In this study we show that IDO acts as a pivotal molecular switch controlling the functional status of Tregs following TLR9 ligation, leading to diametrically opposed counter-regulatory or pro-inflammatory outcomes depending on whether IDO was active or inactive. Counter-regulatory responses manifested only a few hours after CpG administration, and only at high CpG doses that induced pDCs to express IDO. Moreover, IDO-mediated counter-regulation predominated over classic pro-inflammatory and T cell stimulatory responses to TLR9 ligation induced concomitantly. CpG-induced IDO stimulated potent Treg bystander suppressor activity, and simultaneously blocked IL-6 production required to convert Tregs into TH17-like T cells. Conversely, if IDO activity was blocked, CpG treatment elicited purely pro-inflammatory responses, inducing IL-6 expression that drove uniform conversion of mature Foxp3-lineage Tregs into a pro-inflammatory TH17-like phenotype. Thus, high doses of CpG are not intrinsically suppressive; rather, they trigger counter-regulatory responses mediated by IDO that dominantly suppress, or veto their underlying immunostimulatory and inflammatory effects. These findings suggest that under certain circumstances inducible or pre-existing IDO activity at local sites of inflammation may dominantly suppress pro-inflammatory processes, and block effector/helper T cell responses to antigens encountered at such sites. Thus, sufficiently intense inflammation may elicit dominant counter-regulation by IDO-activated Tregs to create local T cell suppression and immune privilege. Conversely, when IDO is absent, even strong pro-inflammatory stimuli do not elicit local Treg suppression, and Tregs are re-programmed to acquire a pro-inflammatory TH17-like phenotype.
Splenic Tregs acquired potent suppressor activity rapidly (12-18 hours) after TLR9 ligation. Activated Tregs blocked T cell proliferation ex vivo
, and clonal expansion of allo-specific effector T cells in vivo
. Rapid responses to CpG treatment were blocked completely by ablating the IDO1 gene, and by pharmacologic inhibition of IDO prior to CpG administration. Thus, intact IDO2 genes, which are closely related and linked to IDO1 genes (29
), did not compensate for loss of IDO1 regulatory functions. Ex vivo
, the CpG-induced form of Treg suppressor activity was dependent on intact PD-1 signaling during suppressor assays. PD-1-dependent Treg suppression was also a distinctive feature of IDO-activated Tregs from inflamed LNs draining sites of melanoma growth (19
), and skin exposed to the pro-inflammatory tumor promoter phorbol ester (PMA, unpublished data). These models of counter-regulation at sites of localized inflammation share with the CpG model the fact that Treg activation was dependent on IDO expression by pDCs in a physiologic setting. The role of PD-1 in these in vivo
systems remains to be elucidated, but the requirement for PD-1 to mediate suppression ex vivo
is a characteristic feature of Tregs activated by IDO in all three models. A recent report identified requirements for PD-L1 to generate Tregs from naïve T cells, implying that the PD-1 pathway is critical for Treg differentiation in vivo
); however, this study did not address if IDO was required to promote Treg differentiation, and to stabilize the Treg suppressor phenotype.
CpG-induced Treg activation in the physiologic setting of the spleen was also dependent on intact IFNAR-signaling, but not IFNγR signaling. These outcomes are consistent with our previous reports showing an obligatory requirement for IFNAR-signaling to induce CD19+
pDCs to express functional IDO following treatment with soluble CTLA4 (CTLA4-Ig), and CpGs, which ligate B7 and TLR9 respectively (17
). We cannot exclude the possibility that IFNAR-signaling may play a direct role in stimulating Treg suppressor activity, but the known role of IFNAR signaling upstream of IDO is sufficient to explain its importance in the high-dose CpG model. However, signaling via IFNAR was not required to re-program Tregs to express IL-17 under IDO-deficient conditions.
IDO mediated inhibition of IL-6 expression was a key novel finding in our study because this provides a plausible explanation for the ability of IDO to prevent Treg re-programming into pro-inflammatory TH17-like cells, which is dependent on a cocktail of cytokines, including IL-6 (8
). Uniform expression of IL-6 and IL-17 by pDCs and Tregs respectively, was induced rapidly (between 6-9 hours) after TLR9 ligation in IDO-deficient mice. The mechanism of IDO-mediated blockade of IL-6 expression is not known, but may involve autocrine and paracrine signaling mediated directly by IDO-expressing pDCs as the patterns of IDO and IL-6 expression induced by CpGs under IDO-sufficient and IDO-deficient conditions, respectively were not identical. Metz and colleagues recently reported that induced IDO activity in transfected cell lines induced expression of liver-enriched inhibitory protein (LIP), an inhibitory isoform of the NF-IL-6 transcription factor required to promote IL-6 gene expression (30
). Thus, molecular pathways exist by which IDO may directly suppress up-regulation of IL-6 gene expression. Whether by this direct mechanism, or an alternative indirect route, our data unambiguously show that IDO blocked TLR9-induced IL-6 expression. CpG is widely used to induce IL-6 production by B cells and myeloid cells expressing TLR9, but our findings identify CpG dose as a critical factor, presumably because IDO expression by pDCs occurs only above a certain signaling threshold, which causes all splenic IDO-competent pDCs to up-regulate IDO simultaneously. Once this threshold was breached the counter-regulatory effects of induced IDO were dominant, and CpG treatment failed to stimulate IL-6 production, unless IDO was absent.
Our finding that IDO predominates over the immunostimulatory and pro-inflammatory effects of CpG treatment has potentially important implications for understanding the role of IDO in clinically significant inflammatory disease processes, and for treatment of such syndromes. We do not know what the human equivalent would be for the ‘high-dose’ CpG used in our murine model. But many natural infections and inflammatory conditions induce IDO in vivo
), and CpGs are often administered locally as a vaccine adjuvant (3
). If local or systemic levels were high enough to induce IDO, then paradoxical immunosuppression might ensue. The corollary of this however, is that blocking IDO at the time of CpG treatment may allow IL-6 production, leading to local reprogramming of Tregs. A further consideration is that, unlike spleen where IDO has to be co-induced by CpG treatment, IDO is constitutively activated in some settings of chronic inflammation, including lymphoid tissues draining local microenvironments where tumors develop, and draining inflamed skin exposed to phorbol esters that promote tumor development (15
). In these settings, constitutive (pre-induced) IDO activity may preclude immunostimulatory and pro-inflammatory responses to a range of insults, including tumors, certain infectious pathogens that activate Tregs (32
) and induce local IDO expression, and artificial immunostimulants such as vaccines and vaccine adjuvants (14
). Moreover, the use of IDO inhibitors to enhance tumor vaccine efficacy in murine models of tumor growth correlated with loss of suppressor activity by Tregs, and concomitant IL-6-dependent conversion of Tregs into TH
17-like T cells in tumor dLNs (33
), implying that the potent effects of IDO in spleens of CpG-treated mice are relevant to settings of chronic inflammation created by local tumor growth that induce constitutive IDO activity. IL-6 is known to synergize with other cytokines such as IL-1, and IL-23 to re-program Foxp3-lineage committed Tregs to express IL-17 (11
). Cytokine induced functional reprogramming of cultured Foxp3-lineage Tregs to become TH
17 T cells has been described, and was observed in mice treated with a novel immune modulator (B7-DC XAb), and in some infectious disease settings (7
). However, the physiologic mechanisms that drive Treg to TH
17 re-programming remain obscure. The findings we report in this study identify IDO as a critical molecular switch that stimulates potent Treg suppressor functions, and simultaneously blocks IL-6-mediated re-programming of Tregs to generate pro-inflammatory effector T cells expressing IL-17, and other pro-inflammatory cytokines such as IFNγ, TNFα and IL-2. Thus, our findings suggest that manipulating IDO may have profound effects on the balance of effector and counter-regulatory suppressor functions during inflammation. This scenario is also consistent with a recent study showing that absence of functional IDO activity contributed to unregulated local pro-inflammatory responses to a pulmonary infection (34
In summary, the striking dichotomy of physiologic responses by splenic Tregs to high dose CpG treatment under IDO-sufficient and IDO-deficient conditions suggests that IDO-competent CD19+
pDCs are pivotal regulators of T cell responses at sites of inflammation. Thus, IDO emerges as a key molecular switch controlling the balance between suppressor and effector functions of T cells. Chronic activation of IDO has been reported in a diverse range of clinically relevant syndromes, including persistent infections, and cancer (14
). In these syndromes, chronic or excessive IDO activity may create paradoxical local immune suppression and privilege that promotes disease progression by blocking innate T cell immunity to pathogens, and tumor antigens. Conversely, a deficiency in IDO promotes excessive autoimmunity in mice prone to type I diabetes (35
), and allows exaggerated pro-inflammatory responses in infected mice genetically predisposed to chronic granulomatous disease (34
). Thus, IDO appears to be a pivotal regulator of inflammation in certain settings: suppressing inflammation and maintaining the suppressive phenotype of Tregs when IDO is active, or allowing unchecked inflammation and re-programming of Tregs when IDO is absent. This has significant implications for the pathogenesis of chronic inflammatory disorders, and also has significant practical implications for the use of artificial ligands as vaccine adjuvants. Recently, IDO induced by treatment with the NKT (CD1d) ligand αGalCer was identified as the reason why αGalCer treatment did not stimulate a vaccine adjuvant effect in a murine model of influenza vaccination (37
), suggesting that IDO-mediated attenuation of immune responses to vaccine adjuvants may not be restricted to TLR ligands. When it is desirable to achieve the maximal pro-inflammatory effect of immune agonists, it may be crucial to block the dominant counter-regulatory effects of IDO. On the other hand, in settings where it is desirable to reduce excessive inflammation and T cell activation, enhancing IDO activity may be beneficial.