Martin Hewison

Vitamin D is required for IFN-gamma-mediated antimicrobial activity of human macrophages.

Vitamin D is required for both innate and adaptive immunity to tuberculosis. The Sunny Side of Antimicrobial Response Nearly one-third of the world’s population is thought to be infected with Mycobacterium tuberculosis, which causes a potentially fatal lung disease in untreated patients. Although most M. tuberculosis infections can be treated by antibiotic therapy, the burden of infection is especially high in immunodeficient (HIV+) patients and individuals from developing nations. Moreover, drug-resistant M. tuberculosis is increasingly prevalent. Yet, most humans with M. tuberculosis infection are asymptomatic, perhaps because of successful immunological control. Understanding the mechanisms behind immune control of M. tuberculosis infection may pinpoint potential new therapeutic avenues. Now, Fabri et al. examine the antimicrobial function of M. tuberculosis–infected human macrophages. The authors found that cells from the adaptive immune system—T cells—governed bacterial control by releasing the cytokine interferon-γ (IFN-γ), which then activated infected macrophages, inciting the cells to attack the invading M. tuberculosis. This activation depended on the presence of vitamin D, a fat-soluble prohormone thought to be beneficial for everything from bone health to cancer therapy. Indeed, this antimicrobial response was not seen with macrophages maintained in human sera from subjects with insufficient vitamin D levels. Vitamin D3 has been used historically to treat M. tuberculosis infection, but its effects have not been thoroughly tested in clinical trials. This study suggests that increasing serum levels of vitamin D, whether through supplementation or increased sun exposure, should improve the human immune response to M. tuberculosis and supports further testing of vitamin D in the clinic. Control of tuberculosis worldwide depends on our understanding of human immune mechanisms, which combat the infection. Acquired T cell responses are critical for host defense against microbial pathogens, yet the mechanisms by which they act in humans remain unclear. We report that T cells, by the release of interferon-γ (IFN-γ), induce autophagy, phagosomal maturation, the production of antimicrobial peptides such as cathelicidin, and antimicrobial activity against Mycobacterium tuberculosis in human macrophages via a vitamin D–dependent pathway. IFN-γ induced the antimicrobial pathway in human macrophages cultured in vitamin D–sufficient sera, but not in sera from African-Americans that have lower amounts of vitamin D and who are more susceptible to tuberculosis. In vitro supplementation of vitamin D–deficient serum with 25-hydroxyvitamin D3 restored IFN-γ–induced antimicrobial peptide expression, autophagy, phagosome-lysosome fusion, and antimicrobial activity. These results suggest a mechanism in which vitamin D is required for acquired immunity to overcome the ability of intracellular pathogens to evade macrophage-mediated antimicrobial responses. The present findings underscore the importance of adequate amounts of vitamin D in all human populations for sustaining both innate and acquired immunity against infection.

Vitamin D-Directed Rheostatic Regulation of Monocyte Antibacterial Responses

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D3 (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D2 twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D “insufficient” conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.

Vitamin D Deficiency in Mice Impairs Colonic Antibacterial Activity and Predisposes to Colitis

Vitamin D insufficiency is a global health issue. Although classically associated with rickets, low vitamin D levels have also been linked to aberrant immune function and associated health problems such as inflammatory bowel disease (IBD). To test the hypothesis that impaired vitamin D status predisposes to IBD, 8-wk-old C57BL/6 mice were raised from weaning on vitamin D-deficient or vitamin D-sufficient diets and then treated with dextran sodium sulphate (DSS) to induce colitis. Vitamin D-deficient mice showed decreased serum levels of precursor 25-hydroxyvitamin D(3) (2.5 +/- 0.1 vs. 24.4 +/- 1.8 ng/ml) and active 1,25-dihydroxyvitamin D(3) (28.8 +/- 3.1 vs. 45.6 +/- 4.2 pg/ml), greater DSS-induced weight loss (9 vs. 5%), increased colitis (4.71 +/- 0.85 vs. 1.57 +/- 0.18), and splenomegaly relative to mice on vitamin D-sufficient chow. DNA array analysis of colon tissue (n = 4 mice) identified 27 genes consistently (P < 0.05) up-regulated or down-regulated more than 2-fold in vitamin D-deficient vs. vitamin D-sufficient mice, in the absence of DSS-induced colitis. This included angiogenin-4, an antimicrobial protein involved in host containment of enteric bacteria. Immunohistochemistry confirmed that colonic angiogenin-4 protein was significantly decreased in vitamin D-deficient mice even in the absence of colitis. Moreover, the same animals showed elevated levels (50-fold) of bacteria in colonic tissue. These data show for the first time that simple vitamin D deficiency predisposes mice to colitis via dysregulated colonic antimicrobial activity and impaired homeostasis of enteric bacteria. This may be a pivotal mechanism linking vitamin D status with IBD in humans.

Vitamin D-binding protein directs monocyte responses to 25-hydroxy- and 1,25-dihydroxyvitamin D.

BACKGROUNDnSerum 25-hydroxyvitamin D (25OHD) is a key factor in determining monocyte induction of the antimicrobial protein cathelicidin, which requires intracrine conversion of 25OHD to 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. Both vitamin D metabolites circulate bound to vitamin D-binding protein (DBP), but the effect of this on induction of monocyte cathelicidin remains unclear.nnnMETHODSnHuman monocytes were cultured in medium containing 1) serum from DBP knockout (DBP(-/-)) or DBP(+/-) mice, 2) serum-free defined supplement reconstituted with DBP or albumin (control), and 3) human serum with different DBP [group-specific component [Gc]] genotypes with varying affinities for vitamin D metabolites. In each case, response to added 1,25(OH)(2)D(3) or 25OHD(3) was determined by measuring expression of mRNA for cathelicidin and 24-hydroxylase. Monocyte internalization of DBP was assessed by fluorescent tagging followed by microscopic and flow cytometric analysis of tagged DBP.nnnRESULTSnMonocytes cultured in DBP(-/-) serum showed more potent induction of cathelicidin by 25OHD(3) or 1,25(OH)(2)D(3) when compared with DBP(+/-) serum. Likewise, DBP added to serum-free medium attenuated 25OHD(3)/1,25(OH)(2)D(3) responses. Fluorescently tagged DBP showed low-level uptake by monocytes, but this did not appear to involve a megalin-mediated mechanism. Human serum containing low-affinity Gc2-1S or Gc2-2, respectively, supported 2.75-fold (P = 0.003) and 2.43-fold (P = 0.016) higher induction of cathelicidin by 25OHD relative to cells cultured with high affinity Gc1F-1F.nnnCONCLUSIONnThese data indicate that DBP plays a pivotal role in regulating the bioavailablity of 25OHD to monocytes. Vitamin D-dependent antimicrobial responses are therefore likely to be strongly influenced by DBP polymorphisms.

T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism.

We investigated the mechanisms by which T-cell cytokines are able to influence the Toll-like receptor (TLR)-induced, vitamin D-dependent antimicrobial pathway in human monocytes. T-cell cytokines differentially influenced TLR2/1-induced expression of the antimicrobial peptides cathelicidin and DEFB4, being up-regulated by IFN-γ, down-regulated by IL-4, and unaffected by IL-17. The Th1 cytokine IFN-γ up-regulated TLR2/1 induction of 25-hydroxyvitamin D-1α-hydroxylase (i.e., CYP27B1), leading to enhanced bioconversion of 25-hydroxyvitamin D3 (25D3) to its active metabolite 1,25D3. In contrast, the Th2 cytokine IL-4, by itself and in combination with the TLR2/1 ligand, induced catabolism of 25D3 to the inactive metabolite 24,25D3, and was dependent on expression of vitamin D-24-hydroxylase (i.e., CYP24A1). Therefore, the ability of T-cell cytokines to differentially control monocyte vitamin D metabolism represents a mechanism by which cell-mediated immune responses can regulate innate immune mechanisms to defend against microbial pathogens.

Vitamin D Induces Innate Antibacterial Responses in Human Trophoblasts via an Intracrine Pathway

Abstract The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)2D (1–100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)2D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% ± 10.9%) or 1,25(OH)2D (45.4% ± 9.2%), both P < 0.001. Treatment with 25OHD (1–100 nM) or 1,25(OH)2D (0.1–10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%–26.9% and 22.3%–40.2% protection, respectively). These observations indicate that 1,25(OH)2D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism for activation of innate immune responses in the placenta.

Vitamin D and the Regulation of Placental Inflammation

The vitamin D-activating enzyme 1α-hydroxylase (CYP27B1) and vitamin D receptor (VDR) support anti-inflammatory responses to vitamin D in many tissues. Given the high basal expression of CYP27B1 and VDR in trophoblastic cells from the placenta, we hypothesized that anti-inflammatory effects of vitamin D may be particularly important in this organ. Pregnant wild type (WT) mice i.p. injected with LPS showed elevated expression of mouse Cyp27b1 (4-fold) and VDR (6-fold). Similar results were also obtained after ex vivo treatment of WT placentas with LPS. To assess the functional impact of this, we carried out ex vivo studies using placentas −/− for fetal (trophoblastic) Cyp27b1 or VDR. Vehicle-treated −/− placentas showed increased expression of IFN-γ and decreased expression of IL-10 relative to +/+ placentas. LPS-treated −/− placentas showed increased expression of TLR2, IFN-γ, and IL-6. Array analyses identified other inflammatory factors that are dysregulated in Cyp27b1−/− versus Cyp27b1+/+ placentas after LPS challenge. Data highlighted enhanced expression of IL-4, IL-15, and IL-18, as well as several chemokines and their receptors, in Cyp27b1−/− placentas. Similar results for IL-6 expression were observed with placentas −/− for trophoblastic VDR. Finally, ex vivo treatment of WT placentas with the substrate for Cyp27b1, 25-hydroxyvitamin D3, suppressed LPS-induced expression of IL-6 and the chemokine Ccl11. These data indicate that fetal (trophoblastic) vitamin D plays a pivotal role in controlling placental inflammation. In humans, this may be a key factor in placental responses to infection and associated adverse outcomes of pregnancy.

Divergence of macrophage phagocytic and antimicrobial programs in leprosy

Effective innate immunity against many microbial pathogens requires macrophage programs that upregulate phagocytosis and direct antimicrobial pathways, two functions generally assumed to be coordinately regulated. We investigated the regulation of these key functions in human blood-derived macrophages. Interleukin-10 (IL-10) induced the phagocytic pathway, including the C-type lectin CD209 and scavenger receptors, resulting in phagocytosis of mycobacteria and oxidized low-density lipoprotein. IL-15 induced the vitamin D-dependent antimicrobial pathway and CD209, yet the cells were less phagocytic. The differential regulation of macrophage functional programs was confirmed by analysis of leprosy lesions: the macrophage phagocytosis pathway was prominent in the clinically progressive, multibacillary form of the disease, whereas the vitamin D-dependent antimicrobial pathway predominated in the self-limited form and in patients undergoing reversal reactions from the multibacillary to the self-limited form. These data indicate that macrophage programs for phagocytosis and antimicrobial responses are distinct and differentially regulated in innate immunity to bacterial infections.

Vitamin D Binding Protein and Monocyte Response to 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D: Analysis by Mathematical Modeling

Vitamin D binding protein (DBP) plays a key role in the bioavailability of active 1,25-dihydroxyvitamin D (1,25(OH)2D) and its precursor 25-hydroxyvitamin D (25OHD), but accurate analysis of DBP-bound and free 25OHD and 1,25(OH)2D is difficult. To address this, two new mathematical models were developed to estimate: 1) serum levels of free 25OHD/1,25(OH)2D based on DBP concentration and genotype; 2) the impact of DBP on the biological activity of 25OHD/1,25(OH)2D in vivo. The initial extracellular steady state (eSS) model predicted that 50 nM 25OHD and 100 pM 1,25(OH)2D), <0.1% 25OHD and <1.5% 1,25(OH)2D are ‘free’ in vivo. However, for any given concentration of total 25OHD, levels of free 25OHD are higher for low affinity versus high affinity forms of DBP. The eSS model was then combined with an intracellular (iSS) model that incorporated conversion of 25OHD to 1,25(OH)2D via the enzyme CYP27B1, as well as binding of 1,25(OH)2D to the vitamin D receptor (VDR). The iSS model was optimized to 25OHD/1,25(OH)2D-mediated in vitro dose-responsive induction of the vitamin D target gene cathelicidin (CAMP) in human monocytes. The iSS model was then used to predict vitamin D activity in vivo (100% serum). The predicted induction of CAMP in vivo was minimal at basal settings but increased with enhanced expression of VDR (5-fold) and CYP27B1 (10-fold). Consistent with the eSS model, the iSS model predicted stronger responses to 25OHD for low affinity forms of DBP. Finally, the iSS model was used to compare the efficiency of endogenously synthesized versus exogenously added 1,25(OH)2D. Data strongly support the endogenous model as the most viable mode for CAMP induction by vitamin D in vivo. These novel mathematical models underline the importance of DBP as a determinant of vitamin D ‘status’ in vivo, with future implications for clinical studies of vitamin D status and supplementation.

Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling

Transcriptional regulation by hormonal 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] involves occupancy of vitamin D response elements (VDREs) by the VDRE binding protein (VDRE‐BP) or 1,25(OH)2D3‐bound vitamin D receptor (VDR). This relationship is disrupted by elevated VDRE‐BP, causing a form of hereditary vitamin D‐resistant rickets (HVDRR). DNA array analysis showed that of 114 genes regulated by 1,25(OH)2D3 in control cells, almost all (113) were rendered insensitive to the hormone in VDRE‐BP‐overexpressing HVDRR cells. Among these was the gene for DNA‐damage‐inducible transcript 4 (DDIT4), an inhibitor of mammalian target of rapamycin (mTOR) signaling. Chromatin immunoprecipitation PCR using 1,25(OH)2D3‐treated osteoblasts confirmed that VDR and VDRE‐BP compete for binding to the DDIT4 gene promoter. Expression of DDIT4 mRNA in these cells was induced (1.6–6 fold) by 1,25(OH)2D3 (10–100 nM), and Western blot and flow cytometry analysis showed that this response involved suppression of phosphorylated S6K1T389 (a downstream target of mTOR) similar to rapamycin treatment. siRNA knockdown of DDIT4 completely abrogated antiproliferative responses to 1,25(OH)2D3, whereas overexpression of VDRE‐BP exerted a dominant‐negative effect on transcription of 1,25(OH)2D3‐target genes. DDIT4, an inhibitor of mTOR signaling, is a direct target for 1,25(OH)2D3 and VDRE‐BP, and functions to suppress cell proliferation in response to vitamin D.—Lisse, T. S., Liu, T., Irmler, M., Beckers, J., Chen, H., Adams, J. S., Hewison, M. Gene targeting by the vitamin D response element binding protein reveals a role for vitamin D in osteoblast mTOR signaling. FASEB J. 25, 937–947 (2011). www.fasebj.org