In recent years, our perception of what constitutes normal vitamin D status has changed dramatically. Vitamin D sufficiency was originally defined as the minimum serum concentration of 25OHD that would prevent rickets in children and osteomalacia in adults, approximately 20 nM (8 ng/ml) (11
). However, the observation that 25OHD concentrations as high as 80–100 nM (32–40 pg/ml) are inversely correlated with serum parathyroid hormone (17
), and continue to enhance gastrointestinal calcium uptake (20
), have led to a revision of these parameters. Specifically, current data suggest that serum 25OHD is biologically optimal above 75 nM (30 ng/ml), with concentrations between 20 and 75 nM constituting sub-optimal levels, or vitamin D ‘insufficiency (21
). As a consequence of these new definitions, it has been estimated that as many as 1 billion people worldwide may be classified as vitamin D insufficient (11
), with specific groups such as the elderly, children and pregnant mothers being particularly vulnerable. Individuals with dark skin pigmentation who are less able to generate vitamin D in the epidermis via photolytic modulation of 7-dehydrocholesterol also have a greater risk of vitamin D insufficiency, particularly those living in geographically less sunny areas (22
The re-classification of optimal vitamin D status raises a key clinical question, namely what are the health consequences of vitamin insufficiency? In common with the rickets/osteomalacia that is characteristic of vitamin D deficiency, sub-optimal levels of 25OHD may also compromise skeletal homeostasis, with osteopenia or osteoporosis being the principal problem (21
). However, the well-documented non-classical effects of active 1,25(OH)2
D suggest that vitamin D insufficiency will have a much broader clinical impact. In particular, expression of CYP27b1 and VDR by macrophages and dendritic cells has highlighted a potential role for intracrine synthesis of 1,25(OH)2
D as a modulator of both innate and adaptive immunity (1
). We have shown recently that intracrine conversion of 25OHD to 1,25(OH)2
D by macrophages is potently stimulated by the TLR2/1 ligand 19 kDa, and this in turn induces expression of the antimicrobial peptide hCAP (5
). Further studies have confirmed that this is a key requirement for host defense against pathogens such as Mycobacterium tuberculosis
). Perhaps most significantly we also demonstrated that the efficacy of this innate immune mechanism is dependent on the availability of substrate for macrophage CYP27b1, namely the pro-hormone 25OHD. Specifically, ex vivo culture of monocytes using serum from 25OHD-sufficient (Caucasian) subjects supported levels of TLR-induced hCAP production that were much higher than observed with serum from 25OHD-deficient (African-American) populations (5
). In data presented here we have expanded these observations to show that the ability of human macrophages to induce antimicrobial hCAP in response to TLR-activation is directly proportional to serum vitamin D status, with this being enhanced in vitamin D-insufficient patients treated with supplementary vitamin D.
D is known to be a direct regulator of hCAP gene expression (25
), to our knowledge data in represent the first assessment of the impact of vitamin D metabolites on circulating levels of the antimicrobial peptide. The lack of association between circulating 25OHD, 1,25(OH)2
D and hCAP levels emphasizes the importance of a local, intracrine mechanism of action. However, it should also be recognized that the patients studied in this trial were relatively vitamin D-replete, with 70% of those used in the ex vivo studies being classified as vitamin D sufficient and no patients technically vitamin D ‘deficient’. Thus, it is possible that circulating levels of hCAP will be more dependent on vitamin D status at very low levels of serum 25OHD. It was also interesting to note that serum levels of hCAP varied considerably independent of either vitamin D metabolite, suggesting alternative determinants of its background expression. The effect of vitamin D on serum hCAP may also be tissue specific: no association was found between serum hCAP protein and its mRNA from PBMCs (see ), suggesting that other cell types contribute to the peripheral reservoir of this antimicrobial peptide (12
In previous studies we showed that activation of TLR2 stimulated expression of CYP27b1 and VDR (5
). Here we show that similar effects are also evident with the TLR4 ligand LPS indicating that induction of localized production of 1,25(OH)2
D may occur in response to gram+VE
bacteria (). ELISA data suggest that, under these conditions, relatively low levels of 1,25(OH)2
D are generated by human macrophages. Following the addition of 100 nM 25OHD, less than 50 pM 1,25(OH)2
D was synthesized (see ). Due to the detection limits of the assay kit employed, we were unable to quantify any significant changes in 1,25(OH)2
D production using 5 nM 25OHD as substrate for CYP27b1, even when the enzyme was induced by TLR-activation. This underlines the relatively low levels of 1,25(OH)2
D produced by this intracrine system but is also consistent with previous reports in which we demonstrated the relative efficiency of intracrine versus endocrine delivery of 1,25(OH)2
D in regulating immune cell function (4
). It was also interesting to note that 100 nM 25OHD facilitated the synthesis of 1,25(OH)2
D without any need for TLR-mediate stimulus. This suggests that there is a low baseline expression of CYP27b1 in macrophages which is capable of synthesizing 1,25(OH)2
D in the presence of high levels of substrate 25OHD, but which requires transcriptional activation via TLRs to effectively metabolize low levels of 25OHD. This proposal was endorsed by hCAP expression studies in which showed that in the absence of TLR ligands, 100 nM 25OHD was able to significantly induce expression of mRNA for the antimicrobial peptide. By contrast, 5 nM 25OHD only increased hCAP expression in conjunction with 19 kDa or LPS.
A key observation from this study is that in the absence of any exogenous 25OHD, treatment of macrophages with ligands to TLR2/1 or TLR4 suppressed expression of hCAP (see ). This is consistent with similar reports of hCAP inhibition in macrophages infected with M. tb
), and other pathogenic agents (28
). As a consequence of these studies it has been proposed that suppression of antibacterial peptides such as hCAP provides a mechanism by which pathogens escape innate immune surveillance and thereby survive in the host (28
). To the best of our knowledge, the data we present here are the first to show specific suppression of hCAP by purified TLR ligands. However, it is important to stress that the 10% culture autologous serum culture conditions employed in this study are effectively equivalent to vitamin D “deficiency” in that the levels of 25OHD to which cells are exposed ex vivo are less than 10 nM (see ). We postulate that in vivo regulation of hCAP by TLR ligands will be different to that observed in vitro because of higher concentrations of serum and concomitantly higher levels of 25OHD. Specifically, in individuals with higher levels of serum 25OHD it is likely that TLR ligands will enhance hCAP expression, whilst those with low serum 25OHD will have less induction or possibly even suppression of hCAP expression. It is noteworthy that addition of 25OHD at concentrations as low as 5 nM was able to rescue TLR-mediated suppression of hCAP in the autologous serum experiments. In cultures with 10% supplementary serum, this represents an effective concentration of 50 nM in undiluted (100%) serum, enough to change all the vitamin D-insufficient sera into vitamin D-sufficient sera. Similar effects were also observed in vitamin D-insufficient patients whose serum 25OHD levels were elevated in vivo from 25 ng/ml (62 nM) to 40 ng/ml (100 nM) (). Collectively, these data emphasize the fundamental importance of 25OHD as a determinant of hCAP expression following TLR-mediate immune challenge. However, it is also important to recognize that supplementation of any culture medium with 10% serum effectively represents an environment which is 25OHD ‘insufficient’, and this may have implications for in vitro experimentation per se.
Vitamin D-mediated induction of hCAP appears to have been a relatively recent evolutionary development, as the gene promoter vitamin D response element (VDRE) required for liganded VDR stimulation of this protein is only present in higher primates (26
). Consistent with this, comparison of macrophage cell lines cultured under identical conditions of vitamin D insufficiency (i.e. 10% FCS-supplemented medium) showed that 25OHD induced hCAP in human cells but had no effect in mouse macrophages (see Appendix 2). A potential explanation for this is provided by the fact that under these culture conditions, TLR ligands suppressed hCAP in human THP-1 cells but conversely stimulated
this protein in mouse J774A macrophages. Both cell types showed induction of IL-1 expression following treatment with 19 kDa or LPS, indicating similar levels of sensitivity to the TLR ligands. Moreover, in autologous 10% serum cultured human macrophages and THP-1, cells TLR activation stimulated the related antimicrobial defensin DEFB4. Although the gene for DEFB4 also has a promoter VDRE (26
), it did not show the same 25OHD-mediated induction observed for hCAP ( and Figure 8A). Thus, we can speculate that the sensitive regulation of hCAP by vitamin D may have arisen in as a mechanism that countered microbial subversion of innate immune responses. The efficacy of such a mechanism is illustrated by fact that sub-human primates have greatly elevated circulating levels of 25OHD when compared to humans (30
) and thus monocyte synthesis of 1,25(OH)2
D in these animals is likely to be an effective way of stimulating hCAP following infection. By contrast, the migration of Homo sapiens
out of Africa and into Europe was accompanied by a significant fall in serum 25OHD, and this may have substantially compromised hCAP innate immunity.
Several other factors factors are also likely to influence the regulation of monocyte hCAP expression following TLR activation including the localized concentration of TLR ligands themselves, and potential modulation by other factors such as cytokines. Moreover, the cohort of donors used in the study were relatively senior (mean age 63.3 ± 14.9), and this may be a significant factor in the defining the interrelationship between TLR-activation, hCAP and vitamin D. In similar studies carried out using monocytes from a younger donor cohort (mean age 33.2 ± 8.2), we observed the same correlation between serum 25OHD concentrations and hCAP mRNA following activation of TLR2/1 (see Appendix 3). However, in this instance suppression of hCAP by 19 kDa was less pronounced despite the fact that donor serum 25OHD levels in this younger cohort (21.1 ± 13.0) were lower than those reported in the current study (35.8 ± 10.2, p<0.001). Thus, it is possible that the ability of monocytes to promote hCAP antibacterial responses via 25OHD metabolism decreases with age, further underlining the need for maintenance of vitamin D status in elderly individuals. This is emphasized by analysis of the serum levels of 25OHD required to support optimal innate immunity in the ex vivo culture model. In the cohort of older subjects (see ) a 2-fold increase in monocyte hCAP levels would require serum 25OHD levels of approximately 200 nM (80 ng/ml). By contrast, monocytes from the younger cohort of donors in Appendix 3 would require 25OHD levels of only 100 nM (40 ng/ml). Irrespective of the potential effects of donor age, what is clear is that vitamin D functions as fundamental rheostatic regulator of macrophage hCAP levels following TLR-activation, with the magnitude of this effect being entirely dependent on the 25OHD status of the individual.
In data presented here we have confirmed the close association between vitamin D (25OHD) status and innate immunity. Consistent with our previous studies (5
), we have shown that ability of physiological levels of serum 25OHD to induce hCAP is dependent on enhanced expression of macrophage CYP27b1, although at relatively high concentrations 25OHD can stimulate hCAP without TLR-mediated activation of macrophages. The upregulation of hCAP by 25OHD appears to counteract TLR-mediated suppression of the antimicrobial peptide, suggesting that a key function of vitamin D in this setting is to prevent pathogen-induced evasion of innate immunity. We have also shown for the first time that vitamin D supplementation in vivo is capable of promoting improved innate immune responses. As such, these data provide further support for prospective clinical trials to assess the effects of vitamin D supplementation in preventing infectious diseases such as tuberculosis.