Cord blood vitamin D status impacts innate immune responses

Abstract

Objectives: Our objectives were to 1) assess cord blood vitamin D concentrations from healthy term newborns, 2) ascertain whether cord blood vitamin D insufficiency precludes optimal induction of the Toll-like receptor (TLR) antimicrobial pathway in monocytes, and 3) determine whether in vitro supplementation with 25-hydroxyvitamin D(3) [25(OH)D(3)] and/or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] restores TLR-induced antimicrobial responses.

Study design: Plasma concentrations of 25(OH)D and 1,25(OH)(2)D were measured from cord blood of 23 newborns. Human monocytes were cultured in cord blood plasma and stimulated with TLR2 and TLR4 ligands, and then antimicrobial gene expression was analyzed using quantitative PCR.

Results: Cord blood 25(OH)D and 1,25(OH)(2)D concentrations were positively correlated to each other (r = 0.78; P <0.0001). Compared with those conditioned in vitamin D-sufficient plasma [25(OH)D > 75 nmol/liter], monocytes cultured in severely vitamin D-deficient plasma [25(OH)D < 30 nmol/liter] exhibited decreased TLR-induced cathelicidin expression (P <0.05). Supplementation in vitro of vitamin D-deficient plasma with 25(OH)D(3) increased antimicrobial peptide gene expression.

Conclusions: Cord blood vitamin D deficiency, by its effects on TLR-induced antimicrobial production, altered in vitro monocyte responses. The observation that exogenous 25(OH)D(3) in vitro recovered TLR-induced antimicrobial responses suggests the need for additional prospective investigations to further delineate the role of vitamin D in the newborn immune response.

Fig. 1.

The relationship between 25(OH)D and 1,25(OH)₂D is unique in cord blood compared with adults. Blood samples taken from cord blood and healthy adults donors had concentrations of 25(OH)D and 1,25(OH)₂D determined by RIA. Cord blood measurements of 25(OH)D and 1,25(OH)₂D demonstrated a strong correlation to each other (r = 0.78; P = 0.0001). This was in contrast to the lack of correlation between 25(OH)D and 1,25(OH)₂D in adult measurements where conversion of 25(OH)D to 1,25(OH)₂D is tightly regulated by PTH and renal 1α-hydroxylase.

Fig. 2.

TLR4L induction of the vitamin D-dependent antimicrobial pathway in monocytes is perturbed in the presence of vitamin D insufficiency. Primary human monocytes were stimulated with TLR4L for 24 h in 10% cord blood plasma. These plasma samples were either vitamin D sufficient [25(OH)D > 75 nm], insufficient [25(OH)D 50–75 nm], deficient [25(OH)D 30–50 nm], or severely deficient [25(OH)D < 30 nm]. Cathelicidin, DEFB4, CYP27B1, VDR, and CYP24 gene expression were determined by qPCR (mean fold change ± sem). A, Cathelicidin gene expression was notably diminished in adherent cells cultured with severely vitamin D-deficient cord blood plasma samples vs. vitamin D-sufficient ones (P = 0.03). When compared with insufficient samples, this finding remained statistically significant (P < 0.02). B, DEFB4 exhibited a trend toward diminished gene expression when monocytes were cultured in the markedly deficient plasma, but it did not approach statistical significance. C, CYP27B1 gene expression was notably less in monocytes cultured in deficient plasma when compared with the sufficient plasma group (P = 0.03). D, However, there were no differences in VDR gene expression. E, After TLR4L stimulation, there was greater induction of CYP24 gene expression in the monocytes cultured with vitamin D-sufficient plasma (P = 0.03).

Fig. 3.

TLR2/1L induction of the vitamin D-dependent antimicrobial pathway in monocytes is also perturbed in the presence of vitamin D insufficiency. Primary human monocytes were then stimulated with a TLR2/1L for 24 h in 10% cord blood plasma of the same varying vitamin D concentrations as described in Fig. 2. Cathelicidin, DEFB4, CYP27B1, VDR, and CYP24 gene expression were again determined by qPCR (mean fold change ± sem). A, Cathelicidin gene expression was notably diminished in adherent cells cultured with severely vitamin D-deficient plasma samples vs. vitamin D-sufficient ones (P < 0.01). B, No difference was detected in DEFB4 expression after stimulation with a TLR2/1L when compared with cord blood 25(OH)D concentrations of the plasma. C, CYP27B1 gene expression was notably less in monocytes cultured in plasma with 25(OH)D below 50 nm. D, There were no differences in VDR gene expression after stimulation with the TLR2L. E, There was a greater induction of CYP24 gene expression in the monocytes cultured with vitamin D-sufficient plasma as previously described with CYP24 gene expression after TLR4L stimulation (P = 0.02).

Fig. 4.

Restoration of the TLR-stimulated antimicrobial pathway is achieved with exogenous 25(OH)D₃ supplementation in vitro. A, Primary monocytes were cultured in vitamin D-deficient cord blood plasma with and without supplementation of exogenous 25(OH)D₃ at the time of stimulation with the TLR2/1L. In the unsupplemented samples, TLR2/1L activation of monocytes did not induce robust amounts of cathelicidin mRNA. However, the addition of 10⁻⁸ m 25(OH)D₃ resulted in a greater than 5-fold induction of cathelicidin mRNA compared with the unsupplemented samples. B, Primary monocytes were cultured in vitamin D-deficient cord blood plasma with and without the addition of exogenous 1,25(OH)₂D₃ at the time of stimulation with the TLR2/1L. Independent induction of cathelicidin gene expression occurred in monocytes that were supplemented with 1,25(OH)₂D₃ in the absence of TLR2/1L stimulation. In monocytes stimulated with a TLR2/1L, the addition of 1, 25(OH)₂D₃ led to only a slightly greater expression of cathelicidin mRNA compared with those without supplementation.