The role that IDO plays in regulating immune responses has been the subject of intense investigation. The bulk of the literature has focused on investigating the suppressive effects of IDO activity, predominantly on the activation of T cells (43
). The prevailing theory is that IDO expressed by dendritic cells inhibits T cell activation, either directly or indirectly by driving the development of Tregs (30
). In contrast to IDO’s effect on T cell responses, the role that IDO may play in B cell responses has not been evaluated. In this study, we show that administration of 1MT to K/BxN mice reduced inflammatory cytokines and autoantibodies, resulting in an attenuated course of arthritis. Surprisingly, no difference was detected in the percentage of Tregs, nor in the levels of TH
17 cytokines. Instead, the main effect of 1MT appeared to be to suppress the autoreactive B cell response. Our findings suggest that IDO is not simply an immunosuppressive enzyme, but rather plays a more complex role that includes supporting the establishment of B cell-mediated inflammatory responses.
RA patients show evidence of elevated IDO activity that correlates with disease activity (18
), but it has been unclear what relevance this has, if any, to the autoimmune response. In K/BxN mice, IDO activity was highest at the initiation of arthritis, and treatment with the pharmacological inhibitor of IDO, 1MT, at this early stage delayed the development of arthritis and reduced disease severity. Importantly, 1MT exposure was required only during the initiation of arthritis to exert its protective effect. In fact, starting 1MT treatment after disease initiation was no longer effective. There is precedence for short-term exposure to 1MT having a lasting effect on immune cell function (20
). Therefore, in K/BxN mice, we suggest that IDO plays an activating role in establishing the autoreactive B cell profile at the onset of the autoimmune response. If IDO activity is inhibited at this critical stage, the autoreactive B cell profile is not established and subsequent joint inflammation and damage is reduced.
Although 1MT was effective at alleviating arthritis, 1MT treatment did not completely prevent arthritis development, as most mice developed an attenuated course of disease. This study, like most in the literature, used a pharmacological agent to inhibit IDO activity. A potential caveat of pharmacological inhibitors is that they may not be fully effective at inhibition or may have off-target effects. Indeed, 1MT can also inhibit the IDO-related enzyme IDO2 (48
). Additionally, there may be an underlying biological difference between constitutive loss of IDO due to genetic ablation versus acute loss through pharmacologic inhibition. This is consistent with studies in pregnancy and tumor models in which compensatory mechanisms for maintaining tolerance that apparently come into play in IDO-deficient mice are not as effectively engaged following IDO inhibitor treatment (49
). To address these possibilities, it will be important to evaluate the impact of genetic loss of IDO and/or IDO2 on the development of arthritis in the K/BxN model.
1MT has been used in several other inflammatory disease models with conflicting results. 1MT exacerbated disease in experimental autoimmune encephalomyelitis (EAE) and trinitrobenzene sulphonic acid (TNBS) induced colitis (14
). In collagen induced arthritis (CIA), one study showed accelerated disease upon administration of 1MT (15
). However, another study showed 1MT had no effect on its own, but did reverse the protective effect of immunotherapy with an antibody to the B7 family molecule 4-1BB (51
). In contrast to the disease exacerbating effect of 1MT in these models, 1MT administration was protective in a mouse model of allergic airway inflammation (17
). In this case, the disease-initiating TH
2 response was inhibited in 1MT-treated mice, suggesting that IDO normally promotes TH
2-mediated inflammatory responses. A similar response was reported in vitro where IDO was shown to inhibit TH
1 responses and promote TH
2 responses (31
). We have likewise shown in this study that 1MT treatment was also protective against joint inflammation in K/BxN mice. However, in this case, the protective effect of 1MT was not due to a skewing of the TH
cytokine profile. The autoimmune response in K/BxN mice exhibited characteristics of TH
2, and TH
17 responses ( and ref. (36
)) and 1MT treatment did not affect this cytokine profile.
In contrast to the TH
17 cytokines, cytokines associated with inflammation, MCP-1, IL-6, and IL-10, were reduced in 1MT-treated mice. MCP-1, a cytokine that plays a key role in recruiting monocytes into sites of inflammation, has been shown to be elevated in RA patients (52
). Likewise, IL-6, a cytokine thought to induce inflammatory joint destruction through the recruitment and induction of inflammatory TH
17 cells, is also elevated in RA patients (53
). MCP-1 and IL-6 were also both elevated in control-treated and reduced in 1MT-treated K/BxN mice. Therefore, the alleviation of inflammation in 1MT-treated K/BxN mice was reflected in a reduction of contributory cytokines. At this point, it is not known whether 1MT treatment caused a reduction in IL-6 and MCP-1 levels directly, or if the reduced levels were simply a consequence of the overall reduced inflammatory response. IL-10 levels have also been shown to increase in response to inflammation, however, unlike MCP-1 and IL-6, IL-10 serves to dampen the response (55
). Although the significance of the reduced IL-10 levels is not clear, it may also be the result of the overall reduction in the inflammatory response in the 1MT-treated mice.
The most dramatic effect of 1MT treatment was the reduction observed in the autoreactive B cell response. Autoantibody secreting B cell numbers were significantly decreased and titers of anti-GPI Ab in the serum were greatly reduced in 1MT-treated K/BxN mice. A role for IDO in driving B cell responses has not been previously appreciated. B cells, like most APCs, express IDO and levels increase upon activation (unpublished observations). Our experiments do not distinguish between 1MT having a direct effect on autoreactive B cells or inhibiting their activation by an indirect mechanism. However, our in vitro experiments demonstrate that 1MT does not directly inhibit the activation of non-autoreactive B cells. IDO expression in another cell type could affect the environment required for efficient B cell activation and Ab secretion. Macrophages and dendritic cells, in particular plasmacytoid dendritic cells, have been implicated in the IDO-mediated suppression of T cells (13
). However, we were unable to detect any difference in the percentage or activation status of these cells in carrier- vs. 1MT-treated K/BxN mice (unpublished observations). Future experiments will be directed at identifying the cell type(s) responsible for the 1MT-mediated suppression of arthritis in the K/BxN model.
Recent evidence has shown that topical application of the pro-inflammatory agent PMA drives IDO activity in the regional lymph nodes and that this was a key component of the inflammatory microenvironment required for supporting tumor outgrowth following carcinogen exposure (20
). The elevation of IDO in response to PMA in these studies was interpreted as paradoxical because IDO was considered to be immunosuppressive and yet no indication that IDO was having a negative impact on the development or severity of PMA-driven inflammation was observed. In light of the current study, it is clear that categorizing IDO strictly as an immunosuppressive enzyme is an oversimplification and that its involvement in disease processes such as cancer and autoimmune disorders will be much more complex. In particular, its role in driving the activation of autoreactive B cells may have broad clinical implications for the future utility of IDO inhibitors as potential therapeutic agents.