Published ahead of print on April 26, 2007, doi:10.1164/rccm.200701-007OC
© 2007 American Thoracic Society doi: 10.1164/rccm.200701-007OC
A Single Dose of Vitamin D Enhances Immunity to Mycobacteria1 Centre for Health Sciences, Queen Mary's School of Medicine and Dentistry, Barts and The London, London, United Kingdom; Newham Chest Clinic, London, United Kingdom; 3 Wellcome Trust Center for Research in Clinical Tropical Medicine, Division of Medicine, Wright Fleming Institute, Imperial College London, London, United Kingdom; 4 Institute of Infectious Diseases and Molecular Medicine and Department of Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa; 5 Tuberculosis Clinic and 6 Department of Clinical Biochemistry, North West London Hospitals NHS Trust, Northwick Park Hospital, Harrow, United Kingdom; and 7 Vitamin D Research Group, University School of Medicine, Manchester Royal Infirmary, Manchester, United Kingdom Correspondence and requests for reprints should be addressed to Adrian R. Martineau, M.R.C.P., Centre for Health Sciences, Queen Mary's School of Medicine and Dentistry, Barts and The London, London E1 2AT, UK. E-mail: a.martineau{at}qmul.ac.uk
Rationale: Vitamin D was used to treat tuberculosis (TB) in the preantibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. Objectives: To determine the effect of vitamin D supplementation on antimycobacterial immunity and vitamin D status. Methods: A double-blind randomized controlled trial was conducted in 192 healthy adult TB contacts in London, United Kingdom. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks.
Measurements and Main Results: The primary outcome measure was assessed with a functional whole blood assay (BCG-lux assay), which measures the ability of whole blood to restrict luminescence, and thus growth, of recombinant reporter mycobacteria in vitro; the readout is expressed as a luminescence ratio (luminescence postinfection/baseline luminescence). IFN-
Conclusions: A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants' whole blood to restrict BCG-lux luminescence in vitro without affecting antigen-stimulated IFN-
Key Words: human • vitamin D • innate immunity • mycobacteria
Tuberculosis (TB) is a global emergency: in 2004, there were an estimated 8.9 million new cases and 1.7 million deaths due to the disease (1). One-third of the global population has latent TB infection (2), providing a reservoir for future reactivation disease well into the present century. An understanding of the factors causing reactivation is therefore of considerable public health significance. Clinical studies suggest that vitamin D enhances antimycobacterial immunity, and that deficiency is associated with susceptibility to active disease. High doses of vitamin D were widely used to treat active TB in the preantibiotic era (3); more recently, case-control studies have demonstrated that a vegetarian diet (low in vitamin D) is an independent risk factor for active TB in South Asians (4) and that patients with TB who are of Gujarati Hindu ethnic origin have significantly higher rates of vitamin D deficiency than ethnically matched tuberculin-positive TB contacts (5).
Vitamin D is synthesized in the skin during exposure to ultraviolet light and is also available in the diet, principally from oily fish. It is readily metabolized in the liver to form 25-hydroxyvitamin D (25[OH]D), the accepted measure of vitamin D status (6). 25(OH)D is then further metabolized by the 1-
1 Randomized controlled trials evaluating the effect of vitamin D supplementation on antimycobacterial host response have yet to be performed. Placebo-controlled prospective studies in tuberculin-positive individuals with the development of active TB as primary outcome measure would require very large sample sizes to detect clinically significant effects. We therefore adopted an alternative approach, conducting a double-blind randomized controlled trial of vitamin D supplementation in a large cohort of TB contacts in London, United Kingdom, using a surrogate primary outcome measure: the BCG-lux assay (15). In this assay, whole blood is cultured with a recombinant Mycobacterium bovis bacille Calmette-Guérin (BCG) expressing luciferase, an enzyme that catalyzes the conversion of aldehyde substrate to produce light detectable in a luminometer. Because this reaction is ATP dependent, light production relates to bacillary metabolic activity and colony forming units (cfu) (16). The ability of whole blood to suppress BCG-lux bioluminescence is determined at both 24 and 96 hours postinfection, to assay innate and acquired components of host response, respectively. This model has yielded intuitive correlates of protection in several previous studies (15, 17, 18).
We combined BCG-lux analysis with a whole blood interferon (IFN)- Some of the results of this study have been previously reported in the form of an abstract (21).
Participants Study participants were recruited from TB contact clinics at Newham and Northwick Park Hospitals, London, United Kingdom. All people over 17 years of age who had been exposed to a patient with active TB were assessed. Individuals were excluded if they had symptoms, clinical signs, or radiographic evidence of active TB; if they had HIV infection, renal failure, sarcoidosis, or hyperparathyroidism; if they were taking corticosteroids, thiazide diuretics, or supplementary vitamin D (either alone or as part of a multivitamin preparation); or if they were breastfeeding or pregnant. Sociodemographic and dietary details were recorded on case report forms. Participants self-classified ethnicity into one of the following five categories (22): black African, South Asian, white, mixed, or other. A blood sample was drawn for baseline assays, a tuberculin skin test was performed according to national guidelines (23), and a urine sample was collected from women of childbearing age for pregnancy testing. TB contacts were reviewed at 1 week, and the grade of the tuberculin reaction was recorded. The study nurse then allocated a study ID number to eligible participants, and administered study medication from a container labeled with that number. These containers had previously been filled with either a single oral dose of 2.5 mg ergocalciferol or lactose placebo of identical appearance, according to a computer-generated sequence of random numbers in blocks of 10. Participants were reviewed at 6 weeks postrandomization, when a second blood sample was drawn for follow-up assays. The study research nurse and all participants and individuals performing laboratory assays were blinded to allocation. The study was approved by the research ethics committees of North East London and Harrow (REC refs. P/02/146 and EC 2759, respectively), and written, informed consent to participate was obtained from all participants.
Whole Blood Assays
The IFNGRA used in this study has also been described elsewhere (19). Triplicate samples of venous blood diluted 1:10 with RPMI 1640 (Sigma) were cultured with 2.5 µg/ml recombinant ESAT-6 (24), 5 µg/ml CFP-10 (Lionex, Braunschweig, Germany), or no stimulus at 37°C in 5% CO2. Supernatants were aspirated at 96 hours for determination of IFN-
Determination of 25(OH)D Concentration
Statistical Analysis
We assessed 364 TB contacts for eligibility between December 16, 2002, and January 31, 2005; 39 were ineligible, and 133 declined randomization (Figure 1). The group of 133 TB contacts who declined randomization had similar sex ratio, age range, and ethnic composition to the 192 participants who were randomized to receive vitamin D or placebo. All received the intended treatment; 43 participants (22 in the placebo group and 21 in the intervention group) were lost to follow-up, and a further 18 (10 in the placebo group and 8 in the intervention group) were excluded from the analysis in the absence of a valid BCG-lux assay result at either baseline or follow-up. Analysis of primary outcome was conducted on the remaining 131 participants, of whom 64 received placebo and 67 received vitamin D.
Baseline characteristics of these 131 participants are compared in Table 1. Control and intervention groups did not differ significantly with respect to age, sex ratio, ethnic composition, BCG immunization rate, tuberculin reactivity, nature of TB exposure, vitamin D status, BCG-lux assay luminescence ratios, or ESAT-6–stimulated IFN-  concentration. The median CFP-10–stimulated IFN- concentration at baseline was higher in the placebo group than in the intervention group (median, 40 vs. 0 pg/ml; p = 0.03).
At follow-up, the mean 24-hour luminescence ratio was 20.4% lower for individuals allocated to vitamin D compared with those allocated to placebo (0.57 vs. 0.71 respectively; 95% confidence interval [CI] for difference, 0.01 to 0.25; p = 0.03; Figure 2). Mean 96-hour luminescence ratio at follow-up was not significantly different for individuals allocated to vitamin D compared with those allocated to placebo (1.34 vs. 1.30, respectively; 95% CI for difference, –0.27 to 0.25; p = 0.94). The median antigen-stimulated whole blood IFN- secretion at follow-up was not significantly different for individuals allocated to vitamin D compared with those allocated to placebo (for ESAT-6: 88 vs. 54 pg/ml, respectively; p = 0.13; for CFP-10: 45 vs. 26 pg/ml; p = 0.23).
Baseline serum 25(OH)D concentrations were determined for all 192 randomized participants plus an additional 10 TB contacts who gave a baseline blood sample but subsequently declined randomization (n = 9) or were found to be ineligible for randomization (n = 1) (Figure 1). Of these 202 TB contacts, 84 (42%) were vitamin D deficient (serum 25[OH]D < 20 nmol/L) (26) and 189 (94%) were vitamin D insufficient (serum 25[OH]D < 75 nmol/L) (27) at baseline. Vitamin D deficiency was associated with black African and South Asian ethnicity, lack of fish in the diet, and enrollment from November to April on univariate analysis (Table 2). Multivariate analysis revealed each of these factors to be independent correlates of risk of vitamin D deficiency (for black African ethnic origin: exp[B] [estimated odds ratio] = 0.16; 95% CI, 0.04 to 0.67; p = 0.01; for South Asian ethnic origin: exp[B] = 0.20; 95% CI, 0.06 to 0.73; p = 0.02; for absence of fish in the diet: exp[B] = 0.42; 95% CI, 0.22 to 0.84; p = 0.01; for enrollment from November to April: exp[B] = 0.45; 95% CI, 0.24 to 0.84; p = 0.01).
Among randomized participants, administration of a single oral dose of 2.5 mg vitamin D induced a 91% increase in mean serum 25(OH)D (mean 25[OH]D pre- vs. postsupplementation, 35.2 vs. 67.4 nmol/L; 95% CI for difference, 26.3 to 38.7 nmol/l; p < 0.0001; Figure 3A) and corrected deficiency in all 23 participants in the intervention arm with baseline serum 25(OH)D < 20 nmol/L. No study participant experienced hypercalcemia or any other adverse event.
Concentrations of 25(OH)D were also determined in BCG-lux assay supernatants performed at baseline and follow-up in a randomly selected subset of 32 participants allocated to the intervention arm of the study. Vitamin D supplementation induced a 95% increase in mean 24-hour supernatant 25(OH)D concentration (pre- vs. postsupplementation, 7.3 vs. 14.2 nmol/L; 95% CI for difference, 4.9 to 8.8 nmol/l; p < 0.0001; Figure 3B) and a 123% increase in mean 96-hour supernatant 25(OH)D concentration (pre- vs. postsupplementation, 6.5 vs. 14.5 nmol/L; 95% CI for difference, 5.4 to 11.8 nmol/L; p < 0.0001; Figure 3C).
We have shown that a single oral dose of 2.5 mg vitamin D enhanced the ability of TB contacts' whole blood to restrict BCG-lux luminescence in vitro without affecting antigen-stimulated IFN- responses. Profound vitamin D deficiency was found in more than 40% of this cohort, and was independently associated with absence of fish in the diet, South Asian and black African ethnic origin, and sampling in winter or spring. Supplementation induced a 91% increase in mean serum 25(OH)D concentration and corrected deficiency in all individuals with baseline 25(OH)D < 20 nmol/L for at least 6 weeks without inducing hypercalcemia in any participant.
Interestingly, vitamin D supplementation suppressed BCG-lux bioluminescence at the 24-hour time point, but did not affect BCG-lux bioluminescence or whole blood antigen-stimulated IFN- A large proportion of TB contacts assessed for eligibility declined randomization (Figure 1); however, the fact that this group did not differ significantly from participants in terms of median age, sex ratio, or ethnic composition suggests that the external validity of this study is unlikely to have been severely compromised by its low recruitment rate. The significant numbers of participants who were lost to follow-up or excluded from the analysis also represent a weakness of the study, primarily by increasing the potential for type II error. The very high rates of profound vitamin D deficiency that we report in this group of otherwise healthy adults are higher than those documented in the institutionalized elderly (28) and are a cause for grave public health concern, given emerging evidence implicating vitamin D deficiency in the pathogenesis of a wide range of chronic diseases (29). The observation that black African and South Asian ethnicity are risk factors for vitamin D deficiency independently of diet may be explained by the effect of increased skin pigmentation in reducing cutaneous vitamin D synthesis (30); however, this possibility cannot be evaluated in the absence of data on participants' skin pigmentation. Our finding that a single oral dose of 2.5 mg vitamin D corrects profound vitamin D deficiency for at least 6 weeks without causing hypercalcemia underlines the potential use of this formulation as a safe, effective, and cheap ($1.20) public health intervention. In conclusion, we have demonstrated that a single oral dose of vitamin D enhances TB contacts' immunity to mycobacteria. Prospective trials to determine the effect of vitamin D supplementation on TB incidence rates should be performed in deficient populations with high rates of latent TB infection.
The authors thank Dr. Barbara Boucher for helpful discussions and review of the manuscript.
Supported by the Wellcome Trust (Refs. 064261, 066321, 072070, and 077273), the Department of Environmental Health, London Borough of Newham, Newham University Hospital NHS Trust Research Fund, and Northwick Park Hospital Tropical Research Fund. A.R.M. is supported by the British Lung Foundation.
* These authors contributed equally to this work. Originally Published in Press as DOI: 10.1164/rccm.200701-007OC on April 26, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form January 2, 2007; accepted in final form April 26, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
-hydroxylase enzyme Cyp27B1 to its biologically active metabolite, the steroid hormone 1-
2 tests for univariate analysis and binary logistic regression analysis for multivariate analysis. Effect of allocation on whole blood assay outcomes was evaluated using analysis of covariance. Mean concentrations of 25(OH)D pre- versus postsupplementation were compared with paired t tests. We analyzed data using SPSS (version 12.0.1, 2003; SPSS, Inc., Chicago, IL) and GraphPad Prism (version 4.03, 2005; GraphPad, Inc., San Diego, CA) software packages. 
