TGF-β induces the expression of the adaptor Ndfip1 to silence IL-4 production during iTreg cell differentiation.

Mice deficient in the adaptor Ndfip1 develop inflammation at sites of environmental antigen exposure. We show here that such mice had fewer inducible regulatory T cells (iT(reg) cells). In vitro, Ndfip1-deficient T cells expressed normal amounts of the transcription factor Foxp3 during the first 48 h of iT(reg) cell differentiation; however, this expression was not sustained. Abortive Foxp3 expression was caused by production of interleukin 4 (IL-4) by Ndfip1(-/-) cells. We found that Ndfip1 expression was transiently upregulated during iT(reg) cell differentiation in a manner dependent on transforming growth factor-β (TGF-β). Once expressed, Ndfip1 promoted degradation of the transcription factor JunB mediated by the E3 ubiquitin ligase Itch, thus preventing IL-4 production. On the basis of our data, we propose that TGF-β signaling induces Ndfip1 expression to silence IL-4 production, thus permitting iT(reg) cell differentiation.

Once in peripheral lymphoid compartments, T cells are poised to activate the immune system in an effort to destroy invading pathogens. These responses play essential roles in pathogen clearance; however, mechanisms exist to ensure that T cell responses are directed towards harmful pathogens while remaining tolerant to self. Furthermore, T cells must remain tolerant not only to self, but also to non-pathogenic (or harmless) environmental antigens. One mechanism that prevents T cells from directing immune responses towards self or environmental antigens is suppression by T regulatory (Treg) cells [1,2].

Treg cells are a specialized subset of T cell that can be generated in one of two ways. Natural Treg cells (nTregs) develop in the thymus and bear T cell receptors (TCR) that primarily recognize self-peptides, whereas iTreg cells, also known as adaptive Treg cells) differentiate from naïve T cell precursors in peripheral lymphoid tissues such as mesenteric lymph nodes that drain the gastrointestinal (GI) tract[1,2]. These two Treg cell subsets differ in their expression of certain genes and their plasticity, but both subsets express a transcription factor known as Foxp3[1,2]. The essential role of Foxp3 in Treg cell development was revealed by genetic mutations leading to the loss of Foxp3 function. The spontaneous Scurfy (sf) mutation in mice results in a loss of function mutation in Foxp3 and death of the mice by 3-4 weeks of age[3], while the loss of Foxp3 function in humans leads to IPEX (immunodysregulation, polyendocrinopathy and enteropathy, X-linked syndrome) [4,5]. In these cases, the result of the genetic mutation is loss of Foxp3 expression and a consequent lack of functional Treg cells[6-8].

Foxp3 expression in Treg cells relies on both TGF-β and IL-2 receptor (IL-2R) signaling[9-11]. Treg cells constitutively express CD25, the IL-2Rα component of the high affinity IL-2 receptor complex[12]. Signaling by IL-2 is important for Treg cell differentiation and maintenance[9,10]. In addition to IL-2, both nTreg and iTreg cells need TGF-β to induce Foxp3 expression[9,11]. Stimulation of naïve T cells by TGF-β promotes the induction of Foxp3 expression and iTreg cell differentiation[13-18]. Additionally, TGF-β dampens IL-4 production and thus suppresses TH2 differentiation[19,20]. Both of these TGF-β mediated outcomes depend on Smad proteins. For example, Smad3 binds to the Foxp3 gene and activate its transcription[21]. In addition to directly regulating Foxp3 transcription, Smad activation downstream of TGF-β signaling also induces the expression of TGF-β induced early gene 1 (TIEG1)[22]. TIEG1 is a transcription factor that binds the Foxp3 gene and induces its transcription[23,24]. Thus, Smad proteins induce Foxp3 expression by both direct and indirect mechanisms. Following TGF-β signaling, TIEG1 is monoubiquitylated by the E3 ubiquitin ligase known as Itch[23]. This monoubiquitylation allows TIEG1 to induce Foxp3 transcription[23] and is proposed to explain why Itch-deficient T cells are defective at differentiating into iTreg cells in vitro[23].

We have identified an adaptor protein, known as Ndfip1, that is required for Itch polyubiquitylation of transcription factors of the Jun family[25]. Jun family members can act with NFAT to induce expression of IL-4[26,27]. Thus, in the absence of either Itch or Ndfip1, levels of Jun family members, such as JunB and c-Jun, accumulate and promote the transcription of IL-4 and TH2 polarization[25,28] leading to TH2-mediated inflammation in the skin, lung, and GI tract[25,28,29] in these mice. Knowing that Ndfip1 is required for Itch polyubiquitylation of JunB, we hypothesized Ndfip1 may also promote Itch mono-ubiquitylation of TIEG1. Indeed, T cells lacking Ndfip1 were much less likely to become iTreg cells in vitro than their wild-type (WT) counterparts. However, we did not see a defect in TIEG1 binding to the Foxp3 promoter in either Ndfip1–/– or Itch-deficient T cells within the first 48 hours of iTreg cell induction. Rather our results demonstrate that their defect in iTreg cell induction was due to overproduction of IL-4. Our data indicate that Ndfip1 is highly expressed in a TGF-β-dependent manner, peaking after 24 hours of iTreg cell induction, to prevent the accumulation of JunB and IL-4 production. Based on these results, we propose that Ndfip1 and Itch dampen IL-4 production and thus provide a window of opportunity for iTreg cell lineage commitment.

RESULTS

Mice lacking Ndfip1 have fewer iTregs in vivo

Ndfip1-deficient mice develop a severe atopic inflammatory disease, characterized by hyperproliferative T cells that produce TH2 cytokines and eosinophilia[25,29]. The disease is reminiscent of the TH2 aspects of the pathology that occurs in Scurfy mice[3] and IPEX patients[4,5], suggesting that Ndfip1–/– mice might have defects in Foxp3+ Treg cells. Thus, we sought to determine whether Ndfip1–/– mice had a block in the development of Foxp3+ nTreg cells in the thymus. We first analyzed numbers and percentages of CD4+CD25+Foxp3+ cells in the thymi of 4-6 week old mice (. This revealed a significant increase in the percentages of Foxp3+ Treg cells in Ndfip1–/– thymi. In contrast, no difference was seen when comparing the total numbers of these cells, possibly due to a decrease in the total number of thymocytes harvested from Ndfip1–/– mice (data not shown). Inflammation can cause both an increase in thymic Foxp3+ cells[30] as well as thymic involution. To test whether the increased percentages were due to inflammation or increased nTreg cell differentiation, we analyzed 9-day old neonatal mice, when Treg cell numbers are increasing[31], and 2.5-week old mice, prior to histologic evidence of inflammation[29]. Flow cytometric analysis of Foxp3+ T cells in the thymi of 9-day old mice revealed, based on both percentages and total numbers, that nTreg cells in mice lacking Ndfip1 are similar to their Ndfip1+/+ littermates (). Analysis of 2.5-week old Ndfip1-deficient mice showed slightly increased numbers and percentages of these cells (). From these data, we conclude that the increase in the percentages of nTreg cells in Ndfip1–/– mice at 4-6 weeks is consistent with changes caused by inflammation. Supporting this, when we analyzed thymi from mixed bone marrow chimeras, the percentages of WT nTreg cells were increased to amounts comparable with Ndfip1–/– cells (). Moreover, while Foxp3 staining of the lymph nodes and spleens showed similar percentages (data not shown), there was an increase in the absolute numbers of Foxp3+ Treg cells in Ndfip1–/– over Ndfip1+/+ littermates (). Taken together, these results demonstrate that there is an age-dependent increase in the percentages of nTreg cells in the thymi of Ndfip1–/– mice due to inflammation. Additionally, since nTreg cells still develop in the absence of Ndfip1, Ndfip1 is not required for the development of Foxp3+ cells in the thymus.

The small bowel is a major site of iTreg cell accumulation[32-34]. Importantly, the small bowel is a major site of inflammation in Ndfip1–/– mice[29], suggesting that iTreg cell differentiation in the GI tract may be defective. Flow cytometry analysis of cells isolated from the small bowel of Ndfip1+/+ and Ndfip1–/– littermates revealed a significant decrease in the percentages and numbers of Foxp3+ Treg cells at this site (). Recently, the transcription factor Helios was described as a marker to differentiate thymically derived nTreg cells from peripherally induced iTreg cells[35]. However, the use of Helios as a marker for iTreg cells remains controversial. While Helios expression is not a useful marker for iTreg cells differentiated in vitro[36], most in vivo models support the original report[37]. Therefore, we sought to determine whether the decrease in Foxp3+ Treg cell in the small bowel was due to a decrease of the Helioslo iTreg cell population. Helios staining of the cells described in , showed a significant decrease in the percentages of Helioslo Foxp3+ population () while the percentages of Helioshi cells were lower but not statistically different from those in Ndfip1+/+ littermates (). These results suggest that in the absence of Ndfip1 there is a defect in iTreg cell differentiation in vivo.

Ndfip1–/– T cells are defective in iTreg cell differentiation

To test whether Ndfip1 is required for iTreg cell differentiation, we next sought to determine whether Ndfip1–/– T cells could differentiate into Foxp3+ iTreg cells in vitro. To do this, we first needed to eliminate TH2-effector T cells from our cultures as these cells can inhibit iTreg cell differentiation[19,38,39]. Thus, we sorted naïve (CD25–, CD44low, CD62Lhi) CD4+ T cells from 5-7 week old Ndfip1–/– and Ndfip1+/+ littermates. To ensure that IL-4-producing effectors were removed, we tested the cells before and after sorting for IL-4 production by ELISA. Although IL-4 was not detectable in cultures of Ndfip1+/+ cells, prior to sorting Ndfip1–/– cells produced 4.1 ng/ml IL-4 after overnight stimulation. Sorting reduced IL-4 production by Ndfip1–/– cells to 0.02 ng/ml. Knowing this, we cultured naïve Ndfip1+/+ and Ndfip1–/– T cells under iTreg cell differentiation conditions for 5 days and then assessed their expression of CD25 and Foxp3. We found that Ndfip1-deficient T cells were severely impaired in their ability to induce Foxp3 even when given a concentration TGF-β sufficient to induce Foxp3 expression in nearly all of the WT T cells ().

We next assessed whether defective iTreg conversion in Ndfip1–/– T cells could also be observed in vivo. For this, we adopted a recently described model of Ovalbumin (Ova) -induced iTreg cell conversion of Ova-specific (OTII transgenic) T cells[33]. To generate Ndfip1–/– Ova-specific T cells, we crossed Ndfip1–/– mice to Rag1–/– OTII. As with Ndfip1+/+Rag1–/–OTII+ T cells, T cells from Ndfip1–/–Rag1–/– OTII+ mice were naïve and Foxp3– when isolated and analyzed directly ex vivo (). To test iTreg cell conversion in vivo, we transferred Ova-specific T cells into congenic recipients and fed animals a low dose of Ovalbumin (Ova) for 5 consecutive days. We found that approximately 13% of transferred WT T cells isolated from the Peyer's Patches, and the mesenteric lymph nodes (mLN) had differentiated into Foxp3+ iTreg cells in response to oral antigen (). In contrast, fewer Ndfip1–/– T cells became Foxp3+ during this period, resulting in slightly reduced percentages in the small bowel () and significantly reduced percentages of Ndfip1–/– iTreg cells in the mLN and Peyer's Patches (). These results demonstrate that Ndfip1–/– T cells are defective at converting into iTreg cells both in vitro and in vivo.

Impaired conversion by Ndfip1- and Itch-deficient cells

Ndfip1 is an adaptor protein that promotes the Itch-mediated ubiquitylation and consequent degradation of JunB and cJun[25]–transcription factors involved in TH2 development. Thus both Itchy mutant and Ndfip1–/– T cells are TH2 biased. Itchy mutant T cells are also impaired in iTreg cell conversion[23]. Considering this, we sought to test whether the defect in iTreg cell differentiation in Ndfip1–/– T cells was due to Ndfip1 regulation of Itch function. We thus compared the iTreg cell differentiation capacity of Ndfip1- and Itch-deficient T cells, using the same sorting and in vitro culture conditions described above. Consistent with what was shown previously[23], Itchy mutant T cells are impaired at converting into iTreg cells (). We found that Ndfip1–/– T cells are even less likely to differentiate into iTregs in vitro than Itch-deficient counterparts (). Combining data from these experiments, we calculated that Ndfip1–/– T cells would need approximately 29 fold more TGF-β for a half-max conversion to Foxp3+ iTreg cells than WT cells, whereas Itchy mutant T cells would need about 2 fold more TGF-β (). This is unlikely to be due to background differences between the two strains as both have been backcrossed more than 9 generations onto C57BL6. Nonetheless, both Itchy mutant and Ndfip1–/– T cells are defective in iTreg cell conversion.

It has been suggested that Itch promotes iTreg cell differentiation via monoubiquitylation of TIEG1[23], a transcription factor that promotes Foxp3 expression. Monoubiquitylation of TIEG1 appeared to promote the association of TIEG1 with DNA elements in the Foxp3 locus[23]. TIEG1 binds two sites in the Foxp3 locus, one within the Foxp3 proximal promoter region[24], and the other in an enhancer region known as CNS2[23]. In Itchy mutant T cells, TIEG1 did not bind to the CNS2 enhancer region[23], but binding of TIEG1 to the proximal promoter region was not described. However, the CNS2 region was recently shown to be irrelevant for iTreg cell differentiation[40]. Thus, to test whether Ndfip1 regulates TIEG1 binding to Foxp3 sequences, we used chromatin immunoprecipitation (ChIP) to analyze TIEG1 association with the Foxp3 proximal promoter region in T cells lacking either Ndfip1 or Itch. For this analysis, cells were analyzed for binding after both 18 and 42 hours of iTreg cell conversion. This was based on data that TGF-β signaling is particularly important during this period[41]. The location of the primers used to detect Foxp3 DNA bound to TIEG1 is illustrated in . TIEG1 associated with the Foxp3 proximal promoter region in WT, Itchy mutant and Ndfip1–/– T cells (). Supporting these results, TIEG1 was also bound to the CNS2 region as determined using previously published primers (data not shown). These results show that impaired iTreg cell differentiation in Ndfip1- and Itch- deficient T cells cannot be explained by a lack of TIEG1 binding to the Foxp3 locus at early time points during iTreg differentiation cell.

Abortive Foxp3 expression in T cells lacking Ndfip1

Based on our results thus far, TIEG1 is bound to the Foxp3 promoter 48 hours after iTreg cell induction. However, these cells do not express Foxp3 after 5 days in these same culture conditions. To resolve this apparent contradiction, we decided to test whether Ndfip1–/– T cells express Foxp3 during the time points tested by ChIP, namely two days after stimulation. Using the same protocol described in , we tested Foxp3 expression by flow cytometry analysis at day 2 and again at day 5 during iTreg cell differentiation. Using this approach, we found that on day 2, Ndfip1–/– T cells express comparable levels of Foxp3 to those in WT cells (). In contrast, but consistent with our previous results, Foxp3 expression is diminished by day 5 in Ndfip1–/– T cells, while it continues to increase in WT T cells. In addition, we see a similar trend in Foxp3 expression with Itch-deficient T cells (). Recently, it was shown that IL-2 can stabilize Foxp3 expression[42]. Thus, we sought to determine whether increased amounts of IL-2 can rescue the loss of Foxp3 expression in Ndfip1–/– T cells that occurred between day 2 and day 5. However, increasing the concentration of IL-2 in our cultures to 100U/ml did not rescue the defect (). Another possible explanation for the decline in Foxp3+ T cells from day 2 to day 5 could be that Foxp3+ T cells lacking Ndfip1 die during this culture. Thus, we assessed the percentage of 7AAD+ cells at day 2 and day 5 of iTreg cell differentiation. While we observed a slight increase in the percentage of Ndfip1–/– T cells that are 7AAD+ at day 2, at day 5 the percentages of 7AAD+ cells are reduced compared to controls (). These data suggest that other mechanisms must account for the loss of Foxp3+ cells in the Ndfip1–/– cultures. Consistent with Foxp3 protein expression, Foxp3 mRNA was induced, albeit to a lesser extent, in cells lacking Ndfip1 (). Ndfip1–/– T cells showed reduced Foxp3 mRNA levels beginning at day one while Itch-deficient T cells began to show a reduction in mRNA induction after 2 days of iTreg cell induction (). These data indicate that Foxp3 expression, and by inference iTreg cell induction, is initiated in Ndfip1- and Itch-deficient T cells, but then is aborted. Knowing that IL-4 can block Foxp3 expression and that Ndfip1–/– T cells are prone to produce IL-4 under other culture conditions, we hypothesized that IL-4 production by Ndfip1-deficient T cells could be aborting the iTreg cell differentiation process.

To begin to test this, we first wanted to determine the amount of IL-4 that inhibits iTreg cell differentiation by adding IL-4 into cultures of WT cells undergoing iTreg cell conversion. Using this approach, we found that iTreg conversion was inhibited by small amounts of IL-4. Graphing this on a logarithmic scale, we could quantify the half maximal inhibitory concentration of IL-4 as 190 pg/ml (). Knowing this, we next sought to determine whether Ndfip1–/– T cells were producing amounts of IL-4 that would block iTreg cell differentiation. To do this, we measured the amount of IL-4 in cultures of Ndfip1–/– and Ndfip1+/+ cells undergoing iTreg cell differentiation using ELISA. While we saw little IL-4 produced from sorted naïve Ndfip1–/– T cells cultured for 24 hours, we found that the amount of IL-4 detected in supernatants increased after 48 hours of stimulation to levels sufficient to inhibit iTreg cell differentiation (). Interestingly, while the amount of IL-4 produced by Ndfip1–/– T cells at 24 hours was not different regardless of whether the cells were stimulated in the presence or absence of TGF-β (data not shown), the amount of IL-4 produced by Ndfip1–/– T cells at 48 hours of iTreg cell culture was lower than Ndfip1–/– T cells stimulated in the absence of TGF-β (). This is consistent with previous data showing TGF-β can attenuate IL-4 production[19,20]. Furthermore, in agreement with the less severe defect in Itchy mutant T cells undergoing iTreg cell differentiation (), T cells lacking Itch produced much less IL-4 than Ndfip1-deficient counterparts ().

To test whether cells were able to detect IL-4 from their environment, we used flow cytometry to analyze levels of the IL-4 receptor (IL-4R). After day 1 in culture IL-4R expression was only slightly elevated compared to levels on naïve T cells (data not shown). In contrast, by day 2 of iTreg cell differentiation, IL-4R had increased (). This elevated expression of IL-4R at day 2 was seen in cells stimulated in the presence or absence of TGF-β, likely due to IL-2R signaling[43]. This implies that there is a ‘window of opportunity’ in iTreg cell differentiation during which T cells express IL-4R to sense cues from their environment. Signals they receive through these receptors likely impact how they proceed in the differentiation process. Furthermore, these data suggest that the impaired iTreg cell differentiation in Ndfip1- and Itch-deficient T cells may be due to IL-4 produced by these cells.

Knowing that Ndfip1–/– T cells cultured under iTreg cell differentiation conditions in vitro produced high levels of IL-4 and were defective in iTreg cell differentiation we tested IL-4 production in Ndfip1–/– T cells using the in vivo model described in . Consistent with our in vitro results (), Ova-specific Ndfip1–/– T cells induced to become iTreg cells in vivo also produced IL-4, suggesting that the in vivo and in vitro iTreg cell defects worked via a similar mechanism ().

IL-4 blocks iTreg differentiation in Ndfip1-/- T cells

To determine whether IL-4 was inhibiting iTreg cell differentiation in Ndfip1- and Itch-deficient T cells, we performed iTreg cell conversion assays in the presence or absence of antibodies that block the binding of IL-4 to its receptor. While addition of anti-IL-4 had no impact on WT cells (data not shown), when IL-4 blocking antibodies were added to the Ndfip1- and Itch-deficient T cells, iTreg cell differentiation was restored to that seen in the WT (). Thus, production of IL-4 is sufficient to explain why both Ndfip1–/– and Itchy mutant T cells are poor at differentiating into iTreg cells in vitro. Additionally, if the Ndfip1–/– T cells were converted into iTreg cells in the presence of anti-IL-4 blocking antibodies, the cells could suppress just as well as WT iTreg cells ().

Furthermore, it is unlikely that other TH2 cytokines, such as IL-5, that are also detectable in the supernatants of Ndfip1- and Itch-deficient T cells undergoing iTreg cell differentiation (data not shown) lead to impaired iTreg cell differentiation since the addition of IL-5 to WT T cells undergoing iTreg cell conversion had no effect on their ability to become Foxp3+ ().

To confirm that IL-4 production by Ndfip1–/– T cells was inhibiting iTreg cell differentiation, we generated mice lacking both Ndfip1 and IL-4. As shown (), iTreg cell differentiation in T cells from Ndfip1–/–Il4–/– mice was similar to T cells from Ndfip1+/+Il4–/– littermates. These data show that IL-4 produced by Ndfip1- or Itch-deficient T cells prevents iTreg cell differentiation, since blocking either IL-4 production or the binding of IL-4 to its receptor restores iTreg cell differentiation in these cells in vitro.

As described above, IL-4 production increases in Ndfip1–/– T cells during iTreg cell differentiation between 24-48 hours at a time when the cells are upregulating IL-4R expression. This suggests that there is an early ‘window’ during Foxp3 induction following the initial stimulation when cells are sensing their environment and that IL-4 signaling during this time would lead to abortive iTreg cell differentiation. To test whether IL-4 was indeed mediating the abrogation of Foxp3 expression during this ‘window’, we repeated iTreg cell conversion assays, blocking IL-4 signaling at various times following the initial stimulation. The delayed addition of IL-4 blocking antibodies after 24 hours restored iTreg cell differentiation in Ndfip1–/– T cells () and this did not occur if the antibodies were added after 48 or 72 hours. These data show that IL-4 can abrogate Foxp3 expression during this early stage of iTreg cell differentiation and that there is a ‘window’ between 24-48 hours following initial stimulation where Foxp3 expression is unable to be rescued by neutralization of IL-4.

IL-4 produced by the Ndfip1-deficient T cells could act preferentially on the cells producing the cytokine, and/or it could act on neighboring cells, preventing their expression of Foxp3. To test which of these occurred in iTreg cell differentiation cultures, we mixed Ndfip1–/– and congenic WT cells together at various ratios prior to initiating iTreg cell differentiation. Using these mixed cultures, we found that IL-4 produced by the Ndfip1–/– T cells was able to inhibit iTreg cell differentiation of WT cells at all ratios tested (). Thus, IL-4 can prevent iTreg cell differentiation in trans. However, Ndfip1–/– T cells were more defective at iTreg cell differentiation than their WT counterparts, particularly when co-cultured at low ratios (25:1) (). This indicates that while IL-4 can act in both an autocrine and paracrine manner, it has a more profound effect on Ndfip1–/– cells. This implies that the there is something intrinsic to T cells lacking Ndfip1 that makes them more sensitive to IL-4 than their WT counterparts. This could be due to enhanced IL-4 receptor signaling in T cells lacking Ndfip1. To test this, we added IL-4, at the half maximal inhibitory concentration (based on ) to Ndfip1–/– and Ndfip1–/– T cells undergoing iTreg cell differentiation (to eliminate the confounding effects of IL-4 production by the cells). As predicted, we saw approximately 50% inhibition of iTreg differentiation in Il4–/– cells and a similar level of inhibition in Ndfip1–/– cells (). Interestingly, the Ndfip1–/– cells showed a modest (but not statistically significant) increase in sensitivity to IL-4. This may be due to the slight increase in IL-4R levels we observe in Ndfip1–/– T cells (data not shown). This might also explain why the Ndfip1–/– cells were more inhibited than their WT counterparts in the co-culture experiments. Nonetheless, this appears to play a minor role in the defective iTreg cell differentiation as this difference is not as profound as their difference in IL-4 production.

Normal frequency of iTreg cells in Ndfip1–/– Il4–/–

To test whether IL-4 production accounts for the reduced numbers of iTreg cells in Ndfip1–/– animals, we analyzed the percentages of Foxp3+Helioslo cells in the small bowel from mice lacking both Ndfip1 and IL-4. Whereas mice lacking Ndfip1 have reduced percentages of iTreg cells (Foxp3+Helioslo) in the small bowel, mice lacking both Ndfip1 and IL-4 showed percentages comparable to WT and IL-4-deficient mice (). Thus, similar to what we observed with iTreg cell differentiation in vitro, iTreg cell differentiation in Ndfip1–/– mice in vivo appears to be due to overproduction of IL-4.

Mice lacking Ndfip1 have increased percentages of activated T cells in their peripheral lymphoid organs[25, 29]. These activated T cells could be the direct or indirect consequence of aborted iTreg cell differentiation in vivo. Thus, having shown that iTreg cell induction was restored in mice lacking both Ndfip1 and IL-4, we next wanted to determine whether the percentages of activated T cells were reduced in mice lacking both Ndfip1 and IL-4. While mice lacking Ndfip1 had twice as many CD44hi cells as Ndfip1+/+ controls, the percentages of these cells in mice lacking both Ndfip1 and IL-4 were comparable to controls (). Supporting this, fewer CD4+ T cells were found in the small bowel of mice lacking both Ndfip1 and IL-4 than in mice lacking only Ndfip1 (). Furthermore, mice lacking both Ndfip1 and IL-4 had reduced GI pathology, as evidenced by reduced eosinophil infiltration, compared to mice lacking only Ndfip1 (). Additionally, mice lacking both Ndfip1 and IL-4 have longer life-spans than their Ndfip1–/– counterparts and have reduced inflammation in their lungs, as evidenced by decreased infiltrating leukocytes (manuscript in preparation). This would be expected, since eosinophil infiltration is likely the result of TH2 cytokine production in this model[29]. Nonetheless, these in vivo data suggest that when Ndfip1–/– T cells cannot make IL-4, more T cells differentiate into iTreg cells, fewer T cells have an activated phenotype, and GI pathology is reduced.

Ndfip1 limits JunB levels during iTreg cell commitment

Taken together, our data show that as WT cells begin to differentiate into iTreg, cells they upregulate both Foxp3 and their IL-4R. This implies that during this time they are acutely sensitive to cues from their environment, such as the presence of IL-4. It seems likely that Ndfip1 is acting at this stage since Ndfip1–/– T cells express Foxp3 early during iTreg cell differentiation and then fail to fully differentiate into iTreg cells. Thus, we analyzed the expression of Ndfip1 at different time points during iTreg cell differentiation. To do this, we cultured naïve T cells under iTreg cell differentiation conditions and extracted mRNA on days 1, 2 and 3, and expression of Ndfip1 was determined using Quantitative real-time PCR (qRT-PCR). Ndfip1 mRNA expression peaked in cells cultured 1 day in the presence of TGF-β (). Expression of Ndfip1 peaks at approximately the time when cells are expressing both Foxp3 and IL-4R, and committing to the iTreg cell lineage. This may explain why Foxp3 expression fails between day 2 and day 5 in Ndfip1–/– T cells. It is worth noting that the induction of Ndfip1 expression was TGF-β-dependent since stimulation without TGF-β showed lower levels of Ndfip1 expression (data not shown). Taken together these data indicate that, in the first 24-48 hours of iTreg cell differentiation, T cells upregulate Ndfip1 in an effort to dampen IL-4 and allow iTreg cell differentiation.

To identify a transcription factor that could account for the increased IL-4 production in Ndfip1- and Itch-deficient T cells we assessed the expression levels of factors that are known to promote early IL-4 production, namely Gata3 and Jun family members. Whereas Gata3 mRNA expression is increased at day 5 after iTreg cell induction, Gata3 expression is comparable to Ndfip1+/+ T cells at day 2 in both Ndfip1- and Itch-deficient T cells (). We next looked at JunB, c-Jun, and JunD levels during iTreg cell differentiation (). We found a considerable increase in JunB protein in Ndfip1–/– T cells (). Itchy mutant T cells also show elevated JunB but Ndfip1-deficient T cells had higher JunB than both Itch-deficient and Ndfip1+/+ T cells (). This is consistent with the increased production of IL-4 by Ndfip1–/– T cells during iTreg cell conversion (). Elevated JunB protein expression in Ndfip1–/– T cells was evident as early as day 2 following iTreg cell induction and increased further over that seen in the control cells at day 3 (). In Ndfip1+/+ cells, JunB protein increased from day 1 to day 2 but then stayed relatively constant at day 3 () when Ndfip1-deficient T cells had increased amounts (). Taken together, these data show that the elevated amounts of IL-4 produced by Ndfip1- and Itch-deficient T cells during iTreg cell differentiation is likely due to the accumulation of JunB in these cells.

To determine whether the elevated amounts of JunB in Ndfip1–/– T cells could be a cause or consequence of the IL-4 production during iTreg cell differentiation, we next tested if there was an increase in JunB levels in Ndfip1–/– T cells in the presence or absence of anti-IL-4. We found that JunB protein was still elevated in Ndfip1–/– T cells undergoing iTreg cell differentiation in the presence of anti-IL-4, suggesting elevated JunB was not a consequence of IL-4R signaling (). Thus, we next tested whether JunB might be the cause of IL-4 production. Supporting that JunB causes IL-4 production, we detected JunB binding to the IL-4 promoter in Ndfip1–/– T cells (). However, we did not observe any binding of JunD to this region ().

Knowing that JunB could bind the IL-4 promoter and was not downstream of IL-4 production, we next sought to determine the signals leading to JunB expression in T cells undergoing iTreg cell differentiation. To do this, we cultured cells under iTreg cell conditions in the presence or absence of TGF-β or after removal of TCR stimulation. TGF-β induced an increase in JunB protein in both Ndfip1+/+ and Ndfip1–/– T cells (). Thus, as has been seen previously in other cell types, TGF-β signaling can induce JunB expression. Furthermore, consistent with previously published data[44], we found that TCR signals are necessary for JunB expression (). When we cultured Ndfip1+/+ and Ndfip1–/– T cells for 24 hours under normal iTreg cell conditions and then removed TCR signaling for the duration of the culture, JunB protein was undetectable. Having found a scenario under which JunB was not expressed, we next tested whether TCR withdrawal affected iTreg cell differentiation in Ndfip1+/+ and Ndfip1–/– T cells. We found that the withdrawal of TCR signals during iTreg cell differentiation had no discernible impact on Ndfip1+/+ T cells. Importantly, TCR signal withdrawal resulted in a loss of IL-4 production (data not shown) and restored iTreg cell differentiation in Ndfip1–/– cells (). Thus, iTreg cell differentiation can be rescued by removal of initial TCR signals concomitant with loss of JunB expression. Taken together these results show that overexpression of JunB is not a consequence of IL-4R signaling and that JunB is likely an active participant leading to IL-4 overproduction in Ndfip1–/– T cells during iTreg cell differentiation.

Previous data has shown that JunB levels are increased in TH2 cells lacking Ndfip1 and that this was due to impaired degradation of JunB[25]. To test whether the elevated levels of JunB were the result of impaired degradation or increased production, we assessed JunB mRNA levels and protein stability during iTreg cell differentiation. JunB mRNA expression in Ndfip1–/– T cells was comparable to that in WT T cells (). In contrast, and consistent with it's known role as an adaptor for E3 ubiquitin ligases, Ndfip1–/– T cells showed impaired degradation of JunB (). Thus, the increased levels of JunB in T cells lacking Ndfip1 were a result of increased stability of JunB. Given these results, we propose a model in which TGF-β induces expression of Ndfip1 to dampen IL-4 production during iTreg cell differentiation ().

DISCUSSION

iTreg cells, generated from naïve T cell precursors in peripheral lymphoid compartments, can attenuate immune responses to either self or environmental antigens[1,2]. These regulatory T cells are characterized by expression of Foxp3[2]. However, expression of Foxp3 is not sufficient to define a regulatory T cell, as activated T cells can transiently upregulate Foxp3[45] along with transcription factors that dictate other T cell fates. This has lead to the proposal that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals that ultimately allow cells to commit towards a particular T cell lineage. Here we report that Ndfip1 helps to regulate this process by dampening TH2 cytokine production during the decision making phase of iTreg cell differentiation.

In contrast to the apparent defect in iTreg cell differentiation in Ndfip1–/– mice, we find elevated percentages of nTreg cells in the thymi of Ndfip1–/– mice, thus nTreg cells do develop in the absence of Ndfip1. This increase in nTreg cells was likely a result of the inflammatory cytokines present in these mice. However, a more precise analysis of nTreg cells in mice 3 to 9 days old would be required to entirely rule out a role for Ndfip1 in nTreg cell development. TGF-β is important for both nTreg cell differentiation and iTreg cell differentiation [9,13,16,17,30]. That we see defects only during iTreg cell differentiation could reflect a reduced capacity of developing thymocytes to produce or respond to IL-4 compared to peripheral naïve T cells. Also, whether IL-4 can affect nTreg cell development in Ndfip1–/– mice is not clear. We do not detect significant amounts of IL-4 in the serum of Ndfip1–/– even when they present with overt signs of inflammation when IL-4 production is detectable in splenocytes (unpublished observation). Thus, it will be important to determine whether IL-4 is produced locally by Ndfip1–/– thymocytes and whether developing nTreg cells respond to IL-4. While it is clear that there are circumstances under which CD4 -single positive (SP) cells in the thymus can make IL-4, it is possible that IL-4 is not produced in the thymi of young Ndfip1–/– mice since the majority of CD4 SP thymocytes in neonatal mice are not functionally competent and do not respond the same as peripheral T cells[46]. These will be the focus of future studies.

Preventing IL-4 production is a particular challenge for cells undergoing iTreg cell differentiation. While iTreg cells are dependent on IL-2R signaling[9,10], these signals are known to promote both IL-4 production and IL-4R expression[47]. Thus, as iTreg cells differentiate, they receive IL-2R signals, increase expression of their IL-4R[43], and inhibit their own IL-4 production to seek cues from their environment. If IL-4 production is not silenced during this period, it could prevent iTreg cell differentiation in both an autocrine and paracrine manner. This could result in enhanced and/or prolonged immune responses with damaging consequences.

Although it is known that iTreg cell differentiation is remarkably sensitive to effector cytokines such as IL-4[19,38,39], the mechanisms that prevent IL-4 production by T cells during iTreg cell differentiation are only partially understood. For example, it is known that TGF-β receptor signaling dampens IL-4 production in WT T cells[19]. In part, this is because TGF-β receptor signaling reduces Gata3 expression[20]. We show here that in the absence of Ndfip1, T cells produce IL-4 at levels that inhibit their own iTreg cell differentiation, and iTreg cell differentiation of other T cells in their vicinity.

Paradoxically, JunB was increased in a TGF-β-dependent manner. While this was surprising, data in other non-immune cell types has shown that TGF-β can induce JunB via a Smad-dependent pathway[48]. Why and how TGF-β induces JunB expression in T cells is not clear. However, JunB expression in WT cells plateaus at day 2 during iTreg cell differentiation. In contrast, in Ndfip1–/– T cells, JunB expression continues to increase and JunB was bound to the IL-4 promoter in Ndfip1–/– T cells undergoing iTreg cell differentiation, demonstrating a causal role for JunB in IL-4 production. Supporting this, in one scenario under which JunB is not expressed in Ndfip1–/– T cells, IL-4 is not produced and iTreg cell differentiation is restored.

TGF-β also induces increased expression of Ndfip1, an adaptor protein that promotes the ubiquitylation and degradation of Jun-family proteins by the E3 ligase Itch. Ndfip1 is particularly important in the first 24-48 hours of iTreg cell differentiation, as cells are increasing expression of Foxp3 and IL-4R. In the absence of Ndfip1, T cells initially increase Foxp3, but also aberrantly express IL-4. This ultimately aborts the iTreg cell differentiation process in these cells. Given these results, we suggest that Ndfip1 promotes Itch ubiquitylation and degradation of JunB to prevent IL-4 production and allow iTreg cell differentiation. While these data support a role for Ndfip1 regulation of Itch, it is also clear that Ndfip1 also regulates iTreg cell differentiation via an Itch-independent mechanism.

Interestingly, it seems that Ndfip1 is not needed once Foxp3+ Treg cells are fully differentiated since cells that had already committed to the Treg lineage have lower Ndfip1 expression than their naïve T cell counterparts (data not shown). Supporting this, Ndfip1–/– cells that differentiate into iTreg cells (in the presence of anti-IL-4) suppress proliferation as well as WT iTreg cells.

Here we define an early ‘window’ where Ndfip1 is expressed to dampen IL-4 production during iTreg cell differentiation. The kinetics of Ndfip1 expression and inhibition of iTreg cell differentiation by IL-4 are consistent with data showing that optimal iTreg cell conversion occurs when TGF-β was added within 1-2 days[41]. Thus, environmental cues received by the T cell early during this time can alter the ability of these cells to differentiate into Foxp3 expressing Treg cells. Supporting this, it is known that Foxp3 (induced by TGF-β receptor signaling) can bind directly to Gata3 (induced by IL-4R signaling) to prevent the induction of TH2 cytokines[19]. On the other hand, if Gata3 levels increase (due to IL-4R signaling) and outcompete Foxp3, iTreg cell differentiation is prevented [38,39]. Based on the data we have presented, we propose that Ndfip1 dampens IL-4 production during TGF-β stimulation to provide a ‘window of opportunity’ for iTreg cell differentiation.