. T-bet-expressing cells, which were used as the marker of Th1 cells, showed no changes in all groups.

Discussion
In this study, we evaluated the therapeutic effects of CM from DPSCs or BMMSCs in a secondary SS mouse model. DPSC-CM contains numerous anti-inflammatory factors (e.g., TGF-b1, IL-10, and IL-13) than BMMSC-CM ( Fig. 1a and b).
The MRL/lpr strain is a well-established mouse model for SLE [21,22]. It is characterized by the inflammation of multiple tissues (e.g., skin, joints, glands, lungs, heart, and kidneys), massive lymphadenopathy, and splenomegaly, which progress in an agedependent manner. Destructive mononuclear infiltrates in the lacrimal and SMGs of MRL/lpr mice are hallmarks of SS: we confirmed lymphocyte infiltration around the salivary duct cells in 12-week-old mice (Fig. 2b); the salivary flow rate declined at 12 weeks in MRL/lpr mice in the non-treatment and DMEM (À) groups than in the DPSC-CM group (Fig. 2d).
Extensive studies have been conducted to elucidate the immunomodulatory properties of MSCs; however, existing clinical trials of cell-based therapy are controversial, and their security is not guaranteed [23]. Many studies have determined that cell paracrine factors have functions similar to those of cells and the study of exosomes in the field of immunology is extensive [24e26]. Paracrine factors are better preserved, have lower risks of tumorigenesis and immune rejection, and can be used as alternative therapies for various immune diseases, comparable to cells [25].
A previous study reported that BMMSC-CM contains cytokines such as vascular endothelial growth factor, monocyte chemoattractant protein (MCP)-1, MCP-3, and hepatocyte growth factor. Many types of biomaterials and stem cell transplantation therapies have recently been proposed to enhance anti-inflammatory effects and functional recovery [19,27]. BMMSC acquisition is a difficult procedure; DPSC-CM is a more accessible resource, but its immunoregulation properties have not been fully studied.
Studies have shown that the Th17/Treg imbalance is a driving factor in the occurrence and development of immune diseases such as SLE and SS [28,29]. We investigated mRNA levels of inflammatory and anti-inflammatory cytokines, which are induced in the Th1, Th2, Treg, or Th17 cells of MRL/lpr mice SMGs. As shown in Fig. 3a, DPSC-CM administration decreased expression levels of Ifng, Il-6, and Il-17a and increased those of Il-10 and Tgf-b1 in the SMGs, indicating Treg cell differentiation in the local environment. Furthermore, IFN-g is primarily secreted by cytotoxic or Th1 T cells and natural killer cells [30]. One study reported that exogenous administration of IFN-g with desiccating stress exposure increased epithelial apoptosis, indicating that IFN-g promotes epithelial apoptosis through the extrinsic apoptosis pathway in SS [31]. We investigated and confirmed that DPSC-CM had an anti-apoptotic effect on the SMGs (Fig. 3b). We evaluated the kidneys of MRL/lpr mice, and the results suggested that DPSC-CM also improved the kidneys (e.g., creatinine level); decreased the expression levels of Ifn-g, Il-6, and Il-17a; and increased the expression levels of Il-10 and Tgf-b1, which can easily promote Treg cells in the kidneys ( Supplementary Fig. 1).
Effector cytokines of Th17 and Treg cells have been increasingly recognized as key players in anaphylaxis, autoimmunity, and inflammation [32]. IL-10 produced by Treg cells resulted in amelioration of the severity of collagen-induced arthritis mice than wild-type mice [33]. Our experimental results confirmed that DPSC-CM induced Treg cells and suppressed Th1 and Th17 cells in splenic lymphocytes relative to other groups (Fig. 4).

Conclusions
This is the first study to report that DPSC-CM alleviates hyposalivation caused by SS by decreasing the number of inflammatory cytokines, regulating the local inflammatory microenvironment, and decreasing apoptosis in the SMGs. DPSC-CM shows promise as a novel option in cell-free therapy for various autoimmune diseases such as SLE and SS.

Declaration of competing interest
None.