Distinct roles of T-bet and STAT4 in suppression of IL-4-producing potential in Th1 cells by IFN-γ signaling

Introduction

In a typical paradigm of type I T helper (Th1) differentiation, interleukin (IL)12 primes Th1 differentiation by activating STAT4 and subsequently up-regulating interferon γ (IFN-γ) expression [1, 2]. Coincidentally to IFN-γ expression, IL-4, a hallmark of Th2 cells that antagonize Th1 immunity, is eventually silenced in Th1 cells [3]. Fully differentiated Th1 cells no longer can be primed to re-express IL-4. This phenotype is critical for Th1 immunity since failure to shut down IL-4-producing potential in IFN-γ-expressing cells led to impaired immunity to parasite infection [4, 5].

Mechanisms that dominate Th1 cell differentiation have been well established. T-bet is the master regulator and STAT1 promotes Th1 cells partly through upregulating T-bet and IFN-γ [6-8]. However, the mechanism that leads to silence of IL-4 expression in Th1 cells has not been fully understood. It is well known that STAT6 signaling as well as GATA3 are critical for maintaining IL-4 expression [9-11]. Interferon γ signaling has been shown to play an important role in silencing IL-4 expression: IFN-γ or its receptor deficient Th1 cells failed to shut down IL-4-producing potential [12]. We have further demonstrated that STAT1 is the downstream molecule that mediated IL-4 inhibition by IFN-γ signaling and STAT6 signaling is essential for maintaining the IL-4-producing potential [13, 14]. Recently a reserved IL-4 silencer has been identified [5]. T-bet and Runx3 formed complex and bound to the silencer to mediate suppression of IL-4 expression [15]. Although T-bet has been known to be activated by IFN-γ/STAT1 in T helper cells, it is not clarified whether IFN-γ/STAT1 function through T-bet or coordinate T-bet in silencing IL-4 expression.

In this study we continued to explore which factors were required for STAT1 to mediate suppression of IL-4 expression. We showed that IFN-γ directly antagonized IL-4 function. Furthermore, IFN-γ not only promoted IFN-γ expression but also efficiently suppressed IL-4 expression in STAT4-deficient CD4+ T cells. However, it failed to do so in T-bet deficient cells. These observations suggest that not T-bet but STAT4 is critical for IFN-γ signaling to inhibit IL-4 expression in Th1 cells.

Material and methods

Animals and cell cultures



WT control mice, Stat1–/–, Stat4–/–, and Tbx21–/– mice were purchased from Taconic (Germantown, NY). Naïve CD4+ T cells and T cell-depleted antigen-presenting cells (APC) were purified from lymph nodes as described previously [12]. For all priming conditions, anti-CD3 (2C11, 3 µg/ml), and anti-CD28 antibodies (3 µg/ml) were commonly used to stimulate purified naïve CD4+ T cells (0.2 × 106) with irradiated antigen-presenting cells (2 × 106) in 2 ml of complete RPMI medium for 2 days. For priming Th1 cells, additional IL-12 (10 ng/ml, BD-Pharmingen, San Diego, CA), anti-IL-4 antibody (11B11, 10 μg/ml) were added. For Th2 cell priming, IL-4 (5 ng/ml, BD-Pharmingen, San Diego, CA), anti-IL-12 (10 µg/ml), and anti-IFN-γ (10 µg/ml) antibodies were added. After two days of stimulation, cells were washed and cultured in the presence of IL-2 (10 ng/ml), IL-12 (2 ng/ml, for Th1 cell priming), or IL-4 (2 ng/ml, for Th2 cell priming) till day 5. Cells were further expanded with IL-2 till day 11.



Intracellular staining



Interleukin 4 or IFN-γ detection by intracellular staining was described previously [12]. In brief, cells were harvested, washed, and stimulated with PMA/ionomycin for 6 h in the presence of IL-2 and monensin. Then cells were permeabilized in saponin (1 × PBS, 0.1% Saponin) buffer and stained with PE- or FITC-labeled antibodies specific to IL-4 or IFN-γ. The production of IL-4 or IFN-γ was measured and analyzed by FACS.



RT-PCR



RNA was isolated from T helper cell cultures using Trizol (Invitrogen, Carlsbad, CA). For semi-quantitative RT-PCR, cDNA was generated using Superscript III Reverse Transcriptase (Invitrogen). Each PCR template was further 10- and 100-fold diluted and used for PCR. PCR amplification condition was as following: 94C for 2 min, 94C for 30 s followed by 60C for 2 min for 25 cycles. PCR products were separated on 1.5% agarose gel. Primer sequences can be provided upon request.

Results

Interferon γ directly antagonizes interleukin 4 signaling



Interferon γ signaling has been shown to stabilize Th1 phenotype and we further found that STAT1 was the downstream molecule that mediated IFN-γ signaling and functions. To test whether IFN-γ directly inhibits IL-4 signaling, we primed CD4+ T cells in the presence of the combination of IL-12, IL-4, or IFN-γ for 5 days. Consistently with previous findings, we found that both IL-12 and IFN-γ effectively promoted IFN-γexpression in WT cell, which was severely impaired in STAT1-null cells (Fig. 1A). More importantly, IFN-γ was able to suppress IL-4-primed IL-4 expression in WT cells but not in STAT1-deficient cells. On the contrary, IL-12 failed to do so in either WT or STAT1 knockout cells (Fig. 1B). The percentages of IL-4 or IFN-γ-producing cells in each quadrant are listed in Table 1. Suppression of IL-4 expression by IFN-γ signaling was confirmed by semi-quantitative RT-PCR (Fig. 1C). These findings suggest that IFN-γ/STAT1 can directly suppress IL-4 signaling.



Abrogation of interferon γ signaling leads to failure to shut down interleukin 4-producing potential



Suppression of IL-4 expression in Th1 cells eventually leads to permanent silence of Il4 gene. Th1 cell differentiation is accompanied by the acquired ability to secrete a high level of IFN-γ that could contribute to the IL-4 expression silencing. To distinguish the effect of IL-12 and IFN-γ signaling in this process, we set to culture two-round T helper cell culture and measure the IL-4-producing capacity. We abrogated IFN-γ signaling in two ways. One was to prime cells in the presence of IL-12 and antibody neutralizing IFN-γ in cell cultures and compare to cells primed in the presence of IFN-γ; the other one was to use the STAT1-null mice. Then, cells from the first round priming were reactivated in the presence of IL-12 or IL-4. As controls, STAT1-deficient cells showed impaired IFN-γ expression and maintained the IL-4-producing potential in either condition: they could be induced to re-express IL-4 under Th2-inducing conditions (Fig. 2). The percentages of IL-4 or IFN-γ-producing cells in each quadrant are listed in Table 2. In WT cells, cells primed in the presence of IFN-γ expressed a high level of IFN-γ and completely repressed IL-4 expression. On the other hand, WT cells that were primed in the presence of IL-12 as well as IFN-γ neutralizing antibody retained the ability to produce IL-4: they could be induced to express IL-4 under Th2-inducing condition. These observations confirmed an essential role of IFN-γ/STAT1 in silencing IL-4 expression in Th1 cells.



STAT4 is dispensable for interferon γ/STAT1 signaling-mediated suppression of interleukin 4 expression in Th1 cells



To further distinguish the effect of IL-12/STAT4 and IFN-γ/STAT1 signaling pathways on silencing IL-4 expression, we used STAT4-null CD4+ T cells to set up two-round T helper cell culture. As shown in Fig. 3, WT cells primed in the presence of IL-12 plus anti-IFN-γ antibody retained the ability to produce IL-4 as expected and STAT4-deficiency severely impaired IFN-γ production. STAT4-null also impaired IFN-γ production in cells primed in the presence of IFN-γ. However, STAT4-deficient cells primed in the presence of IFN-γ lost the potential to produce IL-4 under Th2-inducing condition. These results strongly suggest that STAT4 may not be required for IFN-γ-mediated repression of IL-4. The percentages of IL-4 or IFN-γ-producing cells in each quadrant are listed in Table 3.



T-bet mediates suppression of interleukin 4 by interferon γ signaling



T-bet has been shown to bind the conserved IL-4 gene silencer and coordinate Runx3 to repress IL-4 expression [15]. To test whether IFN-γ function through T-bet or coordinate T-bet, we used T-bet-deficient mice to set two-round T helper cell culture. As shown in Fig. 4, we found that T-bet deficiency not only abolished IL-12 ability to induce IFN-γ expression but also abrogated IFN-γ capacity to do so. Most importantly, IFN-γ signaling failed to repress IL-4 expression in T-bet deficient cells. These findings suggest that T-bet is indispensible for IFN-γ-mediated IL-4 inhibition in Th1 cells. The percentages of IL-4 or IFN-γ-producing cells in each quadrant are listed in Table 4.

Discussion

Acquiring IFN-γ expression and silencing IL-4 expression are two critical processes in Th1 cell differentiation. A defect in either process leads to immunity incompetency. It is not clear how these two processes correlate or coordinate with each other. Present evidence suggests that these two processes may be tightly associated with each other yet can be separated [16]. Our study provides several lines of evidence to support this idea.

First, we have revealed STAT4- and STAT1-dependent mechanisms that are coordinated in IFN-γ expression. It is well established that IL-12 primes Th1 cell differentiation by activating STAT4 and subsequently up-regulating IFN-γ expression while IFN-γ promotes IL-12-driven Th1 cell differentiation in vitro, possibly by making cells respond to IL-12 through inducing the expression of the IL-12R2 chain. Our study here showed that two mechanisms might collectively contribute to IFN-γ expression in Th1 cells. One is IL-12 signaling through STAT4 and the other is IFN-γ signaling through STAT1. Deficiency in either STAT1 or STAT4 almost completely abrogates its correspondent cytokine to induce IFN-γ expression. Yet these two mechanisms may coordinate since STAT4-deficiency only partially impaired IFN-γ-induced IFN-γ expression and vice versa. Furthermore, T-bet is the emerging point since T-bet knockout severely impaired both IL-12- and IFN-γ-induced IFN-γ expression.

Second, priming and maintaining the IFN-γ expression may be controlled by two different mechanisms. It is well known that STAT4 activated by IL-12 signaling can bind to IFN-γ promoter and directly activate IFN-γ expression. And IL-12/STAT4 is not known to regulate T-bet expression. However, T-bet deficiency completely abolished IL-12-induced IFN-γ expression. These findings suggest that IFN-γ/STAT1/T-bet may be required in maintaining IFN-γ expression. It is reasonable to speculate that the critical role of IL-12/STAT4 is to prime the initial IFN-γ which can be maintained through IFN-γ/STAT1/T-bet positive regulatory loop.

Most importantly, we found that silencing IL-4 expression is controlled by IFN-γ/STAT1/T-bet regulatory loop through a STAT4-independent mechanism. We previously showed that T-bet rescued STAT1-deficiency and stabilized Th1 phenotype [14]. In this study we further used T-bet-null mice to confirm an essential role of T-bet in silencing IL-4 expression by IFN-γ signaling. Although STAT4-deficiency impaired IFN-γ-induced IFN-γ expression, it did not affect the ability of IFN-γ to inhibit IL-4 expression. Thus, priming IFN-γ expression and silencing IL-4 expression are two separate processes.

In general, our study has revealed distinct roles of different signaling pathways in Th1 differentiation. Our findings support IFN-γ/STAT1/T-bet regulatory loop as a critical signaling pathway in silencing IL-4 expression in Th1 cells.



This work was supported by the National Natural Science Foundation of China (Grant No. 30700412 and 31170328).

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