Cyclic AMP-Responsive Element-Binding Protein (CREB) is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

Abstract

The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg) cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

Keywords: Autoimmune diseases; CREB; Th17 cells; Treg cells.

Fig. 1

CREB-deficient mice are resistant to EAE. CREB^fl/fl (WT, n = 6) and CREB-CD4KO (KO, n = 7) mice were immunized twice with MOG35-55 for EAE induction. A, Mean clinical scores are shown versus days after second MOG immunization. B, Intracellular staining of IL-17 and IFN-γ in CNS of EAE mice (Gated on CD4⁺ cells). C, Cellularity data in CNS. D, Intracellular staining of FOXP3 in CNS, spleen, and dLN (healthy = never developed EAE diseases; Dis = developed EAE diseases; Recov = recovered from EAE diseases). E, Statistics of FOXP3 staining results. The experiment was repeated twice with consistent results.

Fig. 2

CREB expression in Th17 but not Treg cells is required for EAE development. A, EAE was induced in CREB^fl/fl (WT, n = 6) and CREB-FOXP3KO (KO, n = 5). B, EAE was induced in RAG1 KO mice transferred with WT 2d2 naïve T cells (n = 5) or CREB-deficient 2d2 naïve T cells (n = 5). C, EAE was induced in CREB^fl/fl (WT, n = 8) and CREB^fl/flxIL-17F-Cre mice (n = 8). Left column: EAE disease scores; Middle column: IL-17 and IFN-γ staining in CNS (Gated on CD4⁺); Right column: statistic data of IL-17 and IFN-γ staining. All the experiments were repeated twice with consistent results.

Fig. 3

CREB controls Th17 cell differentiation in vitro. A, Th17 cell differentiation was performed under optimal conditions (TGF-β + IL-6 + IL-β + IL-23) for 6 days, using WT and CREB-IL-17FKO naïve CD4 + T cells, and then restimulated and analyzed for IL-17 and IL-17F staining. The differentiation was performed for > 5 times with consistent results. B, Clustering analysis of CREB regulated genes vs published microarray data of Th17 cells generated under different in vitro culture conditions (b6 = IL-1β + IL-6; b623 = IL-1β + IL-6 + IL-23; T16 = TGF-β + IL-6; T1623 = TGF-β + IL-6 + IL-23; T36 = TGF-β3 + IL-6; T3623 = TGF-β3 + IL-6 + IL-23). C, Left: heat map of selected CREB regulated genes; Right: validated realtime PCR results.

Fig. 4

CREB regulates ROR-dependent IL-17 expression. A, The luciferase activities of the PGL3, IL-17 promoter (17p)-PGL3, CNS2-17p-PGL3, CNS2^RORmu1/2-17p-PGL3 (RORmu1/2) (in which the two RORE sites in CNS2 were mutated), CNS2-17p-CREB^mu1/4 (CREBmu1/4) (in which the two CRE sites in both CNS2 and Il17p were mutated) reporter constructs were performed in EL4 cells in the absence or presence of RORα and RORγt. B, The luciferase activities of the PGL3, Il17p, CNS2-Il17p, CNS2-17p-CREB^mu1/2 (CREBmu1/2) (in which the two CRE sites in CNS2 were mutated), and CREBmu1/4 reporter constructs were performed in Th17 cells. C, ChIP assay was performed in Th17 cells with antibodies against CREB and pCREB, and realtime PCR was used to quantify the relative bindings to the Il17-17f gene locus. The assays were repeated twice with consistent results.

Fig. 5

CREB regulates the survival of iTreg cells. WT and CREB-deficient (KO) naïve CD4⁺ T cells were induced to iTreg cells in the presence of TGF-β with or without exogenous IL-2. A, The expression of FOXP3 was determined by flow cytometry. B, The viable cells were determined by SSC-/FSC-scattering. C, Cell apoptosis was determined by 7-AAD and Annexin V staining. The experiment was repeated three times with consistent results.

Fig. 6

CREB positively regulates p27 expression in iTreg cells. WT and CREB-CD4KO T naïve CD4⁺ T cells were induced to iTreg cells using TGF-β in the absent of exogenous IL-2, and the p27 expression was determined by western blotting (A) and real-time RT-PCR (B) at various time points. ChIP assay was performed in iTreg cells with antibodies against CREB and pCREB, and real-time RT-PCR was used to quantify the relative bindings to the FOXP3 and p27 gene loci. All the assays were repeated twice with consistent results.

Fig. 7

CREB is required for T cell-dependent colitis. WT and CREB-CD4KO naïve CD4 + T cells were first transferred into RAG1 KO mice, and then monitored for developing colitis in recipient mice. A, Percentage of weight loss. B, Staining data of FOXP3, IL-17 and IFN-γ expression in the spleen and mesenteric lymph nodes. C. Statistic data of (B). The results were repeated twice with similar results.