Differential IL-21 signaling in APCs leads to disparate Th17 differentiation in diabetes-susceptible NOD and diabetes-resistant NOD.Idd3 mice

Abstract

Type 1 diabetes (T1D) is an autoimmune disease that shows familial aggregation in humans and likely has genetic determinants. Disease linkage studies have revealed many susceptibility loci for T1D in mice and humans. The mouse T1D susceptibility locus insulin-dependent diabetes susceptibility 3 (Idd3), which has a homologous genetic interval in humans, encodes cytokine genes Il2 and Il21 and regulates diabetes and other autoimmune diseases; however, the cellular and molecular mechanisms of this regulation are still being elucidated. Here we show that T cells from NOD mice produce more Il21 and less Il2 and exhibit enhanced Th17 cell generation compared with T cells from NOD.Idd3 congenic mice, which carry the protective Idd3 allele from a diabetes-resistant mouse strain. Further, APCs from NOD and NOD.Idd3 mice played a central role in this differential Th17 cell development, and IL-21 signaling in APCs was pivotal to this process. Specifically, NOD-derived APCs showed increased production of pro-Th17 mediators and dysregulation of the retinoic acid (RA) signaling pathway compared with APCs from NOD.Idd3 and NOD.Il21r-deficient mice. These data suggest that the protective effect of the Idd3 locus is due, in part, to differential RA signaling in APCs and that IL-21 likely plays a role in this process. Thus, we believe APCs provide a new candidate for therapeutic intervention in autoimmune diseases.

Figure 1. Cells from NOD mice generate Th17 cells more efficiently than cells from NOD.Idd3 mice.

Naive T cells were polarized with TGF-β and IL-6. After 3 days, cells were restimulated with PMA and ionomycin in the presence of monensin for 4 hours before cell surface and then intracellular staining. Values in each quadrant represent the percentage of 7-AAD^–CD4⁺ cells. Cells were activated with (A) plate-bound anti-CD3 and soluble anti-CD28 or (B) with soluble anti-CD3 in the presence of APCs, with or without anti–IFN-γ mAb. Data shown are representative of 4 to 9 experiments.

Figure 2. The Treg/T effector balance is skewed toward T effectors in NOD mice.

LN and spleen cells from Foxp3.gfp KI NOD (prediabetic) and NOD.Idd3 mice were stimulated with PMA and ionomycin for intracellular cytokine staining. Foxp3/GFP⁺ expression on CD4⁺ T cells was assessed in the same samples prior to restimulation. Each dot represents the ratio of Treg/cytokine⁺ 7-AAD^–CD3⁺CD4⁺ T cells per individual mouse, and the means are indicated by the bars.

Figure 3. The defect in Th17 cell generation in NOD.Il21r KO mice is both T cell intrinsic and APC driven.

(A) Naive T cells from NOD or NOD.Il21r KO mice were activated with plate-bound anti-CD3, and (B and C) naive T cells from NOD mice were activated with soluble anti-CD3 in the presence APCs, (A and B) in the presence or (C) absence of IL-6 and TGF-β. Intracellular staining was performed after 3 days. Values in each quadrant or gate represent the percentage of 7-AAD^–CD4⁺ cells. (C) Cell-conditioned media were assessed for IFN-γ production after 48 hours. Data are representative of 2 to 3 independent experiments.

Figure 4. CD11b⁺ APCs regulate Th17 cell differentiation.

(A) NOD-derived naive T cells were polarized with IL-6 and TGF-β in the presence of APCs, with or without depletion of either CD11c^–CD11b⁺ or CD11c⁺ cells, and, after 3 days, intracellular staining was performed to assess IL-17 and IFN-γ production. Values in each quadrant represent the percentage of 7-AAD^–CD4⁺ cells. Data are representative of 3 independent experiments. (B) CD11b⁺ cells were stimulated with PGN for 48 hours, and supernatants were assayed for mediator production. Data represent the mean values from 2 to 3 experiments and are representative of 3 to 4 independent experiments.

Figure 5. Gene profiling of cells from NOD and NOD.Idd3 mice.

RNA was extracted from purified CD11b⁺ cells cultured for 4 hours with or without PGN. (A) Scatter plot shows gene profiling performed on duplicate CD11b⁺ cell samples from NOD and NOD.Idd3 mice cultured with or without PGN stimulation for 4 hours. Red dots indicate transcripts downstream of RA signaling. (B) The biological network showing differentially regulated genes in the RA pathway was produced with Ingenuity Pathways Analysis software. Genes expressed differentially more than 2 fold in CD11b⁺ cells from NOD and NOD.Idd3 mice are shaded pink, whereas more highly expressed genes are depicted by a darker pink and red. Indirect (dashed lines) and direct (solid lines) network interactions are shown. The asterisk denotes that this gene has multiple identifiers on the genechip. (C and D) Expression of RA-regulated genes was determined by real-time PCR and normalized to β-actin expression.

Figure 6. RA signaling in APCs dampens Th17 differentiation in NOD T cells.

Naive T cells from NOD mice were stimulated with soluble anti-CD3 plus TGF-β and IL-6 in the presence of APCs that were pretreated with or without RA. (A) After 3 days, Th17 polarization was assessed by intracellular staining. Values in each quadrant represent the percentage of 7-AAD^–CD4⁺ cells. Scatter plots are representative of 3 independent experiments and summarized in B; the mean of 3 independent experiments are shown (mean ± SEM). *P < 0.05. (C) APCs were pretreated with RA in vivo in NOD/SCID mice. Values are representative of 2 independent experiments (mean ± SEM; n = 7). *P < 0.05.