Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial inflammation, which is orchestrated by both innate and adaptive immune responses. Major hallmarks of autoimmunity in this disease include the appearance of serum autoantibodies and the persistent activation of self-reactive CD4+ cells (1). Although therapeutic strategies targeting cytokines and B or T cells have had a major clinical impact, disease in many patients remains refractory to current biologic interventions, and for patients who show a response to therapy, true remission associated with reestablishment of immune tolerance is rare. This has increased the interest in exploring strategies to reestablish immune tolerance and provide long-term disease suppression (2).
Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that can induce either immunity or tolerance. Myeloid DCs and plasmacytoid DCs represent the 2 major subsets of DCs, with human plasmacytoid DCs defined as CD45+CD123+CD303+CD11c− cells and myeloid DCs defined as CD45+CD1a+ CD11c+CD1c+ cells. Although both subsets exhibit a functional plasticity in directing T cell responses (3), current evidence supports a predominant role of plasmacytoid DCs in the maintenance of tolerance through the expansion/induction of Treg cells (4–7).
To date, 2 major subsets of Treg cells have been described: thymus-derived Foxp3+ Treg cells and adaptive interleukin-10 (IL-10)–producing Treg cells (Tr1 cells) generated in the peripheral blood (8). The importance of Treg cells in the maintenance of tolerance is highlighted by the development of spontaneous multiorgan autoimmunity following Treg cell deletion in rodents (9). In humans, reduced numbers of Treg cells and impaired function of Treg cells have been reported in patients with autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus, and multiple sclerosis (10–12). The molecular pathways involved in prompting plasmacytoid DCs to promote Treg cell development are not fully understood. Expression of the indoleamine 2,3-dioxygenase (IDO) enzyme by plasmacytoid DCs is thought to play a significant role in plasmacytoid DC–mediated Treg cell induction (13–16). Whether human DCs exploit similar mechanisms of Treg cell induction that might facilitate reestablishment of tolerance in a disease setting remains to be seen.
Cytokine-directed therapy may have an impact on Treg cell function. Thus, Treg cells in patients with active RA are functionally defective. Anti–tumor necrosis factor α (anti-TNFα) therapy reverses this defect (10, 17, 18), through conversion of naive T cells into Foxp3+ Treg cells (18). Whether therapy may also have an impact on DCs, in terms of changing their effects on Treg cells, has not been explored. Herein, we present findings demonstrating that plasmacytoid DCs isolated from patients with RA whose disease is in remission induce an additional, distinct population of Treg cells, Tr1 regulatory cells, in a manner that is dependent on the presence of IDO. Unlike the previously described anti-TNFα–induced Treg cells (18), this population of genuine Tr1 cells does not express Foxp3 but, instead, secretes high levels of IL-10. Most notably, these cells suppress the proliferation of autologous naive CD4+ cells, in a dose-dependent manner.
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- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
In this study, we have shown that the remission of RA is associated with the restoration of plasmacytoid DC levels in the peripheral blood, and we have provided evidence of an important role for this DC subset in the maintenance of tolerance through the induction of IL-10–secreting Tr1 regulatory cells. Amelioration of disease activity in RA patients resulted in the reestablishment of the levels of both plasmacytoid DCs and myeloid DCs in the circulation. Because plasmacytoid DCs migrate to the sites of inflammation, we sought to determine whether the lack of plasmacytoid DCs in the peripheral blood of patients with active RA may be due to the recruitment of plasmacytoid DCs at the synovium. Although we were unable to detect CD303+ plasmacytoid DCs in the SF of patients with inactive RA, plasmacytoid DCs may still be present at the synovial membrane (25).
There is ample evidence for the functional plasticity of plasmacytoid DCs in directing T cell responses, which mainly depends on their maturation status (26, 27). Thus, virus-infected plasmacytoid DCs elicit a potent Th1 response (28), whereas IL-3–treated plasmacytoid DCs prime Th2 cells in an OX40 ligand–dependent mechanism (29). In addition, plasmacytoid DCs induce Treg cell–mediated differentiation of naive T cells, which depends on the activation stimulus used (4, 5, 13). Our findings are consistent with those in previous studies in that our results also support the notion that amelioration of an autoimmune disease in humans may be associated with plasmacytoid DC–induced Treg cell development. Based on these observations, we speculate that in patients with active RA, plasmacytoid DCs are exposed to a cytokine milieu and adapt to a tolerogenic phenotype. During disease remission, tolerogenic plasmacytoid DCs are restored in the peripheral blood, where they are able to polarize naive T cells toward IL-10–secreting Treg cells. Although it is not clear whether remission of RA is directly mediated through plasmacytoid DC–primed Treg cells, our results demonstrate that IL-10–secreting Treg cells can potently suppress the activation of naive T cells in vitro, implying a possible role in the homeostatic control of inflammation in vivo.
Both naturally occurring Treg cells and Tr1 cells have a crucial role in the induction and maintenance of tolerance to self and foreign antigens (30). Our results indicate that T cells primed by mature plasmacytoid DCs from patients with RA in remission have the cardinal features of Tr1 regulatory cells (31), since they secrete high levels of IL-10 and low levels of IFNγ, and they proliferate poorly following polyclonal T cell receptor–mediated stimulation. Most notably, they suppress the activation and proliferation of autologous naive CD4+ T cells, in a dose-dependent manner. Previous studies have convincingly shown that treatment of RA patients with infliximab led to the induction of Foxp3+CD25highCD62L− Treg cells that were distinct from natural Treg cells and Tr1 cells (18). Although we confirmed the presence of Foxp3+CD4+CD62L− T cells in patients with inactive RA (Figure 1A), we were unable to detect Foxp3 expression by plasmacytoid DC–primed IL-10–producing Tr1 cells (results not shown). These results suggest that restoration of tolerance during an autoimmune disease may be mediated by distinct types of Treg cells with specialized functions. Support for this hypothesis has been provided in mouse models, in which distinct types of Treg cells could act in concert to suppress autoimmune phenomena (32, 33).
Secretion of high levels of IL-10 by plasmacytoid DC–primed T cells could have a beneficial role during induction and/or maintenance of remission in these patients. Therefore, the plasmacytoid DC–mediated secretion of IL-10 by Tr1 cells in patients with RA in remission may exert pleiotropic effects toward maintenance of immune tolerance. Secretion of IL-10 by plasmacytoid DC–primed Treg cells might also increase the regulatory activity of other Treg cell subsets (34) or restore the suppressive capacity of Foxp3+ Treg cells that were previously defective. In addition, IL-10 can indirectly inhibit immune responses by abolishing the antigen-presenting ability of APCs, either through down-regulation of the expression of the major histocompatibility complex and costimulatory molecules or through inhibition of cytokine secretion by these cells (35). Consistent with our findings, an increase in serum IL-10 levels has been demonstrated in patients who have exhibited a prolonged clinical response upon anti-TNFα treatment (36, 37).
Our results clearly demonstrate that IDO expression by mature plasmacytoid DCs isolated from patients with RA in remission was required for the induction of IL-10–producing Treg cells. Blockage of IDO activity by the inhibitor 1-MT completely reversed the T cell polarization. This is in line with mouse and human studies demonstrating the crucial role of IDO in Treg cell induction (13–15, 38–42). This reversal was IDO-specific, since blockage of the ICOS pathway or neutralization of cytokines such as TNFα and IL-10 did not affect the ability of plasmacytoid DCs to polarize naive T cells toward IL-10–producing Treg cells. Recent study findings have suggested that the ICOS pathway is essential for the induction of anergic T cells with a regulatory phenotype in immature myeloid DCs (43), and therefore it might not be required for the induction of Tr1 cells.
Although the exact molecular mechanism that links Treg cell induction with IDO-expressing plasmacytoid DCs is still unresolved, it has been proposed that interactions between Treg cells and plasmacytoid DCs might initiate a so-called reverse-signaling mechanism that leads to the activation of NF-κB signaling and up-regulation of IDO (24, 44). Subsequently, the combined effects of IDO expression and tryptophan depletion may induce Treg cells from naive T cells (45). What drives the up-regulation of IDO on plasmacytoid DCs in patients with RA who have experienced disease remission remains to be determined. IDO is strongly up-regulated on plasmacytoid DCs that have been exposed to IFNγ, a cytokine present during the course of RA (46). We postulate that plasmacytoid DCs exposed to the cytokine milieu in RA increase the expression of IDO and, upon contact with naive T cells, mediate the conversion of these cells toward Treg cells.
In summary, we have shown that plasmacytoid DCs isolated ex vivo from patients with RA in remission display increased IDO expression and prime naive CD4+ T cells to differentiate in vitro into IL-10–producing Tr1 regulatory cells that potently suppress the proliferation of autologous naive CD4+ T cells. Amelioration of the disease was correlated with the reappearance of circulating plasmacytoid DCs in RA patients, raising the possibility that IDO-expressing plasmacytoid DC–induced Treg cells might be involved, at least in part, in the restoration of immune homeostasis. It is tempting to speculate that plasmacytoid DCs exposed to an autoimmune environment have been conditioned to express a tolerogenic phenotype that induces the de novo generation of Treg cells with the ability to control ongoing immune responses. A better understanding of the pathways involved in the plasmacytoid DC–Treg cell crosstalk will facilitate the exploration of the potential use of cell-based therapies for the induction of tolerance in transplantation and autoimmunity.
- Top of page
- PATIENTS 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. Verginis 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. Verginis.
Acquisition of data. Kavousanaki, Makrigiannakis, Verginis.
Analysis and interpretation of data. Kavousanaki, Makrigiannakis, Boumpas, Verginis.