Vitamin D deficiency impacts on expression of toll-like receptor-2 and cytokine profile: a pilot study

Abstract

Background: Vitamin D is believed to play an important role outside the endocrine system in the regulation of the immune system, and in cellular proliferation and differentiation. The aim of the study was to investigate the impact of vitamin D levels on innate immunity.

Methods: Participants for this prospective, longitudinal study were recruited amongst otherwise healthy staff of a large hospital in Victoria, Australia. Those fulfilling the inclusion criteria, including a vitamin D level of <50 nmol/L, were supplemented. Using flow cytometry, expression of the innate immune receptors TLR2, TLR4 and CD86 was measured on peripheral blood mononuclear cells (PBMCs) collected prior to vitamin D treatment and then at 1 and 3 months. Additonally, PBMCs at each timepoint were stimulated with specific TLR ligands and resultant supernatants were assayed for the cytokines TNFα, IL-6, IFN-α and IP-10.

Results: In participants whose vitamin D level was >100 nmol/L post supplementation (n=11), TLR2 expression on PBMCs increased significantly, with no change noted in TLR4 or CD86 expression. Stimulation of vitamin D deficient samples with TLR ligands produced a number of proinflammatory cytokines, which were significantly reduced upon vitamin D normalisation. In patients whose levels returned to a deficient level at 3 months despite ongoing low-level supplementation, an increase in the pro-inflamamtory state returned. This suggests that vitamin D may play an important role in ensuring an appropriate baseline pro-inflammatory state.

Conclusions: This ex-vivo pilot study adds clinical evidence supporting a possibly important role for vitamin D in innate immunity. If confirmed, this unique clinical study has potentially significant implications for the treatment of a variety of inflammatory conditions, where achieving optimal vitamin D levels may help reduce inflammation.

Figures

Figure 1

Summary of study protocol.

Figure 2

TLR2, TLR4 and CD86 expression on Monocytes. TLR 2, TLR4 and CD86 expression, was measured at baseline, 1 and 3 months post commencement of supplementation, in those who reached peak levels of >100 nmol/L. Geometric mean fluorescence (GMF) was measured by flow cytometry and normalised to an isotype matched control.

Figure 3

Cytokine concentrations upon stimulation with TLR specific ligands, as measured at baseline, 1 and 3 months post start of vitamin D supplementation. PBMCs were stimulated with TLR ligands LPS (TLR4), Pam3Cys (TLR2), CpG (TLR9) for 24 hours and supernatants were assayed by cytokine ELISA for TNF (Figure 3A) and IL-6 (Figure 3B). Results were expressed as fold change compared to unstimulated PBMCs. In Figures 3C and 3D, PBMCs were stimulated with TLR ligands PolyI:C (TLR3) and R848 (TLR7/8) and supernatants were assayed by ELISA for IP-10 (Figure 3C) and IFNalpha (Figure 3D). Results were expressed by fold change for Figure 3C and absolute values for Figure 3D.