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Early Target Genes of IL-12 and STAT4 Signaling in Th Cells¹

Riikka J. Lund^2,*,, Zhi Chen^*,, Joonas Scheinin^* and Riitta Lahesmaa^*

^* Turku Centre for Biotechnology, Turku University and Åbo Akademi, Turku Graduate School of Biomedical Sciences, Turku University, and Drug Discovery Graduate School, Turku, Finland

	Abstract

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IL-12 signaling through STAT4 is essential for induction of optimal levels of IFN- production and commitment of Th1 cells. The molecular mechanism that controls how IL-12 and STAT4 signaling induces Th1 differentiation is poorly described. To identify the early target genes of IL-12 and STAT4 signaling, oligonucleotide arrays were used to compare the gene expression profiles of wild-type and STAT4-knockout murine Th cells during the early Th1 differentiation. According to the results, 20 genes were regulated in an IL-12- and STAT4-dependent manner. Importantly, Ifn was clearly the first gene induced by IL-12 in a STAT4-dependent manner. Most of the other defects in gene expression in STAT4-knockout cells were seen after 48 h of Th1 polarization. In addition to IL-12 signaling mediated by STAT4, STAT4-independent induction of a number of genes was observed immediately in response to Th1 induction. This induction was at least in part driven by IFN- independently of STAT4. Importantly, addition of exogenous IFN- into Th1 cell cultures of STAT4-knockout cells restored the defect in IFN- production further demonstrating the critical role of IFN- in early Th1 differentiation.

	Introduction

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Interleukin-12 is a cytokine involved in regulation of cell-mediated immune responses and induction of Th1 differentiation. The effects of IL-12 are mediated through IL-12R consisting of two subunits IL-12R1 and IL-12R2 (1). IL-12R2, which transmits the signals inside the cell, is not expressed on naive Th cells, but is induced in response to Ag stimulation and is selectively down-regulated during Th2 differentiation (2, 3). Triggering of IL-12R leads to induction of tyrosine phosphorylation and DNA binding of Janus kinase 2, tyrosine kinase 2, and STAT4 (4, 5, 6). Also STAT1, STAT3, and STAT5 are tyrosine phosphorylated in response to IL-12 (6, 7, 8, 9, 10). However, only STAT4 has been shown to be necessary for the long-term commitment of Th1 cells, as mice deficient for STAT4 show impaired Th1 and enhanced Th2 differentiation (10, 11). In addition to IL-12, the only cytokines known to induce STAT4 phosphorylation are IFN- and IL-23 (12, 13, 14, 15, 16).

Although STAT4 has been shown to be required for the long-term Th1 differentiation and IFN- production, cells deficient for both STAT6 and STAT4 can differentiate to functional IFN--producing Th1 cells (17). Transcription factor T-box expressed in T cells (T-bet)³ may be involved in this STAT4-independent Th1 differentiation. T-bet has been shown to be required for Th1 differentiation and its expression is sufficient to induce IFN- production in Th cells (18, 19). Initial expression of T-bet is induced by IFN- and STAT1 signaling during the activation of Th cells independently of STAT4 (20, 21, 22). Activation of T-bet during Th1 differentiation leads to remodeling of the Ifn locus, induction of IFN- production, and IL-12R2 expression essential for STAT4-mediated IL-12 signaling (18, 22, 23).

Although both STAT1 and STAT4 signaling are contributing to the early Th1 polarization, only the requirement of STAT4 for long-term Th1 development has been clearly demonstrated (10, 11). However, the exact role of STAT4 in the early induction of Th1 cell differentiation is still unclear. Recent studies indicate that instead of being the primary factor inducing Th1 differentiation, STAT4 would rather be involved in enhancing initial IFN- production to optimal levels (21, 22, 23). Whether the effects of STAT4 are restricted to regulation of IFN- or whether it also regulates other factors involved in inducing Th1 differentiation is unknown. Previously described target genes of STAT4 include macrophage inflammatory protein-1 (Mip-1, Scya3), macrophage inflammatory protein-1 (Mip-1, Ccl4, Scya4, Act-2), IL-1RA (Il1rn), IFN regulatory factor 1 (IRF1), Ets-related transcription factor (ERM), CCR5, and IL-18R (24, 25, 26, 27). In a recent study, the target genes of IL-12 and STAT4 in already polarized Th1 cells were described (28). The aim of this study was to elucidate the mechanism of early Th1 differentiation by identifying the immediate and early upstream genes regulated in response to IL-12 and STAT4 signaling.

	Materials and Methods

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Mice

STAT4-knockout mice and wild-type controls with a BALB/cJ background were purchased from The Jackson Laboratory (Bar Harbor, ME). The mice used in the studies were sacrificed at the age of 6–8 wk.

Cell cultures

The murine mononuclear cells were isolated from spleen single cell suspension using Lympholyte-M gradient (Cedarlane Laboratories, Hornby, Canada). The CD4⁺ cells were further purified using magnetic MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany). For induction of Th1 polarization pooled CD4⁺ cells were cultured in Iscove’s DMEM containing 10% FCS, nonessential amino acids, and 2-ME (Life Technologies, Paisley, Scotland) in the presence of plate-bound anti-mouse CD3 (315 ng/well), soluble anti-mouse CD28 (500 ng/ml; BD PharMingen, San Diego, CA), recombinant mouse IL-12 (10 ng/ml; R&D Systems, Minneapolis, MN) and rat anti-mouse IL-4 (10 µg/ml; BD PharMingen). Part of the cells were activated with anti-CD3 and -CD28 and cultured in "neutral" conditions in the presence of rat anti-mouse IL-4 (10 µg/ml; BD PharMingen), rat anti-mouse IL-12 (10 µg/ml; BD PharMingen), and hamster anti-mouse IFN- (10 µg/ml; BD PharMingen). The cultures were conducted in parallel for both the cells deficient for STAT4 and the wild-type controls. Samples were collected at the time points of 0, 2, 6, and 48 h. For the real-time RT-PCR or cytokine secretion assay, the cells were cultured in the presence of plate-bound anti-mouse CD3 (315 ng/well), soluble anti-mouse CD28 (500 ng/ml), and indicated combinations of cytokines IL-4, IL-12, IFN- (500 U; BD PharMingen) and neutralizing Abs for IL-12, IFN-, and IL-4.

Oligonucleotide array experiments

The total RNAs were isolated using the TRIzol method (Invitrogen, Carlsbad, CA) and were further purified with the Qiagen RNAeasy minikit (Qiagen, Valencia, CA). For the Affymetrix sample preparation, 5 µg of total RNA was used as starting material. The sample preparation was performed according to the instructions and recommendations provided by the manufacturer (Affymetrix, Santa Clara, CA). The samples were hybridized to MG-U74Av2 arrays containing 12,488 probes for different genes. The data was analyzed with Affymetrix Microarray Suite version 5 (MAS5) software and filtered according to recommendations of the manufacturer. Briefly, at the expression level, the statistical algorithm used by MAS5 defined each gene-specific probe set to be present, absent, or marginal. At the comparison level, each probe set was classified as not changed, increased, marginally increased, decreased, or marginally decreased. The genes that were absent in both treatments compared or the genes that were not changed between the comparisons were excluded from the results. Furthermore, only the genes that presented a consistent change in two or three biological repeats and showed fold changes of 2 or –2 (signal log ratio 1 or –1) in at least one of the time points studied were considered as differentially expressed.

Real-time quantitative RT-PCR

Primers and probes were designed for the genes studied using Primer Express software (Applied Biosystems, Foster City, CA). Gene expression levels were measured for the selected panel of genes (Table I) at each time point using TaqMan real-time quantitative PCR (RT-PCR; ABI Prism 7700; Applied Biosystems) as previously described (29). A TaqMan PCR Core Reagent kit (Applied Biosystems) was used for the preparation of the RT-PCR mixtures. The primers and probes (MedProbe, Oslo, Norway) were used in the final concentrations of 300 and 200 nM, respectively. The steps in the quantitative RT-PCR were 50°C for 2 min, 95°C for 10 min, 95°C for 15 min, and 60°C for 1 min, and altogether 40 cycles were run. All measurements were performed in duplicate in two separate runs using samples derived from at least two or three biological repeats.

For the cytokine secretion assay the cells cultured for 7 days were restimulated at a density of 1 x 10⁶ in 0.5 ml in IMDM + 10% FCS containing PMA (50 ng/ml; Calbiochem, La Jolla, CA) and ionomycin (500 ng/ml; Calbiochem). Part of the cells were maintained as unstimulated controls cultured otherwise identically to stimulated cells. After stimulation, the cells were washed (0.5% BSA + 0.01% azide in PBS) and incubated with anti-CD4-PE (Caltag Laboratories, Burlingame, CA) for 15 min. The cells were washed and fixed with 4% paraformaldehyde for 15 min after which they were permeabilized (0.5% saponin + 0.5% BSA + 0.01% azide in PBS, pH 7) for 10 min and washed again. After the permeabilization, intracellular cytokine staining was performed with anti-IFN--FITC (BD Biosciences, San Jose, CA) for 10–20 min. The cytokine profiles of the cells were studied with FACScan and CellQuest software (BD Biosciences).

	Results

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Genes regulated immediately in response to Th1-polarizing stimuli

Affymetrix murine U74Av2 arrays, representing probes for 10,000 different genes, were used to study the target genes of IL-12 and STAT4 signaling during the early polarization of Th1 lymphocytes. To identify the genes regulated by IL-12, cells cultured in Th1 conditions (anti-CD3 + anti-CD28 + IL-12 + anti-IL-4) were compared with the CD3 + CD28-activated cells cultured in the "neutral" conditions (anti-CD3 + anti-CD28 + anti-IFN- + anti-IL-12 + anti-IL-4).

Altogether 16 genes were immediately up-regulated in the cells polarized to differentiate to Th1 cells for 2 or 6 h compared with the CD3/CD28-activated cells (Table II). These early response genes included Iigp-pending, Icsbp, Irf1, Gbp1, Gbp2, AA816121, Tgtp, Ifi1, Igtp, Gtpi-pending, Ifi47, Irf4, Ly6a, Cxcl9, Il12rb1, and Ifn. The strongest induction by IL-12 was seen in the up-regulation of Ifn.

Genes regulated in response to Th1 polarizing stimuli after 48 h

In addition to the immediately regulated genes, there were 57 genes regulated in response to IL-12 after 48 h of polarization. The genes affected by Th1 induction after 48 h of polarization included 29 up-regulated and 28 down-regulated genes belonging to diverse functional categories (Table III). Ifn and IFN-regulated genes Iigp-pending and Icsbp were the only genes for which the over 2-fold induction by IL-12 was maintained throughout all time points studied (2, 6, and 48 h).

Importantly, the only gene that was clearly regulated by IL-12 in a STAT4-dependent manner at all time points (2, 6, and 48 h) and in all samples studied was Ifn. In the wild-type cells, Ifn was strongly induced by IL-12, but in STAT4-knockout cells the expression levels in Th1 conditions were at the same level as in CD3 + CD28-activated wild-type or STAT4-knockout cells cultured in "neutral" conditions.

A subset of genes becomes up-regulated in response to IL-12 in the absence of STAT4

In addition to the genes that were induced by IL-12 and STAT4 signaling, direct comparison of wild-type and STAT4-knockout cells cultured in Th1-polarizing conditions revealed another group of genes that seemed to become expressed at enhanced levels in response to IL-12 in the absence of STAT4 (Table IV, Genes that become up-regulated in response to IL-12 in the absence of STAT4). The genes Iigp-pending and Tgtp that were immediately induced in response to IL-12 were among these genes. Also, however, new similar genes for which the induction by IL-12 was seen later (after 48 h) were identified. These genes included Ifit1, Trim30, Isg15, Ifit2, Ms4a4c, Mx1, AA959954, Isg20, and Ifi202a.

Validation of the results with quantitative real-time RT-PCR

To validate the results, expression of a subset of genes including Ifn, Irf1, Irf4, Icsbp, Gas5, and Iigp-pending was studied with real-time RT-PCR (Fig. 1). Concordant with the Affymetrix results, Ifn was expressed at a lower level in the absence of STAT4 compared with the wild type. In the wild-type cells, the induction compared with the STAT4-knockout cells was 3.6-times higher already after 2 h and 6-times higher after 6 h of Th1 polarization. In STAT4-knockout cells induced to differentiate to the Th1 direction during the first 6 h, the expression level of Ifn remained at the same level as in wild-type cells cultured in neutral or Th2 conditions. Also, the reduced expression of Gas5 in STAT4-knockout cells, compared with the wild-type cells cultured in Th1 conditions, was confirmed with real-time RT-PCR. A fold difference as high as 12.7 (p < 0.05) in the expression of Gas5 was seen already in Thp cells. Based on Affymetrix results, gene Iigp-pending showed increased (2.7-fold, p < 0.05) expression in the absence of STAT4 after 48 h of Th1 polarization compared with the wild type. In TaqMan analysis, this difference was seen at low levels in only two of three biological repeats (fold changes 1.8, 1.7, and –2.2). Furthermore, at the 2-h time point Iigp-pending was preferentially expressed in wild-type cells (fold change 1.8, p < 0.05). Thus, the increased expression of Iigp-pending in the absence of STAT4 could not be confirmed.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Validation of the results with real-time RT-PCR. CD4+ cells were isolated from spleen of wild-type and STAT4-knockout BALB/cJ mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and cultured for 0, 2, 6, 24, 48 h, or 7 days in the presence of different cytokine combinations or neutralizing Abs as indicated in the figure. The gene expression levels were measured for the selected genes including Ifn, Gas5, Iigp-pending, Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with the levels in Thp cells and are represented as fold changes in the figure. *, Statistically significant differences in gene expression levels between wild-type and STAT4-knockout cells cultured in Th1 conditions (paired t test: p < 0.05).

IFN- induces expression of Irf1, Irf4, and Icsbp during early Th1 polarization independently of IL-12

Irf1, Irf4, and Icsbp were induced in response to IL-12 during early Th1 induction, but regulation by STAT4 was not clear. Therefore, we decided to study further whether early induction could be driven by IFN- instead of IL-12. The expression of Irf1, Irf4, and Icsbp was studied with real-time RT-PCR in the presence and absence of IFN- as indicated in Fig. 2. Interestingly, the RT-PCR analysis revealed that the immediate induction of Irf1, Irf4, and Icsbp was indeed driven by IFN- and IL-12 alone was unable to induce expression of these genes.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. Immediate induction of Irf1, Irf4, and Icsbp is driven by IFN-. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and were cultured for 0, 2, or 6 h in the presence of different cytokine combinations or neutralizing Abs as indicated in the figure. The gene expression levels were measured for the genes Irf1, Irf4, and Icsbp with real-time RT-PCR. The gene expression levels were compared with the levels in Thp cells and are represented as fold changes in the figure. *, Statistically significant differences in the gene expression levels in cells cultured under Th1 conditions in the absence or presence of IFN- (paired t test: p < 0.05).

Addition of IFN- to STAT4-knockout Th1 cultures restores the defect in IFN- production and enhances IFN- production in the absence of IL-12

The most interesting observation of this study was that Ifn was the only gene for which the regulation by IL-12 and STAT4 signaling was seen at all the time points, indicating that it was the earliest target of IL-12 and STAT4. Therefore, our hypothesis also was that the long-term defect in Th1 commitment in STAT4-knockout mice was due to the lack of optimal IFN- levels during early polarization. To test this hypothesis, we cultured wild-type and STAT4-knockout cells in neutral or in Th1 conditions in the presence and absence of exogenous IFN- to see whether IFN- was able to restore the defect in Th1 polarization in STAT4-knockout cells.

In the cells activated with anti-CD3 and anti-CD28 and cultured for one week in neutral conditions (Th0: anti-CD3 + anti-CD28 + anti-IL-12 + anti-IFN- + anti-IL-4), restimulation with PMA and ionomycin induced similar levels of IFN- production both in wild-type and STAT4-knockout cells (Fig. 3a). As expected, in the cells polarized with IL-12 to the Th1 direction (anti-CD3 + anti-CD28 + IL-12 + anti-IL-4), IFN- production in response to PMA and ionomycin was highly increased in wild-type cells, whereas in STAT4-deficient cells the production remained at the basal level (Fig. 3b). Importantly, addition of exogenous IFN- to the cells cultured in Th1 conditions (anti-CD3 + anti-CD28 + IL-12 + IFN- + anti-IL-4) was able to restore the defect in induction of IFN- production in STAT4-knockout cells, whereas in wild-type cells addition of IFN- had no effect (Fig. 3c). Furthermore, in the absence of STAT4 and IL-12, IFN- alone (anti-CD3 + anti-CD28 + anti-IL-12 + anti-IL-4 + IFN-) was able to enhance IFN- production to the similar levels measured in wild-type Th1 cells. Interestingly, in wild-type cells, addition of IFN- alone did not have any effect and the production of IFN- was comparable to that measured in the cells cultured in neutral conditions (Fig. 3d).

View larger version (21K): [in this window] [in a new window]   FIGURE 3. IFN- is able to partly restore its own production in STAT4-knockout mice. CD4+ cells were isolated from spleen of wild-type BALB/cJ mice or STAT4-knockout mice. The cells were activated with plate-bound anti-CD3 and soluble anti-CD28 and cultured for 7 days in the presence of indicated cytokine combinations or neutralizing Abs (a) anti-CD3 + anti-CD28 + anti-IL-4 + anti-IL-12 + anti-IFN-; b, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4; c, anti-CD3 + anti-CD28 + IL-12 + anti-IL-4 + IFN-; d, anti-CD3 + anti-CD28 + IFN- + anti-IL-12 + anti-IL-4). For the intracellular cytokine detection with anti-IFN--FITC, the cells were restimulated with PMA and ionomycin. Isotype controls were used as controls to calculate the number of IFN--producing cells.

	Discussion

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Among the genes that were immediately up-regulated in cells cultured in Th1-inducing conditions, there were three transcription factors, Irf1, Irf4, and Icsbp. These genes were shown to be induced in fact by IFN- and not by IL-12. All these Irf1, Irf4, and Icsbp transcription factors play an essential role in Th1 differentiation. Mice deficient for Irf1 have impaired Th1 differentiation and show a defect in responsiveness to IL-12 (32, 33). Irf4 is required for Th2 differentiation to induce Gata-3 expression in Th2 cells and inhibit Th1 development (34, 35). Icsbp is required for production of IL-12, generation of IFN-producing cells and an optimal amount of CD8⁺ dendritic cells, which preferentially promote Th1 differentiation (36, 37). Thus, early induction of Th1 differentiation involves IFN--mediated up-regulation of transcription factors Irf1, Irf4, and Icsbp, which have essential roles in regulation of Th1 differentiation (21, 22, 23, 32, 33, 34, 35, 36, 37).

The expression profiles between the first hours and 2 days of Th1 polarization were different from each other and the only genes that were up-regulated over 2-fold in Th1 conditions at all time points (2, 6, and 48 h) were Ifn, Iigp-pending, and Icsbp. The number of IL-12-regulated genes was increased after 2 days of polarization probably due to activation of secondary response factors or as a consequence of enhancement of IL-12 signaling in response to IL-12R induction. This is concordant with the previous studies that have shown that the subset of receptor IL-12R2, which transmits the signals inside the cell, is not expressed on naive Th cells, but is induced in response to Ag stimulation (2, 3).

The defects in gene expression in the STAT4-knockout mice were mainly seen after 48 h. Of the genes that were regulated in response to IL-12 after 48 h (Table III), Acadl, Gas5, Furin, Rrad, Gja1, Ctla2b, Serpinb5, and Plac8 were regulated in a STAT4-dependent manner (summarized in Table IV, IL-12/STAT4-induced genes). Acadl and Gas5 showed impaired expression already at the Thp stage and thus could also have implications in the early Th1 differentiation and defective gene expression in the STAT4-knockout mice. The role of Acadl and Gas5 in immune response is unknown. Acadl is implicated in fatty acid metabolism (38, 39). Gas5 is preferentially expressed in growth-arrested cells, but is not believed to encode for a protein (38, 39). Also, the role of these other six genes induced by IL-12 and STAT4 in Th1 differentiation is unknown.

In addition to IL-12- and STAT4-induced genes, expression of a subset of 11 genes was enhanced in response to IL-12 in the absence of STAT4 (Table IV, Genes that become up-regulated in response to IL-12 in the absence of STAT4). Nearly all the genes in this group were known IFN-regulated genes. Interestingly, expression of most of these genes including Iigp-pending, Tgtp, Trim30, Ifit1, Isg15, Isg20, and Ifi202a is repressed by IL-4 during early Th2 polarization. Furthermore, genes Isg15, Isg20, Tgtp, Trim30, and Ifi202a are also repressed by STAT6 (40). Thus, the down-regulation of these genes must be important both for Th1 and Th2 differentiation.

As the major defect in STAT4-knockout cells was in reduced expression levels of Ifn, we studied whether the defect in Th1 polarization was a consequence of the reduced levels of IFN- during the immediate response. The flow cytometric analysis demonstrated that addition of exogenous IFN- to the Th1 cultures of STAT4-knockout cells restored the defect in IFN- production, but in wild-type cells addition of IFN- had no effect. This indicates that in the absence of STAT4, IFN- is able to compensate for the IL-12- and STAT4-mediated induction of its own production, replacing the role that is normally conducted by IL-12. This was further supported by the observation according to which IFN- alone was able to induce normal levels of IFN- production in the Th1 conditions in STAT4-knockout cells, but not in wild-type cells. Thus, it seems that the ability of IFN- to induce its own production in the absence of STAT4 is normally inhibited by STAT4. Interestingly, according to the Affymetrix results in STAT4 knockout cells, a subset of IFN-regulated genes were abnormally induced in response to Th1 induction. It is possible that in the absence of an inhibitory effect of STAT4, the induction of these genes is driven by basal levels of IFN- produced by the knockout cells. The enhancement of IFN- production by itself in STAT4-knockout cells is also consistent with the previous observation demonstrating that IFN- was able to induce expression of T-bet in STAT4-knockout cells (21, 22).

Although the requirement of STAT4 in the development of effector Th1 cells has been demonstrated, the role of STAT4 during early Th1 polarization has been unclear (10, 11, 21, 22). In the current study, we have for the first time examined the role of STAT4 in regulation of gene expression during the first steps of Th1 differentiation in the global scale. The results demonstrate that IFN- indeed is the first gene induced by IL-12 in a STAT4-dependent fashion and thus is likely to be the primary force inducing Th1 polarization in response to IL-12. Importantly, we demonstrate that the defect in Th1 polarization in STAT4-knockout mice can be restored by adding exogenous IFN- to the developing Th1 cells highlighting the importance of the cytokine in Th1 polarization.

	Acknowledgments

 
We thank Marju Niskala, Outi Melin, and Miina Miller for technical assistance and Elizabeth Carpelan for language revision.

	Footnotes

 
¹ This work was supported by the Academy of Finland, Turku Graduate School of Biomedical Sciences, Drug Discovery Graduate School, Ida Montin Foundation, the Finnish Society of Allergology and Immunology, National Technology Agency of Finland.

² Address correspondence and reprint requests to Dr. Riikka J. Lund, Turku Centre for Biotechnology, University of Turku and Åbo Akademi, P.O. Box 123, FIN-20520, Turku, Finland. E-mail address: riikka.lund{at}btk.utu.fi

³ Abbreviations used in this paper: T-bet, T-box expressed in T cells; IRF, IFN regulatory factor; Thp, Th precursor.

Received for publication November 11, 2003. Accepted for publication March 23, 2004.

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