Transplantation of gingiva-derived mesenchymal stem cells ameliorates collagen-induced arthritis

Rheumatoid arthritis (RA) is a chronic, progressive, and inflammatory autoimmune disease involved in multiple systems, and it primarily affects the small arthrodial joints [1, 2]. Pathologically, the hyperproliferation of the synovial membrane and accumulation of activated T cells and macrophages leads to cartilage degradation and erosion of bone in the joints [1]. The precise etiology of RA is still unknown. Extensive evidence suggests it is the result of interactions between genes and environmental factors [3]. The breakdown of the immunologic self-tolerance results in aberrant immune responses directed at self-antigens in the joints. Antigen-driven T cells and B cells are thought to participate in the rheumatoid process. The therapeutic interventions aim to modulate pathogenic cells, neutralize the effector molecules, and restore tolerance. However, current drug therapy for RA is still not curative [1].

Mesenchymal stem cells (MSCs) have been isolated from almost every tissue. They possess the capacity for self-renewal and multipotent differentiation into multiple mesenchymal lineages, including bone, fat, and cartilage [4, 5]. Moreover, MSCs display anti-inflammatory and immunomodulatory capacities, both in vitro and in vivo [6, 7]. Recent studies have demonstrated that MSCs could also be isolated from gingivia tissue (GMSCs), and that they exhibit many advantages over bone marrow-derived MSCs (BMSCs). GMSCs are easy to access from the dental clinic, and they are more morphologically and functionally stable, and hence less tumorigenic. They proliferate rapidly while remaining homogenous. Transplantation of GMSCs has been proven to have therapeutic effects on experimental colitis [8, 9] and mitigating chemotherapy-induced oral mucositis [10], as well as in autoimmune arthritis in mouse models [11].

Collagen-induced arthritis (CIA) is an animal model frequently used to study the effect of new therapeutics for RA, and it shares several clinical, histological, immunological, and genetic features with human RA [12]. In this study, we attempt to test whether one-time transplantation of GMSCs could ameliorate CIA. Our findings suggest that GMSC-mediated T-cell apoptosis via a FasL/Fas pathway results in immune tolerance and ameliorates the severity of CIA in mice.

In recent years, more and more interest has been taken on the immunomodulating property of MSCs. The immunosuppressive capacities of MSCs were evaluated in experimental animal models, as well as in humans, to treat autoimmune diseases [13‐16]. In this study, we isolated and characterized MSCs from the gingival tissues of the mouse, and tested their therapeutic effects using CIA mouse models. We confirmed that GMSCs exhibited similar stem cell properties and immunomodulatory properties as BMSCs. They express a similar profile of surface markers, and exhibit multipotent differentiation into different cell lineages, including mesodermal (adipocytes and osteocytes).

CIA is a reliable model of RA which is widely used in the development of therapeutics. Augello et al. [17] first reported that a single injection of mouse BMSCs prevented the occurrence of severe, irreversible damage to bone and cartilage in CIA mice. We demonstrated that systemic infusion of allogeneic mouse GMSCs also can efficiently ameliorate both clinical and histopathological severity of the CIA inflammation. The clinical score decreased by 44 % on average, and the histological score by 32 %. The serum levels of TNF-α and CII-specific IgG decreased remarkably by 50 % and 63 %, respectively, which further confirmed that GMSC therapy could downregulate the immune response and reduce tissue damage. More recently, several scholars reported that rat BMSCs [18], human AD-MSCs [19], and human GMSCs [11] were also beneficial for CIA. Previous studies demonstrated that MSCs could overcome MHC mismatch; when xenogeneic, allogeneic, or syngeneic MSCs were utilized, each of them may exhibit a therapeutic effect [20]. Although the precise underling mechanism remains to be elucidated, a variety of factors, including transforming growth factor (TGF)-β, interleukin (IL)-10, prostaglandin E2 (PGE2), nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), and FasL have been identified as potential regulators of MSC-based immunomodulation [21, 22].

It is well known that T cells play a key role in induction, maintenance, and relapse of RA, and they are an important target for the development of new anti-arthritic therapies [23]. Previous scholars have regarded RA as a Th1-driven disease, with a predominance of Th1 cytokines and a lack of Th2 cytokines, whereas in recent years the role of Th17 has been emphasized in joint inflammation and destruction [11]. Tregs are involved at the center of immunosuppressive reactions, and suppress several autoreactive responses and maintain self-tolerance in the immune system. Augello et al. [17] found that MSC viability was not required for their long-term immunosuppressant action, and that the prolonged immunosuppressive activity of MSCs could be attributed to the action of Treg clones that can be activated by an antigen-specific stimulus. We found the proportion of Th1 and Th17 to be elevated in CIA mice, along with a reduced level of Treg in CD4⁺. GMSC transplantation can upregulate the Treg level (by ≈ 84 % in spleen, and ≈ 70 % in DLN) and reduce the Th1 and Th17 level, leading to immune tolerance and thereby ameliorating severity of the inflammation. The GMSC/T cell co-culture experiments further reveal that GMSCs could induce apoptosis of activated T cells (Fig. 3). Therefore, GMSCs are a plausible cell source for MSC-based therapy in RA by rescuing the T-cell homeostasis of the recipients.

FasL is a type II transmembrane protein that belongs to the tumor necrosis factor (TNF) family. Its binding with its receptor induces apoptosis. FasL/Fas interactions play an important role in the regulation of the immune system [22, 24]. Previous scholars demonstrated that MSCs (BMSCs or SHED) may induce T-cell apoptosis, which further upregulates the Tregs via a high level of macrophage-released TGF-β, and results in immune tolerance [22, 25]. In this study, we isolated FasL mutant GMSCs from B6Smn.C3-FasL^gld/J mice (FasL^-/- GMSC). We found that WT GMSCs, but not FasL^-/- GMSCs, elevate the Treg levels and reduce the proportion of the Th1 and Th17 subsets, whereas FasL TF GMSCs can rescue the immunosuppressant effects in the treatment of CIA, as well as in vitro co-culture system. Therefore, FasL-induced T-cell apoptosis is required for GMSC-based cell therapy for CIA. Figure 2g shows that the decrease in serum anti-CII tilts in the FasL TF GMSC-treated group has not reach statistical significance (P > 0.05), which may be due to the small sample size. Moreover, the direct/indirect effect of FasL on the humeral immunity may be different from that on the cell-mediated immunity, and the changes in the corresponding indexes are not always synchronized. Figure 3a suggests that, although the FasL level of the FasL TF GMSCs is eminently lower than that of the WT GMSCs, they exhibit similar therapeutic potential. Previous scholars have shown that MSCs can inhibit, in a dose-dependent manner, the proliferation of, and cytokine production by, T cells, B cells, natural killer cells, and dendritic cells via multiple mechanisms; there were also a few studies which failed to demonstrate any improvement in experimental CIA with MSC treatment [26]. The conflicting results emphasize the need for standardizing each step of the MSC therapy. Further studies are required to find the best GMSC dose, and the dose-related effects of GMSCs, as well as the FasL level, should be reassessed in a more standardized manner to achieve the best therapeutic outcomes.

Taken together, the present study demonstrates that systemic infusion of allogeneic mouse GMSCs can efficiently ameliorate severity of the CIA inflammation by inducing T-cell homeostasis and immune tolerance. FasL plays an important role in the mechanism underlying the beneficial effect of the GMSC-based cell therapy.