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The Journal of Immunology, 2004, 173: 3446-3455.
Copyright © 2004 by The American Association of Immunologists

A Critical Regulatory Role of Leucin Zipper Transcription Factor c-Maf in Th1-Mediated Experimental Colitis1

Benno Weigmann*, Andrea Nemetz*, Christoph Becker*, Jan Schmidt§, Dennis Strand*, Hans A. Lehr{dagger}, Peter R. Galle*, I.-Cheng Ho{ddagger} and Markus F. Neurath2,*

* Laboratory of Immunology, I. Medical Clinic, and {dagger} Institute of Pathology, University of Mainz, Mainz, Germany; {ddagger} Harvard School of Public Health and Harvard Medical School, Boston, MA 02115; and § Department of Surgery, University of Heidelberg, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this study, we investigated the role of c-Maf, a transcription factor known to induce IL-4 production, in inflammatory bowel diseases and experimental colitis. Although Crohn's disease (CD) is associated with low IL-4 production by T-bet-expressing Th1 cells in the lamina propria, surprisingly a higher expression of c-Maf in these cells was found as compared with control patients. The relevance of this finding was further evaluated in an animal model of CD induced by adoptive transfer of CD4+CD62L+ T cells in RAG-deficient mice. In this Th1-mediated model, an increase of c-Maf-expressing T lymphocytes in the lamina propria over time was observed. Interestingly, adoptive transfer of c-Maf transgenic CD4+CD62L+ T cells in RAG-1-deficient mice resulted in an IL-4-dependent inability to induce colitis and suppressed colitis activity induced by wild-type CD4+CD62L+ T cells. In contrast, transfer of CD4+CD62L– T cells from c-Maf transgenic, but not wild-type mice induced colitis and augmented colitis induced by CD4+CD62L+ T cells from wild-type mice in an IL-4-independent pathway, as determined by macroscopic, histologic, and endoscopic criteria. This was associated with an accumulation of CD4+ T-bet+ CD25+ effector Th1 cells in the lamina propria of colitic mice. Our results reveal a novel regulatory role of c-Maf in colitis. Although overexpression of c-Maf in naive T cells prevents Th1-mediated colitis, overexpression of c-Maf in memory T-bet+ Th1 cells regulates CD25 expression and augments such colitis. Targeting of c-Maf in memory T cells in CD appears to be an attractive target for therapeutic interventions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Crohn’s disease (CD)3 and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD) in humans, are characterized by a complex chronic inflammatory process (1, 2). Growing evidence indicates the role of a genetically determined dysregulation of the mucosal immune response toward the resident bacterial flora in the pathogenesis of human IBD (3, 4, 5). In IBD, CD4+ T cells represent the vast majority of activated mononuclear cells infiltrating the gut. A large proportion of these lamina propria T cells in IBD bears the phenotypic characteristics of memory T lymphocytes and has been recruited into the intestinal mucosa via adhesion molecules. Recent evidence indicates that differentiation and activation of lamina propria CD4+ T cells play a major role in the pathogenesis of IBD (6, 7, 8, 9, 10).

Naive Th cells can differentiate into several T cell subsets that can be distinguished by their signature cytokine production and functions (11, 12, 13, 14). Th1 cells produce the signature cytokine IFN-{gamma} and are important in macrophage activation as well as inflammatory and organ-specific autoimmune reactions. Whereas Th1 cells are essential for protection against intracellular pathogens such as bacteria, Th2 cells are required to eliminate various extracellular parasites by producing cytokines such as IL-4, IL-5, IL-9, and IL-13, and are mainly involved in controlling humoral and allergic immune responses (13). In IBD, T cell cytokines play a key role in the pathogenesis of chronic intestinal inflammation and tissue damage (5, 15). Whereas CD is associated with increased production of Th1-like cytokines such as IFN-{gamma} and TNF, the cytokine profile in chronic UC is characterized by increased production of the Th2 cytokine IL-5, but not IL-4 (16, 17, 18, 19). Interestingly, both Th1- and Th2-type cytokines have been shown to play an important pathogenic role in various animal models of IBD, suggesting that both Th1 and Th2 cells can induce chronic intestinal inflammation in vivo (20, 21, 22, 23). The pathogenic function of Th1 and Th2 cells can be counteracted by immunosuppressive cytokines such as IL-10 and TGF-{beta}, produced by regulatory T cells or Th3 cells (24).

Recent studies have focused on molecular mechanisms of Th1 and Th2 cell development in peripheral T cells (25). Transcription factors such as c-Maf, STAT-6, GATA-3, JunB, and NF-ATc1 have been shown to induce or augment Th2 cytokine production (12). In contrast, transcription factors such as STAT-1, STAT-4, and T-bet are associated with IFN-{gamma}, IL-12, IL-23, and IL-27 signaling, respectively, and play a key role in Th1-specific cytokine production in peripheral T cells (12, 26). In the mucosal immune system, recent studies have suggested an important role of STAT-4 and T-bet for Th1 T cell effector functions in the gut in experimental colitis and CD (9, 27, 28). However, the functional role of Th2-associated transcription factor such as c-Maf is less well understood.

c-Maf is a member of the AP-1 family of basic leucin zipper transcription factors and is induced in activated T lymphocytes via a recently cloned pleckstrin homology domain-containing protein denoted Tc-mip (29, 30). C-Maf binds to a consensus site (Maf recognition element) in the proximal IL-4 promotor, thereby inducing IL-4 gene transcription in T lymphocytes (25, 29). In peripheral T cells, c-Maf is exclusively expressed in Th2, but not Th1 clones, and is induced during normal differentation of T helper precursor cells along a Th2, but not Th1 pathway. Although c-Maf is a potent trans activator of the IL-4 gene in vitro, the function of c-Maf during Th cell differentation in vivo has been less well defined. However, c-Maf transgenic mice display an increased production of Th2 cytokines and increased serum levels of IgG1 and IgE (31). Consistently, c-Maf-deficient mice have greatly impaired IL-4 production, and their T cells showed a Th1-polarized phenotype (12, 32). However, if c-Maf-deficient T cells are cultivated under Th2 conditions, they are capable of expressing normal levels of Th2 cytokines, suggesting the existence of c-Maf-independent pathways that can induce Th2 development (25). Therefore, c-Maf has been considered as a specific key activator of IL-4 gene expression (25, 33). In addition to its regulatory role in IL-4 gene transcription, c-Maf has been shown recently to control CD25 expression in T cells via activation of STAT-5, suggesting that it modulates Th cell differentiation and activation via both IL-4-dependent and -independent pathways (34).

In the present study, we have analyzed the expression and function of c-Maf in T cell-dependent colitis. Our data demonstrate a novel regulatory role of c-Maf in T-bet+ Th1 cells in the lamina propria that augments Th1-mediated experimental colitis. Taken together with the increased expression of c-Maf in T-bet+ lamina propria T cells in CD, targeting of c-Maf function in memory T cells emerges as an attractive approach to modulate gut inflammation in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Two- to 4-mo-old BALB/c and B6 mice were obtained from the central breeding facility at the University of Mainz or from Charles River Laboratories (Sulzfeld, Germany); c-Maf transgenic mice were described elsewhere (31). RAG-1-deficient mice were obtained from M&B (Ry, Danmark).

Screening of c-Maf transgenic mice

Tail DNA from mice was isolated and subjected to PCR (Eppendorf Thermal Cycler, Hamburg, Germany) using two synthetic primers (primer 1 c-Maf, 5'-TGTTGTGGTGCAGAACTGGAT-3', and primer 2, 5'-GTTTCAGGTTCAGGGGGAGGT-3') obtained from MWG Biotec (Heidelberg, Germany) and ready mix polymerase (Sigma-Aldrich, St. Louis, MO). PCR was performed for 45 cycles (95°C for 1 min, 60°C for 1 min, and 72°C for 1 min), and the resulting PCR product of 409 bp was analyzed on a 1% agarose gel.

Patients

Gut specimens obtained from patients with CD, UC, or control patients were studied. The diagnosis for each patient was made using clinical parameters, radiographic studies, and histologic criteria. The CD group (n = 10) consisted of four men and six women, ranging from 29 to 50 years of age. The control group consisted of colonic specimens from 11 patients ranging from 41 to 78 years of age. In addition, colonic samples from 10 patients with UC were obtained. Collection of surgical samples was approved by the ethical commitee and the institutional review board of the University of Mainz.

Adoptive transfer of CD4+ T cell subsets in immunocompromised hosts

To induce colitis by adoptive transfer of CD4+ T cells, a modification of a previously described protocol was used (22, 35). In brief, CD4+ T cells were purified from spleen mononuclear cells of healthy mice using FITC-conjugated mAbs, anti-FITC immunomagnetic beads, and MACS (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by enzymatic removal of the beads or negative selection by using a T cell purification kit (Miltenyi Biotec). The resulting CD4+ T cells (purity >97%) were further separated by immunomagnetic beads into CD62L+ and CD62L– T cells. The former cells (purity: >95%) showed high expression of CD45RB by FACS analysis. A total of 0.5–1 x 106 CD62L+CD45RBhighCD4+ T cells or 1 x 106 CD62L+CD45RBhigh and CD62L– (ratio 4:1) CD4+ T cells was finally transfered into syngenic RAG-1-deficient mice. Colitis activity was monitored by weight curves, endocopy, and histologic analysis, as specified below. Mice were maintained in isolated cages under specific pathogen-free conditions. No evidence of graft vs host disease, such as skin inflammation and histopathologic evidence of small bowel inflammation, was observed in the reconstituted animals under our experimental conditions.

Treatment of mice with neutralizing Abs to IL-4

In some experiments, colitic mice were treated with neutralizing Abs to IL-4 (11B.11; 7 mg/wk) and corresponding rat control Abs. Mice were given i.p. anti-IL-4 or control Abs every week after reconstitution. To generate large amounts of Ab, supernatants from hybridoma cells were collected and Abs were concentrated with ammonium sulfate precipitation. After washing steps, supernatants were purified with protein G columns (Amersham Biosciences, Braunschweig, Germany). Purified Abs were desalted with Sephadex column (Amersham Biosciences), dissolved in PBS, and i.p. injected into mice.

T cell culture and cytokine assays

To measure cytokine production, 1 x 106 splenic T cells/ml were activated with 10 µg/ml purified hamster anti-mouse CD3{epsilon} (clone 145-2C11) and 1 µg/ml soluble hamster anti-mouse CD28 (clone 37.51) and cultured in complete medium (IMDM supplemented with 3 mM L-glutamine, 100 U/ml penicillin/streptomycin, 10% heat-inactivated FCS) or serum-free medium at 37°C in a humidified atmosphere containing 5% CO2. Lamina propria mononuclear cells (LPMC) were isolated, as previously described (23). Briefly, the colon was opened longitudinally, washed several times in PBS to remove feces and debris, and cut into small pieces. Tissues were incubated at 37°C in PBS supplemented with 0.145 mg/ml DTT and 0.37 mg/ml EDTA for 15 min. The tissue was then digested in RPMI 1640 containing 0.15 mg/ml type II collagenase (Worthington, Munich, Germany) and 0.1 mg/ml DNase (Roche Molecular Biochemicals, Mannheim, Germany) for 75–90 min at 37°C on a shaking platform. LPMC were stimulated, as described above.

After 48 h (96 h for TGF-{beta}), culture supernatants from splenic T cells and LPMC were removed and assayed for cytokine concentration. Cytokine concentrations were determined by specific ELISA using commercially available recombinant cytokines and Abs (BD Pharmingen, San Diego, CA; R&D Systems, Heidelberg, Germany).

Immunohistochemistry

Immunohistochemistry was performed on 7-µm cryosections from gut specimens of both control and CD/UC patients, as previously described (35). Cryosections were analyzed by immunofluorescence or diaminobenzidine (DAB) staining. Immunofluorescence was performed using the tyramide signal amplification Cy3 system (PerkinElmer Life Sciences, Heidelberg, Germany) and a fluorescence microscope (Olympus fluorescence microscope; Olympus Deutschland, Heidelberg, Germany).

Briefly, tissues were fixed in 4% paraformaldehyde/PBS and washed in 0.01 M PBS, followed by sequential incubation with avidin/biotin (Vector Laboratories, Burlingame, CA)-, peroxidase-, and protein-blocking reagent (DakoCytomation, Wiesbaden, Germany) to eliminate unspecific background staining. Samples were then pretreated with 10% of serum (corresponding to the secondary Ab) in PBS/0.1% Triton-X and incubated overnight at 4°C with primary Abs specific for murine or human CD3, CD25, c-Maf, T-bet, Ki-67, or CD4 (BD Biosciences, San Jose, CA; DakoCytomation; Caltag Laboratories, Burlingame, CA; and Santa Cruz Biotechnology, Heidelberg, Germany), in PBS/0.5% BSA/0.2% saponin. Samples incubated with isotype-matched control Abs served as negative control. The following day, samples were rinsed in PBS and incubated with a biotinylated secondary IgG Ab (1/200 dilution; obtained from Dianova, Hamburg, Germany; Caltag Laboratories; and BD Biosciences) or Alexa-488-conjugated Ab (Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature, followed by incubation with streptavidin-conjugated Cy2 or Cy3 (Dianova) (1/500–1/1000 dilution) for 1 h at room temperature or with streptavidin-HRP and stained with tyramide-Cy3, according to the manufacturer’s instructions (PerkinElmer Life Sciences). In some cases, samples were subjected to staining with a second Ab for double staining analysis, as previously described (36). Before examination, the nuclei were counterstained with 4',6'-diamidino-2-phenylindole (DAPI; Vector Laboratories).

For DAB staining, some samples were treated with streptavidin and stained with the DAB chromogen, according to the manufacturer’s instructions (DakoCytomation). Before examination, the nuclei of these cryosections were counterstained with hematoxylin. Slides were mounted with mounting medium (Vector Laboratories) and analyzed with an Olympus microscope. c-Maf-positive cells in 6–10 high power fields were finally counted in all patients per condition. For confocal microscopy, the counterstaining was done with Hoechst 33342, and a Leitz confocal microscope was used. No staining was detected in samples without primary Ab.

Histologic analysis of colon cross sections

Tissues were removed from colitic mice at indicated time points, and cryosections or paraffin sections were made and stained with H&E. For colitis induced by CD4+CD62L+ T cell transfer, the degree of inflammation and epithelial injury on microscopic cross sections of the colon was graded semiquantitatively from 0 to 5 (35). Grading of colitis activity was done in a blinded fashion by the same pathologist (H.A.L.). Small bowel sections were taken from the same animals as an additional control and showed no evidence for inflammation.

In vivo high resolution endoscopic analysis of the colon

A novel method was used to perform endoscopy in mice using a miniendoscope (Karl Storz, Tuttlingen, Germany). This technique was established in >250 mice with and without colitis (C. Becker and M. F. Neurath, manuscript in preparation). In brief, mice were anesthetized with avertine and the colon was flushed with PBS. Prominent endoscopic signs of inflammation in SCID mice were masking of the normal vascular pattern, the presence of mucosal granularity, and the appearance of ulcers. To determine colitis activity in reconstituted RAG-1-deficient mice, the mice were monitored by endoscopy at indicated time points after the cell transfer.

RT-PCR

Colonic tissue was taken from reconstituted RAG-1-deficient mice, followed by RNA extraction. Total RNA was prepared using Trifast reagent (Peqlab, Erlangen, Germany) and Mixer Mill (Qiagen, Hilden, Germany) and purified with Qiagen RNeasy MinElute Cleanup Kit following the manufacturer’s instructions. For RT-PCR, Superscript II RNaseH (Invitrogen Life Technologies, Carlsbad, CA) was used according to the manufacturer’s instructions. cDNA was sythesized from 4–5 µg of total RNA using oligo(dT) primers. The PCR primer sequences used were as follows: IL-4, 5'-CTCGAATGTACCAGGAGCCA-3' and 5'-TTGCARGATGCTCTTTAGGCTT-3'; IFN-{gamma}, 5'-ACACTGCATCTTGGCTTTGC-3' and 5'-CGGATGAGCTCATTGAATGCT-3'; IL-10, 5'-CACTGCTATGCTGCCTGCTCT-3' and 5'-GTAGACACCTTGGTCTTGGAG-3'; {beta}-actin, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'. PCR cycling conditions were as follows: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min. Amplification was performed for 35 cycles. PCR products were analyzed on 1% agarose gels.

Statistical analysis

Statistical analysis was made using Student's t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Enhanced expression of c-Maf in T-bet-positive lamina propria T cells in patients with CD

Pathologic cytokine production by lamina propria T cells has been suggested to be a major mechanism in the pathogenesis of IBD. In particular, increased production of Th1-type cytokines has been found in patients with CD, while UC is associated with the production of the Th2 cytokine IL-5 (16, 17). We focused in an initial series of experiments on the expression of the Th2-associated transcription factor c-Maf in the lamina propria of IBD patients. Immunohistochemical staining showed an increased expression of c-Maf in the lamina propria of patients with UC as compared with control patients (Fig. 1, A and B). Surprisingly, however, there was a strong up-regulation of c-Maf-expressing LPMC in CD that was even more pronounced than in UC, although CD is known to be associated with low IL-4 production by T cells (16). Quantitative analysis showed a significant up-regulation of c-Maf-expressing LPMC in CD as compared with controls (Fig. 1B). Furthermore, staining of colonic sections with Abs against CD3 and c-Maf showed that many c-Maf-expressing cells in the lamina propria were T lymphocytes (Fig. 1, C and D). Quantitative analysis showed a significant increase in the number of CD3/c-Maf double-positive T cells in CD as compared with controls (Fig. 1E). Finally, as demonstrated by confocal laser microscopy, c-Maf was colocalized with T-bet in lamina propria T cells and mainly localized in the nucleus of lamina propria T cells in CD, indicating the existence of T-bet/c-Maf double-positive gut T cells (Fig. 1F). Thus, although CD is known to be mediated by Th1 T cells, lamina propria T lymphocytes in this disease expressed the Th2-associated transcription factor c-Maf.



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  		FIGURE 1. Enhanced expression of c-Maf in LPMC of patients with UC and CD as compared with control patients. A, Colon sections were incubated with a c-Maf Ab, stained by using the DAB-staining kit, and analyzed by microscopy. Cryosections were counterstained with H&E. Increased c-Maf expression was observed in sections from UC and CD patients as compared with control patients. One representative patient per group is shown. B, Quantitative analysis (high power field) of c-Maf-positive cells in patients with CD (n = 5) and UC (n = 6) or control patients (n = 4). Data represent mean values ± SD. C, Staining of consecutive sections with anti-CD3 and c-Maf Abs. Colon sections from control patients (upper panels) and CD patients (lower panels) were incubated with CD3 and c-Maf Abs, followed by incubation with a biotinylated secondary IgG Ab and streptavidin-HRP. Staining was performed with tyramide-Cy3. One representative patient per group is shown. Double-positive cells are indicated by arrows. D, Double staining for CD3 and c-Maf on cryosection from patients with CD and control patients (control). Samples treated with isotype-matched control Abs are shown in the lower panels. Many CD3+ cells coexpressed c-Maf in CD patients. E, Quantification of the number of double-positive T cells in the lamina propria of CD and control patients. There was a significant increase in the number of double-positive cells in CD as compared with control patients. F, Detection of double-positive cells in the lamina propria in CD (upper left panel). Samples from CD patients were stained with anti-c-maf and anti-T-bet Abs, followed by confocal laser microscopy. Nuclei were stained with DAPI. Upper right panel, Shows isotype control Abs as negative control. c-Maf/T-bet double-positive cells were seen in the lamina propria in CD. Further studies showed that the expression of c-Maf in CD3+ lamina propria T cells in CD was mainly nuclear (lower panel).

 
Increased expression of c-Maf in lamina propria T cells in an experimental model of CD mediated by Th1 T lymphocytes

Because the above data were consistent with the possibility of a regulatory role of c-Maf in controlling cytokine production in the inflamed intestine in CD Th1 cells, we focused our attention on the expression of c-Maf in a Th1 cell-mediated animal model of chronic intestinal inflammation. Accordingly, RAG-1 knockout mice were reconstituted with syngenic CD4+CD62L+ T cells from wild-type donor mice. Although T lymphocytes in this model are known to express high levels of T-bet and produce Th1 cytokines (22, 27), c-Maf was found to be highly expressed in lamina propria cells in RAG-1-deficient mice reconstituted with syngenic CD4+CD62L+ T cells (Fig. 2). In additional time course studies, a weak c-Maf expression was observed at 3 wk after cell transfer before development of colitis started. Furthermore, increased c-Maf expression occurred at 6 wk after cell transfer, and maximal expression was noted 9 wk after cell transfer when mice had full-blown colitis (Fig. 2). These findings further suggested that c-Maf might be involved in regulating Th1 cell activation in experimental colitis in vivo.



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  		FIGURE 2. Increased expression of c-Maf in murine experimental colitis in RAG-1 knockout mice reconstituted with syngenic CD62L+CD4+ T cells. Immunohistochemical analysis of the colon of RAG-1-deficient mice for c-Maf expression at indicated time points after reconstitution of RAG-1 knockout mice with CD4+CD62L+ T cells. Cryosections were incubated with anti-c-Maf Abs and consequently stained with the DAB-staining kit. c-Maf-expressing cells were noted as soon as 3 wk after T cell transfer.

 
Overexpression of c-Maf in CD4+CD62L+ T cells protects from Th1-mediated experimental colitis in an adoptive transfer model using RAG-1-deficient mice

To further test the hypothesis that c-Maf regulates T cell activation in the lamina propria, we next assessed in initial studies the effects of c-Maf overexpression on Th1-mediated colitis induced by transfer of naive CD4+CD62L+ T cells into RAG-1-deficient mice. Accordingly, we used transgenic mice overexpressing c-Maf in T lymphocytes as donor mice in adoptive transfer studies. Splenic CD4+ T cells isolated from these transgenic mice produced lower amounts of Th1, but higher amounts of Th2 cytokines than T cells from wild-type mice (Fig. 3A). Whereas transfer of CD4+CD62L+ T cells from wild-type mice resulted in clinical signs of severe colitis, transfer of c-Maf transgenic CD4+CD62L+ T cells failed to induce weight loss and histological signs of colitis (Fig. 3, B–D). Furthermore, transfer of c-Maf transgenic T cells resulted in amarkedly reduced colitis activity in mice reconstituted with CD4+CD62L+ T cells in two independent experiments, as observed by macroscopic, endoscopic, and histologic criteria (Fig. 3, B–E), although such T cells were present in the lamina propria of reconstituted mice and showed evidence for proliferation, as determined by Ki67 and CD3 double staining (Fig. 3, F and G). This finding was associated with a down-regulation in IFN-{gamma} levels and an up-regulation of IL-4 levels in the colon of mice reconstituted with CD4+CD62L+ c-Maf transgenic cells as compared with those given CD4+CD62L+ T cells from wild-type animals (Fig. 3H). In addition, cotransfer of CD4+CD62L+ c-Maf transgenic T cells prevented colitis and weight loss induced by CD4+CD62L+ T cells from wild-type animals (Fig. 3I), suggesting that overexpression of c-Maf in CD4+CD62L+ T cells protects from Th1-mediated experimental colitis.



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  		FIGURE 3. A regulatory role for c-Maf in experimental colitis. A, Cytokine production by splenic CD4+ and CD4+CD62L+ T cells from c-Maf transgenic mice before T cell transfer. T cells were stimulated with Abs to CD3 and CD28, followed by analysis of culture supernatants using ELISA. Data represent mean values of four to eight mice per group. T cells from c-Maf transgenic mice produced lower amounts of Th1 cytokines, whereas an increased production of Th2 cytokines from c-Maf transgenic CD4+ T cells was noted as compared with T cells from control wild-type mice. B, Splenic CD4+CD62L+ T cells from wild-type or c-Maf transgenic mice were isolated and injected into RAG-1-deficient mice, followed by monitoring of the body weight at indicated time points after cell transfer, as described in Materials and Methods. Two independent experiments are shown. Transfer of CD4+CD62L+ T cells from wild-type mice caused colitis in RAG-1 mice, whereas transfer of CD4+CD62L+ T cells from c-Maf transgenic mice protected mice from experimental colitis. Data represent mean value ± SEM from five to seven mice per group and experiment. C, Quantitative histopathologic assessment of colitis activity showed a significant (p < 0.05) protection from inflammation and tissue injury in mice reconstituted with CD4+CD62L+ T cells from c-Maf transgenic mice as compared with mice reconstituted with wild-type T cells. Data represent mean values ± SEM. D, Histologic analysis of colon inflammation in mice reconstituted with T cells from wild-type (lower panel) or c-Maf transgenic (upper panel) mice. Signs of inflammation such as goblet cell depletion, erosions, and accumulation of mononuclear cells were noted in mice reconstituted with wild-type cells only. E, Endoscopic analysis of the colon of RAG-1-deficient mice reconstituted with wild-type or c-Maf transgenic T cells. Marked erosions and ulcers were seen in the group given wild-type T cells, whereas a normal colon architecture was noted in mice reconstituted with T cells from c-Maf transgenic mice. F, Staining for CD3 on colonic cryosections from mice reconstituted with CD4+CD62L+ T cells from c-Maf transgenic mice (upper left panel) and mice given CD4+CD62L+ T cells from wild-type mice (lower left panel). Samples treated with isotype-matched control Abs are shown on the right panels. CD3+ T cells were detectable in the lamina propria of mice given T cells from both wild-type and c-Maf transgenic donor mice. G, Double staining for CD3 and Ki-67 in the lamina propria of mice reconstituted with CD4+CD62L+ T cells from c-Maf transgenic mice (upper panels) and mice given CD4+CD62L+ T cells from wild-type mice (lower panels). H, Analysis of cytokine levels in the colon of six reconstituted mice by RT-PCR. Mice reconstituted with CD4+CD62L+ T cells from c-Maf transgenic mice (c-Maf 1–3) showed higher IL-4, but lower IFN-{gamma} and similar IL-10 mRNA levels as compared with mice given T cells from wild-type mice (wt 1–3). I, Cotransfer of CD4+CD62L+ T cells from c-Maf transgenic mice prevents colitis induced by CD4+CD62L+ T cells from wild-type mice. CD4+CD62L+ T cells from c-Maf transgenic mice were isolated, mixed together with CD4+CD62L+ T cells from wild-type mice, and then injected into RAG-1-deficient mice, followed by monitoring of the body weight at indicated time points. Reconstitution with c-Maf transgenic CD4+CD62L+ T cells served as an internal control. Cotransfer of CD4+CD62L+ T cells from c-Maf transgenic mice prevented diarrhea and weight loss induced by transfer of CD4+CD62L+ T cells from wild-type mice.

 
Neutralizing anti-IL-4 Abs prevent the protective effects of c-Maf overexpression on colitis activity in vivo

To determine whether the observed inability of c-Maf transgenic T cells to cause colitis in the above experiments was due to c-Maf-dependent regulation of IL-4 production, we next administered neutralizing Abs to IL-4 or control rat Ig to RAG-1-deficient mice reconstituted with c-Maf transgenic T cells. Whereas transfer of CD4+CD62L+ T cells from wild-type mice resulted in weight loss and histologic and macroscopic signs of severe colitis, transfer of CD4+CD62L+ c-Maf transgenic T cells followed by treatment with control Abs failed to induce signs of severe colitis as expected (Fig. 4). Remarkably, transfer of CD4+CD62L+ T cells from c-Maf transgenic mice, followed by treatment with neutralizing anti-IL-4 Abs, suppressed the protective effect of c-Maf overexpression in these mice (Fig. 4). Thus, anti-IL-4-blocking Abs overcome the protective effects of c-Maf overexpression on colitis activity in vivo.



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  		FIGURE 4. Neutralizing anti-IL-4 Abs prevent the inability of c-Maf transgenic T cells to induce colitis activity. A, Splenic CD4+CD62L+ T cells from wild-type or c-Maf transgenic mice were isolated and injected into RAG-1-deficient mice, followed by monitoring of the body weight at indicated time points. Mice reconstituted with T cells from c-Maf transgenic mice received either anti-IL-4 Abs or control rat Abs, as described in Materials and Methods. Data represent mean values ± SEM. B, Macroscopic evidence of colitis in RAG-1-deficient mice reconstituted with CD4+CD62L+ T cells from wild-type mice. Little colitis activity was noted in RAG-1-deficient mice reconstituted with CD4+CD62L+ T cells from c-Maf transgenic mice. Anti-IL-4 treatment prevented the effects of c-Maf overexpression on colitis activity. One representative experiment is shown.

 
CD4+CD62L– memory T cells from c-Maf transgenic mice induce colitis and augment colitis activity in CD4+CD62L– T cell-reconstituted RAG-1 knockout mice

Because the above studies suggested a potential regulatory role of c-Maf in T-bet-expressing Th1 memory cells, we tested in subsequent studies the potential capacity of CD4+CD62L– memory T lymphocytes from wild-type and c-Maf transgenic mice to modulate colitis activity induced by naive CD4+CD62L+ T cells from wild-type mice. Accordingly, we determined the potential of CD62L–CD4+ T cells from c-Maf transgenic or wild-type control mice to modulate inflammatory colitis symptoms induced by CD4+CD62L+ in this transfer model using clinical and histopathologic criteria. Therefore, CD4+CD62L– memory T cells from wild-type or c-Maf transgenic mice were transferred together with naive CD4+CD62L+ T cells from wild-type mice into syngenic RAG-1-deficient mice, followed by analysis of colitis scores as well as histologic analyses (Fig. 5). Interestingly, mice reconstituted with CD4+CD62L+ T cells from wild-type mice plus CD4+CD62L– T from c-Maf transgenic mice showed a very severe colitis activity that was much more pronounced than in mice reconstituted with CD4+CD62L+ T cells plus CD4+CD62L– T cells from wild-type mice. This finding was not associated with differences in IL-10 or TGF-{beta} production by isolated LPMC (Fig. 5C), suggesting that the observed differences are not related to changes in the regulatory capacity of c-Maf-overexpressing T cells. Furthermore, this difference was not related to changes in IL-4 production by CD4+CD62L– T cells, because the administration of neutralizing anti-IL-4 Abs had no effects on the potential of c-Maf transgenic CD4+CD62L– T cells to augment Th1-mediated colitis in three independent experiments (data not shown). Similarly to the situation in CD in humans, however, T-bet/c-maf double-positive T cells were detected in the lamina propria of RAG-1 knockout mice reconstituted with CD4+CD62L+ T cells from wild-type mice plus CD4+CD62L– memory T cells from c-Maf transgenic mice (Fig. 5D). Furthermore, a marked accumulation of CD4+CD25+ T lymphocytes was noted in the lamina propria of these reconstituted animals (Fig. 5E), suggesting that c-Maf regulates CD25 expression in T-bet+ lamina propria Th1 cells in experimental colitis in vivo. We thus tested in a final series of experiments the capacity of c-Maf-overexpressing CD4+CD62L– T cells to induce colitis on their own. Indeed, as shown in Fig. 5F, transfer of CD4+CD62L– c-Maf transgenic T cells induced diarrhea, weight loss, and colitis, although such changes were noted at a much later time point upon reconstitution as compared with mice given CD4+CD62L+ T cells from wild-type mice. This finding was associated with increased levels of IFN-{gamma} in the lamina propria of colitic mice (Fig. 5G), suggesting that CD4+CD62L– c-Maf transgenic cells are capable of inducing Th1-mediated colitis in vivo.



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  		FIGURE 5. Cotransfer of CD4+CD62L– T cells from c-Maf transgenic mice causes increased colitis activity. A, Splenic CD4+CD62L+ T cells from wild-type mice were isolated, mixed together with CD4+CD62L– T cells from either wild-type or c-Maf transgenic mice, and then injected into RAG-1-deficient mice, followed by monitoring of the body weight at indicated time points. One representative experiment is shown. The ratio between CD62L– and CD62L+ T cells (see Materials and Methods) was determined in pilot experiments (data not shown) and kept low to avoid potential protective effects of regulatory T cells. Mice reconstituted with CD62L+ T cells from wild-type mice and CD62L– T cells from c-Maf transgenic mice developed severe early colitis symptoms as compared with mice given CD62L+ T cells plus CD62L– T cells from wild-type mice. B, Analysis of colon pathology in mice given CD62L+ T cells plus CD62L– T cells from wild-type mice (left panel) vs mice reconstituted with CD62L+ T cells from wild-type mice plus CD62L– T cells from c-Maf transgenic mice. C, IL-10 and TGF-{beta} production by isolated LPMC from both groups. No significant differences were noted. D, Detection of c-Maf/T-bet double-positive cells (yellow) in RAG-1 knockout mice reconstituted with CD62L+ T cells from wild-type mice plus CD62L– T cells from c-Maf transgenic mice. Samples without primary Abs served as negative control (right panel). E, Immunohistochemical analysis of colonic tissue in both groups. Consecutive colon sections were incubated with CD4 and CD25 Abs, followed by incubation with a biotinylated secondary IgG Ab and streptavidin-HRP. Staining was made with tyramide-Cy3, and the nuclei were counterstained with DAPI. A massive induction of CD4+CD25+ lamina propria T cells was noted in mice reconstituted with CD62L+ T cells from wild-type mice plus CD62L– T cells from c-Maf transgenic mice (wt/c-Maf) as compared with mice given CD62L+ and CD62L– T cells from wild-type mice (wt/wt). F, CD4+CD62L– T cells from c-Maf transgenic, but not wild-type mice are capable of inducing colitis in reconstituted mice with delayed onset. Five mice per group were reconstituted with CD4+CD62L+ T cells or CD4+CD62L– T cells from wild-type or c-Maf transgenic mice, followed by analysis of body weight. The initial body weight before reconstitution was defined as 100%. Whereas transfer of CD4+CD62L– T cells from wild-type mice failed to induce diarrhea and weight loss, transfer of CD4+CD62L– T cells from c-Maf transgenic mice caused diarrhea and weight loss. However, such weight loss was observed with delayed kinetics as compared with mice reconstituted with CD4+CD62L+ T cells from wild-type mice. G, Analysis of cytokine levels in the colon of reconstituted mice by RT-PCR. Transfer of CD4+CD62L– T cells from c-Maf transgenic mice resulted in higher IFN-{gamma} levels in colonic tissue as compared with mice given CD4+CD62L+ T cells from c-Maf transgenic mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inappropriate CD4+ T cell responses against luminal bacterial Ags are considered to cause the two major forms of IBD in humans: UC and CD. Although CD is clearly associated with Th1 T lymphocytes, showing evidence for Th1 cytokine signaling and Th1 cytokine production (9, 16, 17, 27), we found in the present study that lamina propria T lymphocytes in this disease unexpectedly expressed the transcription factor c-Maf that has been associated with Th2 T cells in the periphery (29). This finding led us to further investigate the functional role of c-Maf in naive and memory T cells in an animal model of CD mediated by adoptive transfer of CD4+ T lymphocytes. Whereas c-Maf transgenic CD4+CD62L+ T cells showed an inability to cause colitis and protective effects on colitis activity induced by wild-type T cells in cotransfer experiments, cotransfer of CD4+CD62L– T cells from c-Maf transgenic, but not wild-type mice augmented colitis induced by CD4+CD62L+ T cells from wild-type mice in an IL-4-independent way. Our results therefore reveal a novel pathogenic role of c-Maf in memory T cells in colitis. Taken together with the increased expression of c-Maf in T-bet+ memory T cells in the gut in CD, our data thus suggest that modulation of c-Maf function in memory T cells might be a target for therapeutic interventions in IBD.

Naive T cells require three key signals to differentiate into Th1 and Th2 effector T cells, and various studies have shown that lamina propria T cells respond differently to these signals than peripheral T cells (2, 5, 11, 33, 37, 38, 39, 40, 41). For instance, the first differentiation signal for T cells delivered by the TCR complex induces stronger proliferation of T lymphocytes in the periphery as compared with lymphocytes in the Ag-rich environment of the gut, because normal lamina propria T cells do not proliferate well on TCR/CD3 stimulation in comparison with lymphocytes in the periphery (42). This finding may contribute to the well-known hyporesponsiveness of mucosal T cells in the normal gut (43, 44, 45). Furthermore, the second signal necessary for T cell polarization delivered by costimulatory molecules on APCs via the CD2 and CD28 surface molecules may result in enhanced proliferation and cytokine production of mucosal T cells as compared with peripheral T cells (2, 43). The third differentiation signal is delivered by cytokines, and T cell responses to cytokines may differ between peripheral and mucosal T cells as well, because most T cells in the gut represent memory T lymphocytes (5) that critically depend on effector cytokines such as TNF and IL-6 for their activation and survival (17, 35). Finally, the data in the present work suggest that lamina propria memory Th1 cells may express transcription factors that are not found in Th1 T cells in the periphery, suggesting that lamina propria T cells have unique properties in terms of cytokine signaling. Specifically, we detected c-Maf in T-bet-expressing Th1 cells in CD and Th1-mediated experimental colitis. To our knowledge, this is the first report on c-maf expression in Th1 cells suggesting a novel and potentially unique role of c-Maf in lamina propria Th1 T cells. These findings led us to investigate the functional role of c-maf in memory T cells in the gut. Time course studies of c-Maf expression in Th1-mediated experimental colitis showed an increased expression of c-Maf in lamina propria cells that was correlated with clinical and histological signs of colitis. To obtain further information on the functional role of c-Maf in colonic inflammation, we then performed experiments with genetically altered strains of mice overexpressing c-Maf.

In studies on the role of c-Maf in naive T cells, it was found that transfer of c-Maf-overexpressing naive CD4+CD62L+ T cells failed to induce Th1-mediated colitis in immunodeficient RAG-1-deficient mice. This finding was shown to be mediated by the effects of c-Maf on IL-4 production, because the protective effect of c-Maf overexpression on CD4+CD62L+ T cell-induced colitis could be suppressed by neutralizing IL-4 Abs. Presumably, IL-4 production by such transferred c-Maf transgenic T cells occurs upon activation by bacterial Ags in the colon and suppresses local Th1 differentiation. Furthermore, we observed that such c-Maf transgenic cells prevent colitis induced by CD4+CD62L+ T cells from wild-type mice, suggesting a protective role of c-Maf in naive T cells for the development of Th1-mediated colitis. However, chronic active CD is known to be associated with CD45RO memory T cells, and CD45RA naive T cells are present in blood vessels rather than in the lamina propria in chronic Crohn's colitis (46). Taken together, this suggests that c-Maf expression in naive T cells plays a protective role in the onset rather than in the chronic phase of colitis and that this effect is due to its effects on IL-4 production in early T cell differentiation and blockade of Th1 differentiation by mucosal T cells. Consistent with this hypothesis, early, but not late lesions in CD are known to contain more naive T cells (5) and have been shown to contain IL-4 (47).

In contrast to the above findings on naive T cells, we observed that cotransfer of c-Maf transgenic memory CD4+CD62L– T cells augmented colitis activity induced by CD4+CD62L+ wild-type T cells. We did not find any evidence that c-Maf modulated IL-10 or TGF-{beta} production and regulatory T cell function in lamina propria T cells, as lamina propria cells from mice reconstituted with c-Maf transgenic T cells produced equal amounts of these known anti-inflammatory cytokines as cells from mice given wild-type T cells. Furthermore, the above effect was not mediated by the regulatory effects of c-Maf on IL-4 production in T cells, because neutralizing anti-IL-4 Abs failed to suppress the increased colitis activity seen upon transfer of c-Maf-overexpressing CD62L–CD4+ T cells in several independent experiments. Although c-Maf is present in memory Th1 cells in CD, it is apparently not sufficient to induce Th2 cytokine production in these cells, because CD lamina propria T cells are known to produce lower amounts of IL-4 than T cells from control patients (16). Consistently, Ho et al. (29, 31) found that forced expression of c-Maf in peripheral Th1 cells using transfection methods is not sufficient to induce Th2 cytokine production. Therefore, the colitis-augmenting function of c-Maf in mucosal Th1 cells is IL-4 independent. However, a marked accumulation of CD4+CD25+ T lymphocytes in the lamina propria of reconstituted mice was noted upon transfer of c-maf transgenic CD4+CD62L– T cells. Similarly to the above situation in CD in humans, these cells coexpressed c-Maf and T-bet, suggesting that c-Maf expression induces CD25 expression in memory Th1 cells in the gut. This finding strongly supports the recent observation that c-Maf controls CD25 expression and T cell activation in T cell populations from the periphery (34). Therefore, our data are consistent with a model in which c-maf expression in T-bet-expressing Th1 memory cells controls CD25 expression and subsequently Th1 T cell activation in experimental colitis. This concept would explain the pathogenic potential of c-maf-expressing memory T cells in adoptive transfer studies as well as the high frequency of CD25+ TCR{alpha}{beta} T cells in lesional CD tissue (48) that correlates with disease severity (49).

Taken together, these data define a critical and novel role for c-Maf in regulating colitis activity in vivo. Its functional role, however, strongly depends on the stage of T cell maturation. Although overexpression of c-Maf in naive T cells fails to induce Th1-mediated experimental colitis in an IL-4-dependent pathway, overexpression of c-Maf in memory T cells augments such colitis via an IL-4-independent pathway. Because CD in humans is known to be associated with Th1 cells in the lamina propria and because these memory T cells express large amounts of T-bet and c-Maf, our data suggest that targeting c-Maf expression in lamina propria T cells in CD might be an attractive approach to modulate colonic inflammation in this disease.


   Acknowledgments
 
We thank L. H. Glimcher (Harvard Medical School) for her generous support of this study; E. Schmitt and A. Hobel (Institute of Immunology, University of Mainz) for advice with regard to the Ab purification; and B. Bartsch (I. Medical Clinic, University of Mainz) for technical help with the staining of cryosections.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 M.F.N. was supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich 548, and the Innovationsstiftung Rheinland-Pfalz, Germany. Back

2 Address correspondence and reprint requests to Dr. Markus F. Neurath, Laboratory of Immunology, I. Medical Clinic, University of Mainz, Germany, 55131 Mainz, Langenbeckstrasse 1. E-mail address: neurath{at}1-med.klinik.uni-mainz.de Back

3 Abbreviations used in this paper: CD, Crohn’s disease; DAB, diaminobenzidine; DAPI, 4',6'-diamidino-2-phenylindole; IBD, inflammatory bowel disease; LPMC, lamina propria mononuclear cell; UC, ulcerative colitis. Back

Received for publication November 17, 2003. Accepted for publication June 10, 2004.


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