Vitamin D3 alters Toll-like receptor 4 signaling in monocytes of pregnant women at risk for preeclampsia

Abstract

Vitamin D deficiency during pregnancy is thought to play a role in the development of preeclampsia; however, the underlying mechanism is not fully understood. In this study, a randomized double-blind placebo-controlled clinical trial was performed among 60 pregnant women at risk for pre-eclampsia according to abnormal uterine artery Doppler waveform. Subjects were randomly divided into 2 groups to receive a daily dose of 2000 IU vitamin D3 supplements (n=30) or receive placebo (n=30) between gestational weeks 20-32 for a total of 12 consecutive weeks. Because vitamin D3 supplementation can induce anti-inflammatory cytokine signaling, peripheral blood monocytes were investigated by flow cytometry for expression of toll-like receptor 4 (TLR4), an important mediator of innate immune response. The pro-inflammatory cytokines secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1 from monocytes, which are typically upregulated in preeclampsia, was also assessed. The incidence of preeclampsia was significantly lower in patients treated with vitamin D3 compared to the placebo group. Both the mean fluorescence intensity and the positive percentage of monocytes TLR4 in the vitamin D group were significantly lower compared to the placebo group, as well as the concentrations of secreted TNF-α, IL-6, and IL-1, while the concentration of IL-10 was higher. In the placebo group, the positive frequency of monocytes TLR4 was negatively correlated with the concentration of serum 25-hydroxyvitamin D in preeclampsia patients. Based on these results, we conclude that vitamin D3 supplementation for patients at risk of preeclampsia leads to a decrease in the expression of peripheral blood monocytes TLR4 and a subsequent decrease in pro-inflammatory cytokine secretion. Therefore, inhibiting the expression of monocytes TLR4 through vitamin D3 supplement may be a new approach to preeclampsia prevention.

Keywords: Preeclampsia; Toll-like receptor 4; monocytes; vitamin D3.

Figure 1

Analysis of monocytes TLR4 by flow cytometry. A. Representative flow cytometry plot showing forward and side scatter characteristics with gating used to identify monocytes (R1); B. Percentage of TLR4 positive monocytes. C. Mean fluorescence intensity (MFI) of monocytes TLR4; D. Comparison of the percentage of monocyte TLR4+ between Vitamin D and placebo groups. E. Comparison of the MFI monocyte TLR4+ between Vitamin D and placebo groups. Note: Statistical significance was determined by Mann-Whitney U test.

Figure 2

The concentration of serum 25-hydroxy vitamin D in pregnant women. A. The concentration of 25-hydroxy vitamin D in peripheral blood. B. The correlation of 25-hydroxy vitamin D and monocyte TLR4+.

Figure 3

LPS stimulation alters cytokine secretion of monocytes from patients supplemented with vitamin D.

Figure 4

Comparison of the concentrations of cytokines in serum between vitamin D supplementation and placebo group.