Experiments were performed with age-matched FVB WT and CREMα tg FVB mice (7, 32) All mice were bred in our animal facility and kept under standardized conditions. Animals were housed in the same rooms under pathogen-free and helicobacter-free conditions. The study was approved by the regional government authorities and animal procedures were performed according to German legislation for animal protection. Permission for the projects has been granted by the Regierungspräsident/LANUV Nordrhein-Westfalen.

T cells were obtained from spleens of WT and CREMα tg mice by negative isolation using magnetic cell separation via CD4⁺ MACS Kits (Miltenyi, Germany) according to manufacturer’s instructions. Cells were cultured in RPMI medium supplemented with 1% penicillin/streptomycin and 10% inactivated fetal calf serum (Gibco Life Technologies, Germany) in individual wells of 96-well round-bottom microtiter plates. T cells were stimulated with anti-CD3 and anti-CD28 antibodies (both at 3 μg/ml; BD Bioscience, Germany) or with PMA (20 nM) and ionomycin (2 μM) (both Sigma-Aldrich, USA) as indicated.

Freshly isolated T lymphocytes (5 × 10⁶ cells/ml) were washed in phosphate-buffered saline (PBS), resuspended in RPMI containing 1% bovine serum albumin (BSA), and incubated for 40 min at 37°C with 1.3 μM Fluo-3-AM and 2.75 μM Fura-Red-AM (both from Invitrogen) in the presence of 0.02% Pluronic acid F-127 (Invitrogen). After incubation, cells were washed gently and resuspended in RPMI containing 0.1% BSA and incubated for 10 min at 37°C. Cells were then stimulated with anti-CD3 and anti-CD28, alternatively with PMA and ionomycin. Immediately after addition of the stimulus, Fluo-3/Fura-Red ratio as a measure of the released cytosolic Ca²⁺ concentration was monitored by FACS analysis. The quantification of the measurements was analyzed using FlowJo software (Tree Star, USA). For T lymphocytes stimulated with anti-CD3 and anti-CD28, [Ca²⁺]i was calculated from the equation: [Ca²⁺]i = Kd × Sf380/b380 (R–Rmin)/(Rmax–R), where Kd is 224 nm in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340/380 nm) of the intracellular Fura-2; Rmax and Rmin are the maximal and minimal fluorescence ratios obtained by addition of Digitoxin and EGTA (final concentration 3 mmol/l), respectively.

The 3–4 × 10⁶ splenic CD3⁺ T-cells from CREMα tg and WT mice were stimulated for 45 min for CREM binding or 4 h for Histon 4 acetylation using Phorbol-12-myristat-13-acetat (30 nM) and ionomycin (1.5 µM) (both Sigma-Aldrich, USA) or left unstimulated. T-cells were cross-linked with 1% formaldehyde (Merck, Germany) for 5 min, washed, lysed, and sonicated. Supernatants were diluted in RIPA buffer. A proportion (10%) of the diluted supernatants was kept as input (PCR amplification of the total sample). For each immunoprecipitation reaction, an equal DNA amount was used. Samples were immunoprecipitated with 0.6–3 µg antibody (anti-CREM 2 µg; anti-NFAT 3 µg, both Abcam, UK; anti-Histon H4 Acetyl-K8 0.6 µg, Epitomics, USA) or with its isotype control (mouse monoclonal IgG1 or rabbit polyclonal IgG, both Abcam, UK) at 4°C overnight. Protein G Dynabeads (Novex by Life technologies, Germany) were incubated with the samples for 4 h at 4°C. Protein–DNA beads complexes were washed stringently with wash buffer (X-ChIP protocol from Abcam). Protein–DNA complexes were eluted in 1% SDS, 100 mM NaHCO₃ for 15 min at 30°C, and reverse-cross-linked at 65°C overnight. For protein digestion, Proteinase K (20 mg/ml) was added to the samples and incubated for 2 h at 54°C. DNA was recovered using the Wizard kit (Promega, Germany) and subjected to PCR analysis on an ABI prism 7300 real-time PCR system. Primer sequences used for real-time PCR for the CREM-binding site or H4-acetylation of the IL-21 promoter were 5′-GAAAACTGGAATTCACCCATGTCTCTCTT-3′ and 5′-AGAAGAGGCCAAGCCCTCCCATTG-3′. The immunoprecipitated DNA was calculated as relative to the respective input DNA. For H4-acetylation, the percent of input of the stimulated immunoprecipitated DNA was calculated as n-fold to the respective unstimulated DNA. Primer sequences used for the putative CREM-binding site of the CD3ζ chain promoter were 5′-CTGAATTGAGGCCAAGTTCAGA-3′ and 5′-AAGCATATATCT AAGTTGCTGTGTGG-3′. The immunoprecipitated CREMα tg DNA was calculated as n-fold to the respective WT DNA.

Reporter ChIP technique enables the study of the in vivo binding of transcription factors to a specific binding site on a promoter of a reporter construct (33). To analyze CREM binding to the mutated or not-mutated Il-21-luc promoter, 5 Mio EL-4 culture cells were spread in six-well plates in 3 ml RPMI + 5% FCS (without P/S) and left to rest for 2 h at 37°C. EL-4 cells were transfected with 6 µg Il-21-luc promoter plasmid Il-21-luc promoter mutated plasmid and K2 transfection reagent overnight. Cells were split the next day and incubated for 4 h with PMA (20 nM) and ionomycin (2 µM) or left unstimulated. After 4 h, cells were harvested for R-ChIP and analyzed for CREM binding as described above.

The 267-bb IL-21 promoter region (25) was amplified by PCR from genomic DNA of FVB WT mice using Q5 Hot Start High-Fidelity DNA Polymerase (NEB, USA) and cloned into KpnI and BglII sites of the pGL3 plasmid (Promega, USA) to generate the Il-21-luc construct. Oligonucleotides used for genomic PCR were 5′-GCAGATCTGATGACAGGGCCTTGGTCTG-3′ and 5′-ACGGTACCTCAGACAAA CCAGGTGAGGTG-3′. Mutagenesis of the half-CRE site within the Il-21-luc plasmid was performed with the Q5 Site-Directed Mutagenesis Kit (NEB, USA) according to manufacturer’s instructions. Oligonucleotides used for mutagenesis of the half-CRE site cagt into ccgg were 5′-CTTCAACCTGccggTGCACAGGTTGTCG-3′ and 5′-TGAGAACCAGACCAAGGTG-3′.

EL-4 cells were transfected with the K2 Transfection System (Biontex, Germany) by using 1 × 10⁶ cells and a total of 1.5 µg of indicated reporter plasmids and expression plasmids or pcDNA according to manufacturer’s instructions. Whenever an effector:reporter co-transfection was performed, the molar ratio between the two was 3:1. Before transfection cells were incubated with 20 µl/ml Multiplayer (K2 Transfection System) for 2 h at 37°C. All cells were co-transfected with 0.5 µg of Renilla luciferase promoter plasmid (pRL-TK) as a normalization control for transfection efficiency. Cells were left to rest over night before luciferase activity was measured by using the Dual-Glo Luciferase Assay System (Promega, USA). For experiments with cyclosporin A (CSA), transfected cells were incubated with or without 1 µg/ml CSA for 1 h and stimulated with PMA (20 nM) and ionomycin (2µM) for 5 h before luciferase activity was measured. Expression plasmid NFATc2 was a kind gift from Tobias Bopp (University of Mainz, Institute of Immunology) and the expression plasmid CREMα was a kind gift from Dr. P. Sassone-Corsi (University of California, Irvine, CA, USA).

Acute dextran sodium sulfate (DSS) colitis was induced by giving mice 3.5% (w/v) DSS (molecular mass, 36–50 kDa; MP Biomedicals, USA) in drinking water ad libitum for 6 days. Body weight and fecal status were followed daily from the time of colitis induction. Mice were killed on day 6 when some of the animals had lost more than 15% of their initial body weight. Spleen, mesenterial, and peripheral lymph nodes were harvested for further analysis. The biggest part of the colon was fixed in formalin, and the other small part was washed with PBS and frozen in RNA later (Qiagen, Germany) for mRNA analysis.

For T cell-dependent immunization, groups of age-matched mice were immunized i.p. with 100 µg 4-hydroxy-3-nitrophenylacetyl chicken gamma globulin (NP-CGG) (Biosearch Technologies, USA) in Imject Alum (Thermoscientific, USA). Spleens were harvested 7 days later.

Four micrometers of paraffin sections from the fixed colon were serially cut, mounted onto glass slides, and deparaffinized. The colon sections were stained with hematoxylin and eosin (H&E) by the Core Facility (IZKF) of the RWTH Aachen University. Blinded histological scoring was performed using a standard microscope, based on The Jackson Laboratory Scoring (TJL) method, as described previously (34, 35). Each colon section was scored for the four general criteria: severity, degree of hyperplasia, degree of ulceration, if present, and percentage of area involved. A subjective range of 1–3 (1 = mild, 2 = moderate, 3 = severe) was used for the first three categories. Severity: focally small or widely separated multifocal areas of inflammation limited to the lamina propria were graded as mild lesions (1). Multifocal or locally extensive areas of inflammation extending to the submucosa were graded as moderate lesions (2). If the inflammation extended to all layers of the intestinal wall or the entire intestinal epithelium was destroyed, lesions were graded as severe (3). Hyperplasia: mild hyperplasia consisted of morphologically normal lining epithelium that was two or more times thicker (length of crypts) than adjacent or control mucosa. Moderate hyperplasia was characterized by the lining epithelium being two or three times normal thickness, cells were hyperchromatic, numbers of goblet cells were decreased, and scattered individual crypts developed an arborizing pattern. Severe hyperplastic regions exhibited markedly thickened epithelium (four or more times normal), marked hyperchromasia of cells, few to no goblet cells, a high mitotic index of cells within the crypts, and numerous crypts with arborizing pattern. Ulceration was graded as: 0 = no ulcer, 1 = 1–2 ulcers (involving up to a total of 20 crypts), 2 = 1–4 ulcers (involving a total of 20–40 crypts), and 3 = any ulcers that exceed the previous. A 10% scale was used to estimate the area involved in the inflammatory process: 0 = 0%, 1 = 10–30%, 2 = 40–70%, 3 = >70%.

For surface staining, single-cell suspensions were isolated from spleens and mesenterial lymph nodes (mLNs) after 6 days of colitis induction and stained with anti CD4-FITC (eBioscience, USA) and anti CD4-APC (BD Bioscience, Germany). For measurement of intracellular cytokines, cells were treated with P/I and GolgiPlug (BD) for 5 h and fixed and permeabilized with FoxP3 staining buffer set (eBioscience) following the manufacturers’s instructions. Intracellular cytokines were stained with anti-IL-21-Alexa 647 (eBioscience) and anti IL-17-Alexa 488 (BD) antibodies. Intracellular staining of CD3ζ was performed using Cytofix/Cytoperm Kit (BD) and anti CD3ζ-PE antibody (BD) according to the manufacturer’s instructions. Samples were analyzed using a FACS Canto II (BD) and data analyzed using FCS Express (De Novo Software) software.

Total RNA from cells of mLN, inguinal lymph nodes, and colon tissue after DSS treatment was isolated using RNeasy Mini Kit (Qiagen, Germany). cDNA was then generated from 700 ng total RNA using RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Real-time PCR was performed using SYBR Green PCR kit (Eurogentec, Germany) and data were acquired with the ABI prism 7300 real-time PCR system (Applied Biosystems by Life Technologies, Germany). Each measurement was set up in duplicates, and two independent experiments were performed. After normalization to endogenous housekeeping control gene β-Actin for mice, the relative expression was calculated. The following primers were used: 5′-AAGATTCCTGAGGATCCGAGAAG-3′ and 5′-GCATTCGTGAGCGTCTATAGTGTC-3′ for IL-21 [adapted from Ref. (36)] 5′-AGCTGGACCACCACATGAATT-3′ and 5′-CCACACCCACCAGCATCTTC-3′ for IL-17; 5′-CCAGTGTGAAGATGGTTGTGACC-3′ and 5′-GGTGCTTATAAAAAGCCAGACCTTG-3′ for IL-23; 5′-TGCCAAGTTTGAGGTCAACAACCCA-3′ and 5′-CCCACCCCGAATCAGCAGCG-3′ for IFN-γ; 5′-ACTATTGGCAACGAGCGGTTC-3′ and 5′-TTACGGATGTCAACGTCACACTTC-3′ for β-Actin. mRNA expression levels of IL-21 in mLNs were compared to IL-21 mRNA expression levels in peripheral lymph nodes (n-fold). mRNA expression levels of IL-21, IL-17, IL-23, and IFN-γ in colon tissue of CREMα tg mice were compared to the their respective mRNA expression levels of their untreated WT littermates (n-fold).

mRNA expression of the CD3ζ chain was determined in CD4 positive T cells extracted by MACS isolation with the following primers: 5′-ATTGTAGAGCTGGTTGGGGTCCTG-3′ and 5′-CATCCTCCACGTGCGGTTCC-3′. n-fold of CD3ζ chain mRNA expression in CREMα tg mice was calculated to the CD3ζ chain mRNA expression of their WT littermates.

Expression of cMAF, Bcl-6, and IRF4 was determined in isolated T cells, which were stimulated with P/I for 6 h or left unstimulated. The following primers were used: 5′-GCC CAA CAA GCT AGA AAG-3′ and 5′-TCT CTG AGG GTC TGG AAA CT-3′ for IRF4; 5′-AGG GAC CTG TTC ACG AGA TTA TTG-3′ and GTC CCC GAC CAA GCT CAG T for Bcl-6; 5′-AGCAGTTGGTGACCATGTCG-3′ and 5′-TGGAGATCTCCTGCTTGAGG-3′ for cMAF.

All data are presented as mean ± SEM. Differences between two groups were evaluated using unpaired or paired, when necessary, two-sided (or one-sided, where specifically indicated) Student’s t-test when data were normally distributed. Otherwise, a non-parametric Mann–Whitney test was performed. All statistical analysis and subsequent graphics generations were performed using GraphPad Prism version 5.0 (GraphPad Software, USA). A p-Value <0.05 was considered significant.

Previously, we have shown that mice with a T cell-specific overexpression of CREMα show an enhanced IL-21 production both in vitro after T cell stimulation with anti-CD3 and anti-CD28 and in vivo in a contact dermatitis model (7). To further confirm that CREMα regulates IL-21 expression, we immunized WT and CREMα transgenic (CREMα tg) mice with the T cell-dependent antigen NP-CGG. Untreated CREMα tg mice already showed a higher frequency of IL-21 producing cells within the CD4⁺ T cell population (Figures 1A,B). Seven days after immunization IL-21 production was enhanced in WT and CREMα tg mice compared to untreated mice but to higher extent in CREMα tg mice (Figures 1A,B). These data confirm our previously published observation that CREMα enhances IL-21 expression both in vitro and in vivo.

IL-21 expression depends on increased intracellular calcium (Ca²⁺) levels which activate NFAT that in turn activates Il21 gene transcription (23). Accordingly, we evaluated Ca²⁺ responses in both WT and CREMα tg T cells. Compared to WT T cells, we noted an enhanced-free intracytoplasmic Ca²⁺ response in CREMα tg T cells that had been stimulated with anti-CD3/CD28 antibodies (Figure 2A). No difference was found in T cells that had been stimulated with PMA and ionomycin (Figure S1A in Supplementary Material) suggesting that this difference is related to alterations in the CD3 complex. Interestingly, both enhanced Ca²⁺ signaling and NFAT activity have been reported in T cells of SLE patients (37, 38). Enhanced calcium influx is observed in SLE T cells as well and was linked to a decreased expression of the CD3ζ chain (38). In detail, reduced CD3ζ chain expression in human SLE T cells results in enhanced calcium signaling and vice versa transfection of SLE T cells with the ζ chain construct corrected the heightened calcium response in SLE T cells (39). Previously, we have shown that CREMα binds to the human CD3ζ chain promoter and represses its activity (40). Accordingly, we checked expression of the CD3ζ chain and found it decreased in both CD4⁺ and CD8⁺ T cells of CREMα tg animals compared to WT animals on protein (Figures 2B,C) as well as on mRNA level (Figure 2D), whereas the overall expression of CD3 was not altered (Figure S1B in Supplementary Material). Moreover, we screened the murine CD3ζ chain promoter for CRE sites and identified a putative-binding site of CREMα within this promoter. Congruent to the CRE site within the human CD3 promoter, which we already showed to be regulated by CREMα, the identified CRE site in the murine CD3ζ chain promoter is located about 390 bp upstream of the transcription start. ChIP experiments using an anti-CREM antibody confirmed an up to twofold binding of CREMα to this promoter (Figure 2E). Thus, the transgenic overexpression of CREMα in T cells probably results in a decreased CD3ζ chain expression, thereby causing an enhanced Ca²⁺ influx, which may be responsible for the enhanced Il-21 transcription.

We next stimulated splenic T cells from WT and CREMα tg mice in vitro either with anti-CD3/CD28 antibodies or with Phorbol-12-myristat-13-acetate and ionomycin (P/I) and analyzed Il-21 mRNA expression (Figure 3A). As expected, stimulation with anti-CD3/CD28 antibodies significantly upregulated Il-21 expression of CREMα tg T cells. However, stimulation with P/I resulted in a much higher and significant induction of Il-21 mRNA expression in both WT and CREMα tg T cells. This implies that the IL-21 expression in CREMα tg T cells might in part be due to a low CD3ζ expression, but that pathways downstream of the TCR signaling complex must also be involved, since P/I acts independently from the CD3ζ complex. However, NFAT binding was insignificantly enhanced in CREMα transgenic T cells after stimulation (Figure 3B), which on the other hand suggests that calcium/NFATc2-dependent mechanism at least partially influence IL-21 transcription in CREMα tg T cells. Stimulation of WT and CREMα tg T cells with P/I resulted in enhanced acetylation on histone 4 of the IL-21 promoter in comparable manner (Figure 3C), which means that CREMα does not change acetylation of histone 4. We, furthermore, did not detect differential expression of c-Maf, IRF4, and Bcl-6, which are known to be associated with IL-21 induction (Figure S2 in Supplementary Material) (41–43). We, therefore, assumed that CREMα might also directly regulate IL-21 expression itself. To determine whether Il-21 was a direct target of the transcription factor CREMα, the Il-21 promoter sequence was screened for potential CRE-binding sites. Mehta et al. already identified and characterized a murine IL-21 proximal promoter region, which is located immediately 5′of the first coding exon. This region is highly conserved between men and mice and activates IL-21 transcription (25). This region also contains a binding site matching a consensus half-site for CREM (half CRE-site) (Figure 3D). We next sought to confirm CREM binding to the Il-21 promoter in vivo using ChIP assays with murine splenic CD3⁺ T cells. CREM binding to the proximal IL-21 promoter region was enhanced in WT T cells as well as in CREMα tg T cells after stimulation with P/I; however, we did not find a significant difference in binding between CREMα tg T cells and WT T cells (Figure 3E). To identify if the site is indeed important for binding of CREM, we used the R-ChIP technique, which we recently established and which enables the study of the in vivo binding of transcription factors to a specific binding site on a promoter of a reporter construct. Using this technique, we could show a specific binding of CREM to this CRE site. Transfection of EL-4 cells with a IL-21 promoter construct spanning 267 bp of the Il-21 promoter (Il-21-luc) containing a mutated CRE site almost abolished CREM binding after stimulation compared to the non-mutated IL-21 promoter plasmid (Figure 3F). To further test the specific function of the CREM-binding site in the Il-21 promoter, we used the abovementioned luciferase construct (Il-21-luc). We transfected EL-4 T cells with the Il-21-luc reporter plasmid including co-transfection of expression plasmids for CREMα or NFATc2 and measured luciferase activity compared to an empty control vector (pcDNA) (Figure 3F). NFATc2 plasmid served as a control in our setting as activation of IL-21 transcription by NFATc2 is well known (23, 25). Consequently, NFATc2-transfected cells revealed an about 20-fold increased IL-21-luc activity compared to cells transfected with pcDNA. Transfection with Il-21-luc and an expression vector for CREMα led to an about twofold-enhanced Il-21-luc activity compared to co-transfection with pcDNA suggesting that CREMα is at least partially responsible for the activity of the Il-21 promoter. The mutagenesis of the half-CRE site prevented upregulation of Il-21-luc activity in the presence of exogenous CREMα (Figure 3G), which is in accordance with the reduced binding shown by R-ChIP in Figure 3F. However, promoter activity was also reduced in the presence of exogenous NFATc2. This somehow unexpected finding probably results from chromatin remodeling or CREM/NFAT interactions suggesting crucial importance of this site for the whole promoter activity. To further dissect the direct and Ca²⁺-mediated effects of CREMα on IL-21 expression, we evaluated the luciferase activity of cells transfected with IL-21-luc and CREM or NFATc2 plasmids in the presence of CSA. CSA is an immunosuppressive drug and inhibits calcineurin that, in turn, results in the blockade of the transcription factor NFATc2 (44). As a consequence, NFATc2-mediated Il-21-luc activity was abrogated in stimulated cells in the presence of CSA. However, CREM-mediated Il-21-luc activity was not altered by CSA (Figure 3H). These data show that the direct effect of CREMα on IL21 promoter activity is mediated independently of NFATc2. We conclude from these experiments that CREMα directly binds to the proximal Il-21 promoter and thereby activates IL-21 transcription.

IL-21 supports generation and stabilization of pathogenic Th17 cells (13, 29), which might be one of the mechanisms by which IL-21 expands and sustains ongoing mucosal inflammation. IL-17 expression in CREMα tg mice is probably to some extent regulated by IL-21, as was shown by blockade experiments with IL-21RFc, which prevented RORyt and IL-17 expression in vitro (7). We next asked how CREMα tg T cells affect disease activity and cytokine release in a murine colitis model. We, therefore, used an established murine model of colitis, in which colitis is induced by oral administration of DSS. Wild-type and CREMα tg mice were given 3.5% DSS in their drinking water. While CREMα tg mice suffered from a progressive weight loss, WT mice hardly lost weight (Figure 4A). Histological changes following DSS treatment were detectable in both groups, but the extent of tissue damage was greater in CREMα tg mice compared to WT mice (Figures 4B,C). We conclude from this that overexpression of CREMα in T cells enhances inflammation in DSS-induced colitis.

To further analyze if higher disease activity also involves higher expression of IL-21 and IL-17, we analyzed IL-21 protein expression by flow cytometry in spleen and mLNs of colitic mice. CREMα tg mice showed enhanced T cellular IL-21 production in mLN (Figure 5A) and spleen (Figure 5B) compared to WT animals. Additionally, mRNA levels of Il-21 were also enhanced in mLN (Figure 5C) of CREMα tg mice compared to WT mice and colon tissue also showed enhanced Il-21 mRNA expression (Figure 5D), while there was an insignificant tendency toward enhanced IL-17 transcription (Figure 5E). IL-23 (Figure 5F) and IFN-γ expression was not altered between both groups neither on mRNA (Figure 5G) nor on protein level (not shown). IL-17⁺ T cells were not enhanced in mLN (Figure 5H) from CREMα tg mice, but significantly enhanced in spleens (Figure 5I). These data proof enhanced production of the inflammatory cytokine IL-21 in CREMα tg colitic mice, which potentially accounts for accelerated disease activity.