Foxo1 Promotes Th9 Cell Differentiation and Airway Allergy

T helper 9 (Th9) cells are effector CD4+ T cells that are characterized by the production of interleukin-9 (IL-9) and have been associated with allergic responses. Here, we found that the expression of the transcription factor forkhead box O1 (Foxo1) was induced in Th9 and Foxo1 plays a crucial role in the differentiation of Th9 cells. Pharmacological inhibition of Foxo1 or genetic disruption of Foxo1 in CD4+ T cells caused a reduction in IL-9 expression while upregulating IL-17A and IFNγ production. Furthermore, chromatin immunoprecipitation (ChIP) followed by luciferase assays revealed direct binding of Foxo1 to both the Il9 and Irf4 promoters and induces their transactivation. Lastly, adoptive transfer of Th9 cells into lungs induced asthma-like symptoms that were ameliorated by Foxo1 inhibitor, AS1842856. Together, our findings demonstrate a novel regulator of Th9 cells with a direct implication in allergic inflammation.

T helper 9 (Th9) cells are effector CD4 + T cells that are characterized by the production of interleukin-9 (IL-9) and have been associated with allergic responses. Here, we found that the expression of the transcription factor forkhead box O1 (Foxo1) was induced in Th9 and Foxo1 plays a crucial role in the differentiation of Th9 cells. Pharmacological inhibition of Foxo1 or genetic disruption of Foxo1 in CD4 + T cells caused a reduction in IL-9 expression while upregulating IL-17A and IFNγ production. Furthermore, chromatin immunoprecipitation (ChIP) followed by luciferase assays revealed direct binding of Foxo1 to both the Il9 and Irf4 promoters and induces their transactivation. Lastly, adoptive transfer of Th9 cells into lungs induced asthma-like symptoms that were ameliorated by Foxo1 inhibitor, AS1842856.

Together, our findings demonstrate a novel regulator of Th9 cells with a direct implication in allergic inflammation.
Naive CD4 + T cells differentiate into one of several functional classes of effector cells upon antigen stimulation. T helper (Th) subsets include the classical Th1 and Th2 lineages and Th17 cells that have been described and extensively characterized 1 . Recently, a new subset of interleukin (IL)-9-producing T helper cells, induced in vitro by IL-4 and transforming growth factor (TGF)-β1, has been identified 2,3 . Traditionally associated with the Th2 response, IL-9 is a pleiotropic cytokine that exerts broad effects on a variety of cell types such as mast cells, T cells and epithelial cells 4 . Several transcription factors have been reported to be indispensable for fully differentiated Th9 cells including GATA3 2 , PU.1 5 and IRF4 6 . Recently we demonstrated that RBP-Jκ and Smad3 cooperate to promote Th9 cell development 7 .
Forkhead box O (FOXO) transcription factors are central to many aspects of cell biology 8 . They translate a variety of environmental stimuli, including insulin, growth factors, nutrients and oxidative stress, into specific gene-expression programs. Foxo1, a member of this family, is involved in T cell homeostasis and survival, and is considered as tumor suppressor in various cell systems 8,9 . Foxo1 has been shown to negatively regulate Th17 cell differentiation and pathogenicity by physically inhibiting the transcription factor RORγt activity, the master regulator of Th17 cells 10 . Moreover, Foxo1 is also involved in the development and function of regulatory CD4 + T cells (Tregs) under the control of Akt signaling 11 .
In the present study, we identified Foxo1 as a novel transcription factor required for the differentiation of Th9 cells. We found that Foxo1 expression was induced during Th9 cell polarization in vitro and positively regulated the transactivation of Il-9 and Irf4, a process that was reversed by disrupting Foxo1 expression either genetically or pharmacologically. Consequently, inhibition of Foxo1 ameliorated the disease in an asthma-like mouse model.

Results
Induced Foxo1 Expression in Th9 Cells. To investigate the role of the transcription factor Foxo1 in Th9 cells, we first measured Foxo1 expression in different T helper subsets including Th1, Th2, Th9 and Th17 cells. Naïve CD4 + T cells were polarized in vitro under the abovementioned conditions for 4 days and Foxo1 mRNA and protein levels were measured by quantitative Taqman PCR and Western blot, respectively. We found that Foxo1 protein and mRNA were readily expressed by Th9 cells (Fig. 1A,B; Supplemetary Fig. 3A). Controls for T cell polarization were measured by Luminex assay (Supplementary Figure 1). We also measured the temporal Foxo1 expression in Th9 cells polarized for 1-3 days. The time course of Foxo1 protein expression showed that Foxo1 was induced in Th9 cells starting on day 1 after polarization and was maintained on day 3 suggesting that this transcription factor plays a role in the early stages of Th9 cell development and possibly in the maintenance of this lineage ( Fig. 1C; Supplementary Fig. 3B). Next, we measured the frequency of IL-9 + T cells that co-expressed Foxo1. Using intracellular co-staining of IL-9 and Foxo1 by flow cytometry, we showed that majority of IL-9 + CD4 + T cells (cells that expressed IL-9 in the Th9 pool) that were polarized for four days, co-expressed Foxo1 (8.74% out of 10.51%) supporting our hypothesis of a potential role of Foxo1 in Th9 cell developments (Fig. 1D).
Given that Th9 cells were differentiated in the presence of the combination of IL-4 and TGF-β1, we measured Foxo1 expression in T cells exposed to either IL-4, TGF-β1, or IL-4 + TGF-β1. We found that while IL-4 treatment had no effect on Foxo1 protein expression compared to control TCR-stimulated T cells, TGF-β1 alone or added to IL-4 induced a noticeable increase in Foxo1 protein level suggesting that TGF-β1 signaling may be involved in the regulation of Foxo1 in Th9 cells ( Fig. 1E; Supplementary Fig. 3C). Full-length blots are shown in Supplementary Figure 2. Additionally, quantification of the average of two independent experiments representing Foxo1 protein expression in the T cell conditions outlined in Fig. 1A,C,E, are shown relative to β-actin (Supplementary Figure 3A,B,C).
To test the role of TGF-β1 signaling in Foxo1 expression, we utilized a pharmacological inhibitor of Smad3 activity (SIS3, 10 μM), where Th9 cells were kept untreated or exposed to SIS3 for 4 days. We then measured Foxo1 mRNA level by quantitative Taqman PCR. Interestingly, we found that inhibition of Smad3 activity prevented the increase in Foxo1 expression mediated by TGF-β1 treatment confirming the implication of TGF-β1/ Smad3 signaling in Foxo1 expression (Fig. 1F).

Foxo1
Regulates IL-9 Expression in Th9 Cells. The transcription factor Foxo1 has been involved in the negative regulation of Th17 cell differentiation 12 but the role of Foxo1 in Th9 cell development and function has not been described. To study the role of Foxo1 specifically in CD4 + T cells polarized under Th9 cell condition, we generated CD4 Cre Foxo1 fl/fl mice. Naïve CD4 + T cells purified from spleens of conditional Foxo1 knockout mice (Foxo1−/−) or littermate wild-type (WT) controls were differentiated for 4 days under Th9 cell condition followed by intracellular cytokine staining. Control Th0, Th1 and Th17 cell conditions were also generated. We found that genetic deletion of Foxo1 reduced significantly the percentage of IL-9 + cells and the level of IL-9 production in Th9 cell conditions compared to WT Th9 cell conditions (~6 fold) by flow cytometry (Fig. 2A, left and right panels) and by Luminex bead-based assay (Fig. 2B). These findings suggest that Foxo1 is a positive regulator    of IL-9 expression in Th9 cells. IL-10, another cytokine that is also produced by Th9 cells was not altered in the absence of Foxo1 by Luminex (Fig. 2B), which highlights the specificity of Foxo1 signaling in the regulation of IL-9 transcription under Th9 cell conditions. Interestingly, the decrease in IL-9 expression in Th9 cells lacking Foxo1 was accompanied by an upregulation in IL-17A and IFNγ expression suggesting that Foxo1 may control the plasticity of Th9 cells ( Fig. 2A,B). For Th0 and Th17 cell conditions, we detected a striking increase in IFNγ + T cells in these two conditions in the absence of Foxo1 ( Fig. 2A), which is in agreement with a previous report showing that Foxo1 is a negative regulator of IFNγ in Th17 cells. However, there was a slight reduction in IFNγ in CD4 Cre Foxo1 fl/fl Th1 cells ( Fig. 2A). The differential consequence of Foxo1 deletion on IFNγ among T helper subsets may suggest a unique mechanism adopted by Foxo1 in Th1 cells. To further characterize the effects of Foxo1 on Th9 cells, we measured two transcription factors that are required for Th9 cell development, IRF4 and PU.1. We found that Th9 cells lacking Foxo1 exhibited a significant downregulation of IRF4 and PU.1 gene expression measured by Taqman PCR 4 days following Th9 polarization (Fig. 2C). To test whether changes in IFNγ levels contributed to the observed Th9 phenotype in the absence of Foxo1, an anti-IFNγ neutralizing antibody was added to WT and Foxo1−/− Th9 cells during differentiation and re-stimulation. We found that although neutralizing IFNγ in Foxo1−/− Th9 cells caused a slight but significant (p = 0.04) upregulation in IL-9 expression, it did not reverse the massive downregulation of IL-9 expression in the absence of Foxo1 (Fig. 2D). Thus, IFNγ may play a negative, modulatory role in IL-9 expression.
Pharmacological Inhibition of Foxo1 Suppresses Th9 Cells. The Foxo1 inhibitor AS1842856 binds to Foxo1 and disables its ability to transactivate downstream target genes by preventing its interaction with cAMP response element-binding protein 13 . To determine whether pharmacological inhibition of Foxo1 is an effective, therapeutic approach to dampen IL-9 expression, we analyzed the effects of AS1842856 on Th9 cell differentiation in vitro. Naïve CD4 + T cells were pretreated with AS1842856 or control vehicle for 2 hours followed by cell polarization under Th9 cell condition for four days. At the end of the differentiation, supernatants from these culture conditions were analyzed for cytokine release by Luminex. In agreement with the data collected from Foxo1 deficient mice, we found that targeting Foxo1 pharmacologically was effective in inhibiting IL-9 production by Th9 cells. Moreover, the decrease in IL-9 production correlated with a significant upregulation of IL-17A and IFNγ production while IL-10 level was not changed (Fig. 3A).
Given that both genetic and pharmacological neutralization of Foxo1 inhibits IL-9 production in Th9 cells, we examined whether Foxo1 was required to maintain IL9 production in established Th9 cells. To address this, we exposed Th9 cells 4 days after their differentiation to AS1842856 for four additional days before IL-9 expression was analyzed by Luminex assay. We found that the Foxo1 inhibitor had no significant effect on already differentiated, IL-9 positive cells, suggesting that Foxo1 signaling is mostly required for the early development of Th9 cells (Fig. 3B).

Foxo1 Binds to Il9
Promoter and Induces Its Transactivation. Since Foxo1 has been involved transcriptionally in regulating inflammatory molecules in different immune cells, we hypothesized that Foxo1 may be implicated in the regulation of the Il9 promoter in Th9 cells. Foxo proteins mainly act as potent transcriptional activators by binding to the conserved consensus motifs TTGTTTAC 14 and (T/C)(G/A)AAACAA 15 (Fig. 4A,  left panel). Thus, we searched the Il9 promoter for potential binding sites for Foxo1 using Biobase database. We identified three putative binding sites for Foxo1 at −0.56 kb, −0.76 kb and −0.92 kb upstream of the transcription start site (TSS) of the Il9 promoter. We also found two potential binding sites upstream of the TSS of the Irf4 promoter, a key transcription factor required for Th9 cell differentiation (Fig. 4A, right panel). To determine the Il9 promoter occupancies by Foxo1, the binding motifs were used to design chromatin immunoprecipitation (ChIP) experiments. Primer sets flanking the Foxo1 binding on three sites in the Il9 were designed to amplify the immunoprecipitated ChIP DNA by qPCR. Naive CD4 + T cells were differentiated under Th9 cell polarizing conditions for 24 hours and then analyzed by ChIP-PCR. We detected significant binding of Foxo1 to the −0.76 kb site in the Il9 promoter in Th9 cells that was associated with an increase in the histone 3 lysine 4 monomethylation (H3K4me1), a mark that characterizes active transcription. As expected, Foxo1 inhibitor abolished Foxo1 recruitment to the Il9 promoter in Th9 cells and the active transcription mark (Fig. 4B), which correlates with the suppression of IL-9 expression. We confirmed the specificity of Foxo1 binding by the failure to amplify a region of the Il9 promoter that does not contain Foxo1 binding sites (data not shown). To analyze the functional relevance of the binding of Foxo1 to their target sequence in the Il9 locus, we investigated the ability of Foxo1 to regulate the activity of the Il9 promoter in reporter assays. We used reporter construct pGL3-Il9, containing the firefly luciferase gene under the control of the Il9 promoter. We found that co-transfection of the pGL3-Il9 luciferase reporter construct with a plasmid encoding Foxo1 in 293 T cells resulted in a significant increase in Il9 transcription that was inhibited by pre-incubating the cells with the Foxo1 inhibitor AS1842856 confirming the specificity of the assay (Fig. 4C).
Further, we investigated the hypothesis that Foxo1 also modulates IRF4 function. Using ChIP assay, we found that Foxo1 is indeed recruited to the −0.82 kb and the −1.23 kb sites upstream of the TSS of the Irf4 promoter in Th9 cells (Fig. 4D). Next, we measured the functional effect of Foxo1 on the Irf4 promoter activity. We used a luciferase reporter assay to measure the activity of the Irf4 promoter in cells overexpressing Foxo1. We found that co-transfection of the pGL3-Irf4 luciferase reporter construct with a plasmid encoding Foxo1 in 293 T cells resulted in a significant increase in Irf4 transcription (Fig. 4E). These findings were in agreement with decreased Irf4 mRNA levels in CD4 Cre Foxo1 fl/fl Th9 cells compared to control Foxo1 fl/fl Th9 cells shown in Fig. 2C. Altogether, our findings suggest that Foxo1 exerts a dual regulatory role in Th9 cells by directly binding to and transactivating the Il9 and Irf4 promoters. cells have been implicated in airway inflammation and asthma pathogenesis mainly due to the production of IL-9. To examine the role of Foxo1 in the development of inflammatory Th9 cells in vivo, we used an adoptive transfer model of ovalbumin (OVA)-specific Th9 cells in which OT-II cells were first polarized in vitro under Th9 cell conditions in the presence or absence of Foxo1 inhibitor followed by intra-tracheal (IT) transfer in BALB/C recipients. A third group of mice received PBS treatment only. BALB/C recipients were subjected to OVA nebulization for three consecutive days. On day 4, mice were analyzed for inflammation, mucus overproduction, and changes in airway reactivity. As expected, compared to controls without OT-II cells (PBS only), adoptive transfer of Th9 cells triggered infiltration of eosinophils as analyzed by flow cytometry of lung dissociates stained for cell surface markers SiglecF and CD11c (Fig. 5A). Compared to vehicle-treated Th9 cells, Th9 cells treated with the Foxo1 inhibitor AS184285 reduced the abundance of eosinophils in lungs by ~30% (Fig. 5A). In accordance, the percentages of eosinophils in the bronchoalveolar lavage (BAL) were reduced by half in mice that received AS184285-treated Th9 cells compared to vehicle-treated Th9 cells (Fig. 5B). The decrease in eosinophils by Foxo1 inhibitor AS184285 was associated with a significant reduction in IL-9 but not in IL-4 or IL-13 levels (Fig. 5C). We observed a slight but not significant increase in IFNγ level measured by Luminex assay (Fig. 5C). In addition, vehicle-treated Th9 cells in lungs induced mucus overproduction assayed by PAS staining (Fig. 5D) and by Muc5ac gene expression, a molecule that is associated with mucus hypersecretion in the pulmonary tracts (Fig. 5E). In contrast, AS184285-treated Th9 cells showed a dramatic reduction in mucus formation suggesting protective effects (Fig. 5D,E). Consistent with the above inflammatory activity, mice that received vehicle-treated Th9 cells exhibited airway hyper-responsiveness to methacholine measured using prepared, precision cut lung slices compared to PBS controls, while the Foxo1 inhibitor treatment prevented airway responsiveness in the recipient mice (Fig. 5F).

Conclusion
In the present study, we provide evidence that the transcription factor Foxo1 plays a crucial role in the differentiation of naïve T cells into IL-9-producing T cells. First, Foxo1 is induced in Th9 cells and the majority of IL-9-expressing T cells There is heightened interest in understanding the role of Foxo1 in the regulation of adaptive and innate immune systems [16][17][18] . In primary T cells, Foxo1 is phosphorylated upon TCR ligation, and this process leads to the inhibition of its transcriptional activity by nuclear exclusion 19 . However, Foxo1 posttranslational modification occurs mainly during the early phase of T cell activation to promote cell proliferation clonal cell expansion 20 . In the later stage of TCR engagement, Foxo1 escapes its cytoplasmic retention and translocates to the nucleus to bind to its downstream DNA targets. In the present study, we found that Foxo1 expression is induced during Th9 cell promoter in Th9 cells. Naïve CD4 + T cells from WT mice were polarized under Th9 cell conditions. ChIP-Sybr Green PCR was performed to determine Foxo1 binding to the Il9 promoter. Abs used for immunoprecipitation are anti-Foxo1, anti-H3K4me1 and control IgG. Total input DNA before IP was used for normalization of data. The graphs represent quantitative PCR analysis of the ratio of enriched Il9 promoter with Foxo1 binding sites to the input DNA. Foxo1 binding sites were amplified using Il9-specific promoter primers. Data represent mean ± SE of a representative experiment each performed in triplicate. (C) Il9 promoter Luciferase reporter assay. HEK 293 T cells were transfected with Foxo1 together with a constant amount of Il9 promoter-luciferase vector. Cells were lyzed 48 hrs later and luminescence was measured. (D) ChIP analysis of Foxo1 binding to the Irf4 promoter in Th9 cells. Naïve CD4 + T cells from WT mice were polarized under Th9 cell conditions. ChIP-Sybr Green PCR was performed to determine Foxo1 binding to the Irf4 promoter. (E) Irf4 promoter Luciferase reporter assay. HEK 293 T cells were transfected with Foxo1 together with a constant amount of Irf4 promoterluciferase vector. Cells were lysed 48 hrs later and luminescence was measured. Data represent mean ± s.e.m. of a representative experiment each performed in triplicate. *p < 0.01, **p < 0.005 by Unpaired Student t test.  polarization where it binds both Il9 and Irf4 promoters and induces their activation. Recently, Foxo1 has been shown to bind the DNA binding domain of RORγt to inhibit its activity in Th17 10 . In agreement with these findings, our study shows that the decrease in IL-9 expression was associated with a noticeable upregulation of IL-17A levels in Th9 cells. The observed increase in IFNγ expression in Th9 cells lacking Foxo1 is in line with previous reports demonstrating that Foxo1 represses T-bet function in memory T cells 21 . Additionally, an inverse correlation between Foxo1 and Tbx21 expression in NK cells was reported and confirmed by Foxo1 genetic disruption leading to an increase in TBX21 mRNA expression 22 . However, we did not find any alteration in IFNγ production in Th1 cells differentiated according to standard protocol using recombinant IL-12 where T-bet is the master transcription regulator. This suggests that Foxo1 does not regulate directly T-bet function but rather an alternative pathway upstream of T-bet that is not active in Th1 cells. The fact that IFNγ neutralizing antibodies only slightly reverse the decrease in IL-9 expression in Th9 cells deficient in Foxo1 suggests that Foxo1-mediated regulation of IL-9 expression is not exclusively IFNγ-dependent but rather implicates direct and indirect mechanisms.
Our group and others have demonstrated a crucial role of Smad3 signaling in the differentiation of Th9 cells by inducing Il9 transcription in IRF4-dependent manner 7,23 . Here we found that TGF-β1/Smad3 regulated the expression of Foxo1 in polarized Th9 cells using a pharmacological inhibitor. These findings provide an additional mechanism of action of Smad3 in promoting Th9 cell differentiation that is mediated by Foxo1 signaling.
The role of Foxo1 in the regulating innate immune response in asthma has been recently described. Foxo1 polarizes macrophages into type 2 phenotype by activating IRF4 expression, and inhibition of Foxo1 by AS1842856 attenuates the development of asthmatic lung inflammation 24 . However, to date, Foxo1 was not involved in the regulation of inflammatory T cells in asthma experimental models and particularly Th9 cells. Our present study provides strong evidence demonstrating that Foxo1-IL-9 axis plays a crucial role in the regulation of adaptive immune response in asthma-like mouse models. IRF4 seems to be a common target of Foxo1 in both T cells and macrophages 24 . Together, targeting Foxo1 may have dual impact on the immune responses in asthma by regulating both innate and adaptive immune responses thus ameliorating the disease symptoms. It is intriguing that three recent reports demonstrated a crucial role of Foxo1 in Th9 cell development and function. In one report, the authors investigating the role of mTORC2 in Th9 cells showed that while mTORC2 controls Th9 cell differentiation in a Foxo1/Foxo3a-independent manner, knocking down Foxo1 in CD4 + T cells using shRNA strategy reduced their ability to differentiate in Th9 cells 25 , which is in line with our present findings. In a second study investigating the role of IL-7 in Th9-mediated anti-tumor activity, the authors demonstrated that while Foxo1 and Foxp1 play opposite roles in the context of Th9 cell function in tumor regulation, Foxo1 indeed binds to the Il9 promoter and promotes Th9 cell differentiation and IL-9 production 26 . More recently, and in agreement with our present study, Malik et al. provided a detailed analysis of the role of Foxo1 in IL-9 expression in both human and mouse Th9 cells and their pathogenicity in a mouse model of asthma 27 . The authors found that Foxo1 is not only required for IL-9 production in Th9 cells but also in Th17 cells and this process is negatively regulated by AKT signaling.
In summary, our study introduces a novel transcriptional activator of Th9 cells that interacts with well-known regulators of Th9 cells, IRF4 and Smad3. Inhibition of Foxo1 has double impact on both adaptive and innate immune responses in experimental asthma with promising implications in asthmatic patients.
Expression Analysis by Real-Time PCR. RNA was purified using Stratagene RNA kit and transferred directly into the RT reagent using the Applied Biosystems Taqman reverse transcriptase reagents. Samples were subjected to real-time PCR analysis on an Applied Biosystems PRISM 7000 Sequencer Detection System (Applied