In the preceding studies, we used in vivo
and in vitro
models of T cell differentiation to establish that, downstream of IFN-γ, IL-27 and likely all STAT1-activating cytokines, there are both STAT1- and T-bet-dependent mechanisms capable of suppressing Th17 responses. As evidence for STAT1-mediated inhibition, and consistent with published reports (8
), we demonstrate that STAT1-deficient T cells exhibit a hyper-Th17 phenotype and are refractory to the anti-Th17 effects of IFN-γ and IL-27. As evidence for T-bet-mediated inhibition, we demonstrate that T-bet-deficient T cells also exhibit a hyper-Th17 phenotype, that T-bet-deficiency hinders the ability of STAT1-activating cytokines to suppress Th17 responses and, most importantly, that ectopic T-bet expression can suppress Th17 responses in the complete absence of STAT1. Previous studies have also suggested that STAT1 and T-bet might play independent roles in this process but, since T-bet is both upstream (an inducer) and downstream (induced by) of STAT1, they could not definitively exclude the possibility that T-bet may be arbitrating STAT1-dependent inhibition through its well known ability to drive IFN-γ production (26
). We overcame the ‘chicken-and-egg’ problem by using retroviral vectors to restore T-bet expression in either T-bet or STAT1-deficient T cells, finding that indeed there are two pathways at work, with STAT1 not required for T-bet-mediated inhibition and T-bet not required for STAT1-mediated inhibition.
Aside from those operating through STAT1, other cytokines have been implicated in the regulation of Th17 responses, including IL-2 and IL-4, which are known to act primarily through STAT5 and STAT6, respectively. Similar to STAT1-deficiency, genetic ablation of these STATs is associated with increased Th17 responses but whether this shared outcome is achieved through a common mechanism is yet to be resolved (8
). The most direct way that STATs could limit Th17 responses is by binding to promoter/enhancer regions of Th17-assoctaed genes and thereby obstructing the transcriptional machinery. There is some evidence for this, with STAT5 having been shown to bind the promoter of IL-17A
, but whether this interaction is what determines the ability of STAT5 to suppress IL-17 production was not determined (45
). Likewise, STAT1 has been shown to bind upstream of the RORα
loci in human HELA cells but the nature of this interaction, be it stimulatory or inhibitory, and whether it happens in primary T cells, are questions that remain unanswered (46
). Another, more indirect, way that STATs could impact Th17 responses is by inducing or promoting the function of auxiliary anti-Th17 factors. There is strong evidence for this since cytokines with anti-Th17 activity are already known to induce “Th17 inhibitors’, like T-bet, Ets1 and Gfi-1, and this is not likely to be an exhaustive list of indirect targets (47
). STATs could also influence Th17 responses by interfering with pro-Th17 TFs or signaling pathways, as is the case with the ability of IL-27 to induce expression of SOCS3, which is known to curb STAT3-dependent Th17 responses (50
), the ability of IFN-γ and IL-27 to suppress S1P, a receptor known to promote IL-6-driven Th17 responses (51
), and the ability of several anti-Th17 cytokines to suppress RORγT, which is both necessary and sufficient for Th17 differentiation (29
). Thus, while other regulatory pathways will likely emerge, it is already clear that cytokines limit Th17 responses through both direct and indirect STAT-driven pathways that, together, disable multiple steps in the Th17 differentiation program.
As with the STATs, T-bet could suppress Th17 responses in a variety of ways. A recent genome-wide mapping of T-bet binding sites did not reveal significant enrichment near the IL-17A, IL-17F, IL-22 or ROR loci, making a direct interaction between T-bet and relevant Th17-associated promoters seem unlikely (52
). However, a direct protein-protein interaction between T-bet and pro-Th17 TFs remains a possibility, especially since T-bet is known to interact with and thereby limit the function of other TFs, including GATA-3 and RelA (53
). Consistent with this latter point, our studies demonstrate that T-bet can suppress IL-17 production even in the face of ectopic RORγT, which is driven by a retroviral promoter and, thus, impervious to transcriptional effects. These data suggest a physical interaction between T-bet and elements of the Th17 differentiation machinery, if not RORγT itself, though it should also be noted that T-bet might influence Th17 responses through more indirect means. Adding further complexity, T-bet has a functional homologue, Eomes, which is expressed in T cells and is known to exhibit anti-Th17 activity. Recent studies have shown that ectopic expression of Eomes can suppress IL-17 production but whether this is due to a cell-intrinsic effect or its ability to drive IFN-γ-mediated suppression was not resolved (35
). It is also known that, unlike T-bet and STAT1, genetic ablation of both T-bet and Eomes results in a compound hyper-Th17 phenotype but, again, the increase in IL-17 production was mirrored by a corresponding reduction in Th1 responses, making it unclear whether the phenotype was due to direct effects or a lack of IFN-γ/STAT1-dependent inhibition (26
). Based on the data presented here, we propose that both are true, that T-bet and Eomes can each limit Th17 responses through at least two shared mechanisms, one involving STAT1, with IFN-γ as an intermediary, and the other completely STAT1-independent.
Although our findings establish that STAT1 and T-bet influence Th17-type cytokines through genetically distinct pathways, we noted that T cells lacking both transcription factors did not exhibit an additive, or compound, phenotype. Instead, the hyper-Th17 phenotype of T-bet deficient cells was always more severe than that of STAT1- or double-deficient counterparts which, despite the well-known ability of STAT1 to drive T-bet expression, is also inconsistent with the notion that they operate within the same pathway. Taken together, these contradictory observations suggest epistasis, meaning that the loss of STAT1 affects cellular processes that, while not directly related to Th17 differentiation, impact the overall quality of T cell responses, thereby hindering production of Th17-type cytokines. We found that, beyond Th17 responses, T-bet and STAT1-deficient T cells behaved differently in vivo
, with the latter exhibiting reduced proliferation and increased Th2-type cytokine production. We also found evidence for epistasis during in vitro
differentiation. Those studies confirmed that STAT1 is required for IFN-γ and IL-27 to suppress IL-17 production but also showed that, compared to WT counterparts, expression of many Th17-type mRNAs was not grossly elevated in STAT1-deficient cells which, perhaps, indicates post-transcriptional effects (Supplemental Figure 3
). Thus, while we can still conclude that STAT1 and T-bet influence Th17 responses through both divergent and convergent mechanisms, it must be noted that genetic dissection of these pathways was confounded by the wide-ranging effects of STAT1-deficiency.
The ability of signature Th1-type factors, like IFN-γ, STAT1 and T-bet, to inhibit signature Th17-type factors, like IL-17, IL-22 and RORγT, has led to the idea that there is an inverse relationship between the Th1 and Th17 subsets. However, despite this antagonism, T cells producing IFN-γ and IL-17 are known to occur in multiple inflammatory settings, which suggests a more nuanced relationship (2
). The current work illustrates both sides of this paradox. On one hand, we show that T cells produce either IFN-γ or IL-17 when primed in vitro
and, on the other, that they can produce both when primed in vivo
(in highly immunogenic sOva Rag2−/− mice
). We also report that, when T-bet and RORγT were both highly expressed in the same cells, the result is dichotomous, with some cells expressing one cytokine or the other but rarely both ( & ). Based on this latter finding, and the fact that T-bet is known to promote long-term Th1 lineage commitment, we propose that ‘double-positive’ T cells represent a transitional phase in a linear progression from IL-17-producing Th17 cell to IFN-γ producing Th1 cell. Inherent to this hypothesis is the idea that Th17 cells can convert to other subsets, which has strong experimental support (10
), and that such conversion is a part of normal immune responses, which is now supported by recent studies demonstrating that Th17-type cytokines are required for the development of Th1 responses during infection (55
Although Th17 responses can have important, host-protective functions, it is also widely accepted that, when dysregulated, they can promote autoimmune disease. Given the current and prospective use of STAT1-activating cytokines as therapeutics for Th17-associated pathologies, best exemplified by the use of interferon-β to treat multiple sclerosis, it is critical to understand exactly how they suppress Th17-type inflammation. The studies presented here provide an important piece of mechanistic information, demonstrating that the ability of STAT1 to limit Th17-type responses is intimately linked to T-bet, a transcription factor that is at once a potent anti-Th17 effector and, through its ability to drive IFN-γ production, an essential STAT1 stimulus. Though we have focused on CD4+ T cells, which we interrogated in a select few model systems, this relationship between STAT1 and T-bet is likely to impact other IL-17-producing lineages, such as CD8+ T cells or NKT cells, and is likely to influence Th17 responses in a variety of immune and auto-immune settings, making these pathways acutely relevant in the context of inflammatory disease etiology and cytokine-based drug design.