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GATA3-Driven Th2 Responses Inhibit TGF-beta1-Induced FOXP3 Expression and the Formation of Regulatory T Cells
Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes. FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown. We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway. We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3. This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-beta-mediated FOXP3 induction. This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process. Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response. IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.

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Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently. Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively. A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations. The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa. Specifically interleukin 4 (IL-4)-induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2]. GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3]. Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5]. A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4-dominated responses can break antigen-specific immune tolerance [7]. Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.
The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens. Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery. These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines. Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions. These induced Treg (iTreg) cells were reported to express FOXP3 [10]. The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14]. TGF-beta has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-betaRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell-dependent multiorgan inflammatory disease [16]. Although the effect of TGF-beta on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.
The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively. We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo. We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.

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FOXP3 Induction in T Cell Subsets
It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation. However, it is not known if all CD4+ T cells have the same capacity to express FOXP3. To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1). The CD4+CD25-, CD45RA+, CD45RO+, and CD4+CD45RO+CD25- were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-beta. Th1 cells showed only a 10-fold increase. In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression. The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).
Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-beta signaling and thus prevent FOXP3 induction. However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data). These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B). The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A). Only iTreg cells expressed FOXP3 in resting conditions. Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression. Repeated exposure to TGF-beta did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells. Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4-producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B). Furthermore TGF-beta-mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B). Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS). IL-4-expressing cells were most abundant among CD45RO+CD25- cells, which did not co-express FOXP3 (Figure 3A, left panel). In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel). As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background. The IL-4+ cells were confined to the FOXP3- cells, as shown for the CD45RO+CD25-, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B). In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C). Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap. Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.

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FOXP3 and GATA3 Kinetics in Differentiating Cells
The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs. Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-gamma, and anti IL-12), Th2, and iTreg phenotypes. After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18]. Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel). Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B). Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process. The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B). On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B). Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.

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IL-4 Inhibits TGF-beta-Mediated iTreg Commitment
IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage. We therefore asked whether IL-4 is able to inhibit TGF-beta induction of FOXP3 during the priming of naive T cells. Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta and/or IL-4 and harvested after 5 d. IL-4 efficiently repressed the TGF-beta-mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B). Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3]. However, at low concentration, IL-4 was able to marginally induce FOXP3 expression. Of note, GATA3 was also induced in the presence of TGF-beta at high IL-4 concentration (Figure 5A and 5B). The IL-4-mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-beta, which demonstrates that IL-4 as well as TGF-beta signaling were functional under these conditions (Figure 5C). Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D). Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo. A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25. We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19]. In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2). Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25- cells, but a real decrease of CD4+CD25+ T cells. In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo. To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity. In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C). Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).

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The Role of TGF-beta in T Cell Differentiation
Although TGF-beta-reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-beta-mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A). It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3- cells and thus decrease the relative percentage of FOXP3+ cells. However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3-. In fact both populations showed similarly enhanced proliferation (Figure 6A). Furthermore the TGF-beta-mediated induction of FOXP3 expression was not caused by overgrowth of a CD25-FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells. CD25 was down-regulated in TGF-beta-treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-beta and IL-4.
The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B). In line with the previous findings, the IL-4-producing cells and the FOXP3 expressing cells are nonoverlapping populations. Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression. The expression kinetic of naive T cells exposed to IL-4 and TGF-beta demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4-8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.
These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-beta signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.

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GATA3 Is a Negative Regulator of FOXP3 Expression
FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3. Besides GATA3's well-known positive effect on gene regulation, GATA3's repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells. To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3. After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-beta. TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel). TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-deltaDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A). In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3). The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data). These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22]. To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25- cells were isolated, activated with OVA in the presence or absence of TGF-beta, and Foxp3 expression was analyzed after 4 d. The naive CD4+CD25- cells were Foxp3- (unpublished data). As described for the human cells, TGF-beta dramatically up-regulated Foxp3 in the DO11.10 littermate control mice. In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-beta and OVA (Figure 7B). All mice produced similar amounts of TGF-beta; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-beta signaling.
Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.

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GATA3 Represses the FOXP3 Promoter
To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered. The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24]. This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation. The functional relevance of this site was studied using a FOXP3-promoter construct [24]. We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity. The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A). Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B). To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site. This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3-) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C). Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay. HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site. After the pull-down, GATA3 binding was detected by Western blot. Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach. Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E). These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter. To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F). Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.

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The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells. After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.
In accordance with other studies, we found that CD4+CD25- cells were able to up-regulate FOXP3 [12,27]. Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile. In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-beta to induce iTreg cells [10,28-34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3. This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation. In this competitive process TGF-beta appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38]. In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-beta showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data). Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42-45]. Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.
The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-beta-mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype. To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4. This has been shown to increase the effect of the cytokine in vivo [19]. Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment. Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells. But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression. IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17-producing T cells (Th17) [46,47]. Similar effects have been recently described for IL-6, which, combined with TGF-beta, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49]. Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.
IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25- [50]. In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression. Importantly, these concentrations were not sufficient to induce GATA3 expression. IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].
Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4-dependent inhibition of FOXP3 could be mediated by GATA3. In fact, GATA3-inducing IL-4 concentrations repressed TGF-beta-mediated FOXP3 expression, whereas IL-4 as well as TGF-beta signaling were intact. This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation. Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3. Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells. This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice). In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-beta. Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%-25% lower frequency compared to wild-type DO11.10 mice. Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin. Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52]. In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55]. Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.
Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region. A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24]. Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3. This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments. Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells. It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57-59]. Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.
The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).
In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4. GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter. We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression. These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.

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Mice.
Normal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323-339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8-12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.

expressing


Input:
Isolation of CD4+ T cells.
CD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was >= 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.

secretion

high

secretion


Input:
Quantitative real-time PCR.
The PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5'-GAA ACA GCA CAT TCC CAG AGT TC-3'; FOXP3 reverse primer 5'-ATG GCC CAG CGG ATG AG-3'; EF-1alpha forward primer and reverse primer as described [61]; GATA3 forward primer 5'-GCG GGC TCT ATC ACA AAA TGA-3' and rwd 5'-GCT CTC CTG GCT GCA GAC AGC-3'. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Quantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5'- AGG TCA AAC AGG AAG ACA GAC GTA-3' and 5'-TCACACCCAAGAACAAGCACA-3'; Smad-7, 5'-GAA ACC GGG GGA ACG AAT TAT-3' and 5'-CGC GAG TCT TCT CCT CCC A-3'; TGF-ss1, 5'-TGA CGT CAC TGG AGT TGT ACG G-3' and 5'-GGT TCA TGT CAT GGA TGG TGC-3'. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 degreesC, followed by 40 cycles of 15 s at 95 degreesC and 60 degreesC for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative deltadeltaCT method.


Input:
Inducible murine Treg culture.
Naive CD4+ T cells (CD4+, CD62L+, and CD25-) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 x 105 T cells were co-cultured with 2.5 x 104 bone marrow-derived dendritic cells [62] and 0.01 mug/ml OVA323-339 peptide (Ansynth) in the presence or absence of 20-ng/mul rhTGF-ss1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.

expression


Input:
In vitro T cell differentiation.
CD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 mug/ml, Okt3, IgG1) and anti-CD28 (2 mug/ml) in Th1 conditions: 25 ng/ml IL-12, 5 mug/ml anti-IL-4 (R&D systems); in Th2 conditions: 25 ng/ml IL-4, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12 (R&D systems); or in Treg conditions: 10 ng/ml TGF-beta, 5 mug/ml anti-IFN-gamma, 5 mug/ml anti-IL-12, 5 mug/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).

depletion


Input:
Cloning of the FOXP3 promoter and construction of mutant constructs.
The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 -511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.


Input:
Transfections and reporter gene assays.
T cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 mug of the FOXP3 promoter luciferase reporter vector and 0.5 mug phRL-TK were added to 3 x 106 CD4+ T cells resuspended in 100 muL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.


Input:
RNA isolation and cDNA synthesis.
RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.


Input:
Recombinant TAT proteins.
The cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.

lacking

two zinc fingers


Input:
TAT-GATA3 transduction.
CD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-beta (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.

expression


Input:
Intracellular cytokine staining.
T cells were stimulated with 2 x 10-7 M PMA and 1 mug/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4-8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.


Input:
Flow cytometry.
For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4degreesC in staining buffer with the following antibodies: anti-CD152-PE (CTLA-4; BD), anti-PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcgammaRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4'',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.

expression

expression


Input:
Western blotting.
For FOXP3 analysis on the protein level, 1 x 106 CD4+CD25- cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 degreesC. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3-31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),


Input:
Administration of cytokines and antibodies in vivo.
Age- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 mug rmIL-4, 50 mug anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 mug rmIL-4 plus 50 mug anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R&D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.

expression

expression

expression


Input:
ChIP.
ChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3-31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 degreesC for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region -246 to -511 and was performed using the following primers: 5'-gtgccctttacgagt catctg-3' and 5'-gtgccctttacgagtcatctg-3'. The PCR products were visualized using an ethidium bromide gel.


Input:
Pull-down assay.
CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37degreesC. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 mug of poly(dI-dC) (Sigma) and 70 mul of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 degreesC. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 degreesC for 10 min, and the eluants on a NuPAGE 4-12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).


Input:
Th2 Cells Cannot Induce FOXP3 Expression
(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-beta as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean +/- SD of 4 independent experiments.
(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-beta, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean +/- SD of four independent experiments.

Expression

Induce

Expression


Input:
Th2 Cells Do Not Express FOXP3
(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-beta. FOXP3 expression was measured after 5 d in culture. The dot blots are representative of three independent experiments.
(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed. Data are representative of three independent experiments.

Express

expression

expression


Input:
Th2- or IL-4-Producing Cells Lack FOXP3
(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation. CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25- (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO-CD25- subsets (central panel). A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B). The dotted gray line indicates the IC background level. The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4. Data are representative of three independent experiments.

Producing

expression

expression

expression

expression

secreting

stained

stained

Lack


Input:
FOXP3 Induction During the Differentiation Process
(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-beta (5 ng/ml) or IL-4 (25 ng/ml). The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression. Bars show the mean +/- SD of three independent experiments.
(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure. Data are representative of three independent experiments.

expression

expression

staining

staining

Induction


Input:
Effect of IL-4 on FOXP3 Induction
(A) A statistical analysis was performed with six donors on day 5 (TGF-beta (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors. Statistical analysis was performed using the Dunnett test. Statistical significance is indicated by asterisks (*p <= 0.05, **p <= 0.01, Dunnett).
(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-beta (5 ng/ml) with an increasing concentration of IL-4, as indicated. Cells were harvested for mRNA quantification after 5 d.
(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-beta (10ng/ml), and IL-4 (100 ng/ml) as indicated. After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6. Total STAT6 and GAPDH served as internal control.
(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.

staining

staining

phosphorylated

phosphorylated

Induction

increasing

Effect

Induction


Input:
IL-4 Inhibits TGF-beta-Mediated iTreg Commitment
CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-beta, and IL-4, as indicated. After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B). Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p <= 0.01, ***p <= 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-beta. Data are representative of three independent experiments.

staining

staining


Input:
GATA3 Acts as a Negative Regulator of FOXP3 Expression
(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells. GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel). Data are representative of four independent experiments.
(B) CD4+CD25- T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-beta for 96 h. Surface CD4 and intracellular FOXP3 were measured by FACS. These data are representative of three independent experiments.
(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-beta expression. Bars show the mean +/- SD of three independent experiments.

presence

expression

expression

expression

TGF-beta


Input:
GATA3 Represses the Human FOXP3 Promoter
(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.
(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean +/- SD of three independent experiments.
(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean +/- SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.
(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides-transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.
(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The "input" represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.

binding

promoter

expression

amounts

Represses

Promoter


Input:
Suppression of GATA-3 Nuclear Import and Phosphorylation: A Novel Mechanism of Corticosteroid Action in Allergic Disease
Background
GATA-3 plays a critical role in regulating the expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells and therefore is a key mediator of allergic diseases. Corticosteroids are highly effective in suppressing allergic inflammation, but their effects on GATA-3 are unknown. We investigated the effect of the corticosteroid fluticasone propionate on GATA-3 regulation in human T-lymphocytes in vitro and in vivo.
Methods and Findings
In a T lymphocyte cell line (HuT-78) and peripheral blood mononuclear cells stimulated by anti-CD3 and anti-CD28 in vitro we demonstrated that fluticasone inhibits nuclear translocation of GATA-3 and expression of Th2 cytokines via a mechanism independent of nuclear factor-kappaB and is due, in part, to competition between GATA-3 and the ligand-activated glucocorticoid receptor for nuclear transport through the nuclear importer importin-alpha. In addition, fluticasone induces the expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), the endogenous inhibitor of p38 MAPK, which is necessary for GATA-3 nuclear translocation. These inhibitory effects of fluticasone are rapid, potent, and prolonged. We also demonstrated that inhaled fluticasone inhibits GATA-3 nuclear translocation in peripheral blood lymphocytes of patients with asthma in vivo.
Conclusions
Corticosteroids have a potent inhibitory effect on GATA-3 via two interacting mechanisms that potently suppress Th2 cytokine expression. This novel mechanism of action of corticosteroids may account for the striking clinical efficacy of corticosteroids in the treatment of allergic diseases.
Please see later in the article for Editors' Summary

expression

interleukin (IL)-4

expression

expression

expression

mitogen-activated protein kinase (MAPK) phosphatase-1

Import

Nuclear

translocation

nuclear

transport

nuclear

transport

glucocorticoid receptor

nuclear

translocation

nuclear

translocation

nuclear

Suppression

Import

Suppression

Phosphorylation

inhibits

translocation

competition

transport

inhibits

translocation

inhibitory effect

Phosphorylation

activated

glucocorticoid receptor

induces

expression

necessary

translocation

regulating

expression

effect

regulation

regulation


Input:
Inflammation in allergic diseases such as asthma, rhinitis, and atopic dermatitis is mediated via expression of the cytokines interleukin (IL)-4, IL-5, and IL-13 from T helper-2 (Th2) cells. IL-4 and IL-13 regulate the expression of IgE from B lymphocytes, whereas IL-5 plays a key role in eosinophilic inflammation [1]. Th2 cytokines are regulated by the zinc finger transcription factor GATA-3, which is predominantly expressed in Th2 cells [2],[3]. GATA-3 determines Th2 cell differentiation and selectively activates the promoters of IL-4, IL-5, and IL-13 through chromatin remodelling [4]-[7]. The key role of GATA-3 in allergic airway inflammation has been demonstrated in mice by the reduced release of Th2 cytokines in animals treated with dominant-negative mutants of GATA-3 and by local application of antisense oligonucleotides to GATA-3 [8],[9]. Furthermore, conditional knock-out of the Gata3 gene in mice reduces expression of Th2 cytokines in vitro and in vivo [10], and similar results have been reported in isolated murine CD4+ lymphocytes [11]. Finally, knockdown of GATA-3 expression using siRNA in human T cells results in loss of anti-CD3/CD28-mediated Th2 cytokine expression [12].
In order for GATA-3 to regulate gene expression, it must translocate from the cytoplasm into the nucleus to access its target genes. Enhanced nuclear expression of GATA-3 following T cell receptor activation was first demonstrated in murine T cells [13] and was recently confirmed in human T cells following T cell receptor and co-receptor stimulation [12]. GATA-3 contains a classical nuclear import signal [14] and is transported into the nucleus by the nuclear import protein importin-alpha (also known as karyopherin-alpha) [12]. Deletion of a region encompassing the GATA-3 nuclear localisation sequence (NLS) region in murine and human cells prevents its nuclear localisation [12],[14]. The affinity of the importin-alpha-NLS interaction is regulated by phosphorylation [15], and we have shown that p38 mitogen-activated protein kinase (MAPK) plays a critical role in phosphorylating GATA-3 to enhance its interaction with importin-alpha and subsequent transport into the nucleus [12].
Corticosteroids are highly effective in the treatment of allergic inflammation, with marked suppression of Th2 cytokines in airways of patients with asthma [16]. Corticosteroids mediate their anti-inflammatory effects through binding to glucocorticoid receptors (GRs), which then translocate to the nucleus where they interact with glucocorticoid response elements (GREs) in the promoter regions of steroid-sensitive genes. Alternatively, activated GR interacts with coactivator molecules to suppress the expression of inflammatory genes by inhibiting the action of proinflammatory transcription factors such as nuclear factor-kappaB (NF-kappaB) through the recruitment of co-repressor molecules such as histone deacetylase-2 [17],[18].
Nuclear localisation and retention of GR is mediated through the nuclear localisation sequences NL1 and NL2 [19], by nuclear retention signals [20], and by control of nuclear export via a chromosomal region maintenance 1 (CRM-1) dependent pathway [21]. NL1, which is similar to the SV40 NLS, binds to importin-alpha [22]. NL1 is activated both by glucocorticoid agonists such as dexamethasone and fluticasone propionate (FP) and by glucocorticoid antagonists such as mifepristone (RU486) [23]. NL1 can be mutated, and the resulting GR still translocates to the nucleus in response to ligands, but via interaction with importin 7, an event that requires an as-yet unknown component [23]. NL2 is poorly defined, residing in the ligand-binding domain, and much less is known about its mechanism of GR import [24]. A variety of other factors are also important for the regulation of GR activation and nuclear import including chaperones such as Hsp90 and other immunophilins [24]-[26] and FK506-binding proteins that may be linked to dynein and/or peptidylprolyl isomerase [27],[28].
However, the molecular basis for the inhibition of Th2 cytokines by corticosteroids is not well understood, because the genes encoding IL-4, IL-5, and IL-13 do not have any recognisable GRE sequence [29] and are only partly regulated by NF-kappaB in human cells [30]-[32]. Using overexpression and CAT-reporter genes, Lavender and colleagues [33] have shown that GR reduced GATA-3-mediated IL-5 and -13 promoter activity in human CD4+ T cells. The authors postulated that local recruitment of GR may alter the ability of GATA-3 either to bind to its target site, to cause transcriptional up-regulation, or to maintain an environment that is permissive for transcription.
We therefore investigated the effects of a synthetic corticosteroid, FP, on GATA-3 phosphorylation and nuclear translocation in a T lymphocyte cell line (HuT-78) and in peripheral blood mononuclear cells activated by anti-CD3 and anti-CD28 antibodies in vitro. We also studied the effects of inhaled fluticasone therapy on GATA-3 subcellular localization in peripheral blood mononuclear cells (PBMCs) from patients with asthma.

access

target

interaction

binding

glucocorticoid receptors

interact

glucocorticoid receptors

interacts

GR

recruitment

GR

histone deacetylase-2

binds

GR

interaction

GR

importin 7

bind

expressed

expression

activity

activity

translocate

nucleus

expression

nuclear

transported

nucleus

translocate

glucocorticoid receptors

nucleus

localisation

Nuclear

GR

retention

GR

export

GR

nuclear

translocates

GR

nucleus

import

GR

recruitment

GR

translocation

nuclear

localization

knockdown

expression

reduced

GR

mediated

phosphorylating

phosphorylation

activates

promoters

activates

promoters

activates

promoters

following

expression

following

expression

enhance

phosphorylating

interaction

activated

GR

mediated

localisation

control

mediated

retention

control

in response to

translocates

interaction

requires

interaction

activation

GR

mediated

activity

role

phosphorylating

control

export

dependent

control

chromosomal region maintenance 1

resulting

translocates

important

regulation

regulation

activation

regulated

regulated

regulated

alter

recruitment

bind

effects

phosphorylation

effects

translocation

effects

localization


Input:
Participants and Study Design
We studied patients with mild asthma who were not treated with inhaled corticosteroids who had been included in a previously reported double-blind, placebo-controlled, crossover study with FP [34]. Seven patients with mild asthma entered the study and were randomized to receive a single inhalation of FP (100 and 500 microg) or a matched placebo control via a spacer chamber, and the other treatment was given after a wash-out period of at least 6 d. Blood was taken for preparation of PBMCs at 1 and 2 h after drug administration. All patients gave informed consent and the study was approved by the Ethics Committee of the Royal Brompton and Harefield Hospitals NHS Trust. The clinical study was conducted before the requirement for Clinical Trial Registration.


Input:
Antibodies and Reagents
The monoclonal antibodies against human CD3, CD28, GR, and importin-alpha were purchased from BD Biosciences (Oxford, United Kingdom). Rabbit antibodies against human GATA-3 (H-48) and GR (E-20, sc-1003) were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States), polyclonal rabbit antibodies against phospho-p38 MAP kinase and phospho-ATF-2 from Cell Signaling Technology (New England Biolabs, Hertford, UK), monoclonal antibody against phosphoserine (clone 4H4) from Affiniti Research Products (Exeter, UK), and antibodies against rabbit IgG-conjugated TRITC from Dako Cytomation (Cambridge, UK). The anti-GR antibody (Clone 41) from BD Transduction Laboratories (Oxford, UK) was used for Western blot analysis. All other reagents were purchased from Sigma (Poole, UK).


Input:
Cell Culture and PBMC Isolation
A human T cell line (HuT-78) was purchased from ECACC European Collection of Cell Culture (Wiltshire, UK) were cultured as previously described [12]. PBMCs were isolated by density centrifugation over Ficoll-Hypaque (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) as previously described [34]. Cells were stimulated with anti-CD3/CD28 (1 microg/ml each) for 1 h at 37degreesC to stimulate Th2 cytokine release in the presence or absence of FP (10-12 to 10-8M). Cytospins were prepared and GATA-3 localization determined by confocal microscopy as previously described [12].

localization


Input:
Reverse Transcription PCR
Total RNA was extracted using lysis buffer (RNeasy kit; Qiagen, Crawley, UK). During RNA purification, genomic DNA was digested with RNase-free DNase (Amersham Biosciences). Next, 0.5 microg of total RNA was reversed transcribed using the avian myeloblastosis virus RT (Promega, Southampton, UK). For relative quantification, RT-PCR was carried out using cDNA probes. Primers for IL-4 were from Sigma-Genosys (Cambridge, UK). Sequences of GADPH used are as follows: forward 5'-CCACCCATGGCAAATTCCATGGC, reverse 3'-TCTAGACGGCAGGTCAGGTCCAC.


Input:
Cell Fractionation, Immunoprecipitation, and Western Blot Analysis
Nuclear and cytoplasmic fractions were prepared as previously described [35]. Whole cell lysates were prepared in NP-40 lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl) in the presence of complete protease cocktail inhibitor. Lysates were centrifuged at 4degreesC for 10 min at 12,000 rpm in an Eppendorf microcentrifuge to remove cellular debris. Samples were then immunoprecipitated with either 10 microl of antibody against GATA-3 or importin-alpha using A/G agarose slurry in the presence of protease inhibitor using the Catch and Release methodology (Upstate Biotechnology, Lake Placid, New York, USA). Western blot analysis was performed using anti-GATA-3, anti-importin-alpha, anti-GR, anti-p-p38 MAP kinase, anti-p-ATF-2, and anti-p-serine. Immunoreactive proteins were detected using an enhanced chemiluminescence ECL kit (Amersham Biosciences).


Input:
Chromatin Immunoprecipitation
Chromatin immunoprecipitation (IP) was performed as previously described [12] in HuT-78 T-cells with 2 microg of anti-GATA-3 (Santa Cruz Biotechnology), or isotypic immunoglobulin G as a non-specific control (Santa Cruz Biotechnology) overnight at 4degreesC. Promoter sequences were detected with PCR primers for the IL-5 promoter (-445 to +4): forward 5'-TTAATCTAGCCACAGTCATAG-3' and reverse: 5'-TCATGGCTCTGAAACGTTCTG-3'. PCR was performed using a Hybaid Omnigene thermal cycler (Hybaid, Ashford, UK) with cycling parameters of 72degreesC for 10 min, 35 cycles at 94degreesC for 45 s, 52degreesC for 45 s, 72degreesC for 45 s.


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GR-GATA-3 In Vitro Competition Assay for Importin-alpha (Far-Western ELISA)
Immunoprecipitated importin-alpha (anti-importin-alpha, Santa Cruz Biotechnology) from HuT-78 cells was separated by SDS-PAGE and purified from the excised gel by electroelution [36]. Similarly, GATA-3 was isolated from nonstimulated HuT-78 cells or cells stimulated for 30 min with anti-CD3/CD28 and GR was isolated from FP (10-8 M, 30 min) stimulated HuT-78 cells. These proteins were subsequently refolded in glycine solution. 100 microl of importin-alpha solution (100 ng/ml in TBS) was added to 96-well plates coated with goat anti-importin-alpha antibody. After 1 h incubation, GATA-3 (100 ng/ml in TBS) from stimulated or unstimulated cells were added to the importin-alpha-coated wells. GR (10 or 100 ng/ml in TBS) from stimulated or unstimulated cells were added to some wells. After a further 1 h incubation, the plate was washed and incubated with primary antibodies (a mixture of rabbit anti-GATA-3 and mouse anti-GR, Santa Cruz Biotechnology) for 1.5 h, and then incubated with secondary antibodies. FITC swine anti-rabbit IgG (Dako, Cambridge, UK) was used for the detection of GATA-3, and rhodamine donkey anti-mouse IgG (Novus, Littleton, Colorado, USA) was used for the detection of GR. FITC and rhodamine levels were measured with a fluorescent micro-plate reader (Bioline, London, UK).


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NF-kappaB Activation
NF-kappaB binding activity in nuclear extracts was determined using an ELISA-based kit (Trans-AM p65, Active Motif, Rixensart, Belgium). In brief, 5 microg of nuclear extracts were incubated with a plate coated with an NF-kappaB consensus oligonucleotide. Plates were washed before addition of an anti-p65 antibody. Antibody binding was detected with a secondary HRP-conjugated antibody and developed with TMB substrate. The intensity of the reaction was measured at 450 nm.


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Immunofluorescence Staining
Immunofluorescence staining was performed as previously described [34]. All staining was performed at room temperature and under humidification. Cells were collected and cytospins prepared in a cytocentrifuge (Shandon II, Shandon, Runcorn, UK). Cells were permeabilized, blocked, and then incubated with GR (1:50 E-20 Santa Cruz Biotechnology) or anti-GATA-3 (H-48; Santa Cruz Biotechnology) antibody for 1 h at room temperature. After three washes in phosphate-buffered saline (PBS), cells were incubated with tetrarhodamine isothiocyanate-conjugated goat anti-rabbit antibody (Dako). Cytospins were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), a fluorescent blue nuclear indole chromatin stain, and mounted in PBS:glycerol (50:50). The immunopositive signal was characterised using laser scanning confocal microscopy on a Leica TCS NT/SP interactive laser cytometer equipped with confocal optics (Leica Microsystems, Wetzlar, Germany). To determine the specificity of the antibodies, rabbit serum immunoglobulin (Dako) and secondary antibodies without the primary were used as controls. Positively stained nuclei and total cells were counted (500) on each slide with the observer blinded to the treatment.


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GATA-3-GFP Construct
The GATA-3 clone (BC003070) complete cDNA was obtained from Invitrogen Life Technologies as a 5'- EcoRI/3'-XhoI insert of GATA-3 in the pOTB7 vector. GATA-3 was excised from pOTB7 using XhoI digestion and pEGFP-C2 (Clontech, Saint-Germain-en-Laye, France) was digested with BamHI. DNA was recovered by phenol extraction and ethanol precipitation, and both the GATA-3 fragment and the pEGFP-C2 vector blunt-ended by incubation with Klenow (Bioline Bio-27029) for 30 min at 37degreesC. Klenow was inactivated by incubation for 10 min at 75degreesC. DNA was recovered by phenol extraction and ethanol precipitation, and both the blunt-ended GATA-3 fragment and the GFP vector were subsequently digested with EcoRI before the 5'-EcoRI/3'-blunt end GATA-3 fragment was inserted into the 5'-EcoRI/3'-blunt ended GFP vector. Positive clones were confirmed by digestion and size analysis by 1% agarose gel electrophoresis and by sequencing.


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Transfection
HuT-78 cells were transfected with either EP8 or GFP vector only DNA using solution R, programme V-001 at a ratio of 3x106 cells/4 microg DNA for 7-8 h in complete medium (10% bovine serum in RPMI1640+15 L-glutamine) according to the general Amaxa protocol for nucleofection. The medium was subsequently changed to 1% RPMI for 24 h before transfected cells were added to anti-CD3/CD28 treated wells and live cell videomicroscopy performed.


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Time-Lapse Microscopy
HuT-78 cells expressing GATA-3-GFP were maintained at 37degreesC in growth medium in a closed FCS2 perfusion chamber (Bioptechs, Butler, Pennsylvania, USA) combined with an objective heater (Bioptechs) on the stage a Zeiss Axiovert 200 microscope (Thornwood, New York, USA). Observations were made by 40x1.0 NA oil-immersion objective lens, and fluorescence and phase contrast images were gathered using a Hamamatsu ORCA-ER charged coupled device camera (Bridgewater, New Jersey, USA) driven by Openlab software (Improvision, Coventry, UK). Photographs were taken at 0, 30, 60, 120, and 240 min.

expressing


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Densitometric Analysis
Densitometry of ECL immunoblots was performed using Gelworks ID intermediate software (Ultraviolet Products, Cambridgeshire, UK). Briefly, immunoblots were scanned and gates were drawn tightly around each band. Background values from each lane were subtracted to normalize each measurement. The bands were quantified using the Gelworks software. All Western blots were exposed to film for varying lengths of time, and only films generating subsaturating levels of intensity were selected for densitometric evaluation.


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Statistical Analysis
Data from three or more independent experiments are presented as the mean+/-standard error of the mean (SEM), except where stated and were compared using GraphPad Prism 4 (GraphPad Software, http://www.graphpad.com). Results were analysed using one-way ANOVA with Newman-Keuls post test except for the data from the in vivo inhaled FP study, which was analysed by Friedman's test with subsequent Wilcoxson matched pair signed rank sum test. Data from this analysis are presented as a box-and-whiskers plot. Friedman's test was used as three matched measures were obtained using placebo, 100 microg, and 500 microg of inhaled FP, which had variable baseline levels. We did not assume a Gaussian distribution of the data due to the limited numbers of participants analysed (seven).The null hypothesis was rejected at p<0.05.


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The Effect of Corticosteroids on GATA-3 Nuclear Translocation and IL-4 mRNA
Corticosteroids are effective in inhibiting GATA-3-regulated IL-4 gene expression in vitro and in vivo [32]. We therefore investigated whether corticosteroids affect anti-CD3/CD28-stimulated nuclear import of GATA-3. Stimulation of cells with anti-CD3/CD28 resulted in a rapid cytoplasmic/nuclear GATA-3 translocation (Figure 1A), confirming our previous results [12]. We also confirmed a clear separation of nuclear and cytosolic fractions as indicated by histone H1 and MEK-1 markers (Figure 1B). The potent topical corticosteroid FP caused sustained loss of nuclear GATA-3 expression and cytoplasmic retention of GATA-3 at concentrations ranging from 10-12 to 10-8 M, which cover the therapeutic range [37]. This effect was concentration- and time-dependent, with a peak effect of 11.6-fold at 30 min at a concentration of 10-8 M (Figure 1C) and was associated with marked reductions in anti-CD3/CD28-stimulated IL-4 and IL-5 mRNA expression (Figure 1D) and a loss of GATA-3 binding to the native IL-5 promoter (Figure 1E).

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Ligand-Activated GR Competes with GATA-3 for Importin-alpha
We confirmed and extended previous data [20] to show that ligand-activated GR as well as GATA-3 uses importin-alpha for its nuclear import (Figure 2A and 2B). This interaction between GR and importin-alpha was significant at concentrations as low as 10-12 M and was maximal with 10-8 M FP. Subsequent GR nuclear translocation was rapid and sustained at significant levels for at least 14 h (Figure 2B). Using IP-Western blotting we showed that FP at 10-12-10-8 M decreased the association between GATA-3 and importin-alpha induced by anti-CD3/CD28 stimulation in a concentration-dependent manner (Figure 2C). In addition, using GFP-labelled GATA-3 and confocal microscopy we demonstrated that GATA-3 nuclear import following anti-CD3/CD28 stimulation for 30 min was attenuated by pretreatment with FP (10-8 M) (Figure 2D).

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Effect on MKP-1
Dexamethasone inhibits p38 MAPK function in a cell type-specific manner through the rapid induction of the dual kinase phosphatase MKP-1 (MAPK phosphatase-1), and this effect lasts for up to 24 h [28]. FP (10-8 M) treatment of HuT-78 cells activated by anti-CD3/CD28 in vitro significantly decreased p38 MAPK phosphorylation (Figure 3A) and activity measured by phosphorylation of the downstream target ATF-2 (Figure 3B). This effect was detected at 30 min and lasted for at least 14 h (Figure 3B). FP (10-8 M) also significantly reduced GATA-3 serine phosphorylation induced by anti-CD3/CD28 stimulation in both a time- and concentration-dependent manner (Figure 3C). This reduction in GATA-3 phosphorylation was also seen with lower concentrations of FP. We found that FP significantly induced MKP-1 mRNA in both a time- and concentration-dependent manner, reaching a plateau at 10-8 M after 10 min (Figure 3D and 3E). However, the effects of FP on GATA-3 nuclear import, importin-alpha association and IL-4 mRNA expression are seen at 10,000-fold lower concentrations (10-12 M, see Figure 2).
Using an in vitro competition assay (Figure 4A) utilizing purified activated GATA-3, importin-alpha, and activated GR, we demonstrated that activated GR significantly increased GR-importin-alpha association in the presence and absence of activated GATA-3 (Figure 4B). This effect is not mutual, since activated GATA-3 did not block GR-importin-alpha association (Figure 4C). These data also suggest that both activated GR and phospho-GATA-3 can directly associate with importin-alpha (Figure 4D) and that activated GR attenuates the phospho-GATA-3/importin-alpha interaction in a concentration-dependent manner (Figure 4E). Together, this suggests that ligand-activated GR may compete with phospho-GATA-3 for importin-alpha and thereby limit GATA-3 nuclear import.
Other possible interpretations of our results could include an effect of FP on GATA-3 nuclear export and/or degradation. Leptomycin B, which inhibits nuclear export, did not affect the ability of FP to block GATA-3 nuclear localization (Figure 5A). Additionally, FP had no effect on whole cell GATA-3 expression during the time course of these experiments (Figure 5B). Nor did addition of FP subsequent to anti-CD3/CD28 nuclear translocation affect GATA-3 nuclear residency (Figure 5C), suggesting that activated GR does not enhance GATA-3 nuclear export. Finally, the effect of FP on GATA-3 nuclear import was not nonspecific, since FP (10-8 M) had no effect on p65 nuclear translocation measured at 60 min (Figure 5D).

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The Inhibitory Effect of Corticosteroids on GATA-3 Nuclear Localization in Primary T Lymphocytes Ex Vivo and In Vivo
Treatment with FP ex vivo demonstrated a concentration-dependent decrease in the direct interaction between phospho-GATA-3 and importin-alpha in PBMCs from patients with asthma (Figure 6A and 6B), which was significantly inhibited at 10-12 M FP (p<0.001, ANOVA and Newman-Keuls test) and completely attenuated by 10-8 M FP (p<0.001, ANOVA and Newman-Keuls test).
Our previous T cell line studies indicated that 10-12 M FP suppresses IL-4 and -5 gene expression and attenuated the interaction of GATA-3 with importin-alpha (see Figures 1D and 2). This concentration is close to peak plasma levels obtained from asthmatic patients treated with inhaled FP (500 microg) [27]. Inhaled FP (500 microg) treatment of seven steroid-naive asthma patients significantly reduced GATA-3-importin-alpha interaction in vivo in a time-dependent manner. This produced a >90% decrease in GATA-3-importin-alpha association at 2 h (median [95% CI], 13,494 [6,828-17,829] versus 879 [597-1,165]; p<0.05 Friedman's analysis). However, this did not reach significance using Wilcoxon's post-test analysis (W = 6.00) probably due to low numbers of participants. Similar results were observed when GATA-3-importin-alpha association was measured (Figure 6C and 6D). The lower dose of FP (100 microg) was not effective. The attenuated interaction of GATA-3 did not result from the defective recycling of importin-alpha, as a significant decrease in the abundance of importin-alpha in the cytoplasmic pool was not detected (Figure 6E).
We further examined whether inhaled FP could affect cellular localization of GATA-3 in peripheral blood T cells. Treatment with inhaled FP (500 microg) for 2 h significantly increased GR nuclear translocation (Figure 7A) and concomitantly decreased the number of nuclear GATA-3 immunoreactive peripheral blood T cells (37%+/-4.2% versus 58.2%+/-4.95%, p = 0.016, W = 28.0, Wilcoxon's rank test) compared with placebo as measured by immunocytochemistry (Figure 7A and 7B). This was confirmed by Western blotting, which also indicated that this effect was both time- and dose-dependent (Figure 7C and 7D). Thus, inhaled FP (500 microg) induced significant loss in nuclear GATA-3 at 2 h (median [95% CI], 0.40 [0.27-0.53] versus 0.14 [0.11-0.19], p<0.05, W = 21.00, Wilcoxon's rank test) (Figure 7C) and cytoplasmic GATA-3 levels were enhanced by inhaled FP in a dose-dependent manner (median [95% CI], 0.0032 [0.0026-0.0039] versus 0.658 [0.592-0.720], p<0.05, W = -21.00, Wilcoxon's rank test) (Figure 7D). In addition, FP (500 microg) inhibited p38 MAPK phosphorylation in primary T cells in vivo at 2 h in samples from two patients (Figure 7E).
Taken together, our data suggest that inhaled FP reduces nuclear localization of GATA-3 in vivo by acutely inhibiting phospho-GATA-3-importin association. This effect may be direct, through competition for importin-alpha or associated molecules, or secondary to an effect on p38 MAPK-mediated GATA-3 phosphorylation via rapid induction of MKP-1. The combination of these two interacting effects can result in complete suppression of GATA-3 nuclear import and thus Th2 cytokine gene expression.

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Input:
Here we have demonstrated, to our knowledge for the first time, that corticosteroids may inhibit GATA-3 function and therefore the transcription of Th2 genes via two distinct but interacting molecular mechanisms. Firstly, corticosteroid-activated GR appears to compete with activated GATA-3 for nuclear import via importin-alpha, which is required for the nuclear transport of both GATA-3 and GR. Secondly, corticosteroids at higher concentrations increase the expression of MKP-1, a potent inhibitor of p38 MAPK activity and thereby prevent T cell receptor/co-receptor activation of p38 MAPK to prevent the phosphorylation of GATA-3 that is necessary for interaction with importin-alpha and subsequent nuclear import.
We have previously shown that translocation of GATA-3 from the cytoplasm to the nucleus involves the nuclear transporter protein importin-alpha, which interacts with phosphorylated GATA-3 [12]. We have also previously reported that GATA-3 knockdown using siRNA results in suppression of anti-CD3/CD28-stimulated IL-4/IL-5 mRNA induction, thus implicating an essential role for GATA-3 in the transcription of these genes [12]. We now confirm, in human T cells, that GR also uses the same nuclear import mechanism as GATA-3 [22]. We therefore propose that there is competition between ligand-activated GR and phospho-GATA-3 for nuclear import. Furthermore, we have shown that there is preferential binding of importin-alpha to activated GR over phospho-GATA-3, so that corticosteroids would preferentially reduce GATA-3 entry and thus rapidly switch off Th2 gene transcription without any need for any intermediate steps. Furthermore, there was some degree of specificity for GATA-3, as nuclear translocation of the p65 subunit of NF-kappaB was not affected by corticosteroid exposure.
We tested some alternative explanations for this effect of FP on GATA-3 nuclear exclusion and failed to show that FP either enhances GATA-3 nuclear export directly or induces GATA-3 degradation. The evidence from the in vitro competition assays does, however, suggest that purified activated GR can clearly attenuate purified phospho-GATA-3-importin-alpha association and that the converse does not occur. Furthermore, we have shown that only phospho-GATA-3 can associate with importin-alpha. This mechanism is sensitive to very low concentrations of corticosteroid and would be rapid in onset as no changes in protein synthesis are required. This acute mechanism may also contribute to the reduction in GATA-3 nuclear import and may play a major role at low corticosteroid concentrations and/or at early time points prior to MKP-1 induction.
Corticosteroids can modulate p38 MAPK activity through the induction of MKP-1, a potent endogenous inhibitor of MAPK function [38],[39]. We report here a rapid induction of MKP-1 mRNA following stimulation of cells with relatively high concentrations of FP. We hypothesize that this rapid induction of MKP-1 can reduce GATA-3 nuclear import by attenuating p38 MAPK activity and subsequent GATA-3 phosphorylation, thus preventing nuclear translocation. The location of the serine residue(s) of GATA-3 that are phosphorylated by p38 MAPK are currently unknown, but a bioinformatics search (Motif Scanner, http://scansite.mit.edu/motifscan_seq.phtml) indicates at least three potential p38 MAPK-sensitive serine residues.
As predicted from these in vitro data, impairment of GATA-3 nuclear import by FP may, at least in part, underlie the efficacy of corticosteroids in suppressing allergic inflammation. Although we did not assess the acute inhibitory effect of FP on the expression of IL-4 mRNA in vivo, a single inhalation of FP (500 microg) may have comparable effects, as it provides plasma levels within a relevant range of concentrations used to suppress IL-4 transcription in our in vitro system [37]. A lower dose of inhaled FP (100 microg) was not effective, but plasma concentrations may be below those required for GATA-3 inhibition. However, it is likely that the higher concentrations of FP in the airways after inhaled administration would be effective in inhibiting GATA-3 in airway T cells of asthma patients. The study of PBMCs from asthma patients treated with inhaled corticosteroid therapy clearly demonstrates that these molecular mechanisms are likely to also occur in patients at therapeutic doses of inhaled corticosteroids. In addition, previous studies have shown that corticosteroids can suppress IL-4 and IL-5 release from peripheral blood cells of asthma patients in vitro and in vivo [40]-[42].
In summary, our data provide evidence for a novel action of corticosteroids: suppression of allergic inflammation through a rapid inhibitory effect on GATA-3 nuclear translocation by preferential binding to the shared nuclear import protein importin-alpha and by a second mechanism involving increased synthesis of MKP-1, which inhibits p38 MAPK, thus preventing the phosphorylation of GATA-3 that is necessary for nuclear translocation of GATA-3. These two mechanisms are likely to be synergistic, accounting for the rapid and potent effect of corticosteroids on allergic inflammation. This is exemplified by the rapid inhibitory effect of topical corticosteroids on nasal Th2 cytokine release after allergen provocation in individuals with seasonal allergic rhinitis (hay fever) [43]. Prevention of phospho-GATA-3 interaction with importin-alpha may provide a new approach for the development of novel therapies for the treatment of allergic diseases.

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Fluticasone propionate down-regulates Th2 cytokine gene expression and inhibits GATA-3 nuclear import.
(A) Anti-CD3/CD28 treatment of HuT-78 cells results in translocation of GATA-3 from the cytoplasm to the nucleus within 30 min. (B) Histone H1 and MEK-1 were used to confirm distinct separation of cytoplasmic and nuclear extracts in three separate experiments. (C) Western blot analysis of FP-treated HuT-78 cells demonstrated impaired nuclear localization of GATA-3 induced by anti-CD3/CD28 co-stimulation in a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent manner. Cells were pretreated with FP for 30 min prior to stimulation. MEK1 and histone H1 were used to demonstrate equal cytoplasmic and nuclear loading respectively. Results are presented graphically below as mean+/-SEM of at least three independent experiments. *** p<0.001 compared to t = 0. (D) RT-PCR showing that FP inhibits IL-4 and IL-5 mRNA expression in CD3/CD28-costimulated cells. GAPDH was used as a loading control. Lower panels show graphical analysis of results presented as mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to anti-CD3/CD28-stimulated. (E) FP (10 nM) reduces the ability of anti-CD3/CD28-stimulated GATA-3 to associate with the native IL-5 promoter 60 min after stimulation. Data are also shown graphically as mean+/-SEM of three independent experiments. All data were analysed by ANOVA followed by Newman-Keuls post-test.

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Fluticasone propionate reduces GATA-3 association with importin-alpha and GATA-3 nuclear import.
(A) Western blot analysis demonstrates a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR interaction with importin-alpha (Imp-alpha). A positive control for GR association with importin is shown. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. *** p<0.001 compared to control, ### p<0.001. (B) Western blot analysis demonstrated a time- (at 10-8 M FP) and concentration- (at 60 min after stimulation) dependent induction of FP-activated GR nuclear translocation measured by IP. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. ***p<0.001 compared to control. (C) Western blot analysis of HuT-78 cells treated with FP and anti-CD3/CD28 co-stimulation demonstrated a concentration-dependent decrease in GATA-3-importin-alpha association at 20 min. Quantification of the densitometry data is shown below. Each bar represents mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to alphaCD3/CD28-stimulated cells. (D) GFP-tagged GATA-3 was overexpressed and cells stimulated (b, c) or not (a) for 30 min with anti-CD3/CD28. The effect of 30 min pretreatment of cells with FP (10-8 M, c) is also shown. All data were analysed by ANOVA followed by Newman-Keuls post-test.

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Fluticasone propionate-mediated inhibition of p38 MAP kinase phosphorylation and activation is associated with a marked down-regulation of GATA-3 serine phosphorylation.
(A) Western blot analysis shows that FP (10-8 M, 30 min) treatment reduced dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK in anti-CD3/CD28-co-stimulated HuT-78 cells. (B) Time course of the effect of FP (10-8 M) on phosphorylation of activated transcription factor 2 (ATF-2), a measure of p38 MAPK activity. (C) FP-induced inhibition of p38 MAPK activity is associated with the decrease of anti-CD3/CD28 co-stimulation-induced serine phosphorylation (P-Ser) of GATA-3. For (A-C), quantification of the densitometry data is also shown. Each bar represents mean+/-SEM of at least three independent experiments. ###p<0.001 compared to control, ***p<0.001 compared to alphaCD3/CD28-stimulated cells. (D) FP induced MKP-1 mRNA in a concentration-dependent manner. All results are representative of at least three independent experiments and where appropriate expressed as means+/-SEM, *p<0.05. (E) FP induces MKP-1 mRNA in a time-dependent manner. Results are representative of two independent experiments. All data except (E) were analysed by ANOVA followed by Newman-Keuls post-test.

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Fluticasone propionate competes with phospho-GATA-3 for importin-alpha.
(A) schematic representation of the in vitro binding competition assay. (B) GR isolated from FP (10-8 M) stimulated cells enhances GR-importin-alpha binding in the presence (*) and absence (==blacksquare, square, filled==) of activated GATA-3. * p<0.05 compared to no activated GR. (C) GATA-3 isolated from anti-CD3/CD28-stimulated cells does not attenuate GR-importin-alpha association. *p<0.05 compared to control. (D) Activated GR blocks the ability of purified phospho-GATA-3 isolated from anti-CD3/CD28-stimulated cells interacting with immobilised importin-alpha in an in vitro binding assay. *p<0.05 compared to GATA-3 isolated from unstimulated cells. #p<0.05 compared to stimulated GATA-3-importin binding. (E) The effect of activated (*) versus unstimulated (o) GR on attenuation of GATA-3-importin-alpha association was concentration-dependent. *p<0.05, **p<0.01 between groups. All results are expressed as mean+/-SEM of three independent experiments and analysed by ANOVA followed by Newman-Keuls post-test.

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unstimulated

GR

dependent

attenuation


Input:
Fluticasone propionate does not affect GATA-3 nuclear export.
(A) Western blot analysis showing that the nuclear export inhibitor leptomycin B (2 nM) does not affect the ability of FP (10-8 M) to prevent anti-CD3/CD28-stimulated GATA-3 nuclear localization measured at 60 min. ***p<0.001 compared to unstimulated cells, ###p<0.001 compared to anti-CD3/CD28-stimulated cells. (B) Western blot analysis showing that FP (10-8 M) does not affect whole-cell GATA-3 degradation over 17 h. (C) GFP-tagged GATA-3 is overexpressed and cells stimulated (b-j) or not (a) with anti-CD3/CD28. The effect of treating cells with FP (10-8 M, f-j) after 30 min stimulation with anti-CD3/CD28 is also shown. (D) FP (10-8 M) does not prevent anti-CD3/CD28-stimulated p65 nuclear translocation at 60 min after stimulation. **p<0.01 compared to unstimulated cells. All results are representative of at least four independent experiments and are shown as mean+/-SEM. Results were analysed by ANOVA followed by Newman-Keuls test.

export

localization

nuclear

translocation

nuclear

prevent

localization

prevent

translocation

stimulated

localization

stimulated

translocation

degradation

affect

export

affect

prevent

affect

degradation


Input:
Fluticasone propionate impairs GATA-3 interaction with importin-alpha and GATA-3 nuclear localization in vivo and ex vivo.
(A and B) Co-immunoprecipitation analysis of PBMCs from steroid-naive asthma patients treated with FP in vitro demonstrated impaired interaction between GATA-3 and importin-alpha measured at 60 min. Each bar represents the mean+/-SEM of at least three independent experiments; *** p<0.001 compared with control as determined by ANOVA/Newman-Keuls analysis. (C and D) Co-immunoprecipitation analyses of PBMCs from steroid-naive asthma patients treated with inhaled FP (500 microg via a spacer) in vivo demonstrated decreased association between GATA-3 and importin-alpha. The individual values for each treatment are presented graphically. (E) Representative Western blot showing that importin-alpha expression was unaffected by inhalation of FP. Blot is representative of gels from three participants.

interaction

interaction

association

localization

nuclear

impairs

interaction

impairs

localization

impaired

interaction

decreased

association


Input:
Inhaled fluticasone propionate impairs GATA-3 nuclear localization in PBMCs.
(A) Representative immunocytochemistry of showing the effect of inhaled FP (500 microg) on GR and GATA-3 nuclear localisation. (B) Nuclear GATA-3 immunoreactivity in PBMCs from seven steroid-naive asthma patients 2 h following inhaled FP treatment (100 or 500 microg via spacer). The median and interquartile ranges for each treatment are presented as a box-and-whiskers plot (n = 7); * p<0.05 Wilcoxon's rank test compared with placebo. (C) Immunoblotting analyses of PBMCs demonstrated a time-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression after inhalation of FP. (D) Immunoblotting analyses of PBMCs demonstrated a dose-dependent decrease in nuclear expression of GATA-3, and increased cytoplasmic GATA-3 expression 2 h after inhalation of FP. Histone H1 and MEK-1 immunoblotting confirmed equivalent total protein loading for the nuclear and cytoplasmic fractions respectively. Quantification of the densitometry data in (C) and (D) is shown as a box-and-whiskers plot of results from n = 6 participants for which data were available. *p<0.05 compared to control. (E) Western blot analyses of PBMCs demonstrated a time-dependent decrease in dual phosphorylation (threonine-180 and tyrosine-182) of p38 MAPK after inhalation of FP (500 microg). The results shown in (E) are representative of samples from two participants.

localization

nuclear

localisation

GR

nuclear

localisation

nuclear

expression

nuclear

expression

cytoplasmic

expression

nuclear

expression

cytoplasmic

impairs

localization

decrease

expression

decrease

expression

increased

expression

dependent

decrease

dependent

increased