GM-CSF and IL-4 synergistically trigger dendritic cells to acquire retinoic acid-producing capacity

Abstract

Retinoic acid (RA) produced by intestinal dendritic cells (DCs) imprints gut-homing specificity on lymphocytes and enhances Foxp3(+) regulatory T-cell differentiation. The expression of aldehyde dehydrogenase (ALDH) 1A in these DCs is essential for the RA production. However, it remains unclear how the steady-state ALDH1A expression is induced under specific pathogen-free (SPF) conditions. Here, we found that bone marrow-derived dendritic cells (BM-DCs) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) expressed Aldh1a2, an isoform of Aldh1a, but that fms-related tyrosine kinase 3 ligand-generated BM-DCs did not. DCs from mesenteric lymph nodes (MLN) and Peyer's patches (PP) of normal SPF mice expressed ALDH1A2, but not the other known RA-producing enzymes. Employing a flow cytometric method, we detected ALDH activities in 10-30% of PP-DCs and MLN-DCs. They were CD11c(high)CD4(-/low)CD8alpha(intermediate)CD11b(-/low) F4/80(low/intermediate)CD45RB(low)CD86(high)MHC class II(high)B220(-)CD103(+). Equivalent levels of aldehyde dehydrogenase activity (ALDHact) and ALDH1A2 expression were induced synergistically by GM-CSF and IL-4 in splenic DCs in vitro. In BM-DCs, however, additional signals via Toll-like receptors or RA receptors were required for inducing the equivalent levels. The generated ALDH1A2(+) DCs triggered T cells to express gut-homing receptors or Foxp3. GM-CSF receptor-deficient or vitamin A-deficient mice exhibited marked reductions in the ALDHact in intestinal DCs and the T cell number in the intestinal lamina propria, whereas IL-4 receptor-mediated signals were dispensable. GM-CSF(+)CD11c(-)F4/80(+) cells existed constitutively in the intestinal tissues. The results suggest that GM-CSF and RA itself are pivotal among multiple microenvironment factors that enable intestinal DCs to produce RA.

Fig. 1.

MLN-DCs and PP-DCs express Aldh1a2 and possess ALDHact. (A) Expression of ALDH1A2 in CD11c⁻ or CD11c⁺ cells from MLN or SPL of B10.D2 mice was analyzed by immunoblotting. Data are representative of two independent experiments. (B) Expression of Aldh1a1, Aldh1a2, Aldh1a3, Aldh8a1, Cyp1b1, Cyp2j6 and Rplp0 as a loading control in CD11c⁻ or CD11c⁺ cells from MLN, PP or SPL was analyzed by RT-PCR. Data are representative of two independent experiments. (C) Expression of Aldh1a2 in CD103⁺ or CD103⁻ DCs from MLN or PP was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of three independent experiments. Statistical significance was determined by the one-way analysis of variance; ***P < 0.001. (D) Cells from MLN or PP were incubated with ALDEFLUOR, co-stained for CD11c and CD3ε, CD19, CD49b, F4/80 or Gr-1 and analyzed by flow cytometry. Pre-gated CD11c⁻ cells are shown in the dot plots except those for CD11c staining. Data are representative of two independent experiments. (E) MLN-DCs, PP-DCs or SPL-DCs were incubated with ALDEFLUOR in the presence (bottom) or absence (top) of the ALDH inhibitor DEAB and stained for CD11c expression. Numbers adjacent to gates indicate percentage of ALDHact^high cells (upper number) or ALDHact^intermediate cells (lower number). Data are representative of three independent experiments. (F) ALDEFLUOR-treated MLN-DCs were co-stained for CD11c and B220, CD4, CD8α, CD11b, CD45RB, CD86, F4/80, MHC class II or CD103 expression. ALDHact^high and ALDHact^{−/intermediate} cells are shown as red and blue dots, respectively. Data are representative of two independent experiments.

Fig. 2.

GM-CSF and IL-4 synergistically induce ALDH1A2 expression in DCs. (A) Expression of Aldh1a2 in GM-CSF-induced or Flt3L-induced BM-DCs was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of three independent experiments. Statistical significance was determined by the Student's t test; ***P < 0.001. (B) Expression of Aldh1a2 in Flt3L-induced BM-DCs (left) and SPL-DCs (right) cultured with graded concentrations of GM-CSF was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of three independent experiments. Statistical significance was determined by the one-way analysis of variance (ANOVA). ***P < 0.001 versus untreated. (C) Flt3L-induced BM-DCs were cultured with IL-3 (10 ng ml⁻¹), IL-4 (10 ng ml⁻¹), IL-5 (10 ng ml⁻¹), IL-6 (10 ng ml⁻¹), IL-10 (10 ng ml⁻¹), IL-12 (10 ng ml⁻¹), IL-13 (10 ng ml⁻¹), IFN-α (10⁴ U ml⁻¹), IFN-β (10⁴ U ml⁻¹), IFN-γ (10 ng ml⁻¹), TNF-α (10 ng ml⁻¹), TGF-β1 (10 ng ml⁻¹), TGF-β2 (10 ng ml⁻¹), TSLP (10 ng ml⁻¹), CX3CL1 (10 ng ml⁻¹), prostaglandin E₂ (PGE₂; 10⁻⁶ M) or leukotrien B₄ (LTB₄; 10⁻⁷ M). The expression of Aldh1a2 was assessed by semi-quantitative real-time PCR. The mRNA expression of treated cells is expressed as the ‘fold induction’ relative to that of untreated cells. Data are presented as mean ± SEM and are representative of two independent experiments. (D–F) Flt3L-induced BM-DCs (left) or SPL-DCs (right) were cultured with medium alone (none), GM-CSF (10 ng ml⁻¹), IL-4 (10 ng ml⁻¹) or GM-CSF and IL-4 (10 ng ml⁻¹ each). (D) Expression of Aldh1a2 was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of more than three independent experiments. Statistical significance was determined by the one-way ANOVA. ***P < 0.001 versus all samples. (E) The cultured DCs were incubated with ALDEFLUOR in the presence (solid lines) or absence (shaded histograms) of DEAB and were analyzed by flow cytometry. Numbers in histogram plots indicate percentage of ALDHact⁺ cells. Data are representative of more than three independent experiments. (F) Expression of ALDH1A2 in DCs cultured with GM-CSF and IL-4 was analyzed by immunoblotting. Numbers below immunoblot indicate the signal intensity of treated DCs. Data are representative of two independent experiments.

Fig. 3.

TLR-mediated maturation signals enhance Aldh1a2 expression and ALDHact in BM-DCs. Flt3L-induced BM-DCs (A–C) or SPL-DCs (D and E) were cultured with medium alone (none), sPGN (10 μg ml⁻¹), poly(I:C) (1 μg ml⁻¹), LPS (1 μg ml⁻¹), R837 (1 μg ml⁻¹) or CpG ODN 1826 (CpG; 1 μM) in the presence or absence of GM-CSF and/or IL-4 (10 ng ml⁻¹ each). (A and D) Expression of Aldh1a2 was assessed by semi-quantitative real-time PCR. The mRNA expression of treated cells is expressed as the ‘fold induction’ relative to that of untreated cells (cultured with medium alone without GM-CSF or IL-4). (B and E) DCs were cultured with medium alone or LPS in the presence or absence of GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and CD86 expression. (C) BM-DCs were cultured with LPS, GM-CSF and IL-4, incubated with ALDEFLUOR and co-stained for CD11c and B220, CD4, CD8α, CD11b, CD45RB, CD80, CD103, F4/80 or MHC class II expression. ALDHact⁺ cells are shown as red dots. All data are representative of two independent experiments.

Fig. 4.

DCs treated with GM-CSF or IL-4 and/or LPS induce the expression of gut-homing receptors on CD4⁺ T cells. Flt3L-induced BM-DCs were treated with medium alone (none), GM-CSF (10 ng ml⁻¹), IL-4 (10 ng ml⁻¹) or GM-CSF and IL-4 (10 ng ml⁻¹ each). To promote full maturation of BM-DCs, LPS (1 μg ml⁻¹) was added to their cultures. The treated DCs were pulsed with OVA peptide P323-339 (1 μM) and co-cultured with naive DO11.10 CD4⁺ T cells at a ratio of 1:5 in the presence or absence of DEAB (100 μM). On day 5 of culture, cells were stained for CCR9 (top), α4β7 (middle) and E-selectin ligands (bottom) and analyzed by flow cytometry. (A) The expression levels are presented as ΔMFI. Data are presented as mean ± SEM and are representative of more than three independent experiments. Statistical significance was determined by the one-way analysis of variance; ***P < 0.001. (B) Histogram plots of each staining of T cells co-cultured with untreated or GM-CSF + IL-4-treated DCs are shown. Shaded histograms and solid lines indicate specific staining and isotype control, respectively. Numbers in histogram plots indicate ΔMFI.

Fig. 5.

DCs treated with GM-CSF and/or IL-4 enhance the differentiation of Foxp3⁺ T cells, but suppress the differentiation of T_h17 cells. (A and B) SPL-DCs were treated with medium alone (none), GM-CSF (10 ng ml⁻¹), IL-4 (10 ng ml⁻¹) or GM-CSF and IL-4 (10 ng ml⁻¹ each) and co-cultured with naive DO11.10 CD4⁺ T cells at a ratio of 1:10 in the presence of OVA peptide P323-339 (1 μM) and IL-2 (100 U ml⁻¹) with or without TGF-β1 (5 ng ml⁻¹). DEAB (100 μM) was added to some culture wells. On day 5 of culture, cells were stained for intracellular Foxp3 and analyzed by flow cytometry. (A) The graph shows the mean ± SEM of percentage of Foxp3⁺ T cells generated in each culture condition. (B) Histogram plots of T cells co-cultured with untreated or GM-CSF + IL-4-treated DCs are shown. Shaded histograms and solid lines indicate specific staining and isotype control, respectively. The number shown in each panel indicates the percentage of Foxp3⁺ cells. (C and D) The treated DCs were co-cultured with naive DO11.10 CD4⁺ T cells as described in (A and B), but IL-6 and IL-23 (20 ng ml⁻¹ each) were also included in the culture. On day 5 of culture, cells were re-stimulated for 5 h with phorbol 12-myristate 13-acetate and ionomycin and stained for intracellular IL-17 and IFN-γ. (C) The graph shows the mean ± SEM of percentage of IL-17⁺IFN-γ⁻ cells generated in each culture condition. (D) Dot plots of T cells co-cultured with untreated or GM-CSF + IL-4-treated DCs are shown. The number shown in each panel indicates the percentage of IL-17⁺IFN-γ⁻ cells. All data are representative of two independent experiments. Statistical significance was determined by the one-way analysis of variance; ***P < 0.001.

Fig. 6.

GM-CSF receptor-mediated signals are critical for MLN-DCs to acquire ALDHact and the capacity to imprint gut-homing specificity on T cells. (A) Cells from MLN, PP or SPL of wt or Beta-c^−/− mice were incubated with ALDEFLUOR and stained for CD11c expression. Values indicate ΔMFI of ALDEFLUOR in CD11c⁺ cells. Data are presented as mean ± SEM (four mice in each genotype). Statistical significance was determined by the Student's t test. ***P < 0.001 versus wt mice. (B) MLN-DCs were isolated from Beta-c^−/− or wt mice and co-cultured with naive CD4⁺ T cells from wt mice at a ratio of 1:5 in the presence of soluble anti-CD3ε mAb (1 μg ml⁻¹). DEAB (100 μM) was added to some wells. On day 5 of culture, the expression levels of CCR9 (left) and α4β7 (right) on T cells were analyzed by flow cytometry. Data are presented as mean ± SEM and are representative of two independent experiments. Statistical significance was determined by the one-way analysis of variance; ***P < 0.001. (C–E) Immunohistochemical analysis was performed on CD4⁺ cells or CD8⁺ cells in PP, villi of small intestine, liver or lung from wt or Beta-c^−/− mice. Frozen sections were stained for CD4 (C and E, red) or CD8 (D, red). Cell nuclei were visualized with TO-PRO-3 (blue). Data are representative of two independent experiments. Scale bars, 200 μm (PP); 50 μm (villi, liver and lung). (F) Cells from PP, MLN or SPL of wt or Beta-c^−/− mice were stained for B220 and IgA. The graph shows the mean ± SEM (four mice in each genotype) of percentage of IgA⁺ cells in B220⁺ cells. Statistical significance was determined by the Student's t test. **P < 0.01 versus wt mice. Dot plots of pre-gated B220⁺ cells from PP are shown. Numbers adjacent to gates indicate percentage of IgA⁺ cells.

Fig. 7.

GM-CSF⁺CD11c⁻F4/80⁺ cells constitutively exist in MLN, PP and the intestinal LP. (A) IECs were isolated from the jejunum and ileum of B10.D2 mice. CD11c⁻F4/80⁺ cells were isolated from SPL, PLN and MLN of the same mice. The mRNA expression of Csf2 was assessed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of two independent experiments. (B) Frozen sections of MLN, PP or the intestinal LP were co-stained for GM-CSF (red) and CD11c (left, green) or F4/80 (right, green). (C) Frozen sections of MLN or PLN (brachial LN) were stained for GM-CSF (red) and F4/80 (green). Cell nuclei were visualized with TO-PRO-3 (blue). Data are representative of two independent experiments. Scale bars, 50 μm.

Fig. 8.

Vitamin A is essential for GM-CSF-induced Aldh1a2 expression in DCs and Csf2 expression in CD11c⁻F4/80⁺ cells in MLN. (A) Expression of Aldh1a2 in MLN-DCs from control or vitamin A-deficient (ΔVit.A) mice was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of two independent experiments. Statistical significance was determined by the Student's t test; ***P < 0.001. (B) Cells from MLN of control or vitamin A-deficient mice were incubated with ALDEFLUOR and stained for CD11c expression. Values indicate ΔMFI of ALDEFLUOR in CD11c⁺ cells. Data are presented as mean ± SEM (four mice in each group). Statistical significance was determined by the Student's t test; ***P < 0.001. (C) Expression of Aldh1a2 in Flt3L-induced BM-DCs (left) or SPL-DCs (right) cultured with medium alone (none), all-trans-RA (1 μM) or GM-CSF (10 ng ml⁻¹) was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of two independent experiments. Statistical significance was determined by the one-way analysis of variance (ANOVA); ***P < 0.001. (D) Expression of Aldh1a2 expression in Flt3L-induced BM-DCs (left) or SPL-DCs (right) cultured with GM-CSF (10 ng ml⁻¹) in the presence or absence of LE540 (1 μM) was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of two independent experiments. Statistical significance was determined by the one-way ANOVA; ***P < 0.001. (E) Expression of Csf2 in CD11c⁺, CD11c⁻F4/80⁺ or CD11c⁻F4/80⁻ cells from MLN of control or vitamin A-deficient mice was analyzed by quantitative real-time PCR. Data are presented as mean ± SEM and are representative of two independent experiments. Statistical significance was determined by the one-way ANOVA. ***P < 0.001 versus control mice.

Fig. 9.

Proposed model for the mechanism of GM-CSF-mediated induction of RA-producing DCs in the intestinal tissues. GM-CSF plays a critical role to induce the ALDH1A2 expression in DCs. In the intestinal tissues, CD11c⁻F4/80⁺ cells (i.e. macrophages or granulocytes) produce GM-CSF, which induces the ALDH1A2 expression in DCs. DC maturation signals induced by intestinal stimuli including TLR ligands from intestinal bacterial flora enhance the ALDH1A2 expression. The ALDH1A2⁺ DCs produce RA and imprint gut-homing specificity on T cells upon antigenic stimulation. These DCs also promote the differentiation of naive T cells to Foxp3⁺ Treg cells in the presence of TGF-β. RA is required not only for the GM-CSF-induced expression of ALDH1A2 in DCs but also for the GM-CSF production by CD11c⁻F4/80⁺ cells or their proper migration. Thus, ALDH1A2⁺ DCs as well as ALDH1A1⁺ IECs may provide RA to DCs and CD11c⁻F4/80⁺ cells and enhance the positive feedback loop for RA production in the intestine.