Role of the p38 MAPK/C/EBPβ Pathway in the Regulation of Phenotype and IL-10 and IL-12 Production by Tolerogenic Bone Marrow-Derived Dendritic Cells

Abstract

Dendritic cells (DCs) play a major role in innate and adaptive immunity and self-immune tolerance. Immunogenic versus tolerogenic DC functions are dictated by their levels of costimulatory molecules and their cytokine expression profile. The transcription factor C/EBPβ regulates the expression of several inflammatory genes in many cell types including macrophages. However, little is known regarding the role of C/EBPβ in tolerogenic versus immunogenic DCs functions. We have previously reported that bone marrow-derived DCs generated with GM-CSF (GM/DCs) acquire the signature of semi-mature tolerogenic IL-10-producing DCs as opposed to immunogenic DCs generated with GM-CSF and IL-4 (IL-4/DCs). Here, we show that tolerogenic GM/DCs exhibit higher levels of phosphorylation and enhanced DNA binding activity of C/EBPβ and CREB than immunogenic IL-4/DCs. We also show that the p38 MAPK/CREB axis and GSK3 play an important role in regulating C/EBPβ phosphorylation and DNA binding activity. Inhibition of p38 MAPK in GM/DCs resulted in a drastic decrease of C/EBPβ and CREB DNA binding activities, a reduction of their IL-10 production and an increase of their IL-12p70 production, a characteristic of immunogenic IL-4/DCs. We also present evidence that GSK3 inhibition in GM/DCs reduced C/EBPβ DNA binding activity and increased expression of costimulatory molecules in GM/DCs and their production of IL-10. Analysis of GM/DCs of C/EBPβ^-/- mice showed that C/EBPβ was essential to maintain the semimature phenotype and the production of IL-10 as well as low CD4⁺ T cell proliferation. Our results highlight the importance of the p38MAPK-C/EBPβ pathway in regulating phenotype and function of tolerogenic GM/DCs.

Keywords: CCAAT/enhancer-binding protein beta (C/EBPβ); IL-12); Interleukins 10 and 12 (IL-10; tolerogenic bone marrow-derived dendritic cells (BMDCs).

Figure 1

Increased C/EBPβ expression and DNA binding activity in LPS-stimulated GM/DCs. (A) Bone marrow-derived GM/DCs and IL-4/DCs were generated from NOD and BALB/c mice and left unstimulated or stimulated with LPS (1 μg/mL for 48 h). Nuclear extracts were subjected to EMSA assays to assess C/EBP binding activity. (B) Bone marrow-derived GM/DCs and IL-10/DCs (generated with GM-CSF + IL-4 + IL-10) from C57BL/6 and NOD mice were left unstimulated or stimulated with LPS (1 μg/mL for 48 h). Nuclear extracts were prepared and C/EBP binding activity was determined by EMSA assays (C) Supershift analyses were performed with nuclear extracts obtained from LPS-stimulated GM/DCs of NOD and BALB/c mice, as described in the Material and Methods section of the body of the text. (D) C/EBPβ DNA binding activity was monitored by EMSA, using nuclear extracts from LPS-stimulated GM/DCs and IL-4/DCs from NOD mice at the indicated times. (E) Western blot analysis of pC/EBPβ (Thr188) and C/EBPβ levels in cell lysates from LPS stimulated GM/DCs and IL-4/DCs. (F) Quantitative analysis of data in Figure 1E normalized to loading control (β-actin). Data are representative of a minimum of 2–3 independent experiments.

Figure 2

Role of p38 MAPK/CREB and GSK3b/CREB axis in the activation and DNA binding activity of C/EBPβ in GM/DCs. Bone marrow-derived GM/DCs were preincubated with inhibitors of p38 MAPK (SB203580, 10 μM), p38 MAPK (SB202190, 1 μM), GSK3 (SB 216763, 10 μM), or vehicle (DMSO) for 1 h before stimulation with LPS (1 μg/mL for 8 h). (A) C/EBPβ DNA binding activity in nuclear extracts were analyzed by EMSA. (B) Western blot analysis of the levels of phosphorylated (Ser129, Ser133) and total CREB in whole cell lysates at the indicated times (hours) after LPS stimulation (1 μg/mL). (C) CREB DNA binding activity in nuclear extracts were analyzed by EMSA. (D,E) Western blot analysis of the levels of phosphorylated (Thr188) and total C/EBPβ in whole cell lysates after 8 h of LPS stimulation (1 μg/mL). Data are representative of two independent experiments.

Figure 3

Role of p38 MAPK/CREB and GSK3β/CREB axis in regulating IL-10 and IL-12 in GM/DCs. Bone marrow-derived GM/DCs were preincubated for 1 h in the presence of a p38 MAPK (SB203580, 10 µM) or a GSK3 (SB216763, 10 µM) inhibitor or, vehicle (DMSO) before being exposed to LPS (1 μg/mL for 4 h or 12 h). Total RNA was isolated and the expression of IL-10 (A), IL-12p35 (B) and IL-12p40 (C) was determined by real-time PCR. Fold increases are calculated with reference to vehicle (DMSO) + LPS conditions. Data are representative of five or six independent experiments. In the case of cytokine release essays, GM/DCs were preincubated for 1 h with SB203580 (10 µM), SB216763 (10 µM) or, with vehicle (DMSO), before being exposed to LPS (1 μg/mL for 24 h). IL-10 (D) and IL-12p70 (E) production was quantified in cell supernatants by ELISA. Data are representative of three independent experiments. Results are shown as the mean ± SEM. The asterisks (*) correspond to p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***), one-way ANOVA with post hoc Bonferroni’s test.

Figure 4

Role of p38 MAPK/CREB and GSK3b/CREB axis in regulating GM/DCs maturation status. Bone marrow-derived GM/DCs were preincubated with vehicle (DMSO, Blue thin line) or (A) p38 MAPK inhibitor (SB203580. 10 µM, red line) or (B) LiCl (10 mM, red line) before being exposed to with LPS (1 μg/mL for 48 h). Cells were harvested and the expression of CD80, CD86, and CD40 molecules was analyzed by gating on CD11c⁺ cells using flow cytometry. (C) Bone marrow-derived IL-4/DC were left unstimulated (blue line) or were exposed to LPS (1 μg/mL for 48 h) (red line) and analyzed by flow cytometry. For all the panels, isotype controls are shown (dashed thin line). Expression levels of CD80, CD86, and CD40 cell surface markers are illustrated as mean fluorescence intensities (MFIs) are shown at right-hand sides of panels. Data are representative of two to four independent experiments. * p < 0.05, nonparametric unpaired Mann–Whitney test.

Figure 5

Effect of C/EBPβ deficiency on the maturation status of GM/DCs. Bone marrow-derived GM/DCs generated from C/EBPβ^+/+ mice (Blue line) and C/EBPβ^−/− mice (red line), were left unstimulated (A) or exposed to LPS (1 µg/mL for 48 h) (C) and were stained and analyzed for CD80, CD86, and CD40 expression on CD11c⁺ cells using flow cytometry. Dashed thin lines represent labeling with isotype control antibodies. (B and D) Expression levels of CD80, CD86, and CD40 cell surface markers on unstimulated (B) and LPS exposed (D) GM/DCs are illustrated as mean fluorescence intensities (MFIs). The results are representative of three independent experiments. * p < 0.05, nonparametric unpaired Mann—Whitney test.

Figure 6

Effect of C/EBPβ deficiency on gene expression and production of IL-10 and IL-12 by GM/DCs. Bone marrow-derived GM/DCs generated from C/EBPβ^−/− or C/EBPβ^+/+ mice were left unstimulated or were stimulated with 1 µg/mL of LPS for 4 h and 12 h. The expression of IL-10 (A), IL12p35 (B), IL-12p40 (C), and IL-6 (D) was determined by real-time PCR. Data are shown as fold increases with reference to unstimulated GM/DCs of C/EBPβ^+/+ (t = 0) and are representative of four independent experiments. For cytokines release, bone marrow-derived GM/DCs generated from C/EBPβ^−/− or C/EBPβ^+/+ mice were left unstimulated or stimulated with LPS (1 µg/mL for 24 h). Cell supernatants were collected and IL-10 (E), IL-12p70 (F), and IL-6 (G) production was analyzed by ELISA. Data are representative of three independent experiments and are shown as the mean ± SEM. The asterisks (*) correspond to p < 0.01 (**) and p < 0.001 (***), one-way ANOVA with post hoc Bonferroni’s test.

Figure 7

Elevated CD4⁺ T cell proliferation and IFNγ production induced by the p38 MAPK inhibitor-treated or C/EBPβ-deficient GM/DCs. (A) Proliferative response and IFNγ secretion by autoreactive BDC2.5-CD4⁺ T cells that had been cultured with GM/DCs treated with p38 MAPK inhibitor SB203580 or with vehicle (DMSO) and pulsed or not with BDC2.5-peptide. (B) Proliferative response and IFNγ secretion by naïve CD4⁺ T cells purified from C/EBPβ^+/+ littermate control mice that have been activated with anti-CD3 and anti-CD28 Abs in the presence of C/EBPβ^+/+ or C/EBPβ^−/− GM/DCs before (−) and after (+) LPS stimulation. Quantification of IFNγ released in the supernatant. Data are representative of three independent experiments and are shown as the mean ± SD. The asterisks (*) correspond to p < 0.01 (**), one-way ANOVA with post hoc Bonferroni’s test.