Collagen-induced arthritis (CIA) is a well-established mouse model of rheumatoid arthritis (RA), a chronic inflammatory joint disease. This model is frequently used to develop and test new therapies. Like RA, CIA is characterized by severe inflammation and cellular infiltration of synovial tissue, leading to cartilage and bone destruction (1). Furthermore, T cells play a pivotal role in the pathogenesis of both RA and CIA. Therapeutic T cell costimulation blockade with CTLA-4Ig reduces disease activity in patients with RA (2), and depletion of cytokine-producing T cells inhibits the progression and severity of established arthritis in the CIA model (3).
Recent evidence suggests that Th17 cells are key players in the pathogenesis of CIA (4). In mice deficient for the Th17 cell–associated molecules interleukin-17 (IL-17), IL-17 receptor, or IL-23p19, arthritis is markedly suppressed compared with that in their wild-type counterparts (5–7), and neutralizing antibodies to IL-17 have a therapeutic effect in CIA (8). In humans, some evidence supports the involvement of Th17 cells in the pathogenesis of RA. For instance, the proportion of Th17 cells is increased in the peripheral blood of patients with RA compared with healthy control subjects (9), and the expression of IL-17 messenger RNA in RA synovial tissue is predictive of the progression of joint damage (10). In addition to Th17 cells, γ/δ T cells are a major source of IL-17, and it has been shown that γ/δ T cell–derived IL-17 exacerbates the severity of CIA (11, 12). Contrary to observations in the CIA model, however, there is no evidence to suggest a role for IL-17 production by γ/δ T cells in RA (11, 12).
A new promising immunotherapeutic strategy for the attenuation of pathogenic T cell responses is treatment with autologous dendritic cells (DCs). DCs are antigen-presenting cells that initiate immune responses to invading pathogens while maintaining tolerance to self antigens (13). DCs with potent and stable tolerogenic activity can be generated in vitro by genetic or pharmacologic modification (14). For instance, DCs transduced with FasL or IL-4 have been used to prevent CIA and to inhibit arthritis symptoms in mice with established disease (15–17). Tolerogenic DCs modified by drugs (dexamethasone; Bay 11-7082) or cytokines (tumor necrosis factor [TNF]) have been used successfully to prevent the onset of CIA (18–22) or to suppress established arthritis in a different model, the antigen-induced arthritis model (23).
Our group is in the process of developing tolerogenic DC therapy for RA and has opted for pharmacologic modification of DCs, because it is a robust, simple, and effective method that is ideal for clinical application. Treatment of DCs with 1α,25-dihydroxyvitamin D3 (vitamin D3) in combination with dexamethasone has been shown to synergistically inhibit lipopolysaccharide (LPS)–induced maturation of DCs (24). Previously, we used these immunosuppressive drugs to generate human tolerogenic DCs with potent immunoregulatory function in vitro (25, 26). An important outstanding question is, however, whether these pharmacologically modified tolerogenic DCs can inhibit pathogenic IL-17–mediated responses in vivo, and whether they will be effective at reducing the progression and severity of arthritis when administered after disease onset. The aim of this study was to determine the therapeutic and immunomodulatory actions of murine dexamethasone/vitamin D3–modified tolerogenic DCs in mice with established CIA.
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Our results show that tolerogenic DCs generated by pharmacologic modulation with dexamethasone and vitamin D3 have a clear therapeutic effect in established arthritis in the CIA mouse model. Injecting tolerogenic DCs after the onset of disease significantly reduced the severity and progression of arthritis, whereas treatment with mature DCs exacerbated arthritis. The beneficial effects of tolerogenic DCs required loading with type II collagen, suggesting that tolerogenic DCs modulated the immune response in a type II collagen–specific manner. Tolerogenic DCs did not promote the expansion of FoxP3-positive Treg cells. However, tolerogenic DC treatment resulted in a decrease in type II collagen–specific T cell proliferation and a decrease in the proportion of Th17 cells. Tolerogenic DC therapy also increased the proportion of IL-10–producing T cells, suggesting that a shift from pathogenic toward suppressive T cells may contribute to the suppression of arthritis.
It has been shown that TNF-treated DCs prevent experimental autoimmune encephalitis through the enrichment of IL-10–producing T cells in vivo (31). Previously, our group demonstrated that human dexamethasone/vitamin D3–treated tolerogenic DCs polarize naive T cells toward high IL-10 production in vitro (25). Here, we show for the first time that tolerogenic DCs can promote IL-10–producing T cells in vivo in the setting of inflammation, such as that associated with severe arthritis. These IL-10–producing CD4+ T cells did not produce detectable IFNγ or IL-17 and resemble T regulatory type 1 (Tr1) cells. Tr1 cells are phenotypically different from FoxP3+ Treg cells but have comparable regulatory function and are capable of inhibiting pathogenic T cell responses (32, 33).
Although it has been shown that treatment with recombinant IL-10 or IL-10–producing T cells is effective for preventing arthritis in mouse models (34, 35), contradictory observations have been made regarding the therapeutic effect of IL-10 in established arthritis. Intraarticular administration of IL-10 did not reduce the severity of arthritis (34), whereas systemic injection with recombinant IL-10 effectively inhibited disease (36). We detected a higher proportion of IL-10–producing T cells in the spleen after tolerogenic DC treatment, suggesting a systemic increase in IL-10. This increased IL-10 production could therefore be one of the mechanisms by which tolerogenic DCs inhibit the progression and/or severity of established arthritis.
Th17 cells play an important role in CIA (4). Our study is the first to show a decrease in the number of Th17 cells after tolerogenic DC therapy in mice with established CIA. However, tolerogenic DC treatment did not reduce the proportion of IL-17–producing γ/δ T cells. Because γ/δ T cells are known to exacerbate the severity of CIA (11, 12), a different strategy may therefore be needed to inhibit this pathogenic T cell subset.
Previous studies focused on the ability of tolerogenic DCs to inhibit Th1 cell responses (15, 17–19, 30). In contrast, we observed an improvement in arthritis without an effect on Th1 cell responses. We did, however, also observe a significant increase in IFNγ production by CD8+ T cells. Interestingly, there is some evidence for a regulatory role of CD8+ T cells in arthritis. CD8-knockout mice are more susceptible to a second induction of arthritis after remission of disease (37), and type II collagen–specific CD8+ T cell hybridomas inhibited established disease in a CIA model (38). It has also been shown that CIA develops more readily in IFNγ receptor–knockout mice, and that IFNγ inhibits the development of Th17 cells from naive precursor T cells (39, 40). Therefore, IFNγ-producing CD8+ T cells could contribute to the inhibition of CIA progression by decreasing de novo induction of Th17 cells.
Several studies have shown that prophylactic treatment with tolerogenic DCs (e.g., IL-4–transduced DCs and TNF-treated DCs) is associated with a reduced type II collagen–specific IgG2a:IgG1 ratio, indicative of a switch from a Th1 cell–driven toward a Th2 cell–driven type II collagen–specific immune response (17, 18). In contrast, we did not observe such a change in the type II collagen–specific antibody isotype ratio after tolerogenic DC treatment. A possible explanation for this discrepancy is that our tolerogenic DCs had no inhibitory effect on Th1 cells. Salazar et al have reported similar results in this respect. The tolerogenic DCs used by those investigators, which were generated by short-term LPS treatment, also inhibited established arthritis without changing the IgG2a:IgG1 ratio; however, their tolerogenic DC treatment did inhibit IFNγ production (30). Earlier studies of anti-TNF treatment of established arthritis also did not show a change in the IgG2a:IgG1 ratio (41, 42). It could therefore be contended that a reduction of type II collagen–specific IgG2a is important for the prevention of arthritis but may be of less importance after disease onset.
We have clearly shown that for tolerogenic DCs to be effective, it is necessary to pulse them with type II collagen, suggesting that the targeting of type II collagen–specific T cells by tolerogenic DCs is important for their therapeutic effect. This is consistent with results published by Salazar et al (30) but in contrast to the results of other studies using IL-4–transduced DCs (15, 16). A possible explanation for this discrepancy is that the IL-4–transduced DCs were not matured with LPS. Because immature DCs are likely to have higher endocytic capacity than mature DCs, it is possible that these DCs were effective at taking up relevant antigen in vivo (43).
It has been shown previously that after DCs are injected intravenously, they migrate to the spleen, liver, and lungs (44). In the setting of inflammation, as in CIA, DCs are likely to migrate to draining LNs as well (15). Here, we show that intravenously injected DCs can also migrate to arthritic feet. TNFα and IL-1β, which are major mediators of chronic inflammation, could play a role in recruiting DCs to arthritic feet. These cytokines increase the expression of cellular adhesion molecules, enhancing leukocyte–endothelial cell interactions (45). As expected, because both tolerogenic DCs and mature DCs can modulate disease severity, we did not observe a difference in migratory capacity between these DC types. Future studies, tracking tolerogenic DCs in vivo in real time, will determine in more detail the location of tolerogenic DCs within arthritic feet and other tissues, as well as their interactions with other immune cells. Such studies would be helpful for elucidating where and how tolerogenic DCs exert their inhibitory action(s).
Because vaccination with 1 × 106 tolerogenic DCs was not sufficient to cure disease, we increased the dose of tolerogenic DCs to 2.5 × 106 per vaccination, a dose that is sufficient to prevent CIA using TNF-treated tolerogenic DCs (18–20). However, there is a risk involved in increasing the dose of tolerogenic DCs: another study showed that a dose of 2.5 × 106 TNF-treated DCs was pathogenic (22). Although our study shows that increasing the dose of tolerogenic DCs does not enhance the therapeutic effect, no adverse effects of the higher dose were observed, indicating that our dexamethasone/vitamin D3–treated tolerogenic DCs are safe to use even at higher doses. The data shown in Table 1 demonstrate that, based on the various doses, routes, and number of injections tested, 3 intravenous injections with 1 × 106 tolerogenic DCs was the optimal tolerogenic DC treatment regimen. However, data from other studies indicate that optimal treatment regimens are different for other types of tolerogenic DCs (15, 16, 23, 30).
In conclusion, this study is the first to show that the therapeutic effect of pharmacologically modified tolerogenic DCs in CIA requires pulsing with type II collagen and is associated with a decrease in the number of Th17 cells and an increase in the number of IL-10–producing T cells in arthritic mice.
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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. Hilkens 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. Stoop, von Delwig, Isaacs, Robinson, Hilkens.
Acquisition of data. Stoop, Harry.
Analysis and interpretation of data. Stoop, Robinson, Hilkens.