Epigenetic reduction in invariant NKT cells following in utero vitamin D deficiency in mice

Abstract

Vitamin D status changes with season, but the effect of these changes on immune function is not clear. In this study, we show that in utero vitamin D deficiency in mice results in a significant reduction in invariant NKT (iNKT) cell numbers that could not be corrected by later intervention with vitamin D or 1,25-dihydroxy vitamin D(3) (active form of the vitamin). Furthermore, this was intrinsic to hematopoietic cells, as vitamin D-deficient bone marrow is specifically defective in generating iNKT cells in wild-type recipients. This vitamin D deficiency-induced reduction in iNKT cells is due to increased apoptosis of early iNKT cell precursors in the thymus. Whereas both the vitamin D receptor and vitamin D regulate iNKT cells, the vitamin D receptor is required for both iNKT cell function and number, and vitamin D (the ligand) only controls the number of iNKT cells. Given the importance of proper iNKT cell function in health and disease, this prenatal requirement for vitamin D suggests that in humans, the amount of vitamin D available in the environment during prenatal development may dictate the number of iNKT cells and potential risk of autoimmunity.

Figure 1

Reduced numbers of iNKT cells in 1,25D3 deficient mice. (A) In all cases iNKT cells are defined as being TCRβ⁺ and αGalCer-CD1d tetramer⁺. The empty tetramer and isotype control staining can be found in Supplementary Fig. (S)1. The frequency of iNKT cells in the thymus, spleen and liver from WT, VDR KO and Cyp27B1 (Cyp)KO mice. Values are the mean ± SEM of n=10 mice per group. The percentage of iNKT cells in VDR KO and CypKO thymus and liver are significantly different from those in WT mice (p<0.01). (B) Mice were injected with αGalCer in vivo followed by intracellular staining ex vivo as described in the methods. Histograms show production of IL-4 by iNKT cells from one representative mouse. IL-4 isotype control staining shown in S1. Mean ± SEM values of n=10 per group. The percentage of IL-4-producing iNKT cells in VDR KO mice is significantly lower than that in WT and CypKO mice (p<0.01). (C) Thymocytes from WT and 1,25D3 deficient mice were incubated with or without (control, Ctrl) the CD1d-restricted NKT cell hybridoma and IL-2 production was measured. No IL-2 was produced from the WT and 1,25D3 deficient thymocytes cultured alone (Ctrl). Results shown are from one representative of three independent experiments with thymocytes from n=3 mice each per group.

Figure 2

Decreased iNKT cell numbers in vitamin D deficient mice. (A) The percentage of iNKT cells in thymus and liver from D+WT, D+ CypKO, D−WT and D− CypKO mice (n=10–15 mice per group). Values are the mean ± SEM. (B) Serum cytokine production in D+WT and D− mice induced by systemic administration of αGalCer. The values from the D− CypKO and D−WT mice overlap. Levels of IFN-γ and IL-4 in the serum were determined at different times following injection (n=9 per group). Values are mean ± SEM.** p<0.0001, * p<0.05 (C) Frequency of cytokine-producing iNKT cells from D+ WT and D− livers. A representative histogram from each group shows production of IFN-γ by iNKT cells. IFN-γ isotype control staining shown in S1. Values are mean ± SEM of 10 mice per group. (D) Dot plots showing expression of CD44 and NK1.1 on TCRβ and CD1d-αGalCer tetramer double positive thymocytes. The percentage of CD44⁻NK1.1⁻ iNKT cells and CD44⁺NK1.1⁺ iNKT cells in D−WT or D− CypKO mice are significantly different from D+WT mice (p<0.05). Values are mean ± SEM (n=8 per group).

Figure 3

Epigenetic effects of vitamin D deficiency on iNKT cell numbers. (A) Effect of vitamin D intervention on iNKT cell numbers. Cyp ko/+ breeders started on D− diets and littermates were switched to D+ diet at 3wks of age and continued until 8wks (D+). Values are mean ± SEM of n=5 mice per group. (B) Effect of 1,25D3 intervention on iNKT cell numbers. Cyp ko/+ breeders started on D− diets and littermates were switched to 1,25D3 at 3wks of age and continued until 8wks (1,25D3 late). Values are mean ± SEM of n=8 mice per group. (C) Effect of early 1,25D3 intervention on iNKT cell numbers. Cyp ko/+ breeders started on D− diets and intervention with 1,25D3 started just before birth (embryonic d20) and continued until 8wks (1,25D3 early). Values are mean ± SEM of n=12–15 mice per group. (D) Effect of continuous supplementation of 1,25D3 on iNKT cell numbers. Breeders and offspring were fed 1,25D3 throughout (1,25D3). Continuous supplementation of 1,25D3 resulted in normalization (D+ WT numbers) of the iNKT cell numbers in both the WT and Cyp KO mice. Values are mean ± SEM of n=6–8 mice per group. * p<0.001, ** p<0/05.

Figure 4

Intrinsic defect of iNKT cell precursors in the absence of 1,25D3. BM transplants were done using D+, 1,25D3, and D−WT donor mice into WT recipients (donor BM → recipient). (A) Reconstitution of the thymus and spleen of WT (CD45.1) recipients with donor BM (CD45.2) of WT, 1,25D3, D−, 1,25D3KO, and D-KO mice. Values are mean ± SEM of n=5 mice per group. (B). Percentage of donor CD45.2 gated iNKT cells in the thymus, and spleen are shown for the same groups of mice shown in A. The results from the 1,25D3 WT were identical to those from 1,25D3 KO and the results from the D− WT were identical to those from the D- KO. Data shown are one representative of 5 mice per group and the mean ± SEM is given for all five mice.

Figure 5

1,25D3 deficiency results in an intrinsic defect of iNKT cells. Competitive BM chimeras were generated using a 1:1 ratio of WT CD45.1 and Cyp KO CD45.2 BM into WT CD45.1 recipients. (A) Lymphocyte chimerism was checked by flow cytometry in the thymus, spleen, liver and lymph nodes. Half of the cells are of WT CD45.1 origin and half of the cells are of Cyp KO CD45.2 origin. (B) Dot plots showing percentage of donor-derived iNKT cells in the thymus, spleen, liver, and lymph nodes. Data shown is from 8 mice (mean ± SEM).

Figure 6

Increased cell apoptosis in D− iNKT cell precursors. (A) Annexin V staining of iNKT cells in the thymus from D+ and D− mice. TCRβ and αGalCer-CD1d tetramer positive cells were gated. Data shown is one representative of 8 mice per group. Values are mean ± SEM. The percentage of annexin V⁺ iNKT cells is significantly different between D+WT and D−WT or D− CypKO mice (p<0.05). (B) Gating strategy for identifying DPdull iNKT cell precursors. The DPdull cells in the top panel are gated and evaluated for tetramer+ iNKT cells (bottom panel). The DPdull/tetramer+ cells are then gated. (C) CD24 and annexinV staining of iNKT cell precursors gated in B. Negative control staining for annexinV is shown in S1. Data represent eight mice per group. The percentage of CD24+DPdulltetramer+ cells is significantly different between D+WT and D− WT mice (p<0.01). The apoptosis is significantly higher in the D− iNKT cell precursors (CD24−DPdulltetramer+ cells, p<0.001).