Metabolism of 25-Hydroxy-Vitamin D in Human Macrophages Is Highly Dependent on Macrophage Polarization - PubMed (original) (raw)

Metabolism of 25-Hydroxy-Vitamin D in Human Macrophages Is Highly Dependent on Macrophage Polarization

Rie H Nygaard et al. Int J Mol Sci. 2022.

Abstract

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages.

Keywords: 3-epi-25-hydroxy-vitamin D; immune system; inflammation; liquid chromatography-mass spectrometry; monocyte-derived macrophages; qPCR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Flow cytometry measures of MDM surface markers. MDMs were polarized into M0, M1, M2a and M2c. Cells were harvested and the surface markers CD80 (A), CD206 (B) and CD163 (C) were measured with flow cytometry. The numbers above the horizontal lines indicate the different _p_-values.

Figure 2

Figure 2

Vitamin D metabolites in polarized MDMs after vitamin D stimulation. MDMs were polarized into M0, M1, M2a and M2c MDMs and stimulated with 1000 nM 25-hydroxy-vitamin D. Concentrations of vitamin D metabolites in the different subtypes of MDMs were measured in the media after 24 h: 25-hydroxy-vitamin D (A), 1,25-dihydroxy-vitamin D (B), 24,25-dihydroxy-vitamin D (C) and 3-epi-25-hydroxy-vitamin D (D). * Indicates statistically significant differences between groups (subtypes) on each graph.

Figure 3

Figure 3

Expression of CYP27B1 in polarized MDMs with and without vitamin D stimulation. MDMs were polarized into M0, M1, M2a and M2c MDMs and stimulated with either 0 nM (A) or 1000 nM (B) 25-hydroxy-vitamin D for 24 h. The mRNA expression of CYP27B1 is shown relative to the household gene (YWHAZ). * Indicates a statistically significant difference between groups.

Figure 4

Figure 4

Expression of CYP24A1 and CYP24A1-SV in polarized MDMs after vitamin D stimulation. MDMs were polarized into M0, M1, M2a and M2c MDMs and stimulated with 1000 nM 25-hydroxy-vitamin D for 24 h. The mRNA expression of CYP24A1 is shown in (A) and the mRNA expression of its splice variant, CYP24A1, is shown in (B). Both expressions are shown relative to a household gene (YWHAZ). * Indicates a statistically significant difference between groups.

Figure 5

Figure 5

This figure shows the cell isolation and polarization protocol. First, we performed monocyte isolation (A), then macrophage differentiation (B), then macrophage polarization (C) and last the actual experiments (D). The figure was created using Biorender.com (accessed on 25 August 2022).

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