CREMα overexpression decreases IL-2 production, induces a TH17 phenotype and accelerates autoimmunity
Year: 2012
Type: article
Abstract: Dear Editor, External cytokines produced by cells of the innate immune system induce the differentiation of CD4+ T cells into helper T cell subsets with distinct functions and cytokine profiles. Apart from TH1 and TH2 cells a third subset of helper T cells has been described, which is characterized by increased expression of IL-17A, IL-17F, IL-21 and IL-22 (TH17 cells). TH17 cells are involved in the clearance of infectious agents and contribute to the expression of tissue damage in patients with autoimmune diseases including multiple sclerosis and systemic lupus erythematosus (SLE) (Korn et al., 2009). External cytokines like IL-6, IL-1β, IL-23 and TGF-β induce the generation of IL-17-producing cells, while IL-21 acts to perpetuate the production of IL-17 in T cells (Korn et al., 2007). Vice versa, IL-2 is able to suppress IL-17 production by orchestrating the balance between STAT5 and STAT3 phosphorylation and binding to the IL-17A promoter (Yang et al., 2011). The above-mentioned cytokines activate several transcription factors such as RORγt, RORα and aryl hydrocarbon receptor as lineage transcription factors as well as IRF-4, BAFF, and T-bet, which are involved in the control of IL-17 expression (Korn et al., 2009). Cyclic AMP responsive element modulator (CREM) belongs to a family of transcription factors that also includes CREB and ATFs. They share high structural similarities characterized by a basic region and leucine zipper domains (Montminy, 1997) and bind to palindromic DNA sequences (TGACGTCA) as homo- or heterodimeric complexes (Sassone-Corsi, 1995). CREM proteins are encoded by a multiexonic gene that yields several isoforms through alternative splicing. The exon composition determines the functional propensities of the various isoforms as transcriptional activators or repressors. Since the α-isoform (CREMα) lacks the transactivation domains present in other isoforms, thus far it is considered an inhibitor of transcriptional activation (Asahara et al., 2001). Cellular levels of CREMα mRNA and protein are higher in T cells from patients with SLE than in healthy individuals (Juang et al., 2005). Likewise, CREMα shows increased binding to the −180 site of the IL-2 promoter in patients with SLE (Solomou et al., 2001) and thereby represses IL-2 production (Tenbrock et al., 2002). Apart from IL-2, increased CREMα levels account for decreased expression of multiple other genes in T cells from patients with SLE such as CD3 ζ chain (Tenbrock et al., 2005) and c-fos (Kyttaris et al., 2004). Thus, CREMα overexpression has been proved to be a central mediator responsible for several of the defects known to alter the phenotype of T cells from patients with SLE. In order to determine whether CREMα independently contributes to the expression of autoimmunity in vivo, we generated a transgenic mouse to express CREMα in T cells under control of a CD2 promoter (Zhumabekov et al., 1995). Expectedly, CREMα overexpression in T cells resulted in enhanced binding of CREM to the IL-2 promoter (Supplementary Figure S1E), decreased IL-2 production and T cell proliferation, which is not related to enhanced apoptosis in these cells (Figure 1A and Supplementary Figure S1C and Supplementary Data). These results confirm previous studies performed in T cells from patients with SLE and underscore the importance of CREMα in suppressing IL-2 production (Tenbrock et al., 2002). Although CREMα affects IL-2 production, the expression of CD4+CD25+Foxp3+ regulatory T cells as well as other T cell subpopulations did not differ between CREMα transgenic and wild-type mice (Supplementary Figure S2). Unexpectedly, an enhanced production of IL-17 and the TH17 lineage transcription factor RORγt was noticed after stimulation of isolated CD4+ T cells with anti-CD3 and anti-CD28 in the absence of TH17-inducing cytokines IL-6 or IL-21 for 4 days (Figure 1B and Supplementary Figure S3A). This could not be reversed by the addition of external IL-2 in vitro (data not shown), and the exact mechanism is currently under investigation. Vice versa CREM−/− CD4+ T cells display decreased expression of IL-17-producing T cells when differentiated in vitro into TH17 direction (Supplementary Figure S4). CREM binds directly to the IL-17A promoter as identified by ChIP assays (Figure 1C) resulting in the activation of the IL-17A promoter as measured by luciferase reporter assays (Figure 1D) which was unexpected in a way that CREMα usually acts as a transcriptional repressor. Moreover, IL-17 expression is sustained by an enhanced expression of IL-21 in the CREM transgenic mice, since treatment with an IL-21RFc fragment substantially decreased IL-17 and RORγt expression (Supplementary Figure S5). T cell-specific CREMα overexpression decreases IL-2 transcription and enhances IL-17 expression in vitro and in vivo resulting in accelerated inflammation and autoimmunity. (A) Isolated T cells of CREMα transgenic (CREM tg) and wild-type mice (wt) were stimulated with anti-CD3 and anti-CD28 Abs for 5 days. Supernatants were used for cytometric beads assay (CBA) to detect IL-2 protein levels. Bars show n-fold levels compared with unstimulated cells (n= 4 experiments with 3 animals each, mean ± SD, *P<0.05, **P<0.01, ***P<0.001 vs control). (B) CD4+ T cells were stimulated with anti-CD3 and anti-CD28 Abs for 4 days and mRNA was extracted. Real-time PCR was performed with primers specific for IL-17 and RORγt (n=3 experiments with 2 animals each, mean ± SD). (C) Splenic cells were isolated and a ChIP assay was performed with anti-CREM antibody and IL-17A promoter-specific primers by real-time PCR, bars show n-fold levels of CREM binding in CREM transgenic T cells compared with wild-type T cells (set as 1) related to an unspecific control antibody (n=4 experiments with 2 animals each, mean ± SD). (D) EL4 T cells were transfected with a 153-bp spanning mouse IL-17A promoter luciferase construct and either an empty vector or a CREMα expression plasmid, graph shows relative luciferase activity compared with the empty vector plasmid (n= 4 experiments). (E) Contact dermatitis. Mice were sensitized with DNFB on day 0 on the abdominal surface and challenged on day 6 by painting 10 µl of 0.3% DNFB in acetone/olive oil 4:1 on the ventral and dorsal surfaces of the left ear. CHS was determined by the degree of ear swelling measured with a micrometer of the hapten-exposed ear minus that of the vehicle-treated contralateral ear up to 72 h after challenge. Y-axis shows ear swelling from transgenic and wild-type mice (n= 3 experiments with 5 animals each). (F) Twenty-four hours after induction of CHS mice were sacrificed, mRNA was extracted from whole tissue lysates of affected and unaffected ears and real-time PCR was performed with indicated primers (n= 3 experiments with 2 animals each, mean ± SD). Fold change in mRNA levels of indicated genes in affected ear compared with unaffected contra-lateral ear. (G) A genetic deletion of CD95 (fas−/−) was introduced into CREMα transgenic and wild-type animals. Animals were checked regularly for clinical signs of lymphadenopathy. Lymphadenopathy in the fas−/− CREM transgenic animals occurred as early as 5 weeks of age. Animals at the picture were sacrificed at 16 weeks of age. (H) fas−/− CREMα transgenic and fas−/− wild-type mice were sacrificed at 32 weeks of age and dsDNA antibodies were measured in serum by ELISA (n=6 fas−/− wt and 8 fas−/− CREM tg animals, mean ± SD). To explore the effect of CREMα overexpression in vivo, we induced a contact dermatitis in the CREMα transgenic and wild-type mice and found enhanced ear swelling as a marker of enhanced inflammation in the CREM transgenic mice (Figure 1E) and enhanced in situ expression of the TH17 cytokines IL-17, IL-21, IL-22 and IL-23 in the inflamed ears of CREMα transgenic animals as compared with expression levels in the untreated contralateral ear (Figure 1F and Supplementary Figure S3B). However, despite the fact that T cells of mice overexpressing CREMα show phenotypic similarities to human SLE T cells and express pro-inflammatory cytokines when stimulated in vitro, the mice remained healthy without signs of autoimmunity when followed by up to 52 weeks. Thus, overexpression of CREMα in T cells is insufficient to induce an SLE phenotype by itself. We therefore introduced a genetic deletion of CD95 (fas−/−) into the CREMα transgenic and the wild-type mice. fas−/− CREM transgenic mice display massively enhanced lymphadenopathy (Figure 1G), an enhanced IL-17 production compared with the fas−/− wild-type mice (Supplementary Figure S3C) and higher titers of anti-DNA autoantibodies compared with the wild type fas−/− mice (Figure 1H). These final experiments suggest a critical amplifying role of CREMα in autoimmunity and underscore the relevance of this transcription factor with regard to SLE. [Supplementary material is available at Journal of Molecular Cell Biology online. This work was supported by IZKF Aachen (E-2) and by DFG (TE 339/4-3) to Klaus Tenbrock.]
DOI: 10.1093/jmcb/mjs004
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2144568386