Gingival mesenchymal stem cell‐derived exosomes are immunosuppressive in preventing collagen‐induced arthritis

Abstract Due to the unsatisfied effects of clinical drugs used in rheumatoid arthritis (RA), investigators shifted their focus on the biotherapy. Although human gingival mesenchymal stem cells (GMSC) have the potential to be used in treating RA, GMSC‐based therapy has some inevitable side effects such as immunogenicity and tumorigenicity. As one of the most important paracrine mediators, GMSC‐derived exosomes (GMSC‐Exo) exhibit therapeutic effects via immunomodulation in a variety of disease models, bypassing potential shortcomings of the direct use of MSCs. Furthermore, exosomes are not sensitive to freezing and thawing, and can be readily available for use. GMSC‐Exo has been reported to promote tissue regeneration and wound healing, but have not been reported to be effective against autoimmune diseases. We herein compare the immunomodulatory functions of GMSC‐Exo and GMSC in collagen‐induced arthritis (CIA) model and in vitro CD4+ T‐cell co‐culture model. The results show that GMSC‐Exo has the same or stronger effects compared with GMSC in inhibiting IL‐17A and promoting IL‐10, reducing incidences and bone erosion of arthritis, via inhibiting IL‐17RA‐Act1‐TRAF6‐NF‐κB signal pathway. Our results suggest that GMSC‐Exo has many advantages in treating CIA, and may offer a promising new cell‐free therapy strategy for RA and other autoimmune diseases.


| INTRODUC TI ON
Rheumatoid arthritis (RA) is a severe inflammatory autoimmune disease (AID) characterized by the infiltration of activated immune cells and the production of inflammatory factors that lead to chronic synovitis and progressive destruction of cartilage and bone in multiple joints. 1 Though the pathogenesis of RA is not fully understood, studies show that it involves the abnormal activation of various immune cells, such as macrophagocyte, T and B cells, dendritic cells, and NK cells. Among these cells, CD4 + T cells play a key role in the development of inflammation, especially the helper T cell (Th cells) subsets Th1, Th17 cells and regulatory T cells (Treg). 1 Furthermore, it has been previously reported that the imbalance in Th17/Treg is a decisive factor. [2][3][4] Th17 cells secrete various pro-inflammatory cytokines, mainly IL-17, which could activate related signal transduction pathways (such as NF-κB) and induce the production of proinflammatory cytokines (such as tumour necrosis factor [TNF]α, IL-6 and IL-1β), chemokines and matrix metalloproteinases, leading to tissue invasion and destruction as well as damage of articular cartilage and bone. 5 Currently, RA patients are normally treated with disease-modifying antirheumatic drugs (DMARDs), including synthetic (or chemical) DMARDs (sDMARDS) and biological DMARDs (bDMARDs). The former is divided into conventional synthetic and targeted synthetic DMARDs (csDMARDs and tsDMARDs), while the latter is divided into biological original and biosimilar DMARDs (boDMARDs and bsDMARDs). 6 But the traditional drug treatment can only relieve the symptoms, none is curative. In addition, some adverse reactions occur when these drugs are used for a long time. 1 Therefore, it is urgent to seek novel approaches to improve present status for RA treatment.
Cell-based therapeutic approaches are currently developed in RA, mainly mesenchymal stem cells (MSCs)-based approaches.
Human gingival mesenchymal stem cells (GMSC) exist in human gingiva and have been attracting increased attention due to their easier isolation, faster proliferation, stable phenotype and immunomodulatory capacities. [7][8][9] In addition, GMSC have been reported to have preventive or therapeutic effects in several AIDs animal models and humanized animal models. [10][11][12][13][14][15][16][17] However, a particular problem in MSCs field is that after systemic transplantation, MSCs are rapidly trapped in the pulmonary vascular bed because of their large size. 18,19 Usually less than 1% of MSCs reach and are implanted in the target sites. Despite this, the therapeutic effect still be observed and was mainly attributed to paracrine actions and MSCs apoptosis. [20][21][22] Indeed, apoptotic cells have been shown to treat ongoing experimental arthritis. 23 As one of the most important paracrine mediators, MSC-derived exosomes exhibit therapeutic effects via immunomodulation in a variety of disease models, bypassing potential shortcomings of the direct use of MSCs. [24][25][26][27][28] Furthermore, exosomes are resistant to freezing and thawing, and can be readily available for use. GMSC-derived exosomes (GMSC-Exo) have been reported to promote tissue regeneration and wound healing, 24,29,30 but have not been reported to be effective against autoimmune diseases. Importantly, it is reported that exosomes produced by MSCs pre-stimulated by inflammatory factors have stronger immune regulation capabilities. [31][32][33] Coincidentally, the microenvironment where GMSC are located is rich in food residues, microbial flora and saliva, which provides a natural inflammatory microenvironment for GMSC. This is the unique advantage of GMSC, so the GMSC-Exo naturally have stronger immunoregulatory capabilities than other MSCs and could be the best choice for the treatment of RA.
In the present study, we demonstrate that administration of GMSC-Exo/GMSC significantly alleviate the inflammation and bone erosion in collagen-induced arthritis (CIA) model, and GMSC-Exo have the same or stronger effect compared with GMSC. We further observed that GMSC-Exo play a role by regulating the imbalance of Th17/Treg in vitro or in vivo, and inhibiting IL-17RA-Act1-TRAF6-NF-κB signal pathway in vivo. In conclusion, our results suggest that GMSC-Exo can be applied as a promising therapeutic approach for patients with RA and other autoimmune diseases.

| Isolation, culturing and characterization of human GMSC
Human tissue samples were obtained from discarded tissues of patients who had relatively healthy periodontium undergoing routine dental procedures and who provided informed consent in the Dental Division of the Third Affiliated Hospital at Sun Yat-sen University.
This study was carried out in accordance with the recommendations of the ethical review committee of clinical research of the Third Affiliated Hospital of Sun Yat-sen University. Human GMSC were obtained by following the protocol described previously. 10 We characterized GMSC by detecting the stem cell phenotypic markers and multipotent differentiation properties. Sub-cloning cultures were used to purify GMSC, and cells from the third passages were used in experiments. For GMSC characterization markers detection, GMSC were stained with mAbs for human CD34, CD44, CD45, CD73, CD90 and CD105 (Biolegeng) and assessed by flow cytometry.
For GMSC multipotent differentiation properties detection, osteogenic differentiation and adipogenic differentiation were used.
After 14 days, the cultured cells were stained with Oil Red-O (Sigma-Aldrich).

| Production and characterization of GMSC-Exo
Gingival mesenchymal stem cell were seeded at 5 × 10 5 cells/ml on 10 cm dish with complete growth medium. When GMSC reached 80%-90% confluence, they were cultured in conditioned medium with exosomes-free foetal bovine serum for 24 h, and then, the supernatants containing GMSC-Exo were harvested. The GMSC-Exo were extracted by differential ultracentrifugation. In brief, the supernatants were centrifuged at 300 g for 10 min and 2000 g for 10 min to remove the detached cells and cell debris/apoptotic bodies respectively. Then, the supernatant was centrifuged for 30 min at 10,000 g to remove microvesicles (MVs). After that, the clarified supernatant was centrifuged at 100,000 g for 70 min and the pellet on the bottom of the tube was GMSC-Exo. Then, the pellet was washed with phosphate-buffered saline (PBS) by centrifuging at 100,000 g for 70 min ( Figure 1A). 34 Finally, the concentrated GMSC-Exo were suspended in PBS and determined using a BCA protein assay kit

| In vitro proliferation assay
To examine and compare the effects of GMSC-Exo and GMSC on the proliferation of CD4 + T cells in vitro, we applied CFSE Cell Division Tracker Kit (Biolegend). Mouse CD4 + T cells were isolated from splenocytes by a CD4 + T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's protocol and labelled with CFSE, then cultured alone or with GMSC-Exo at the ratio of 10 µg/40 µg/90 µg: Mice were monitored twice weekly for signs of arthritis based on arthritis scores and paw swelling. Each paw was evaluated and scored individually using a 0 to 4 scoring system: 0 = no damage; 1 = paw with detectable swelling in a single digit; 2 = paw with swelling in more than one digit; 3 = paw with swelling of all digits and instep; and 4 = severe swelling of the paw and ankle. 15 On Day 56, all mice were euthanized, and peripheral blood, spleen, axillary and mesenteric lymph nodes (LN), and limbs were collected for further studies.

| Histology and immunohistochemical staining
The excised paw was fixed in 4% paraformaldehyde, decalcifi-

| ELISA assays
The peripheral blood from CIA mice was stood for 20 min and centrifuged at 1000 g for 10 min to obtain the serum. The levels of cytokines (IFNγ, IL-17A, IL-10, IL-6 and TNFα) in culture supernatants or serum were quantified by enzyme-linked immunosorbent assay (ELISA) kit (Biolegend) according to the manufacturer's instructions.

| Western blot analysis
Total proteins were extracted from the paw of CIA mice, and their concentration was determined using a BCA assay (Beyotime  We used flow cytometry to further verify the stem cell phenotypic markers of GMSC. The results depicted that GMSC were negative for CD34 and CD45, the surface marker of hematopoietic stem/progenitor cell and leukocyte, but positive for CD44, CD73, CD90 and CD105 ( Figure 1D).

| Isolation and characterization of GMSC-Exo
Gingival mesenchymal stem cell-Exo were successfully isolated using the differential ultracentrifugation method as previously described 34 (Figure 2A), and different tools were used to analyse them as recommended by the International Society for Extracellular Vesicles. 41 TEM analysis showed that the round shaped vesicles were surrounded by a bilayer membrane ( Figure 2B). The result of NTA analysis revealed the synthesis of three test data, all of which had single peaks (~100 nm) ( Figure 2C). Our isolated GMSC-Exo had the mean diameter of 106.88 ± 19.28 nm ( Figure 2D). The results of Western blot analysis showed that GMSC-Exo expressed the exosomal markers CD9, CD63 and CD81 ( Figure 2E).

| GMSC-Exo exhibited stronger immunosuppressive effects than GMSC in vitro
We investigated the immunosuppressive properties of GMSC-Exo and GMSC in a proliferation assay. Interestingly, the proliferation of CD4 + T cells increased after GMSC/GMSC-Exo treatment ( Figure 3A,D). However, GMSC-Exo, but not GMSC, increased the percentages of CD4 + IL-10 + T cells in a dose-dependent manner ( Figure 3B,E). Both GMSC-Exo and GMSC inhibited the percentages of CD4 + IFNγ + Th1 and CD4 + IL-17A + Th17 subsets significantly ( Figure 3C,F,G). We also measured cytokines in supernatant of coculture. After GMSC/GMSC-Exo treatment, IL-10 level was significantly up-regulated and the change trend was consistent with that of CD4 + IL-10 + cells ( Figure 3H)

| GMSC-Exo have stronger effects than GMSC in preventing bone erosion in CIA mice
Bone erosion may develop as a result of cytokines released by inflammatory cells in the marrow. We used micro-CT to evaluate the degree of bone erosion because it was better than histologic analysis for this aspect. The 3D images of PBS-treated group showed that the surface of the cortical bone was rough, the bone structure of metatarsophalangeal and toe joints was irregular, and bone dissolu-   and NF-κB p65/p105 compared to PBS treatment ( Figure 7F). In addition, GMSC-Exo showed a better effect in suppressing TRAF6 and NF-κB p65/p105. All differences are statistically significant ( Figure 7G). In addition, the binding of nucleic acid, drug and RNA were also ranked as top functions ( Figure 8B). Interestingly, GMSC-Exo-treated group shared most of molecular functions of GMSC-treated group, indicating the potential of GMSC-Exo in replacing GMSC.

| DISCUSS ION
This study provides the first evidence that GMSC-Exo exert immunomodulatory effect in CIA model. Moreover, this is one of the few studies reporting the role of GMSC-Exo in vivo and in vitro. 24,29,30,48,49 In order to verify whether GMSC-Exo has the same immuno- One of the key mechanisms of MSCs' anti-inflammatory effects is the secretion of soluble factors with paracrine actions, which, at least in part, is mediated by exosomes. 26,58,59 Exosomes are mainly released from the endosomal compartment and contain cargo including miRNA, mRNA and protein from the cell of their origin. 60 In this study, we analysed the components of paws by LC/MS-MS and found that GMSC-Exo-treated group shared most of DEPs and molecular function with GMSC-treated group, which indicated that GMSC-Exo has the potential to replace GMSC (Figure 8). which allows it to be recognized and killed by CD8 + cells, thereby reducing its number. Therefore, GMSC-Exo may have higher safety and more advantages in comparison with GMSC for preventing RA.
However, the arthritis score is not significant at the time of treatment in our experiment, which means that mice have not developed arthritis when mice were treated. This is not really relevant in a clinical point of view, so the clinical application of GMSC-Exo still needs further research.

| CON CLUS ION
This study compared the immunoregulatory effects of GMSC and GMSC-Exo in CIA mice for the first time, and then explored the underlying mechanisms. The results showed that GMSC-Exo have the same or better effects in comparison with GMSC in reducing the incidence of arthritis and bone erosion in CIA mice by regulating the imbalance of Th17/Treg and inhibiting IL-17RA-Act1-TRAF6-NF-κB F I G U R E 6 GMSC-Exo therapy influences the polarization of Th cells in vivo. (A, C, E) Representative flow cytometry images show the expression of CD4 + INFγ + Th1, CD4 + IL-17A + Th17 cells and CD4 + CD25 + FoxP3 + Treg in the spleens and LNs of three groups of CIA mice. (B, D, F) GMSC-Exo and GMSC significantly decreased the percentage of CD4 + INFγ + Th1 and CD4 + IL-17A + Th17 cells in the mouse spleen and LN, and increased the percentage of CD4 + CD25 + FoxP3 + Treg, whereas PBS did not significantly alter the frequencies of the subset Th cells in the CIA mice. n = 6 in each group. The animal experiments were performed in three independent experiments. *p < 0.05, **p < 0.01. Values are given as mean ± SD. CIA, collagen-induced arthritis; GMSC, gingival mesenchymal stem cells; GMSC-Exo, gingival mesenchymal stem cell-derived exosomes; LNs, lymph nodes; NS, not significant; PBS, phosphate-buffered saline F I G U R E 7 GMSC-Exo reduces Th1 and Th17 cytokine production and increases Treg cytokine production in vivo, partially via IL-17RA-Act1-TRAF6-NF-κB signal pathway. (A-E) The expression of serum cytokines IFNγ, IL-17A, IL-10, TNFα and IL-6 in CIA mice was determined by ELISA. Data are mean ± SD, n = 3 mice. (F) Total proteins were extracted from the paw of CIA mice after mouse sacrifice. The experiments were performed in three independent experiments. The main component proteins of IL-17RA-Act1-TRAF6-NF-κB signal pathway expression were analyzed by Western blot. (G) The relative density of IL-17RA, TRAF6, Act1 and NF-κB p65/105 to GAPDH is shown by bar graph. One-way ANOVA was used to compare different groups. Data are mean ± SD, n = 3 independent experiments. *p < 0.05, **p < 0.01. CIA, collagen-induced arthritis; ELISA, enzyme-linked immunosorbent assay; GMSC, gingival mesenchymal stem cells; GMSC-Exo, gingival mesenchymal stem cell-derived exosomes; NS, not significant; PBS, phosphate-buffered saline signalling pathway. Our studies have the potential to provide a promising cell-free therapy for treating RA and other autoimmune diseases.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors declare the data are available.