Nutr Res Pract.  2021 Dec;15(6):673-685. 10.4162/nrp.2021.15.6.673.

Effects of in vitro vitamin D treatment on function of T cells and autophagy mechanisms in high-fat diet-induced obese mice

• Kang MS
• Park CY
• Lee GY
• Cho DH
• Kim SJ
• Han SN

• KMID: 2522649
• DOI: http://doi.org/10.4162/nrp.2021.15.6.673

Abstract

BACKGROUND/OBJECTIVES
Obesity is associated with the impaired regulation of T cells characterized by increased numbers of Th1 and Th17 cells and the dysregulation of vitamin D metabolism. Both obesity and vitamin D have been reported to affect autophagy; however, a limited number of studies have investigated the effects of vitamin D on T cell autophagy in obese mice. Therefore, we aimed to determine whether in vitro treatment with vitamin D affects the proliferation, function, and autophagy of T cells from obese and control mice.
MATERIALS/METHODS
Five-week-old male C57BL/6 mice were fed control or high-fat diets (10% or 45% kcal fat: CON or HFDs, respectively) for 12 weeks. Purified T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies and cultured with either 10 nM 1,25(OH)₂D₃ or 0.1% ethanol (vehicle control). The proliferative response; expression of CD25, Foxp3, RORγt, and autophagy-related proteins (LC3A/B, SQSTM1/P62, BECLIN-1, ATG12); and the production of interferon (IFN)-γ, interleukin (IL)-4, IL-17A, and IL-10 by T cells were measured.
RESULTS
Compared with the CON group, T cell proliferation tended to be lower, and the production of IFN-γ was higher in the HFD group. IL-17A production was reduced by 1,25(OH)2D₃ treatment in both groups. The LC3 II/I ratio was higher in the HFD group than the CON group, but P62 did not differ. We observed no effect of vitamin D treatment on T cell autophagy.
CONCLUSIONS
Our findings suggest that diet-induced obesity may impair the function and inhibit autophagy of T cells, possibly leading to the dysregulation of T cell homeostasis, which may be behind the aggravation of inflammation commonly observed in obesity.

Keyword

Obesity; vitamin D; T lymphocytes; autophagy

Figure

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  Fig. 1 The experimental design and cell culture process.CON, control; HFD, high-fat diet.

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  Fig. 2 Effects of HFD and in vitro 1,25(OH)2D3 treatment on T cell proliferation. Total T cells purified from splenocytes of CON and HFD groups were stimulated with 5 μg/mL plate-bound anti-CD3 mAb and 2 μg/mL soluble anti-CD28 mAb and treated with 10 nM 1,25(OH)2D3 or vehicle CON for 72 h. Cells were pulsed with 0.5 μCi of [3H] TdR for the last 8 h of culture. [3H] TdR uptake was determined by liquid scintillation counting. The data are presented as mean ± SE (n = 6 to 8 per group). A student's t-test and a paired t-test were performed to determine the effects of dietary fat amount and in vitro 1,25(OH)2D3 treatment.Vit D, vitamin D; CON, control; HFD, high-fat diet; mAb, monoclonal antibody.

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  Fig. 3 Effects of HFD and in vitro 1,25(OH)2D3 treatment on the expression of surface marker and transcription factors related to Th17 and Treg in CD4+ T cells. CD4+ T cells purified from splenocytes of CON and HFD groups were incubated with 1,25(OH)2D3 (10 nM) or vehicle CON and stimulated with plate-bound anti-CD3 mAb and soluble anti-CD28 mAb for 72 h. Cells were harvested and analyzed by flow cytometry. Intracellular expression of RORγt was used as the marker related to Th17 cell. Intracellular expression of Foxp3 among cells positive for surface CD25 was used for identification of Treg cells. Values are presented as mean ± SE (n = 4 to 10 per group). Student t-test and paired t-test was used to determine the effects of dietary fat intake and in vitro 1,25(OH)2D3 treatment.CON, control; HFD, high-fat diet; mAb, monoclonal antibody.

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  Fig. 4 Effects of HFD and in vitro 1,25(OH)2D3 treatment on production of cytokines by T cells. Total T cells purified from splenocytes of CON and HFD groups were cultured with 10 nM 1,25(OH)2D3 or vehicle (0.1% ethanol) and activated with anti-CD3 mAb (5 μg/mL) and anti-CD28 mAb (2 μg/mL) for 72 h. Supernatants were collected and assayed to determine the production of (A) IFN-γ, (B) IL-4, (C) IL-17A, and (D) IL-10 using ELISA. Data are presented as mean ± SE (n = 15 to 17 per group). A paired t-test was performed to confirm the effects of in vitro 1,25(OH)2D3 treatment. A student's t-test was used to determine the significant effects of dietary fat intake.IFN, interferon; IL, interleukin; CON, control; HFD, high-fat diet; mAb, monoclonal antibody.*P < 0.05, **P < 0.01, ***P < 0.001.

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  Fig. 5 Effects of HFD and in vitro 1,25(OH)2D3 treatment on expression of proteins associated with autophagy in total T cells (A) Immunoblotting for LC3, ATG12, BECLIN-1, SQSTM1/P62, and β-ACTIN, and (B) densitometric analysis of protein expression. Total T cells purified from splenocytes of CON and HFD groups were incubated with 10 nM 1,25(OH)2D3 or vehicle CON, and stimulated with anti-CD3 mAb (5 μg/mL) and anti-CD28 mAb (2 μg/mL) for 72 h. Cells were harvested, and analyzed on western blot. The intensity of LC3, ATG12, BECLIN-1, SQSTM1/P62 in T cells was densitometrically measured and normalized with β-ACTIN. Data are presented as mean ± SE (n = 5 per group). Student t-test and paired t-test was used to determine the effects of HFD and in vitro 1,25(OH)2D3 treatment.CON, control; HFD, high-fat diet; mAb, monoclonal antibody.*P < 0.05.

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