The CREB/CRTC2 pathway modulates autoimmune disease by promoting Th17 differentiation

Abstract

Following their activation in response to inflammatory signals, innate immune cells secrete T-cell-polarizing cytokines that promote the differentiation of naive CD4 T cells into T helper (Th) cell subsets. Among these, Th17 cells play a prominent role in the development of a number of autoimmune diseases. Although regarded primarily as an immunosuppressant signal, cAMP has been found to mediate pro-inflammatory effects of macrophage-derived prostaglandin E2 (PGE2) on Th17 cells. Here we show that PGE2 enhances Th17 cell differentiation via the activation of the CREB co-activator CRTC2. Following its dephosphorylation, CRTC2 stimulates the expression of the cytokines IL-17A and IL-17F by binding to CREB over both promoters. CRTC2-mutant mice have decreased Th17 cell numbers, and they are protected from experimental autoimmune encephalitis, a model for multiple sclerosis. Our results suggest that small molecule inhibitors of CRTC2 may provide therapeutic benefit to individuals with autoimmune disease.

Figure 1. Decreased Th17 differentiation in CRTC2-mutant mice

(a and b) Wild type and CRTC2 mutant naïve CD4 T cells skewed towards Th1, Th2, Treg, and Th17 cells. Cytokine levels determined by flow cytometry. Plots are gated on live CD4 T cells. Number in each quadrant indicates cell frequency. Data are representative of 3 experiments. Bar graphs represent the mean of percentage (right) +/− SEM, n = 3, from 3 independent experiments with similar results. (c) Decrease in IL-17A and IL-17F secreting Th17 cells from CRTC2-mutant or wild type littermates under Th17 differentiation conditions. IL-17A and IL-17F levels determined by flow cytometry. The electronic gate indicates the frequency of IL-17A (top) or IL-17F (bottom) CD4 T cells. Data are representative of 3 experiments. Bar graphs represent the mean percentage (right) +/− SEM, n = 3, from 3 independent experiments with similar results. (d) mRNA amounts for IL-17A,IL-17F, and NR4A2 in CRTC2-mutant T cells under Th17 differentiation conditions. Treatment with PGE₂ indicated. Data represent the mean +/− SEM2, n = 3, from 3 independent experiments with similar results. (e) Immunoblot showing effect of PGE₂ and forskolin (FSK) on CRTC2 dephosphorylation and CREB phosphorylation in CD4 T cells under Th17 differentiation conditions. Statistical analysis were performed with unpaired Student's t-test. Differences were considered statistically significant at P < 0.05 (* P < 0.05; ** P < 0.005 and *** P < 0.0005).

Figure 2. CREB and CRTC2 regulate the expression of IL-17A and IL-17F

(a) Location of CRE sites on the mouse IL-17A and IL-17F promoters. (b) Effect of retrovirally encoded dominant negative CREB polypeptide (ACREB) on Th17 differentiation. IL-17A levels were determined by flow cytometry. The number in each quadrant indicates the frequency of cells. Data are representative of 3 experiments. Bar graphs represent the mean of percentage (right) +/− SEM, n = 3, from 3 independent experiments. (c) Effect of phosphorylation-defective constitutively active CRTC2 on expression of IL-17A and IL-17F reporters in 293T cells. Effect of Forskolin (FSK) shown. Bar graphs represent the mean of relative luciferase activity relative to co-transfected RSV β-gal reporter +/− SEM, n = 3, from 3 independent experiments. (d) Co-expression of A-CREB and constitutively active CRTC2 (CRTC2AA) on expression of IL-17A and IL-17F reporters in 293T cells. Bar graphs represent the mean of relative luciferase activity relative to co-transfected RSV β-gal reporter +/− SEM, n = 3, from 3 independent experiments. (e) Chromatin Immunoprecipitation (ChIP) assay of sorted naïve CD4 T cells cultured under Th17 polarization conditions. Effect of PGE₂ on recruitment of CRTC2 to IL-17A and IL-17F promoters shown. Bar graphs represent the mean +/− SEM, n = 3, from 3 independent experiments. Statistical analysis were performed with unpaired Student's t-test. Differences were considered statistically significant at P < 0.05 (* P < 0.05; ** P < 0.005 and *** P < 0.0005).

Figure 3. CRTC2-mutant mice are resistant to EAE

(a) Clinical scores of wild type and CRTC2-mutant littermates following MOG_35-55 immunization. Wild type (n = 8) and CRTC2-mutant (n = 10) animals received 400 μg of MOG_35-55 peptide plus Complete Freund's Adjuvant (CFA) on days 0 and 7 and 250 ng pertussis toxin on days 0 and 2. Each line represents the average score of the mice. Similar results were obtained in three independent studies. Data represent the mean +/− SEM. (b) Decreased Th17 cells in CRTC2-mutant mice following MOG_35-55 immunization. T cells were isolated from pooled brain and spinal cord samples from mice in panel a on day 28 as described in methods. T cells were then stimulated with PMA and Ionomycin for 5 hours. IL-17A (top) and IFN-γ (bottom) levels were determined by flow cytometry. Histograms are gated on live CD4 T cells. The electronic gate indicates the frequency of IL-17A+ (top) or IFN-γ (bottom) CD4 T cells. Bar graphs represent the mean of percentage (left) and cell number (right) +/− SEM; wild type (n = 8) and CRTC2 mutant (n = 10) animals. (c) The spinal cord was dissected at day 28 and stained with H&E (top) or Luxol fast blue (LFB) (bottom), and both cell infiltration and demyelination were scored. Arrow indicates infiltrating cells. (scale bars, 100 μm). Bar graphs represent the mean +/− SEM; wild type (n = 4) and CRTC2 mutant (n = 5) animals. (d) Decreased Th17 cells in draining lymph nodes of CRTC2-mutant mice 7 days after MOG_35-55 immunization. T cells were stimulated with PMA and Ionomycin for 5 hours. Cytokine levels determined by flow cytometry. Plots are gated on live CD4 T cells. Number in each quadrant indicates cell frequency. Bar graphs represent the mean of percentage +/− SEM; wild type (n = 5) and CRTC2 mutant (n = 5) animals. Similar results were obtained in two independent studies. Statistical analysis were performed with unpaired Student's t-test. Differences were considered statistically significant at P < 0.05 (* P < 0.05; ** P < 0.005 and *** P < 0.0005).

Figure 4. Decreased Th17 differentiation and EAE in Crtc2^ΔCD4 mice

(a) Immunoblot blot showing CRTC2 protein amounts in control and Crtc2^ΔCD4 Th17 cells. (b and c) Relative differentiation of Th17 cells from CRTC2 floxed^CD4cre or control (CRTC2fl/fl) CD4+ T cells. Frequency of IL-17A (top) and IL-17F (bottom) secreting Th17 cells from CRTC2-deficient or CRTC2fl/fl T cells under Th17 differentiation conditions. Data are representative of 3 experiments with similar results. Bar graphs represent the mean of percentage (left) and cell number (right) +/− SEM, n = 3, from 3 independent experiments. (d) il17a and il17f mRNA amounts from CRTC2floxed^CD4cre T cells under Th17 differentiation conditions. Cells treated with vehicle or PGE2 indicated. Data represent the mean +/− SEM, n = 3, from 3 independent experiments with similar results. (d) Crtc2^ΔCD4 mice are resistant to EAE. CRTC2fl/fl (n = 6) and Crtc2^ΔCD4 (n = 9) mice received 400 μg of MOG35-55 peptide plus CFA on days 0 and 7 and 200 ng pertussis toxin on days 0 and 2. Mice were scored as previously described in 3a. Each line represents the average score of the mice. Similar results were obtained in two independent studies. Data represent the mean +/− SEM, from 2 independent experiments with similar results. (e) Th17 cell numbers in the CNS of CRTC2fl/fl or Crtc2^ΔCD4 mice following MOG_35-55 immunization. Histograms are gated on live CD4+ T cells. The electronic gate indicates the frequency of IL-17A+ (top) or IFN-γ (bottom) levels. Bar graphs represent the mean of percentage (left) and cell number (right) +/− SEM; CRTC2 fl/fl (n = 6) and Crtc2^ΔCD4 (n = 9). Statistical analysis were performed with unpaired Student's t-test. Differences were considered statistically significant at P < 0.05 (* P < 0.05; ** P < 0.005 and *** P < 0.0005).