1,25D3 prevents CD8(+)Tc2 skewing and asthma development through VDR binding changes to the Cyp11a1 promoter

Abstract

Effector CD8(+) T cells convert from IFN-γ(+) (Tc1) to IL-13(+) (Tc2) cells in the presence of IL-4. Underlying regulatory mechanisms are not fully defined. Here, we show that addition of 1,25D3, the active form of vitamin D3, during CD8(+) T-cell differentiation prevents IL-4-induced conversion to IL-13-producers. Transfer of 1,25D3-treated CD8(+) T cells into sensitized and challenged CD8(+)-deficient recipients fails to restore development of lung allergic responses. 1,25D3 alters vitamin D receptor (VDR) recruitment to the Cyp11a1 promoter in vitro and in vivo in the presence of IL-4. As a result, protein levels and enzymatic activity of CYP11A1, a steroidogenic enzyme regulating CD8(+) T-cell conversion, are decreased. An epistatic effect between CYP11A1 and VDR polymorphisms may contribute to the predisposition to childhood asthma. These data identify a role for 1,25D3 in the molecular programming of CD8(+) T-cell conversion to an IL-13-secreting phenotype through regulation of steroidogenesis, potentially governing asthma susceptibility.

Figure 1. IFN-γ and IL-13 expression in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in the presence or absence of 1,25D3 at 100 nM or 1 μM.

Representative results of intracellular staining of IFN-γ and IL-13 expression in CD8⁺ T cells with or without SIINFEKL (T-cell receptor, TCR) restimulation.

Figure 2. 1,25D3 treatment of CD8⁺ T cells alters gene expression of transcription factors and the functional activity of CYP11A1.

Gene expression of (a) Gata3, (b) Tbx21, (c) Cyp11a1 and (d) Vdr was measured by quantitative PCR (qPCR) in CD8⁺ T cells in IL-2 or IL-2+IL-4 in the presence or absence of 100 nM or 1 μM 1,25D3. Results (relative fold change+s.e.m.) are from three independent experiments. (e) CYP11A1 protein levels (mean+s.e.m.) detected by immunoblot analyses and densitometry of autoradiographs in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in the presence or absence of 100 nM or 1 μM 1,25D3. Results are from three independent experiments, * Denotes calculated molecular weight (MW) for CYP11A1 (13363-1-AP, Proteintech, Chicago, IL): 60 kDa, observed MW: 49 kDa, (f) Pregnenolone levels (mean+s.e.m.) determined by ELISA in supernatants from CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in the presence or absence of 100 nM or 1 μM 1,25D3. Results are from six independent experiments. Linear mixed models were employed; pairwise comparisons were performed using t-tests derived from these models. *P<0.05, **P<0.01, ***P<0.001 compared with the IL-2 group, ###P<0.001 compared with the IL-2+IL-4 group, these P values remained significant after correction for multiple comparisons (Benjamini–Hochberg correction); P values that did not reach the threshold P value after adjustment for multiple comparisons are shown numerically.

Figure 3. 1,25D3-mediated VDR recruitment to the Cyp11a1 promoter region is altered in CD8⁺ T cells differentiated in IL-2 or IL-2+IL-4 in the presence or absence of 100 nM or 1 μM 1,25D3.

(a) Localization of VDR-binding sites and qPCR primers in the Cyp11a1 promoter region. (b) qPCR was performed using five Cyp11a1 promoter-specific primers covering seven VDR-binding sites. Data were analysed via the percent input methodology: (2^{CT of total input−CT of specific IP}) × 100 and relative percent input ratios using CD8⁺ T cells stimulated with IL-2 as baseline. Data (relative fold change+s.e.m.) are from three independent experiments. Linear mixed models were employed; pairwise comparisons were performed using t-tests derived from these models. *P<0.05, **P<0.01, ***P<0.001 compared with the IL-2 group, these P values remained significant after correction for multiple comparisons; (Benjamini–Hochberg correction). No statistical significance was detected compared with the IL-2+IL-4 group.

Figure 4. Adoptive transfer of 1,25D3-treated CD8⁺ T cells into CD8-deficient recipients fails to induce AHR.

Recipient mice were sensitized (two intraperitoneal, i.p.) and challenged (four nebulizations, Neb) using secondary allergen challenge model and received no cells, CD8⁺ T cells differentiated in IL-2 alone (CD8) or in the presence of 100 nM (CD8+100 nM 1,25D3) or 1 μM 1,25D3 (CD8+1 μM 1,25D3). (a) Changes in airway resistance (RL) were measured in response to increasing concentrations of methacholine. (b) Cell composition in BAL fluid. Data (mean+s.e.m.) are from two to three experiments with three to four mice per experiment. *P<0.05, **P<0.01, ***P<0.001; compared with sensitized and challenged CD8-deficient recipients that received no cells. General linear models were employed; pairwise comparisons were performed using t-tests derived from these models. #P<0.05, ##P<0.01, ###P<0.001 compared with sensitized and challenged CD8-deficient recipients that received CD8⁺ T cells differentiated in IL-2 alone, these P values remained significant after correction for multiple comparisons (Benjamini–Hochberg correction); P values that did not reach the threshold P value after adjustment for multiple comparisons are shown numerically.

Figure 5. Adoptive transfer of 1,25D3-treated CD8⁺ T cells into CD8-deficient recipients decreased cytokine levels in the BAL.

Cytokine levels in BAL fluid (mean+s.e.m.): (a) IL-4, (b) IL-5 and (c) IL-13. *P<0.05, **P<0.01; compared with sensitized and challenged CD8-deficient recipients that received no cells. Data (mean+s.e.m.) are from two to three experiments with three to four mice per experiment. General linear models were employed; pairwise comparisons were performed using t-tests derived from these models. #P<0.05, ##P<0.01, ###P<0.001 compared with sensitized and challenged CD8-deficient recipients that received CD8⁺ T cells differentiated in IL-2 alone, P values remained significant after correction for multiple comparisons (Benjamini–Hochberg correction).

Figure 6. Adoptive transfer of 1,25D3-treated CD8⁺ T cells into CD8-deficient recipients prevents goblet cell metaplasia.

(a–d) Representative photomicrographs of lung histology (original magnification × 3,200, scale bar, 200 μm). (e) Quantitative analysis of PAS-positive goblet cells was determined in cross-sectional areas of the airway wall. Data (mean+s.e.m.) are from two experiments with three mice per experiment. Linear mixed models were employed; pairwise comparisons were performed using t-tests derived from these models. **P<0.01 these P values remained significant after correction for multiple comparisons; P values that did not reach the threshold P value after adjustment for multiple comparisons (Benjamini–Hochberg correction) are shown numerically.

Figure 7. 1,25D3-mediated VDR recruitment to the Cyp11a1 promoter region is altered in CD8⁺ T cells after adoptive transfer into sensitized and challenged CD8-deficient mice.

CD8⁺ T cells were differentiated in vitro in IL-2 in the presence or absence of 1 μM 1,25D3. CD8⁺ T cells from the lungs were recovered after adoptive transfer into sensitized and challenged CD8-deficient mice (n=3 mice per group). qPCR was performed using five Cyp11a1 promoter-specific primers covering seven VDR-binding sites. Data were analysed via the percent input methodology: (2^{CT of total input−CT of specific IP}) × 100.