Rheumatoid arthritis (RA) is an autoimmune disease caused by chronic joint inflammation, with subsequent cartilage destruction and bone erosion. Although the precise etiology of RA remains unclear, proinflammatory cytokines and autoreactive T cells are crucial in its pathogenesis. Th1 cell and/or Th17 cell activation has been implicated in the development of cell-mediated autoimmune arthritis (1, 2). In contrast, Th2 cells and/or Treg cells are protective in RA and in animal models of collagen-induced arthritis (CIA) (3, 4).
Transforming growth factor β (TGFβ) is a multifunctional cytokine that regulates cell proliferation, differentiation, and migration, and wound healing and tissue repair (5). It also plays critical roles in the induction of FoxP3+ Treg cells (6, 7), interleukin-6 (IL-6)–induced differentiation of Th17 cells (8), and Treg cell homeostasis (9). However, TGFβ must be activated to exert its regulatory effects, and there is controversy regarding the exact mechanism(s) involved.
Treg cells that express the transcription factor FoxP3 play a critical role in controlling autoimmune responses and maintaining peripheral tolerance (10, 11). Deficiencies in Treg cell function have been identified in autoimmune diseases including RA (12), psoriasis (13), and myasthenia gravis (14); these studies suggest that Treg cells control autoimmunity. Th17 cells, a new subset of CD4+ T helper cells, may play a central role in the pathology of autoimmune disease. Genetic deletion of IL-17 in mice inhibited the development of CIA (15). Increased levels of IL-17 have been detected in RA synovial fluid (16) and synovial tissue memory T cells (17).
Mesenchymal stem cells (MSCs) are multipotent progenitor cells. They can be isolated from a number of adult tissues capable of giving rise to adipogenic, osteogenic, and chondrogenic lineages, and have the ability to differentiate to lineages of mesenchymal tissues, including bone, cartilage, and adipose tissues (18). These cells have potent immunosuppressive and antiinflammatory effects, through direct cell–cell contact or by secreting soluble factors, and have the potential for clinical application in the repair of damaged tissue. MSCs also suppress T cell proliferation induced by alloantigens or mitogens (19–21). Because of their immunomodulatory effects, MSCs were proposed as a treatment of acute graft-versus-host disease after allogeneic stem cell transplantation (22) as well as experimental encephalomyelitis (23) and diabetes (24). However, the specific mechanisms involved in the immunoregulatory activity of bone marrow MSCs remain unknown, and the therapeutic effects of MSCs have varied in different CIA models (25, 26).
We used adenoviral TGFβ gene transfer to mouse bone marrow MSCs to achieve more potent therapeutic efficacy for autoimmune arthritis. We observed that systemic delivery of TGFβ-transduced MSCs reduced disease severity, osteoclastogenesis, and bone erosion. Moreover, TGFβ-transduced MSCs regulated type II collagen–reactive T cell responses and proinflammatory cytokines by inducing type II collagen–specific Treg cells and inhibiting Th17 cell differentiation. These data demonstrate the ability to provide effective treatment in established arthritis models through systemic administration of TGFβ-transduced MSCs. Thus, these MSCs may represent a new and powerful approach to the treatment of RA and other autoimmune diseases.
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MSCs are increasingly being used to treat autoimmune and systemic inflammatory diseases. Although considerable progress has been made in understanding the mechanisms by which MSCs exert immunomodulatory functions in autoimmune disease, these mechanisms are still not fully understood. A balance of Treg cells and Th17 cells may be critical for maintaining immune tolerance and for the treatment of autoimmune diseases. In the present study, we demonstrated a novel mechanism of action: use of TGFβ-transduced MSCs to control progression of CIA by regulating Treg/Th17 cells. We showed that MSCs, when given systemically after disease onset, reduce both inflammatory and antigen-reactive T cell responses. Interestingly, we observed that infused TGFβ-transduced MSCs migrated to inflamed joints and inhibited osteoclast differentiation. We believe that these results could help shape future clinical strategies for the treatment of autoimmune diseases.
The immunomodulatory and reparative antiinflammatory properties of MSCs have been evaluated in an experimental autoimmune disease model. Previous studies using bone marrow MSCs in the CIA model have generated conflicting results. Although Augello et al (25) reported that a single injection of MSCs had a therapeutic effect on disease onset by decreasing serum levels of proinflammatory cytokines, another study showed a negative effect of MSCs on CIA, because MSCs alone did not inhibit proinflammatory cytokines such as TNFα (26). The mechanisms underlying the beneficial effect described by Augello et al need to be explored further, because no convincing increase in the number of Treg cells was observed in vivo, despite in vitro evidence of T cell inhibition by MSCs.
In an attempt to augment the therapeutic efficacy of MSCs in suppressing RA, we developed genetically modified MSCs using an adenoviral vector encoding TGFβ. Compared with MSCs, TGFβ-transduced MSCs exerted more potent antiproliferative activity and induced Treg cell generation in vitro (Figure 1). These data suggest that TGFβ-transduced MSCs enhanced therapeutic efficacy in the CIA model.
We demonstrated that compared with systemic administration of MSCs, systemic administration of TGFβ-transduced MSCs had greater long-lasting effects in established CIA. We investigated changes in the cytokine secretion profiles induced by these cells to understand the mechanisms involved in the observed TGFβ-transduced MSC–mediated immunosuppressive effects. Mice with CIA treated with TGFβ-transduced MSCs showed lower production of proinflammatory cytokines. The secretion of TNFα decreased, and the secretion of IL-10 increased in the supernatant of the mixed lymphocyte reaction, suggesting that TGFβ-transduced MSC–mediated suppressive activity in vitro is partly attributable to soluble mediators. Antigen-induced IL-10 secretion is involved in the induction of specific T cell tolerance (37).
Polyclonal or antigen-specific activated TGFβ-induced Treg cells are potent suppressors of both organ-specific (38) and systemic autoimmune diseases (39). There is growing evidence that the immunosuppressive effects of bone marrow MSCs are associated with CD4+CD25+FoxP3+ Treg cell expansion (40, 41). However, it has also been reported that bone marrow MSCs ameliorate autoimmune enteropathy in vivo, an effect that is independent of Treg cells (42). We observed that, compared with mice with CIA treated with placebo or MSCs, mice with CIA treated with an intraperitoneal injection of TGFβ-transduced MSCs had a significantly higher percentage and absolute number of Treg cells in the peritoneum and spleen and a smaller increase in the mesenteric LNs (Figure 4). Treatment with TGFβ-transduced MSCs expanded Treg cells in both the peritoneum and spleen 7 days after the injection, producing a Treg phenotype with potent suppression of type II collagen–reactive effector T cells. We also observed that depletion of Treg cells from TGFβ-transduced MSCs substantially abolished the therapeutic effects on arthritis (data not shown). Thus, the expanded Treg cells prevented the proliferation and trafficking of pathogenic effector T cells, resulting in an absence of inflammatory arthritis.
Our results suggest that expanding Treg cells using TGFβ-transduced MSCs in vivo could play a key role in the maintenance of peripheral tolerance. Interestingly, treatment with TGFβ-transduced MSCs resulted in increased FoxP3 levels with a concomitant decrease in IL-17 production (Figure 5). We propose that treatment with TGFβ-transduced MSCs enhanced FoxP3+ Treg cells and reduced serum levels of IL-6, TNFα, and IL-17. We observed a dramatic increase in the ratio of Treg cells to IL-17–expressing effector T cells following antigen stimulation in mice treated with TGFβ-transduced MSCs compared with MSC-treated mice. Reciprocal regulation of Treg/Th17 cells could control the pathogenesis, onset, and progression of CIA.
Although TGFβ is well known for its immunoregulatory properties, it also promotes angiogenesis (43) and inflammation (44). Previous studies have shown not only that TGFβ synergizes with TRANCE to induce osteoclast-like cells from bone marrow precursors and monocytes, but also that osteoclast-like cell formation is abolished by recombinant soluble TGFβ receptor type II (45). In contrast, TGFβ is a potent inhibitor of osteoclast-like cell formation in human bone marrow cultures (46). Bone marrow MSCs have the capacity to differentiate into bone and cartilage and thus have attracted interest as potential therapeutic tools for tissue repair. Several reports have suggested that TGFβ-transduced MSCs improve cartilage repair (47). We demonstrated that TGFβ-transduced MSCs negatively regulate RANKL-mediated osteoclastogenesis, using in vitro and in vivo analyses (Figure 6). This inhibitory effect was related to MSC migration to inflamed joints. The antiosteoclastogenic effect of TGFβ-transduced MSCs is likely related to OPG expression.
The mechanisms by which MSCs are recruited to tissues and cross the endothelial cell layer are not yet fully understood, but it is probable that chemokines and their receptors are involved. It is thus likely important that TGFβ-transduced MSCs expressed high levels of the chemokine receptor CXCR4, and that the ligand stromal cell–derived factor 1 is produced by synovial fibroblasts in joints. Treatment with TGFβ-transduced MSCs that express CCR1, CCR4, and CCR7 may result in MSC engraftment and improvements in injured tissue. Thus, we suggest that TGFβ-transduced MSCs may be useful for recruiting MSCs to inflamed tissues.
In conclusion, therapy with TGFβ-transduced MSCs is a highly beneficial method for specifically suppressing the immune response. We demonstrated that a systemic infusion of genetically modified TGFβ-transduced MSCs effectively ameliorated autoimmune arthritis through reciprocal regulation of Treg/Th17 cells. Our observations explain the beneficial effects of TGFβ-transduced MSCs in autoimmune diseases, because the balance between Treg cells and Th17 cells is pivotal for controlling the inflammatory response and autoreactivity. Our data also suggest a beneficial effect of treatment with TGFβ-transduced MSCs on CIA-induced osteoclastogenesis. Thus, we conclude that TGFβ-transduced MSCs are highly active components of inflammation modulation and tolerance induction that may offer new therapeutic applications for the treatment of autoimmune arthritis or other T cell–mediated autoimmune diseases.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. S-G. Cho had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. M-J. Park, M-L. Cho, Min, S-G. Cho.
Acquisition of data. M-J. Park, H-S. Park, Oh.
Analysis and interpretation of data. M-J. Park, H-S. Park, M-L. Cho, Oh, Y-G. Cho, Min, Chung, Lee, Kim, S-G. Cho.